HEMATOPOIETIC STEM CELL (HSC) TRANSPLANTATION (HSCT / HCT)

Table of contents :

Sources of HSCs :

• bone marrow transplantation (BMT) (BMSC)

History : in 1939 Osgood unsuccessfully infused a few milliliters of marrow into patients with aplastic anemia. In 1949, Jacobson discovered that shielding the spleen protected mice from lethal irradiation. Lorenz performed allogenic BMT in animals protected by total irradiation. His findings, and research by Barnes and colleagues, demonstrated that a cellular rather than a humoral factor played a role in graft survival. Vanbekkum, DeVries, and other scientists performed important experiments to study immunological reactions in transplantation recipients. They demonstrated that successfully grafted allogeneic marrow cells could later produce an immunological reaction against host tissues; this phenomenon is now known as GVHD. Experimental canine studies during the 1960s resulted in important discoveries that were applicable to human transplantation. Total body irradiation did not seem to prevent GVHD; mismatched canine leukocyte antigen always resulted in graft rejection or GVHD. Methotrexate was also found to be an immunosuppressive agent that could prevent or ameliorate the graft versus host reaction. Although initial attempts at allogeneic BMT in the 1950s and 1960s were unsuccessful, renewed attempts occurred after additional knowledge of the HLA system was obtained. Getty performed a successful allogeneic marrow graft in a patient with severe combined immunologic deficiency using a sibling donor with identical HLA. 2 similar successes were reported around the same time. These patients did not require immunosuppressive therapy and survived for > 25 years. In the 1970s and 1980s, clinical BMT required matched HLA haplotypes between the donor and recipient. Although earlier BMT procedures occurred between siblings, current molecular techniques have enabled the precise characterization of MHC genes and have allowed the possibility of matching unrelated individuals. In the 1970s, researchers performed a large number of BMT procedures using HLA-matched sibling donors for patients with end-stage leukemia or aplastic anemia. These endeavors resulted in mixed success because BMT was only considered for patients with advanced leukemia after conventional therapy failed. In the 1980s and 1990s, researchers noted that BMT performed at first remission or after first relapse resulted in significantly prolonged survival. BMT procedures for treating other disorders, such as thalassemia major and sickle cell disease, were also successful when the procedure was performed at an earlier stage. Because stem cells have been shown to be present in peripheral blood, these cells have now been used for autologous grafting. Extensive studies reviewed by Korngold and Brent have established the role of both T cells and histocompatibility systems in the GVHD reaction. Even with HLA-matched sibling donors, GVHD occurs half of all patients. Although corticosteroids and methotrexate partially ameliorated GVHD in early studies, cyclosporine in combination with other drugs have proven superior in subsequent studies. Following major efforts to determine the effectiveness of autologous BMT in the 1970s and 1980s, autologous BMT has become the therapy of choice for patients with malignant lymphoma and for selected patients with acute myelocytic leukemia and other malignancies. Recent research has also shown that a large number of stem cells from peripheral blood could be harvested following cytotoxic chemotherapy and specifically after growth factor administration. Following high-dose chemotherapy, PBSCT results in more rapid recovery of platelets than BMT. In the future, stem cell transplantation may allow many patients to attain a state of minimal residual disease so that other treatments, such as chemotherapy maintenance, monoclonal antibodies, vaccines, nonspecific immune modulators, and dendritic cell infusions, may be performed^{ref1, ref2, ref3}
Removing RBCs from BM buffy coat products :
• inverted spin procedure removes a median value of 81.2% of the RBCs, while still recovering a median of 94.3% of the mononuclear cells (median: 0.35 x 10⁸/kg; range: 0.17-0.9 x 10⁸/kg). The median volume of RBCs remaining in the product is 15.0 mL (range: 7.3-21.9 mL). The CD34⁺ cell dose of the final product ranged from 1.0 x 10⁶/kg to 4.8 x 10⁶ cells/kg (median: 1.9 x 10⁶/kg). Granulocyte recovery (defined as ANC count >or=500/mL for a period of 3 consecutive days) ranged from 18-30 days post-infusion of the allograft (median: 24.0 days)^ref.
Only 2-3 ml of blood should be sampled per bone marrow aspiration : if 5-10 ml are samples, the number of T lymphocytes increases
• peripheral blood stem cell transplantation (PBSCT) (peripheral blood progenitor cell (PBPC)) : easier accessibility and availability, faster engraftment of neutrophil and platelets when compared to standard bone marrow transplant^ref. PBPC collection in children weighing </=25 kg is hampered by technical and clinical problems related to vascular access, low total blood volume, anticoagulation, side effects, and psychological impact but large-volume leukapheresis (LVL) is a safe and effective procedure^ref

Mobilization of HSCs into PB : recruitment of hematopoietic stem and progenitor cells to the blood after administration of treatment.
• cytokine-mobilized PBSCs (expecially in elderlies):
• G-CSF mobilization (30-fold increase) coincides in vivo with a decrease in BM SDF-1 concentration related to SDF-1 cleavage by marrow serine proteases^{ref1, ref2} and dipeptidyl peptidase IV (CD26)^{ref1, ref2}. Furthermore, treating HSPCs by pertussis toxin (PT), which causes an interruption of Ga_i-protein signaling, did not affect their BM engraftment^ref but induced their release in the peripheral blood^ref, suggesting that a Ga_i-protein signaling identical to that mediated by CXCR4 receptor or other chemokine receptors must be required for HSPC retention in BM microenvironment^{ref1, ref2, ref3}. Consistent with the hypothesis that the CXCR4/SDF-1 axis plays a major role in the retention of hematopoietic cells, it has been shown that elevated numbers of granulocytic precursors were present in the peripheral blood of mice reconstituted with CXCR4^–/– FL cells^{ref1, ref2}. The ribosomal protein SA (RPSA) / 67 kDa laminin receptor (67LR) is a non-integrin cell-surface receptor with high affinity for laminin which plays a key role in tumor invasion and metastasis. The role of 67LR in G-CSF-induced mobilization of CD34⁺ HSCs from 35 healthy donors was investigated. G-CSF-mobilized HSCs, including CD34⁺/CD38^-- cells, showed increased 67LR expression, as compared with unstimulated marrow HSCs; noteworthy, the level of 67LR expression in G-CSF-mobilized HSCs significantly correlated with mobilization efficiency. During G-CSF- induced HSC mobilization, the expression of laminin receptors switched from 6 integrins, which mediated laminin-dependent adhesion of steady-state human marrow HSCs, to 67LR, responsible for G-CSF-mobilized HSC adhesion and migration toward laminin. In vitro G-CSF treatment, alone or combined with exposure to marrow-derived endothelial cells, induced 67LR up-regulation in marrow HSCs; moreover, anti-67LR antibodies significantly inhibited transendothelial migration of G-CSF-stimulated marrow HSCs. Finally, G- CSF-induced mobilization in mice was associated with 67LR up-regulation both in circulating and marrow CD34⁺ cells, and anti-67LR antibodies significantly reduced HSC mobilization, providing the first in vivo evidence for 67LR involvement in stem cell egress from bone marrow after G-CSF administration. In conclusion, 67LR upregulation in G-CSF-mobilized HSCs correlates with their successful mobilization and reflects its increase in marrow HSCs, which contributes to the egress from bone marrow by mediating laminin-dependent cell adhesion and transendothelial migration^ref. A total of 138 patients whose stem cell mobilization failed following chemotherapy and G-CSF at a dose of 5 mg/kg/d were given a higher dose of G-CSF (10 mg/kg/d) for 5 days after a 7-day resting period. HSC mobilization was successful in 90 patients, who yielded a median of 3.5x10⁶ CD34⁺ cells/kg, partially successful in 17 patients (1-2.4x10⁶ CD34⁺ cells/kg) and failed in the remaining 31 patients^ref. Anyway high-dose G-CSF alone was inefficient as second mobilization regimen in fludarabin-cyclophosphamide-treated CLL patients who failed to mobilize after chemotherapy and G-CSF^ref. The HSC mobilization-associated granulocytosis severely worsens acute renal failure^ref
• pegfilgrastim, in combination with salvage chemotherapy, is a predictable and highly effective CD34⁺ stem cells mobilization regimen in pretreated lymphoma patients. This was an open-label phase II study including 25 pretreated patients (Hodgkin's disease=4; aggressive NHL=21). The primary end-point of the study was the successful mobilization of a target cell dose of 2x10⁶ CD34⁺ cells/kg in lymphoma patients receiving ifosfamide, epirubicin and etoposide (IEV) chemotherapy and a fixed dose (6 mg) of pegfilgrastim given as single subcutaneous injection. Following chemotherapy, all patients had grade 4 neutropenia that lasted a median of 1.5 days (1-3). Pegfilgrastim treatment was well tolerated and only 2/25 patients required pain-control medication. CD34⁺ cells were mobilized in all patients. The median (range) peak value of peripheral blood CD34⁺ cells after IEV chemotherapy and pegfilgrastim was 141x10⁶/L (12.8-386) and occurred almost invariably on day +14 (13-16). 23 of the 25 patients underwent a single standard volume leukapheresis to collect a median of 8.7x10⁶ CD34⁺ cells/kg (1.78-17.3). 24/25 patients (96%) reached the target cell dose of 2x10⁶ CD34⁺ cells/kg. High concentrations of circulating CD34⁺ cells (> 50x10⁶/L) were observed for several days after the achievement of the peak value. All the study patients were transplanted with their pegfilgrastim-mobilized CD34⁺ cells and showed a rapid and sustained engraftment after high-dose chemotherapy^ref. Pegfilgrastim effectively mobilizes PBSC in a poor mobilizer multiple myeloma patient^ref
• in allogeneic HSC donors^ref
• in rhesus monkeys, rhPlGF-1 alone had no mobilization effect, whereas rhPlGF-1 (260 mg/kg/day) plus rhG-CSF (100 mg/kg/day) increased rhG-CSF-elicited mobilization of CFCs, HPP-CFCs and LTC-ICs per ml blood by 5-, 7- and 15-fold, respectively^ref
• G-CSF + SCF in poor mobilisers : a predictive model [PBSC expected = 0.6+(G-CSF-based total collection)+2 (rHuSCF-based day 1 collection)] to calculate the cumulative total of PBSC expected following a maximum of five leukaphereses^{ref1, ref2}. Failure has been reported^ref
• G-CSF + GM-CSF : there is no difference between high-dose G-CSF (32 mg/kg/day) or G-CSF(10 mg/kg/day)+GM-CSF (5 mg/kg/day)^ref
• G-CSF + GH^ref
• FLT3L^{ref1, ref2, ref3}
• CXCR4 antagonists^{ref1, ref2, ref3, ref4, ref5}
• induction of CXCL12 / SDF-1 plasma elevation^{ref1, ref2}
• endotoxins serve as cofactors in cytokine-induced mobilization. Modification of the endotoxin content by antibiotic treatment may affect the yield of cytokine-induced mobilization^ref
• polyanions^ref
• combination mobilization (200-fold increase) : PBSC peak at day +12 (collect when CD34⁺ cells > 20/ml)
• low-dose cyclophosphamide (1.5 g/m²) plus lenograstim is a safe and effective mobilizing regimen^ref
• ifosfamide (1.8 g/m² per day for 5 days) + carboplatin (400 mg/m² per day for 2 days) + etoposide (100 mg/m² per day for 5 days) (ICE) /IL-11 (50-100 mg/kg per day) + G-CSF (5 mg/kg per day) is successful in mobilizing large numbers of CD34⁺ PBSC cells with a limited number (one) of apheresis collections in patients that have previously been heavily pretreated with chemotherapy/radiotherapy
• high doses of cyclophosphamide (600 mg/m² twice daily for 10 doses) and etoposide (200 mg/m² twice daily for 10 doses (continuous; n=57) or 2 g/m² over 10 hours on day 5 of etoposide (bolus; n=22)) + filgrastim (5 mg/kg/d beginning day 14)  to mobilize autologous hematopoietic progenitor cells in 79 patients with high-risk or relapsed/primary refractory NHL, MM, and Waldenstrom macroglobulinemia. 59% of patients achieved the primary end point (a CD34 cell dose of 5 million/kg with a single leukapheresis). More bolus etoposide patients achieved the primary end point (86%) compared with continuous etoposide patients (47%; P<.0001). The CD34 cell dose collected was greater in bolus etoposide patients (44 million/kg) than in continuous etoposide patients (10.9 million per kilogram; P<.0001). Patients took 3 weeks to recover >500/ml neutrophils and >20000/ml platelets after cyclophosphamide and etoposide. The overall response rate was 69% for NHL patients and 71% for MM/WM patients. The TR; was 2.5%. 16% of surviving patients experienced grade>or=3 nonhematologic toxicity. Patients receiving bolus etoposide had significantly less grade>or=2 oral mucositis, less use of TPN, and less need for red blood cell and platelet transfusions. 64 patients (81%) underwent autologous HSCT, with prompt engraftment. 4 patients (5%) did not undergo autologous HSCT because of toxicity from high-dose cyclophosphamide and etoposide^ref
The numbers of T cells, CD34+ progenitor cells and monocytes, along with many other cell subsets, have been higher in PBSC grafts compared with marrow grafts (Dreger et al, 1994; Korbling et al, 1995; Ottinger et al, 1996; Theilgaard-Monch et al, 1999; Storek et al, 2001). Several studies have suggested that the cellular composition of PBSC grafts affected engraftment kinetics and clinical outcomes after allogeneic HCT. For instance, T-cell depletion of PBSC grafts resulted in higher relapse rates after myeloablative HCT compared with unmanipulated grafts (Barge et al, 2001; Chakraverty et al, 2001), and graft rejection was recently reported with T-cell depleted PBSC grafts after non-myeloablative conditioning (Kreiter et al, 2001). Furthermore, higher doses of CD34+ cells within PBSC grafts were shown to increase the risk of chronic graft-versus-host disease (GVHD) after conventional and reduced-intensity conditioning for human leucocyte antigen (HLA)-identical sibling HCT (Zaucha et al, 2001; Perez-Simon et al, 2003). In addition, improved overall disease-free survivals have been reported with PBSC compared with marrow after both myeloablative and non-myeloablative HCT in patients with advanced disease (Bensinger et al, 2001; Maris et al, 2003). Another study noted that higher numbers of CD14+ cells (monocytes) with decreased expression of co-stimulatory molecules within the PBSC grafts might impair the proliferation rate of donor T cells (Tanaka et al, 1998). This was cited as a possible reason for similar rates of acute GVHD after PBSC compared with marrow grafts despite the approximately 10-fold higher numbers of T cells given with PBSC products.

Both T cells and natural killer (NK) cells from HCT donors have been implicated in the graft-versus-tumor (GVT) effects after allogeneic HCT (Truitt & Atasoylu, 1991; Glass et al, 1996; Giebel et al, 2003). The GVT effect of T cells has been indirectly confirmed by the results of T-cell depletion trials (Link, 1999; Barge et al, 2001; Chakraverty et al, 2001). Preclinical studies in the canine HCT model showed that grafts created their own marrow space via subclinical graft-versus-host (GVH) reactions, thereby ensuring stable engraftment (Storb et al, 1999). Data from clinical trials in patients showed that a low degree of donor T-cell chimaerism on day +28 after non-myeloablative HCT was associated with an increased risk of rejection and that high donor T-cell chimaerism was beneficial in mediating GVT effects (Antin et al, 2001; McSweeney et al, 2001; Maris et al, 2003; Niederwieser et al, 2003). The level of donor T-cell engraftment (chimaerism) is currently being used as a surrogate marker for donor engraftment after non-myeloablative HCT (Childs et al, 1999; Antin et al, 2001; McSweeney et al, 2001).
This study confirms the substantial variation in PBSC cellular subset composition as reported in previous studies of conventional myeloablative allogeneic HCT recipients (Dreger et al, 1994; Ottinger et al, 1996). Our analysis showed positive correlations between almost all cellular subtypes within the PBSC product.

Results from this study are the first published indication in the truly non-myeloablative setting that infused quantities of some cellular subsets of PBSC products from HLA-matched siblings are associated with important post-HCT outcomes of engraftment (T-cell chimaerism) and survival. The cellular components that were associated with higher donor T-cell chimaerism on day +28 were TNC and CD56+ cell numbers. Given the observation that TNC numbers were highly correlated with CD3+ cell numbers in this study, the relationship of TNC on donor T-cell chimaerism possibly reflected the known role of T cells in establishing donor engraftment after myeloablative and non-myeloablative HCT (Champlin, 1993; Drobyski, 2000; Kreiter et al, 2001). NK and NKT cells analysed separately were marginally associated with T-cell chimaerism; however, the association was stronger when the two values were combined. CD56 has been used as a surrogate marker to describe NK cells, and it was known that NK-cell alloreactivity could influence engraftment and relapse rates after HLA-mismatched myeloablative HCT (Bornhauser et al, 1999; Ruggeri et al, 1999, 2001). This alloreactivity was thought to be the result of the missing expression of HLA-class I epitopes on haematopoietic cells (Ciccone et al, 1992). Whether donor NK cells can exert anti-host activity after HLA-identical related donor HCT is presently unknown (Dupont & Hsu, 2004). Interestingly, a recent report showed that patients who rejected their unrelated grafts after a non-myeloablative conditioning regimen had significantly lower donor NK-cell chimaerism compared with patients who did not experience rejection (Bornhauser et al, 2001). However, it was unclear if, and to what level, these patients were HLA-matched in this report, and no data were given about the actual number of donor cells transplanted.

We suspected that certain cellular subsets in PBSC grafts would be associated with better progression-free and overall survival after non-myeloablative HCT, as this approach relies mainly on graft anti-tumour responses mediated by donor cells (Barrett & Malkovska, 1996; Appelbaum, 2001). In fact, only B cells and CD34+ cells were associated with improved outcomes. Specifically, higher doses of B cells appeared to be associated with a greater occurrence of chronic GVHD, better progression-free survival, and improved overall survival, while the effect of higher numbers of CD34+ progenitor cells was associated only with better overall survival. The finding that higher B-cell numbers were associated with an increase in chronic GVHD in the current study was unexpected, two previous reports had shown that high numbers of CD34+ cells were associated with an increased risk of chronic GVHD after both myeloablative and reduced-intensity-conditioning HCT (Zaucha et al, 2001; Perez-Simon et al, 2003). The association of B cells with chronic GVHD is also the probable explanation for the positive effect of B cells on progression-free and overall survival found in this study, possibly through GVT effects. In addition, humoral responses of B cells against malignant host cells, which have been described for potential CML-associated antigens (Ling et al, 1998; Wu et al, 2000), might have been responsible for the anti-tumour responses. However, it seems most likely that the donor CD20+ cells could have functioned as antigen-presenting cells (Chesnut & Grey, 1986; Pernis & Weber, 1989), thereby facilitating the donor T cells to exert more GVT and GVHD effects.

Higher numbers of CD14+ cells (monocytes) were associated with worse overall survival in our analysis. As CD14+ cells in PBSC might decrease allogeneic T-cell responses (Mielcarek et al, 1997), one might speculate that GVT effects may have been diminished in patients who received a higher CD14+ cell dose in the PBSC graft.

As expected, patients categorized before HCT as having aggressive malignant disease had a higher risk for disease relapse/progression and worse progression-free and overall survival. These results confirmed the prognostic value of this clinical categorization, similar to the high dose conventional HCT setting; patients classified as having aggressive diseases had worse outcomes after non-myeloablative conditioning as well.

Another finding consistent with previous studies was that patients who had received 2 Gy TBI only as conditioning showed lower donor T-cell chimaerism on day 28 compared with patients who had received additional fludarabine or a combination of autologous and allogeneic transplantation (McSweeney et al, 2001). As fludarabine is known to be immunosuppressive and to enhance the effect of radiation (Gregoire et al, 1999; Van Den Neste et al, 2001), it is likely that additional suppression of host immune-competent cells caused by fludarabine enabled the achievement of more rapid donor T-cell chimaerism. Our data were consistent with previous studies showing that donor T-cell chimaerism can be used as a surrogate marker for engraftment (Childs et al, 1999; Antin et al, 2001; McSweeney et al, 2001; Niederwieser et al, 2003), although there were too few rejections in this set to confirm this hypothesis.
In summary, we have shown that the doses of CD34+ cells, B cells and monocytes in the HLA-matched related PBSC graft influenced outcomes after non-myeloablative HCT and confirmed that variations in conditioning regimens, disease status, and age of donors and recipients were important. Evaluations in patients receiving PBSC from alternative donors (e.g. HLA-matched unrelated or HLA-mismatched donor recipient pairs) are needed to support and further exploit these findings. Furthermore, prospective clinical studies with larger numbers of patients will be required. If these results are confirmed, then clinical studies evaluating graft engineering for selective cellular enrichment or depletion may be important in improving clinical outcomes after non-myeloablative conditioning for allogeneic HCT.
 

This process mimics enhancement of the physiological release of stem cells and progenitors from the bone marrow (BM) reservoir in response to stress signals during injury and inflammation. Currently, mobilized cells are the preferable and major source of stem and progenitor cells harvested for autologous and allogeneic transplantations because of the higher yield of these cells, leading to faster engraftment and decreased procedural risks compared with harvested BM cells. The emerging picture of stem cell mobilization involves interfering with the physiological interplay between mesenchymal stromal and hematopoietic cells regulating both bone and BM remodeling processes, which also mediate stem/progenitor cell proliferation and migration. The mobilization process is initiated by stress-induced activation of neutrophils and osteoclasts by chemotherapy and repeated stimulation with cytokines such as G-CSF, resulting in shedding and release of membrane-bound SCF, proliferation of progenitor cells, as well as activation and/or degradation of adhesion molecules such as VLA-4 and P/E selectins. The dynamic secretion and inactivation of the chemokines SDF-1/CXCL12 and IL-8/CXCL8, and multiple cycles of inactivation and degradation of BM components by proteolytic enzymes, such as elastase, cathepsin G, proteinase 3, CD26, and various MMPs, are implicated as major players in progenitor mobilization. These mechanisms will be reviewed in this section. The road to stem cell mobilization began in the 1960s, with publication of a few reports documenting the presence of hematopoietic stem cells in the peripheral blood of mice, dogs, and monkeys, followed by reports revealing low levels of progenitors in the human circulation during steady-state homeostasis. However, steady-state progenitors in the circulation of mice were shown to be inferior to BM as a source of repopulating stem cells. More important, although sporadic clinical transplantations documented successful hematopoietic recovery, in most cases graft failure was detected in autologous, syngeneic, and fully matched related allogeneic transplantations, using steady-state peripheral blood cells. Moreover, the first successful human blood leukocyte transplantations were not better than BM as a source of repopulating stem cells (reviewed in Korbling and Fliender (Korbling M, Fliender TM. History of blood stem cell transplants. Blood stem cell transplants. In: Gale RP, Juttner CA, Henon P eds. Peripheral blood stem cell autographts. New York: Cambridge University Press; 1994:9) and To et al^ref). While the levels of human progenitors in the circulation during steady-state homeostasis are low, they are significantly elevated in patients with myeloproliferative disorders^ref. Similarly, a transient increase in circulating progenitors was documented in dogs treated with dextran sulfate and humans treated with endotoxin or other stress-inducing mobilization agents. These encouraging results were followed by preliminary reports in the late 1970s documenting increased levels of progenitors in the circulation of patients after chemotherapy treatment with cyclophosphamide and other drugs. Technical improvements including in vitro colony assays and the availability of large-scale harvesting of human progenitors by continuous-flow leukopheresis enabled closer examination of chemotherapy-/druginduced mobilization in treated patients^ref (Korbling M, Fliender TM. History of blood stem cell transplants. Blood stem cell transplants. In: Gale RP, Juttner CA, Henon P eds. Peripheral blood stem cell autographts. New York: Cambridge University Press; 1994:9). In the early 1980s, To and colleagues reported high levels of progenitors in the circulation of leukemia patients in early remission^ref. Clinical studies by this group were the first to document the beneficial faster repopulation in patients transplanted with autologous mobilized peripheral blood (PBL). Initially, the mobilization protocols were based on chemotherapy alone. However, after the discovery of human G-CSF by Welte et al^ref, mobilization protocols began to include G-CSF, which today is the standard mobilizing agent^ref.  During late embryonic development, both human and murine stem cells migrate via the blood circulation from the fetal liver to the BM and repopulate this tissue with high levels of immature and maturing cells. These in turn are released into the circulation, while a small pool of undifferentiated stem and progenitor cells within the BM is maintained throughout life. Prior to their localization in the murine fetal liver, hematopoietic stem cells can be isolated from the aorta-gonad-mesonephros (AGM) region and the yolk sac even before completion of blood circulation development^ref. However, some of these stem cells are not definitive, since they cannot rescue and repopulate lethally irradiated recipients because of their inability to migrate in the host circulation and home to and repopulate the BM. These predefinitive stem cells isolated from the murine yolk sac can still engraft the liver of newborn mice preconditioned as fetuses with chemotherapy if the predefinitive stem cells are directly injected into this organ^ref. Furthermore, an additional maturation step provided to predefinitive progenitors isolated from the AGM region, in the form of in vitro incubation on stromal cells and stimulation with cytokines, shifts these cells into definitive stem cells with functional migration, homing, and repopulation potential^ref. Murine embryos that lack the chemokine SDF-1 or its receptor CXCR4 have multiple lethal defects, including impaired seeding of the fetal BM^ref. The essential roles of SDF-1/CXCR4 interactions has been shown in a functional, preclinical model for human stem cells using non-obese diabetes severe combined immune deficient (NOD/SCID) mice as recipients. Homing and repopulation in both primary and serially transplanted mice with human CD34 stem cells was dependent on CXCR4 signaling^{ref1, ref2}, which is dynamically expressed by human progenitors^ref. Moreover, SDF-1-mediated migration of human CD34 cells in vitro correlated with their in vivo repopulation potential in transplanted patients^ref. In conclusion, stem cell motility (migration, homing, and release) is essential for BM repopulation and for the development of the hematopoietic system. The role of circulating stem cells in steady-state homeostasis is currently unknown. While some progenitors are needed for seeding of the thymus, which requires migration of lymphocyte precursors from the BM, this process is markedly reduced in adult life^ref. Stem cells migrate also to nonhematopoietic organs such as the liver, especially during liver injury/inflammation, creating an alarm situation and transmitting stress signals that mobilize and recruit stem cells as part of organ repair^ref. Ramshaw et al suggest that circulating stem cells can reengraft the BM, documenting successful engraftment of unconditioned murine recipients transplanted with high doses of BM cells^ref. In support of this approach, Wright et al elegantly demonstrated rapid clearance of intravenous transplanted mouse stem cells from the murine circulation within a few minutes. By using genetically marked parabiotic mice with a shared circulation, they showed that the low levels of circulating stem cells can durably reengraft the BM, suggesting continuous release and reengraftment of the BM as sequential events taking part in physiological processes^ref. In parallel, Abkowitz et al, using the same parabiotic model, revealed only low levels of reengrafted stem cells with secondary recipient repopulation potential (1–2.5%) in the partner BM as opposed to high levels of progenitors in the spleen. These investigators suggest the release of stem cells into the circulation functions also as an apoptotic pathway for the BM to regulate excess amounts of progenitors because of limited numbers of stem cell niches^ref. Of interest, these investigators also applied G-CSF- and SCF-induced mobilization to the parabiotic mice pairs, documenting a dramatic 4- to 10-fold increase (from 1–2.5% to 10.1%) in the levels of reengrafted stem cells with secondary repopulation potential in the partner BM. These results demonstrate that inducing the release of stem cells in response to stress signals also increases their homing back and reengraftment of the stimulated/stressed BM, confirming the repopulation potential of mobilized progenitors harvested for clinical protocols. These results also support the hypothesis that tissue repair of damaged BM can be supported by intensified homing and reengraftment of circulating hematopoietic stem cells, in response to stress signals, which is similar to migration of mature leukocytes to sites of inflammation as part of the immune system host defense. The concept of BM niches occupied by stationary, quiescent stem cells may be an oversimplification of a more dynamic situation. Higher proliferative and migratory turnover of definitive, repopulating stem cells involves their release into the circulation, migration, homing, and reengraftment of the BM as part of their physiological development. Bradford et al revealed that even the most primitive murine stem cells in the BM must cycle at least once every 30 days^ref. Proliferation and release of hematopoietic stem and progenitor cells require dynamic cycles of BM destruction/restructuring, which seem to be linked to bone remodeling by osteoclast/osteoblast interactions. Moreover, both processes are likely to be orchestrated by the same factors. For example, continuous G-CSF treatment to prevent neutropenia has also been shown to induce osteoporosis in some juvenile patients with severe congenital neutropenia^ref and in transgenic mice that overexpress this cytokine^ref. More important, there is a dramatic increase in the number of murine osteoclasts in response to repetitive G-CSF stimulation, leading to osteoclast-mediated bone resorption during stem cell mobilization and calcium release, which can induce detachment of hematopoietic progenitors from fibronectin in vitro^ref. Unexpectedly, treatment of mice with pamidronate, an inhibitor of osteoclast-mediated bone resorption, together with G-CSF increased the levels of mobilized progenitors in the murine circulation^ref. Osteoclasts secrete high levels of IL-8, a mobilization-inducing chemokine^{ref1, ref2}. Another chemokine, SDF-1, which also participates in G-CSF–mediated mobilization, induces osteoclasts to secrete the metalloproteinase MMP-9^ref. This enzyme is involved in stem cell mobilization by shedding the membrane-bound cytokine SCF within the BM and releasing this cytokine into the circulation^ref. MMP-9 also induces migration of osteoclasts to the cortical bone, adjacent to the SDF-1 and stem cell–rich endosteum region, to form resorption pits^{ref1, ref2}. These results suggest that the role of osteoclasts in mobilization could be related to the rapid increase in their numbers and to the secretion of chemokines and proteolytic enzymes. Activation and secretion of proteolytic enzymes lead to cleavage and degradation of the BM extracellular matrix, adhesion molecules, cytokines, and chemokines and therefore facilitate transendothelial migration and stem cell release from the BM.7 These processes mediate both peripheral bone and medullar BM remodeling as well as stem cell proliferation and release by inactivating the BM microenvironment. Subsequently, stem cells migrate via the circulation, home back to the BM, and repopulate the damaged/destructured sites in this organ as part of tissue repair and the continuous replenishment of the blood with new immature and maturing hematopoietic cells of all lineages, while maintaining a small pool of undifferentiated stem and progenitor cells within their renewed BM niches. In support of this hypothesis, by applying complementary DNA array technology, 2 recent studies revealed high expression of the proteolytic enzyme proteinase 3 and low expression of a proteinase 3 inhibitor in isolated human BM CD34 cells compared with enriched mobilized CD34 cells isolated from G-CSF-treated healthy donors^{ref1, ref2}. These results suggest that proteinase 3, with its ability to cleave connective tissue in the BM, is needed for maintaining a niche for hematopoietic stem and progenitor cells. Recent findings document that proteinase 3 also cleaves the cell cycle checkpoint p21, inducing cells to enter the cell cycle^ref. This could explain the higher levels of cycling BM CD34 cells compared with the more quiescent mobilized PBL CD34 cells^{ref1, ref2}. Immature mesenchymal bone-forming osteoblasts lining the stem cell–rich endosteum region secrete high levels of SDF-1^ref. In response to stimulation with this ligand, hematopoietic stem cell–derived osteoclasts secrete MMP-9^{ref1, ref2}. Thus, peripheral periosteal bone and medullar BM destruction and remodeling are sequential events mediated by the same factors, i.e. MMP-9, G-CSF, SCF, IL-8, and SDF-1, which regulate both new bone formation and stem cell self-renewal, migration, and development. Applying single or multiple stress signals interferes with steady-state homeostasis, creating an alarm situation leading to increased release of immature and maturing hematopoietic cells from the BM into the blood circulation, which occurs naturally during inflammation and injury. For example, increased levels of G-CSF and other cytokines secreted by long-distance runners during marathons cause blood leukocytosis, in particular higher levels of CD34/CD38⁺ committed progenitor cells, but not more primitive CD38^- stem cells compared with control individuals, reflecting adaptation of BM activity to stress induced by extensive exercise^ref. Increased cell release from the BM reservoir is part of the immune system host defense during inflammation as a result of infection- or injury-mediated release of stress signals. This release is induced clinically or in animal models by a wide range of molecules and/or treatments: DNA damaging, single chemotherapeutic drugs such as cyclophosphamide, or combined chemotherapy regimens such as iphosphamide, carboplatin and etoposide (ICE) and etoposide, methylprednisolone, ara-c and cisplatin (ESHAP); cytokines such as G-CSF, GM-CSF, SCF, and flt-3 ligand; and chemokines such as IL-8, Mip-1, Groß, and SDF-1^ref. These molecules differ in their mode of administration, the time frame needed to achieve mobilization, the type of cells mobilized, and the efficiency. G-CSF, the most commonly used mobilization agent, is usually administered daily at a dose of 5–10 µg/kg for 5–10 days, alone or after chemotherapy^ref. However, a substantial number of patients, especially patients having extensive chemotherapy that leads to BM aplasia, older patients, and a minority of healthy individuals, are poor mobilizers^ref. For example, increasing age and longer intensive chemotherapy treatment in old multiple myeloma patients inversely correlate with successful mobilization^ref. These patients are usually treated with higher doses of G-CSF, GM-CSF followed by G-CSF, or a combination of G-CSF and SCF. Recently, it was reported that some chronic myeloid leukemia (CML) patients treated with high doses of imatinib (Glivec, STI 571) suffer hematological toxicity and can develop drug-induced neutropenia^ref. In addition, some treated patients failed to mobilize well, requiring discontinuation of the drug during the mobilization process and the addition of SCF to G-CSF, to successfully overcome the poor mobilization^ref. However, while SCF stimulation increases the levels of mobilized CD34⁺ progenitors, this treatment also has side effects such as allergic reaction due to activation of mast cells. These studies stress the need to identify and characterize the mechanisms of stem cell mobilization in order to develop better strategies for poor mobilizers. Chemotherapy followed by repetitive G-CSF–induced mobilization is a multistep process. Anchorage of stem and progenitor cells to the BM microenvironment by activation of adhesion interactions, which are facilitated by membrane-bound SCF or SDF-1, needs to be disrupted^ref. Papayannopoulou elegantly demonstrated that the integrin VLA-4 plays a critical role in stem cell mobilization since anti-VLA-4 antibodies prevent adhesion molecules expressed on the surface of hematopoietic progenitors from binding their ligand VCAM-1 expressed by stromal cells. This induces rapid mobilization of primate and murine stem cells, which also involves signaling via the c-kit receptor. Moreover, as a result of inactivation during the mobilization process, mobilized CD34⁺ cells have lower levels of VLA-4 and c-kit expression compared with BM progenitors^{ref1, ref2}. In addition, sulfated polysaccharides such as Fucoidan lead to rapid mobilization of progenitors^ref since they compete for adhesion interactions with selectins and for SDF-1 binding to the BM endothelium via its nonsignaling C-terminus, inducing inactivation of selectin interactions as well as release of SDF-1 into the circulation. Levesque et al revealed increased release of elastase and cathepsin G within murine BM during Cy- and G-CSF–induced mobilization; peak levels were reached during stem cell egress^ref. These enzymes cleaved VCAM-1 expressed by stromal cells, preventing adhesion of progenitors via VLA-4 as part of the mobilization process. In addition to their distinctive adhesive properties, mobilized CD34 progenitors differ from their BM counterparts in several ways. Besides reduced VLA-4, c-kit, and CXCR4 expression, a significantly higher percentage of mobilized progenitors are noncycling quiescent cells^{ref1, ref2}. Prior to mobilization, hematopoietic progenitors proliferate within the BM, and this feature could involve proteinase 3–mediated cleavage of p21 in response to neutrophil stimulation^ref. Mobilized progenitors have higher levels of the proapoptotic genes caspase 3, 4, and 8 and reduction in inhibitors of apoptosis such as antiproteinase 2, compared with human BM CD34 cells^ref, supporting the hypothesis of Abkowitz et al that release into the circulation may also serve as an apoptotic pathway for stem cells^ref. Enhanced repopulation documented with mobilized progenitors is due to significantly higher cell doses, in particular committed progenitors, which lead to faster neutrophil and platelet recovery^ref. However, better leukemia-free survival and overall survival were recently documented in acute myeloid leukemia (AML) patients transplanted with increased cell doses of matched BM cells compared with lower doses of BM cells and more important, also compared with high cell doses of matched mobilized PBL, suggesting BM as a superior source of stem cells for HLA-identical allogeneic transplantations^ref. New protocols aimed at increasing the levels of long-term repopulating stem cells as the major source while maintaining short-term repopulating cells need to be developed. Mobilized human CD34 progenitors express reduced levels of the SDF-1 receptor CXCR4, which correlates with improved mobilization^{ref1, ref2}, suggesting involvement of SDF-1/CXCR4 interactions in the mobilization process. Overexpression of SDF-1 in the murine circulation leads to stem cell mobilization^ref. In functional preclinical animal models for human stem cells such as preimmune sheep and immune-deficient NOD/SCID mice, mobilized CD34⁺ cells are inferior in their repopulating potential compared with similar cell doses of CD34 cells obtained from human BM. This is most probably due to their reduced levels of surface CXCR4, which is needed for SDF-1–mediated directional homing and repopulation in transplanted mice and increased levels of short-term repopulating cells^ref. These results should be interpreted with caution, since short-term stimulation with human SCF upregulated surface CXCR4 expression on human CD34⁺ cells and murine SCF is much less potent in stimulating human progenitors^{ref1, ref2}. In addition, other factors such as complement C3a improve SDF-1–mediated directional migration of human progenitors and the in vivo homing of murine progenitors. However, some of these factors can be species-specific or absent in immune-deficient mice^ref. Of interest, priming of mobilized human progenitors by several inflammatory molecules improves their chemotactic responses to SDF-1 (Wysoczynski M, Reca R, Kucia M, et al. Mobilized peripheral blood stem/progenitor cells are primed by several inflammatory molecules for their chemotactic responses to sdf-1: a molecular explanation as to why mobilized peripheral blood cells engraft faster than bone marrow cells after transplantation. Paris, France -July 5–8, 2003; 32nd Annual Scientific Meeting of the International Society for Experimental Hematology (ISEH). abst 277. 2003). We have demonstrated increased levels of SDF-1 production in the murine BM in response to DNA-damaging chemotherapy drugs, including Cy and 5-fluorouracil (5-FU), most probably to prevent cell death since this chemokine is also a survival factor for stem cells^{ref1, ref2, ref3}. Rafii and Hattori et al demonstrated that the increased levels of SDF-1 in response to stress-mediated 5-FU mobilization in mice induce release of MMP-9, which cleaves and releases membrane-bound SCF, leading quiescent stem cells to proliferate in the BM prior to their mobilization into the circulation^ref. SDF-1 mediates secretion of MMP-2 and MMP-9 from human CD34⁺ cells^ref, and these proteolytic enzymes inactivate SDF-1 by cleaving a few amino acids in the N terminus^ref. We demonstrated that each injection of G-CSF stimulates mesenchymal cells such as immature osteoblasts to secrete SDF-1, leading to a transient increase in SDF-1 levels within the BM. This oscillating increase is followed by a profound decrease due to inactivation by proteolytic enzymes, mostly by neutrophil elastase, with the lowest levels of this ligand released during cell mobilization^ref. Levesque et al reported that in addition to inactivation of BM SDF-1 by elastase and cathepsin G, these proteolytic enzymes can also cleave part of the CXCR4 receptor N terminus in the BM^ref, partially inactivating SDF-1 signaling and directional migration. Of interest, neutrophils secrete the proteolytic enzymes elastase, cathepsin G, and proteinase 3 in response to G-CSF, and neutrophil elastase also inactivates G-CSF^ref, which is essential for regulating release of neutrophils from the BM. However, expression of G-CSF receptors by neutrophils is not required because of an indirect mechanism that involves proteolytic reduction of SDF-1 within the murine BM^ref. Of interest, the central role of elastase in regulating release of BM leukocytes is suggested by inherited genetic mutations in the elastase gene, leading to 2 forms of neutropenia in juvenile patients (severe congenital neutropenia, or Kostmann disease, and cyclic neutropenia) that require continuous treatment with G-CSF^ref. CD26, another proteolytic enzyme that inactivates SDF-1, is expressed by human CD34 progenitors and is involved in G-CSF–induced mobilization in mice^ref. A role for CXCR4 signaling in cell egress from the BM to the circulation emerges from several studies. In parallel to gradual reduction of BM SDF-1 during G-SCF administration, CXCR4 expression is elevated within the human and murine BM, reaching peak levels at the time of mobilization^ref. While some CXCR4 inhibitors also lead to release of human progenitors^ref, and treatment with pertussis toxin, which blocks Gi-mediated signaling in 7 transmembrane G coupled receptors such as CXCR4, induced mobilization in mice^ref, SDF-1/CXCR4 interactions are also needed for cell egress. Treatment of mice with G-CSF and neutralizing anti-CXCR4, anti-SDF-1 Ab, or inhibitors of CD26 reduced the mobilization levels^{ref1, ref2}. Finally, warts, hypogammaglobulinemia, immunodeficiency and myelokathexis (WHIM) syndrome, a rare inherited immunodeficiency disease associated with mutations in CXCR4, is characterized by neutropenia and B-cell lymphopenia. In one case, an affected individual was born with cardiac malformation, resembling the murine model in which knocking out CXCR4 or SDF-1 results in a lethal phenotype associated with lack of BM seeding, B-cell development, and cardiac septum formation^ref. Thus, SDF-1/CXCR4 interactions may have a role in the regulation of the routine and active egress of progenitor and maturing cells from the BM into the circulation. Taken together, these results decipher key mechanistic insights into stress-induced mobilization that mimic and amplify naturally occurring recruitment of progenitors during alarm situations. We suggest the BM as a reservoir for immature and maturing hematopoietic cells to be released into the circulation upon stress signals, to migrate to injured sites, and to contribute to host defense and tissue repair. The steady-state balance in the BM is disrupted, leading to transient increased production of SDF-1, and proliferation and activation of neutrophils and osteoclasts. Release of proteolytic enzymes is followed by shedding of membrane-bound SCF, proliferation of hematopoietic progenitors, increasing surface CXCR4 expression and inactivation of SDF-1, G-CSF, the BM adhesion machinery, and extra cellular matrix (ECM). These events are intensified in each cycle of stimulation by G-CSF, eventually leading to release of progenitors into the circulation. A better understanding of the process by which progenitors egress from the BM will eventually lead to the development of improved mobilization protocols, in particular for patients who are poor mobilizers.

• mobilizing the older patient with multiple myeloma
• individually optimized collection of HPCs
• enumeration of HPCs

Variables having an impact on the ability to collect hematopoietic progenitor cells (HPCs) may be donor related or procedure related. Donor-related variables include age, previous chemotherapy, mobilization regimen, and platelet count at the time of mobilization. Procedure-related variables include central access devices as well as variables inherent to the different cell separation devices used. Nonetheless, at present, the basis for optimizing HPC collection is the ability to determine when to start collection, based on the ability to enumerate CD34⁺ cells in the peripheral blood of a donor/patient on a daily basis. It has been demonstrated repeatedly that the best currently available predictor of an adequate collection is the number of CD34⁺ cell/µL in the blood on the morning of collection, both for good mobilizers and for poor mobilizers^{ref1, ref2, ref3, ref4, ref5}. A number of authors have recommended starting collection when a particular number of CD34⁺ cells/µL is present (usually a number between 8 and 20) in order to increase the likelihood of collecting at least 2–4 x 10⁶ CD34⁺ cells/kg in a single apheresis, i.e., an acceptable number of HPCs for either one or two autologous transplants or a single allogeneic transplant (Weaver C, Birch R, Schwartzberg L, et al. CD34+ content of peripheral blood progenitor cells is the single most powerful predictor of recovery kinetics in patients receiving myeloablative high-dose chemotherapy and PBPC infusion. Blood. 1994;84:1388a)^ref. In the past it was important that each center determine a number of its own as the starting point for apheresis, given the historic difficulty in comparing flow cytometry results from center to center^ref. At present, however, there are 2 commercially available single platform tests available for measuring CD34⁺ cells in blood (ProCount from Becton-Dickinson, Mt View, CA, and StemKit from Beckman-Coulter, Fullerton, CA), so that any center using the same technology should have a reasonable chance of success using the same numbers. More importantly, it is possible to use the number of CD34⁺ cells/µL obtained using single platform technology in a predictive formula which allows the clinician to know when to start apheresis in order to optimize collection and how many liters of blood need to be processed in order to collect a given number of CD34⁺ cells (O’Connell B, Ng Y, Rapoport A, Fassas A, Tricot G, Cottler-Fox M. Maximizing collection of human peripheral blood progenitor cells while minimizing the number of aphereses. Blood. 1999;94:4749a) using the formula in Figure 3. While results of collection using this formula have only been reported for the COBE Spectra cell separators, it seems likely that it would work with any continuous flow apheresis device if the appropriate value for machine collection efficiency is used.
Predictive formula that allows the clinician to know when to start apheresis in order to optimize collection and how many liters of blood need to be processed in order to collect a given number of CD34⁺ cells : # of L of blood processed = # CD34⁺ cells desired / [CD34⁺ cells/ml blood x machine collection efficiency)/patient weight in kg]
Sadly, despite the consensus that daily CD34⁺ cell numbers in the blood are the best currently available predictor for achieving an acceptable HPC collection,8 the practice has not been universally adopted because it is both time consuming and relatively expensive. Alternatives that have been reported to be useful include measuring the number of CD34⁺ cells in the blood the day before starting collection, with or without the total white cell count, or change in white cell count from the day before apheresis to the day of apheresis^ref, and the rapidity of rise in white cell count and platelet count. More recently, the Food and Drug Administration (FDA) has approved the use of an HPC window on an automated cell counter (Sysmex, Kobe, Japan) and this number may be used to predict when to start apheresis^ref. The HPC number measured by the Sysmex does not correlate well with CD34⁺ cell number in the blood (nor does CD133, an antigen expressed by the more primitive CD34⁺ cells^ref, but since it is now recognized that not all HPCs express measurable CD34^ref it has been presumed that the HPCs measured by the Sysmex include some that are CD34^-. Thus, the HPCs measured by Sysmex are available rapidly and less expensively, and may serve as a guide for starting apheresis, but they are difficult to use in the above predictive formula.
HPCs expressing high levels of the enzyme aldehyde dehydrogenase (ALDH) are believed to be pluripotent and generally express CD34 on their surface^ref. A commercial assay has now been developed for this intracellular enzyme (Aldecount, Stemco Biomedical, Durham, NC). As this assay detects both CD34⁺ and CD34^- cells, and appears to differentiate viable from non-viable cells (Balber A, Gentry C, Pritchard C, Baucom C, Deibert E, Smith C. ABC-mediated efflux of ALDH reaction product: implications for enumeratioin and isolation of blood progenitor cells. Biol Blood Marrow Transplant. 2003;9:231a), its use may eventually lead to a major change in how HPCs are enumerated in transplant grafts.
Timing of apheresis : there is a fall in circulating leukocytes immediately after G-CSF has been given, with the peak of CD34⁺ cell mobilization approximately 3–6 hours after each dose is given subcutaneously, paralleling its half-life^{ref1, ref2, ref3}. To optimize collection, it seems reasonable therefore, to wait at least 1 hour after giving the injection, trying to time the duration of the collection for the period which includes the rise in leukocytes and as much of the peak as possible. For example, at least one center routinely waits for 2 hours after the injection, since their average collection lasts about 2–3 hours^ref. It is possible that the apparent ability of large volume leukapheresis lasting 4–5 hours to "recruit" CD34⁺ cells into the blood, reported by several groups but not others^{ref1, ref2}, in fact simply reflects the timing of the apheresis collection relative to the dose of G-CSF. With the arrival of pegylated filgrastim (Neulasta, Amgen, Thousand Oaks, CA) it is possible that the relationship of collection yields relative to the time of dosage will cease to be a factor in collection.
Predicting who will mobilize poorly : the effect of age has been shown to be a continuous, incremental variable in the myeloma population, with no threshold past which there is an accelerated decline in mobilization of CD34⁺ cells^ref. That is, the older the patient, the fewer stem cells are likely to be collected, but there is no fixed age past which it is impossible to collect HPCs. Over and above age, however, 2 other variables are statistically significant: number of months of previous chemotherapy and platelet count at the time of mobilization^ref. In 85% of myeloma patients over age 70 who had < 12 months of therapy and a platelet count > 200 x 10⁹/L, it was possible to obtain  4 x 10⁶ CD34⁺ cells/kg in a single apheresis using individually optimized collection conditions after combined chemotherapy and growth factors. Those patient over 70 years of age with > 12 months of prior therapy and platelets < 200 x 10⁹/L, however, were poor mobilizers.
Choosing a mobilization regimen : mobilization with chemotherapy and growth factors has been shown in a number of settings to be significantly more effective than growth factors alone. Nonetheless, it is often the case that the toxicity of chemotherapy makes this combination less attractive in the fragile older patient. It is therefore important to know that in the subgroup of myeloma patients over the age of 70 who were predicted to be poor mobilizers (> 12 months prior therapy and platelets < 200 x 10⁹/L), as many stem cells were collected with growth factors alone as with chemotherapy and growth factors together. Thus, for this group of patients it seems worthwhile trying to mobilize with growth factors alone in order to avoid toxicity, assuming the disease itself does not require chemotherapy at the time of attempted mobilization.
• current standard agents :
• filgrastim has become the standard against which all other mobilization agents are measured. This is because it has been shown to both mobilize more CD34⁺ cells and have less toxicity than any other single agent against which it has been tested to date. It is not completely without toxicity, however, given that there have been deaths attributed to thrombosis (acute myocardial infarction and stroke) in sibling donors^ref, possibly related to receptors on platelets for G-CSF^ref. Also, a recent study of serial ultrasounds in donors receiving G-CSF prompted by 4 reports of splenic rupture related to G-CSF demonstrated universal enlargement of the spleen during mobilization, with regression of size after discontinuing the growth factor^{ref1, ref2}. Other effects of G-CSF which are shared with GM-CSF include pain, nausea, vomiting, diarrhea, insomnia, chills, fevers, and nightsweats^{ref1, ref2}
• sargramostim as a single agent is used less often today for mobilization than G-CSF, because it mobilizes somewhat less well than G-CSF^ref and because of a relatively higher incidence of both mild and severe side effects^ref. However, the fact that it can be more cost effective, and that there are reports of improved immune reconstitution with GM-CSF relative to G-CSF^{ref1, ref2, ref3, ref4}, has caused some clinicians to reconsider its use. Further, for the patient, or normal donor who has failed to mobilize adequately on G-CSF alone, the combination of GM-CSF with or followed by G-CSF has been shown to be efficacious^{ref1, ref2, ref3}. (Law P, Young D, Peterson S, Lane T, Ho A. Mobilization and collection of peripheral blood progenitor cells from normal subjects treated sequentially with GM-CSF and G-CSF. Blood. 1996;88:1578a)
• erythropoietin, now commonly used among cancer patients undergoing chemotherapy to maintain hemoglobin in the near normal range, also has some ability to mobilize CD34⁺ cells^ref. In the hard to mobilize patient, its use may therefore be doubly beneficial.
• stem cell factor (SCF) has been shown to be an excellent mobilizing agent, particularly when used in combination with G-CSF^ref. Unfortunately, the high incidence of allergic reactions to this agent and the need for observation after it is given have made it difficult to move into standard clinical practice. It is not currently available commercially in the US.
• pegfilgrastim and darbopoietin are now available and are in clinical trials as mobilizing agents. They have the benefit of very long half-lives and so add an important measure of patient convenience and the probability that timing of collection may be more flexible without sacrificing optimal collections.
• a new factor (AMD3100, AnorMed, Vancouver, Canada), which is a reversible inhibitor of the binding of stromal derived factor (SDF-1a) to its cognate receptor CXCR4, is currently in clinical trials as a mobilizing agent. It is the first agent to be tried for mobilization based on a rational understanding of its mechanism of action relative to HPC-stromal cell interactions. While it mobilizes CD34⁺ cells adequately on its own, it significantly improves the mobilization capacity of G-CSF when used in combination with G-CSF in mice. Clinical trials in humans with various diseases are in progress, including myeloma.
Remobilization : for the patient who fails to mobilize the necessary number of cells for transplant on the first attempt, but for whom it is clear that transplant is the best option, two decisions need to be made simultaneously: when to remobilize, and with what? When to remobilize is still a subject of debate, and depends to some extent on whether the failed mobilization was with chemotherapy plus growth factor or growth factor alone. Although at least one set of authors recommend immediate remobilization with growth factors for a patient who has not mobilized adequately after chemotherapy plus growth factor (Miclea J, Makki J, Lefrere F, et al. Successful PBPC harvesting with G-CSF alone a short time after previous mobilizataion failure by both chemotherapy and hematopoietic growth factors. Blood. 1999;94:1465a), others feel that at least 2–3 weeks off growth factor prior to remobilization offers the best chance of success.40–42 If adequate cells are given for an autologous transplant, it is even possible to collect enough for a second transplant during the period of white blood cell recovery following the first transplant (Schenkein D, O’Connor C, Morelli J, et al. A randomized trial of two different priming methods for stem cell mobilization in patients with relapsed Hodgkin’s disease and Non-Hodgkin’s lymphoma. Blood. 1999;94:1460a). It is also possible to collect HPCs at least 1 year after a prior transplant: of 38 myeloma patients who attempted such a collection, 36 achieved an adequate number of cells for transplant (Cottler-Fox et al, in preparation). What to use for a repeat mobilization attempt for a specific patient and disease may be a complex decision. However, some general guidelines may be found in the literature. First, mobilization with chemotherapy plus growth factor will generally yield more CD34⁺ cells than growth factor alone (for the single exception see #2 in the Algorithm for Mobilizing Myeloma Patients below)^{ref1, ref2}. Second, chemotherapy plus G-CSF and SCF is more effective than chemotherapy + G-CSF alone^ref. Further, chemotherapy with either sequential or concurrent GM-CSF plus G-CSF may be more effective than G-CSF alone^ref, as erythropoietin may improve the response to G-CSF^ref. Finally, retrospective analysis of data in myeloma patients from a single institution has shown that for patients who have undergone as many as 4 attempts at mobilization, only 1 of the 4 may yield an adequate collection. It is possible that AMD3100 will find a niche in this hard to mobilize population: in an ongoing trial at the University of Arkansas for Medical Sciences in myeloma patients who have previously failed to collect at least 5 x 10⁶ CD34⁺ cells/kg, it provides adequate mobilization in a significant number of patients. Although many variables may affect the decision of how and when to mobilize a myeloma patient, the following is a generally useful approach :
• attempt first collection relatively early, i.e., with < 12 months prior therapy.
• attempt collection first with chemotherapy plus growth factor (except for patients over 70 with > 12 months prior therapy and platelets < 200 x 10⁹/L, for whom growth factors alone may be tried first). The choice of growth factor (G-CSF versus GM-CSF) may depend on data under development regarding the importance of early immune reconstitution on time to progression and long-term disease-free survival. Pegylated filgrastim may replace standard G-CSF if studies show it to be equivalent or better than standard G-CSF.
• if mobilization is inadequate and
• a. patient is in CR or near-CR: wait at least 3 weeks, then remobilize with combination growth factors. In the future, AMD3100 may be a possibility.
• b. patient is not in CR or near-CR
: give planned chemotherapy and combine it with sequential GM-CSF and G-CSF. Consider adding erythropoietin. In the future, AMD3100 may be a possibility.
• if patient is not progressing, the procedure above may be repeated until adequate cells are collected.
• if patient is progressing, and adequate cells are available for autologous transplant (>= 3 x 10⁶ CD34⁺ cells/kg), monitor CD34⁺ cells in the blood at the time of leukocyte recovery, and consider collecting HPCs if there is adequate mobilization.
• if patient achieves a CR with transplant but does not mobilize adequately to collect during the leukocyte recovery phase post-transplant, consider attempting collection with combination growth factors at least 1 year after transplant.
• mobilization of autologous HSCs :  although early attempts to use unmobilized peripheral blood stem cells (PBSCs) for autologous stem cell transplantation were problematic, the use of cytokines such as G-CSF to enhance the peripheralization of CD34⁺ cells and the collection of these stem cells using leukapheresis procedures has become the standard for autologous stem cell transplantation around the world^ref. IBMTR and EBMT data suggest that over 80–90% of all autologous stem cell transplants in the world are performed using cytokine or chemotherapy/cytokine mobilized PBSCs as a source of stem cells^ref. In addition to reducing patient morbidity, the use of mobilized PBSCs has resulted in higher CD34 content of grafts, shorter hospital stays, and reduced engraftment times for both neutrophils and platelets as well as improved lymphocyte recovery resulting in enhanced immunologic reconstitution when compared to patients receiving autologous BM^{ref1, ref2, ref3, ref4}. These beneficial effects of mobilized PBSCs as a source of stem cells for autologous stem cell transplantation have been confirmed in a number of randomized trials^{ref1, ref2, ref3, ref4, ref5}.
• mobilization of allogeneic HSCs : based on the sustained success of using mobilized PBSCs for autologous stem cell transplantation, investigators began to pilot the use of PBSCs for allogeneic stem cell transplantation. Initial concerns focused on the possibility of increased risk of acute and chronic GVHD due to the presence of 10- to 50-fold increased numbers of T cells present in mobilized PBSC products. It was not clear if the function of mobilized allogeneic T cells might be qualitatively altered resulting in even greater risk of GVHD or relapse. In addition, the risk of infusing increased numbers of CMV⁺ granulocytes, dendritic cells, and monocytes into both CMV^- and CMV⁺ recipients remained unknown and potentially posed an increased risk to the recipient. On the other hand, mobilized PBSCs contain 3- to 4-fold more CD34⁺ cells, which might result in faster engraftment and more efficient transformation to complete donor chimersim. Early Phase 2 studies demonstrated that G-CSF had a generalized effect on the peripheralization of many different types of allogeneic peripheral blood cells, not just CD34⁺ cells. These data are consistent with the notion that G-CSF has a generalized effect on remodeling the BM microenvironment, which results in the egress of many types of cells including T cells and monocytes. Activation of neutrophils by cytokines such as G-CSF results in the release of proteases that facilitate the egress of HSCs from the BM microenvironment. Interruption of the G-CSF signal through genetically "knocking out" the G-CSF receptor results in not only the expected elimination of G-CSF-induced mobilization of HSCs but also IL-8- and chemotherapy-induced HSC mobilization in these G-CSF receptor knock-out mice^{ref1, ref2}. Although it is not completely clear which is the most important tether binding HSCs to the microenvironment, LFA-1, VLA-4, CXCR4, and c-kit have all been implicated as critical stem cell adhesion molecules^{ref1, ref2, ref3, ref4}. Likewise, a number of neutrophil-specific enzymes have been implicated in mediating critical cleavages that result in stem cell egress from the microenvironment. These include neutrophil elastase, cathepsin G, proteinase 3, gelatinase B (MMP-9), and other metalloproteinases^{ref1, ref2, ref3, ref4}. Recent evidence has strongly implicated CD26, a CD34-associated protease, as the prime protease that may cleave SDF-1 off the marrow microenvironment resulting in the release of CD34⁺ HSCs into the periphery^ref. Of interest, a collaborative effort of the Link, Simmons and Levesque laboratories have shown that mice deficient in MMPq, neturophil elastase and cathepsin-G and mice deficient in dipeptidyl peptidase I (CD26) all mobilized hematopietic precursors in response to G-CSF normally. These data question the role of neutrophil specific proteins in stem cell egress
• effects of G-CSF mobilization on allograft content : although a number of cytokines and cytokine combinations have been used to mobilize autologous HSCs, only filgrastim and sargramostim have been approved by the Food and Durg Administration (FDA) for use as autologous stem cell mobilizing agents. Thus, these have been the only cytokines used to mobilize allogeneic PBSCs. The majority of the initial Phase 2 studies using mobilized PBSCs in an allogeneic setting utilized G-CSF (10–16 µg/kg/day for 5 days). Leukapheresis was performed on day 4 or day 5 after G-CSF treatment. Fischmeister et al^ref followed CD34⁺ in the peripheral blood after either G-CSF or GM-CSF treatment and showed that CD34⁺ cells peaked in the blood between days +4 and +5 for G-CSF and days +5 and +6 after GM-CSF treatment. Data from Seattle suggested that larger doses of G-CSF (16 µg/kg/day) may result in even higher CD34 yields at the time of pheresis on day 5^ref. We have assessed the effect of 5 days of of G-CSF (10 µg/kg) on the numbers of leukocyte subsets in the peripheral blood of 100 consecutive normal allogeneic PBSC donors. It is clear from these data that G-CSF has a pleiotropic effect of increasing the numbers of circulating neutrophils and monocytes (WBC) as well as T cells (both CD4 and CD8), NK cells and B cells. Korbling and Anderlini compared the allograft content after G-CSF mobilization to cellular contents of BM harvests. These data suggest a 3- to 4-fold enhancement of CD34⁺ cells and a 10- to 20-fold increase in the number of CD3+ T cells in PBSC products compared to BM harvests^ref

It is well known that certain factors may help predict those autologous stem cell recipients who might be expected to be "poor mobilizers." These include extent of previous treatment, treatment with certain drugs such as nitrosoureas and certain diseases such as Hodgkin’s disease, non-Hodgkin’s lymphomas and preleukemic syndromes. No such data exist for allogeneic donors. We have examined stem cell mobilization from over 400 HLA-matched sibling donors since 1995. Using G-CSF as the sole mobilizing agent, only 2.0% of normal donors mobilized with G-CSF (10 µg/kg/day; 20 L exchange on day 5) did not achieve > 2 x 10⁶ CD34/kg and 25% did not achieve > 5 x 10⁶ CD34/kg after a single collection. These data have been recapitulated by other groups. We have studied the few normal donors who did not achieve > 1 x 10⁶ CD34/kg after 3 collections (1.0% of all normal donors in our data set). All of these donors underwent BM harvests as well but these all yielded < 1.0 x 10⁶ CD34 cells consistent with the notion that these poor allogeneic PBSC mobilizers were not defective in cytokine induced mobization per se but had low levels of BM stem cell reserves. Brown et al26 correlated premobilization PB CD34/mL with G-CSF-induced mobilization. None of the normal allogeneic donors who had < 2000 CD34/mL prior to mobilization yielded > 5 x 10⁶ CD34⁺ cells/kg while 95% of those normal donors with > 4000 CD34/mL PB yielded > 5 x 10⁶ CD34/kg after G-CSF mobilization. Although provocative, this has not been widely accepted as a method of identifying poor autologous or allogeneic PBSC donors. In the mid- and late-1990s, a large number of small Phase 2 studies were performed using mobilized PBSCs as a source of HSCs for allogeneic stem cell transplantation. All of these studies yielded similar results. Although neutrophil and platelet recovery was enhanced using cytokine mobilized allogeneic PBSCs, rates of acute GVHD were similar or less than that documented for BM as a source of allogeneic HSCs. The majority, but not all of these early studies, demonstrated increased actuarial rates of limited and extensive^{ref1, ref2, ref3} chronic GVHD (cGVHD). Cost and hospitalization appeared to be reduced compared to patients transplanted using allogeneic BM in these small Phase 2 studies. Similar to many autologous PBSC studies, the number of allogeneic CD34 cells infused correlated well with both neutrophil and platelet engraftment. Brown demonstrated that those allogeneic PBSC recipients who had > 5 x 10⁶ CD34/kg infused had a 95% chance of both neutrophil and platelet engraftment by day +15^ref. In this study, no correlation could be found between GVHD or survival and the number of CD3⁺ cells infused. In one retrospective study by the MD Anderson group, infusion of > 8 x 10⁶/kg CD34 resulted in decreased survival presumably due to increased rates of cGVHD associated complications^ref. These data have not been corroborated by other groups. Therefore, the infusion of high numbers (> 8 x 10⁶ CD34/kg) of allogeneic stem cells remains a controversial negative predictor for outcomes after allogeneic PBSC transplantation.
A major question is whether allogeneic stem cell harvesting results in less morbidity than BM harvesting for allogeneic stem cell donors. Anderlini et al^ref reviewed 1448 mobilized allogeneic PBSC collections from the IBMTR and EBMT registries. G-CSF was used in > 99% of donors. Similar to multiple Phase 2 studies, 20% of normal donors required placement of a central line for apheresis. 11% required > 2 leukapheresis procedures and "serious complications" occurred in 1.1% of allogeneic PBSC collection versus 0.5% after BM harvest. Rowley et al^ref utilized an 11-point scale (0 = minimum and 10 = maximum) for 23 different symptoms occurring during the first 14 days after either BM or PBSC collection. There were no statistical differences between the PBSC and BM groups for any of the symptom complexes or for the severity of any symptoms.
Many of the Phase 2 studies assessing the role of mobilized allogeneic PBSCs on GVHD, relapse and overall survival are limited by design (Phase 2), paucity of patients, short-term follow-up, and heterogeneity of diseases for which allogeneic PBSC transplantation was performed. Several trials have provided insight into the relative effect of PBSC versus BM on GVHD, relapse, and survival. These include randomized studies, case control retrospective studies, and meta-analyses. Table 1 summarizes the results of all randomized trials comparing BM and PB as a source of stem cells for allogeneic stem cell transplantation^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. Half of these trials suffer from low numbers of patients. The 3 largest studies^{ref1, ref2, ref3} demonstrate no significant difference in overall survival when peripheral blood is compared to BM as a source of allogeneic stem cells. These studies, similar to the smaller randomized trials, did demonstrate a significant enhancement in both neutrophil and platelet recovery consistent with the significantly increased numbers of CD34 cells that are harvested in mobilized allogeneic PBSC products compared with BM. Randomized trials comparing allogeneic peripheral blood to bone marrow :

study	n	absolute neutrophil count (ANC) (engraftment (days) ANC > 500/mm3)		PLT (engraftment (days) Plt > 25000/mm3)		TRM		aGvHD		cGVHD		OS
		PB	BM	PB	BM	PB, %	BM, %	PB, %	BM, %	PB, %	BM, %	PB, %	BM, %
Vigoritoref	37	16	18	12	17	78	63	27	19	100	50	47	51
Blaiseref	101	15	21	13	21	23	21	44	42	50	28	61	61
Powlesref	39	17.5	23	11	18	31	35	68	58	44	40	70	68
Heldalref	61	17	23	13	21	17	10	21	10	56	27	80	73
Schmitzref	350	12	15	15	20	ND	ND	52	39	74	53	ND	ND
Coubanref	228	19	22	16	22	7.5	16	40	40	71	55	68	55
Bensingerref	172	16	21	13	19	21	30	64	57	46	35	66	54

In one of the largest and best-designed studies, Bensinger et al^ref, using identical conditioning regimens, GVHD prophylaxis (cyclosporine and methotrexate), and post-transplant growth factor support (no G-CSF), found a slight advantage in both disease-free and overall survival in those patients receiving mobilized allogeneic stem cells versus BM (P = .03 and P = .06, respectively). Although there was no difference in probability of 2-year overall survival in the subgroup of patients with less advanced disease (75% for PB and 72% for BM), those patients with more advanced disease demonstrated a significantly enhanced overall survival when mobilized allogeneic PBSCs were used as a source of stem cells (57% for PB and 33% for BM; P = .04). With a median follow-up for all surviving patients of 26 months (9–47 months), the cumulative incidence of grade III–IV acute GVHD at 100 days was 64% in the PB group and 57% in the BM group (P = .35). The cumulative incidence of grade II–IV acute GVHD was 15% in the PB group and 12% in the BM group (P = NS). Although the follow-up was relatively short, the cumulative incidence of extensive cGVHD was 46% in the PB group and 35% in the BM group (P = .54). These results were inconsistent with many of the other smaller Phase 2 and Phase 3 studies, which all showed no difference in rates of acute GVHD and increased rates of cGVHD in recipients of allogeneic PBSC.
A recent meta-analysis was performed by Cutler et al^ref summarizing 15 Phase 2 and Phase 3 trials assessing the risk of GVHD in recipients of allogeneic PBSC and BM. This analysis demonstrated a modest increased relative risk of acute GVHD (RR = 1.2) and a significant increased risk of developing cGVHD (RR = 1.8) in recipients of allogeneic PBSCs. It also demonstrated a modest reduction in relative risk of relapse in recipients of allogeneic PBSCs compared to BM (RR = 0.8). Champlin et al and the IBMTR40 performed a retrospective case controlled study comparing the outcomes of recipients of allogeneic PBSCs and BM. Median follow-up was 1 year, and this study focused on 1-year outcomes. A total of 288 HLA-identical sibling PBSC recipients was compared with 536 case control allogeneic BM recipients. All patients received T-replete stem cell products. There was no significant difference in the incidence of grades II–IV acute GVHD (40% for PB and 35% for BM; P = NS) or grades III–IV acute GVHD (13% for PB and 19% for BM; P = NS). There was less variability in recovery times of both platelets and neutrophils after allogeneic PBSC compared to BM and statistically faster neutrophil and platelet recovery after allogeneic PBSC compared to allogeneic BM. Treatment-related mortality (TRM), disease-free survival, and overall survival were similar for patients with acute leukemia in first remission. In contrast, acute leukemia patients in second remission and patients with CML in accelerated phase experienced lower TRM, improved DFS and overall survival when allogeneic PBSCs were used as a source of stem cells. There was no apparent difference in the risk of relapse after allogeneic PBSC versus BM transplantation. There was a trend toward lower relapse rates in patients with high-risk leukemia (acute leukemia in second remission and CML in accelerated phase). The relative risk of both limited and extensive cGVHD was increased in recipients of allogeneic PBSCs (RR = 1.3). Mohty et al^ref have performed the only long-term follow-up of allogeneic PBSC and BM recipients focusing specifically on the rates of cGVHD. At a median follow-up of 45 months (range 31–57 months), the 3-year cumulative incidence of cGVHD was 65% in the PBSC group (n = 53) and 36% in the BM group (n = 48) (P = .004). Extensive chronic GVHD was also more frequent in the PBSC group (44% versus 17%; P = .004). These data suggest that although hematopoietic recovery is increased in recipients of allogeneic PBSC, there appears to be no increased risk of acute GVHD and a modest increase risk of cGVHD including extensive cGVHD. Overall survival in recipients of allogeneic PBSCs may be improved modestly but only in those patients with more advanced hematologic malignancies. Although no study has correlated rates of either acute or chronic GVHD with the number of CD3 cells/kg in the stem cell products, several studies have suggested that CD34 cells in excess of 8 x 10⁶/kg found in allogeneic PBSC products are associated with a greater risk of both acute GVHD and GVHD.
Impact of G-CSF mobilization on graft content and immune reconstitution : very little data exist on the relative impact of allogeneic PBSCs versus BM on immunologic reconstitution after allogeneic stem cell transplantation. Storek et al^ref analyzed the incidence of documented and suspected infections after transplantation of mobilized allogeneic PBSCs and BM in the randomized trial carried out by Bensinger et al^ref. The cumulative incidence of infections was higher in the allogeneic BM group (120 versus 90 at 1 year). Since rates of acute GVHD after allogeneic PBSC infusions are similar to BM in spite of these products having 10- to 50-fold increased CD3 cells/kg over allogeneic BM products, a number of investigators have tried to understand the reason for this. To date, no studies have clearly shown a difference in either B-cell or T-cell recovery after allogeneic PBSC transplantation compared to BM transplantation. A number of reports have emphasized the role of cytokines as mediators of GVHD. Cytokines produced by both CD4 and CD8 T cells can be segregated into two patterns: type I cytokines such as interferon- and IL-2 and type 2 cytokines, such as IL-4 and IL-10. Type 1 cytokines are proinflammatory and type 2 cytokines are considered anti-inflammatory. Multiple studies have shown that T cells that elaborate T_h1 cytokines  mediate GVHD whereas those T cells that elaborate type 2 cytokines (T_h2 cells) inhibit GVHD. Pan et al^ref demonstrated that splenocytes from mice mobilized with G-CSF were polarized toward the T_h2 phenotype. Those mice who received splenocytes from G-CSF-mobilized donor mice demonstrated significantly longer survival and less GVHD that those allogeneic transplant recipient mice who were infused with splenocytes from naïve unmobilized donor mice. T cells from G-CSF treated mice showed a significant increase in IL-4 production with a simultaneous decrease in IL-2 and interferon- production. This polarization persisted in secondary mixed lymphocyte reactions (MLR) despite the absence of G-CSF during in vitro MLR. Arpinati et al hypothesized that G-CSF-mobilized PBSC contained antigen-presenting cells which prime T cells to produce T_h2 cytokines^ref. 2 distinct lineages of dendritic cells (DC) have been described in humans. DC1 cells or myeloid DCs express HLA-DR, CD11c, CD13, and CD33 and require GM-CSF for their survival. These cells are negative for both myeloid and lymphoid specific markers (Lin-), produce high levels of IL-12 when stimulated with tumor necrosis factor (TNF) or CD40 ligand and drive the differentiation of naïve T cells into the T_h1 phenotype. DC2 or lymphoid DC are HLA-DR⁺/CD11c^-/CD4⁺/IL-3Ra⁺ express high levels of T-cell receptor  chain and depend on IL-3, and not GM-CSF, for their survival and differentiation. After appropriate activation, they can induce T-cell differentiation into T_h2 cells. These investigators studied the effects of G-CSF mobilization (10–16 µg/kg/day for 5 days) on DC content in the peripheral blood in these allogeneic donors. G-CSF treatment was found to mobilize DC2 but not DC1. Although the numbers of donors and controls studied were very small, the median number of DC1 per liter in the G-CSF group was not different (11 versus 10 x 10⁶/liter; P = .52) than in the control premobilization group. In contrast, the numbers of DC2 were significantly increased in the G-CSF mobilization group compared with control (median 24.8 versus 4.9 x 10⁶/liter; P = .0009). As expected, a proliferative response of naïve allogeneic T cells could be detected in vitro to fresh DC1 but not to fresh DC2. Activation of DC2 in vitro with TNF, GM-CSF, and IL-3 resulted in the rapid maturation of these precursors to mature DC2 cells overexpressing costimulatory molecules such as CD80 and CD86 restoring their ability to induce a proliferative response to naïve CD4⁺/CD45RA allogeneic T cells. Incubation of naïve allogeneic T cells with DC1 resulted in polarization of these T cells toward the T_h1 phenotype as measured by restimulation of these T cells with PMA and ionomycin and detecting primarily IL-2 and interferon- as the major intracellular cytokines produced after restimulation. In contrast, incubation of naïve T cells with DC2 cells results in the polarization of these T cells toward the T_h2 phenotype as noted by the intracellular accumulation of IL-4 and IL-10 after restimulation in vitro with PMA and ionomycin. In conclusion, G-CSF mobilization results in stem cell products with 10- to 50-fold more T cells and 4- to 6-fold more DC2 cells. The increased numbers of DC2 in G-mobilized products may reduce the relative risk of acute GVHD as seen in preclinical murine allogeneic transplant studies described above and observed in the initial Phase 2 and 3 clinical trials in humans comparing BM versus mobilized PBSCs as sources of allogeneic stem cells. It is of interest that cord blood stem cell products, which are associated with a low risk of severe acute GVHD, contain primarily DC2 and negligible DC1^ref. Consistent with this notion, Waller found a strong inverse correlation with the number of precursor DC2 infused in allogeneic bone marrow and the incidence of both cGVHD and, more importantly, relapse^ref.
Alternative allogeneic PBSC mobilization regimens : although other cytokines, in addition to G-CSF, have been used to mobilized autologous PBSCs from humans including GM-CSF, Flt-3 ligand, stem cell factor (SCF), Daniplestim (IL-3 agonist), thrombopoietin agonists, chimeric cytokines including Leridistim (IL-3 agonist-G-CSF chimeric molecule) and Progenipoietin-1 (Flt-3 ligand-G-CSF chimeric molecule), peg-filgrastim (NeulastaTM) and SDF-1 antagonist (AMD 3100), only G-CSF and GM-CSF have been approved by the FDA and only G-CSF has been studied extensively for the mobilization of allogeneic PBSCs in humans. We have performed several sequential nonrandomized trials to determine the comparative effects of allogeneic PBSCs mobilized with G-CSF at 10 µg/kg/day (n = 96), G-CSF (10 µg/kg/day) combined with GM-CSF at 5 µg/kg/day (n = 102) or GM-CSF alone (10 or 15 µg/kg/day (n = 32). All donors underwent leukapheresis (20 liter) on the fifth day of cytokine administration. The target CD34⁺ content was 5.0 x 106 CD34/kg with a minimum of 2 x 10⁶/kg. PBSCs mobilized with G/GM resulted in collection of grafts with similar CD34 content compared with G alone. Grafts obtained following mobilization with GM alone contained significantly fewer CD34⁺ cells, but sufficient numbers for rapid engraftment. Grafts mobilized with GM or G/GM contained significantly fewer T and NK cells. There were no obvious differences in donor toxicities including bone pain. All recipients of GM mobilized cells engrafted with kinetics similar to recipients of G and G/GM, although neutrophil recovery was delayed about 1 day. Rates of neutrophil and platelet recovery and cGVHD for the G and GM groups are shown in Table 3. In 30 evaluable recipients of peripheral blood grafts mobilized with GM alone, the actuarial risk of grades 2–4 acute GVHD was only 0.13 ± 0.05 and 0.00 (0/31) for grades 3–4 acute GVHD. In a multivariate analysis including patient and donor age, sex mismatching, conditioning regimen received, CD3⁺ cell dose and CD34⁺ cell dose, only the receipt of PBSCs mobilized with GM-CSF alone correlated with a lower risk of grades 2–4 acute GVHD. These data suggest that altering the mobilization regimen and cytokines used may alter the functional aspects of the graft thereby modifying outcomes such as GVHD. Randomized Phase 3 studies will need to be performed to more accurately dissect the phenotypic differences in grafts mobilized by different cytokines/chemokines or combinations and to more accurately assess the impact on important endpoints such as multilineage engraftment, disease-free survival, overall survival, GVHD, and relapse. Comparison of G versus G/GM versus GM mobilization on allograft content :

parameter	G alone	G/GM	GM alone	P value
# donors	96	102	32
Total # LP procedures required	1.26 ± 0.5	1.20 ± 0.5	1.6 ± 0.7	G vs G/GM: .5; G vs GM: .002
CD34 (x 106/kg)	9.8 ± 7.2	10.9 ± 7.9	3.7 ± 2.0	G vs G/GM: .28; G vs GM: < .001
CD3 (x 108/kg)	3.5 ± 2.4	1.8 ± 1.5	1.8 ± 1.8	G vs G/GM: < .0001; G vs GM: < .001
CD4 (x 108/kg)	2.3 ± 1.4	1.3 ± 1.0	0.9 ± 0.8	G vs G/GM: < .0001; G vs GM: < .001
CD8 (x 108/kg)	1.1 ± 0.7	0.4 ± 0.4	0.3 ± 0.26	G vs G/GM: .56; G vs GM: .002
CD19 (x 108/kg)	0.97 ± 0.9	0.89 ± 0.63	0.45 ± 0.4	G vs G/GM: .56; G vs GM: .002
CD16/56 (x 107/kg)	4.63 ± 3.7	2.98 ± 2.36	1.66 ± 1.18	G vs G/GM: < .001; G vs GM: < .0001

Effect of cytokine mobilization regimen on graft-versus-host disease (GVHD)/survival :

	G-CSF, % n = 40	G-CSF and GM-CSF, % n = 66	GM-CSF, % n = 30	P Value
acute grade 2–4	51	65	13	G vs GM: .003 GM vs G/GM: < .0001 G vs G/GM: .11
acute grade 3–4	11	13	0	.07
chronic GVHD	86	88	65	NS
survival (2 years)	62 high 38 low 88	56 high 28 low 5	59 high 35 low 69	NS

Chemokines such as IL-8 have been used to induce the egress of HSCs into the peripheral blood of mice and nonhuman primates. This effect is rapid (30 minutes-4 hours) and may result from the ability of these chemokines to induce the release of a protease from mature myeloid cells resulting in a decrease in the intramedullary concentration of SDF-1, the ligand for the receptor CXCR4, which is expressed in many cells including hematopoietic stem cells^{ref1, ref2}. Increasing evidence points to the critical role of the CXCR4/SDF-1 axis in both murine and human stem cell mobilization. The bicyclam molecular AMD 3100 was first clinically developed for its potent and selective inhibition of HIV type 1 and 2 replication through binding to the chemokine receptor, CXCR4. Initial clinical trials in AIDS patients demonstrated that AMD-3100 induced a rapid (within 1 hour) increase in both WBC and circulating progenitor cells. Broxmeyer and colleagues demonstrated a 40-fold increase in the mobilization of hematopoietic progenitors within 1 hour of AMD-3100 injection in mice^ref (Broxmeyer HE, Hangoc G, Cooper S, Bridger G. Interference of the SDF-1/CXCR4 axis in mice with AMD3100 induces rapid high level mobilization of hematopoietic progenitor cells and AMD3100 acts synergistically with G-CSF and MIP-1 alpha to mobilize progenitors. Blood. 2001;96:3371a). Studies in human volunteers and the first Phase 1 and 2 clinical trials in patients undergoing autologous stem cell transplantation are currently underway (Devine S, Flomenberg N, Vesole D, et al. Safety and effect on WBC count and CD34 cell mobilization following administration of AMD3100 to patients with multiple myeloma or non-Hodgkins lymphoma. 8th Congress of the European Hematology Association, Abstract 0406, 2003). These studies have shown a consistent and rapid impact on stem cell mobilization when given alone and a synergistic effect on CD34⁺ mobilization when coadministered with G-CSF. Since CXCR4 is expressed on different types of cells including CD34⁺ progenitor and T cells, the impact of AMD-3100 on both multilineage engraftment and GVHD will need to be appropriately explored in preclinical animal models before it can be safely used as a rapid mobilizing agent for allogeneic PBSC transplantation in humans.
• umbilical cord blood stem cell transplantation (UCBSCT / UCBT)^{ref1, ref2}
• in 1972 Mark Ende first used cord blood to treat a patient with leukemia (Ende M, Ende N. Hematopoietic transplantation by means of fetal (cord) blood. Va Med Mon (1918) 1972;99:276-80)
• in 1989 Gluckman first successfully transplanted cord-blood stem cells, which are immature and less likely to cause GVHD. It was observed that the numbers of progenitor cells present in the low-density (< 1.077 gm/ml) fraction after Ficoll/Hypaque separation typically fell within the range that has been reported for successful engraftment by bone marrow cells. Another observation of practical importance is that procedures to remove erythrocytes or granulocytes prior to freezing, and washing of thawed cells before plating, entailed large losses of progenitor cells, the yield of unwashed progenitor cells from unfractionated cord blood being many times greater. The provisional inference is that human umbilical cord blood from a single individual is typically a sufficient source of cells for autologous (syngeneic) and for MHC-matched allogeneic hematopoietic reconstitution^ref.
• cord blood banks (CBBs) are not always closely located to a GMP-licensed microbiology laboratory, resulting in time delays for transport of specimens prior to microbiological testing : inoculation of culture bottles as soon as possible after sample preparation is preferable. Bacteria can maintain viability in BacT/ALERT bottles inoculated and held at RT for up to 7 days prior to automated culture testing. Bacteria can be successfully recovered from cryopreserved UCB^ref
• liquid storage of cord blood for 24 h due to delays prior to RBC depletion and cryopreservation does not adversely influence the post-thaw cell recovery but the post-thaw mononuclear cell recovery and viability of cord blood stored for 72 h was significantly lower. The use of dilution with a storage solution (STM-Sav with 1:1 ratio) enhances the retention of colony-forming capabilities post-thaw^ref
• a small proportion of  high proliferative potential (HPP)-Q cells recovered from 7-day expansion cultures of of UCB HSCs in the presence of TPO, SCF, flt-3 ligand (FL) and IL-6 retain their multilineage potential: longer culturing of these cells results in the loss of multilineage potential while they maintain quiescent behavior and high proliferative potential^ref.
• as a source of HSCs, UCB has the advantages of speed of availability, tolerance of 1-2 antigen HLA mismatch, and a low incidence of severe GVHD. Thus, UCB represents a highly convenient HSC source that may significantly extend the HSC donor pool. UCBT has become a standard practice in the treatment of pediatric malignancies. Now, UCBT is being investigated in adults using both conventional and non-myeloablative preparative regimens. As graft cell dose is the major factor determining hematopoietic recovery and survival in URD UCBT and cord blood has a limited number of stem cells (each sample is < 200 mL (10-20-folds less than a conventional bone marrow sample) and is unlikely to contain enough stem cells for an efficient transplant (usually 1 x 10⁹ nucleated cells = 0.5 x 10⁶ CD34⁺ cells/kg), its use has largely been restricted to children : methods to increase cell dose such as multiple-unit transplant and ex vivo expansion are being pursued^ref. CD26/dipeptidylpeptidase IV (DPPIV) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N terminus of polypeptide chains. The N terminus of chemokines is known to interact with the extracellular portion of chemokine receptors, and removal of these amino acids in many instances results in significant changes in functional activity. CD26/DPPIV has the ability to cleave the chemokine CXCL12 / SDF-1a at its position Pro². CXCL12/SDF-1a induces migration of HSCs and progenitor cells, and it is thought that CXCL12 plays a crucial role in homing/mobilization of these cells to/from the bone marrow^ref. In mice upon treating mice bone marrow HSCs for 15 minutes with a CD26 inhibitor (Diprotin A), a 9-fold increase in homing efficiency is measured. Similarly, cells in which CD26 had been deleted (CD26^-/-) showed a 11-fold increase. The treatment of low-density bone marrow donor cells, which more accurately resemble stem cells used in a clinical setting, also increased homing efficiency. Furthermore, CD26^-/- cells made a more significant contribution to long-term donor HSCs engraftment in lethally irradiated mice, with fewer CD26^-/- HSCs being required for mouse survival compared with normal HSCs^ref.
• cell dose is a critical determinant of outcomes in unrelated cord blood (CB) transplantation. A strategy was investigated in which CB units should contain at least 2 x 10⁷ total nucleated cells/kg of recipient weight, otherwise a second unit had to be added. In a study that was prematurely closed owing to toxicity, patients with advanced hematologic malignancies without a human leukocyte antigen-matched sibling or unrelated donor were eligible. Conditioning regimen consisted of fludarabine and 12 Gy of total body irradiation (n=11), or melphalan (n=4), with antithymocyte globulin. GvHD prophylaxis was tacrolimus and methotrexate. 15 patients with acute leukemia (n=9), CML (n=2), MM (n=2) and lymphoma (n=2) were treated; 60% had relapsed disease at transplantation. 3 patients received double CB transplants. The 100-day and 1-year treatment-related mortality rates were 40 and 53%, respectively. Median time to neutrophil and platelet engraftment was 22 days (n=10) and 37 days (n=10), respectively. 1 patient had secondary graft failure and 5 patients failed to engraft. 2 patients are alive and disease free; 4-year actuarial survival is 33 versus 0% for patients transplanted in remission versus in relapse. Disease status was the main determinant of treatment failure in this study^ref
Indications :
• Arteriocyte (Cleveland, Ohio) : allogeneic cord stem cell transplant for ischemic disease (preclinical studies)
• Chemokine Therapeutics (Vancouver, Canada) : CTCE-0214 (chemokine agonist for ex vivo cord-blood stem-cell expansion) for blood disorders (phase 1 trial)
• Gamida-Cell (Jerusalem) : StemEx (allogeneic ex vivo expanded cord-blood stem cells combined with unexpanded cells Blood cancers Phase 1/2
• Histostem (Seoul, Korea) : developing technologies for expanding cord-blood stem-cells & for mixing cord blood for transplant Multiple indications including spinal cord injury, type 2 diabetes, liver cirrhosis (phase 1/2 trial)
• Saneron CCEL Therapeutics (Tampa, Florida) U-Cord-Cell: umbilical cord-blood stem-cell platform for myocardial fraction (preclinical studies)
• ViaCell (Cambridge, Massachusetts) : CB001: allogeneic ex vivo expanded cord-blood stem cells combined with unexpanded cells for blood cancers (phase 1/2 trial)
UCB cells coinfused through the jugular vein with a blood-brain barrier (BBB) permeabilizer (mannitol) into a stroke animal model in the first hours and days following a stroke, decrease stroke size by 40% and resulting disability is significantly reduced (source : Saneron CCEL Therapeutics)^ref
The first known attempt at UCBT occurred in 1970 when a young 16-year-old boy with acute lymphoblastic leukemia received cord blood units from 8 different donors. He was receiving 6-mercaptopurine and prednisone and did not receive a preparatory regimen. One unit engrafted as demonstrated by red cell antigens and the graft lasted for 38 days^ref. However, it was the success of the allogeneic UCBT approximately 16 years ago in a child with Fanconi anemia that opened a new source of allogeneic HSCs^ref. UCB is now accepted as an alternative source for hematopoietic stem cells for transplantation in children.
Advantages :
• the relative ease of procurement and availability (given the ability to store fully tested and HLA-typed cord blood available for immediate use)
• the absence of risk for mothers and donors
• the reduced likelihood of transmitting infections, particularly cytomegalovirus
• potential reduced risk of GVHD
• less stringent criteria for HLA matching for donor-recipient selection (with the potential of finding donors for minority populations)
• absence of donor attrition.
Contraindications to UCB stem cell collection :
• preterm delivery
• dystocic delivery
• newborn malformations of hereditary diseases
• previous maternal HCV, HBV, HIV or syphilis infection
• fever of unknown origin in the mother
• vaginal sampling positive for Streptococcus
• rupture of amniotic bag for > 24 hrs
UCB banks have been established for related and unrelated UCBT with about 100,000 units currently available^{ref1, ref2, ref3, ref4}. While the clinical data are encouraging for pediatric patients, the limited number of hematopoietic stem cells in UCB has been a cause for caution for its use in adult patients. Moreover, the use of myeloablative preparatory regimens with their known toxicities together with the known delays in engraftment of all cell lineages and the enrollment of high-risk patients with advanced disease all contributed to this caution. Recently, older allogeneic recipients have been treated successfully following a variety of less intense nonmyeloablative conditioning regimens. On the basis of these encouraging observations, it has been hypothesized that a reduced-intensity preparative regimen would allow engraftment of the UCB stem cells. While the total numbers of mononuclear cells are limited, the progenitor content and the proliferative potential of cord blood cells are high. This nonmyeloablative approach could diminish some of those concerns raised with the use of ablative regimens
• myeloablative preparative regimens : in comparison to the published studies on pediatric patients, the clinical data on the use of UCBT in adult patients are relatively limited, although growing^{ref1, ref2, ref3, ref4, ref5} (Rocha V, Arcese W, Sanz G, et al. Prognostic factors of outcome after unrelated cord blood transplant (UCBT) in adults with haematologic malignancies [abstract]. Blood. 2000;96:587a; Goldberg SL, Chedid S, Jennis AA, Preti RA. Unrelated cord blood transplantation in adults: a single institution experience [abstract]. Blood. 2000;96:208a; Cornetta K, Laughlin M, Carter S, et al. Umbilical cord blood transplantation in adults: results of a prospective, multi-institutional, NHBLI sponsored trial [abstract]. Blood. 2002;100:42a). As can be gleaned from the composite data, these patients received standard total body irradiation or busulfan-based myeloablative regimens. GVHD prophylaxis consisted of cyclosporine alone or in combination with steroids or methotrexate. The degree of HLA disparity was not associated with outcome in any of these studies, although the numbers in each were small. However, in a recent review update with 861 unrelated UCBT recipients from the Placental Blood program of the New York Blood Center, which includes 181 (21%) patients with age  18 years and 170 patients (20%) weighing  60 kg, Rubinstein et al (Rubinstein P, Carrier C, Carpenter C, et al. Graft selection in unrelated placental/umbilical cord blood (PCB) transplantation: influence and weight of HLA match and cell dose on engraftment and survival [abstract]. Blood. 2000;96:588a) have demonstrated in a multivariate analysis that HLA match is an independent predictor of event-free survival. Studies of unrelated umbilical cord blood (UCB) transplantation in adults :

		Laughlin et alref	Sanz et alref	Rocha et alref (Rocha V, Arcese W, Sanz G, et al. Prognostic factors of outcome after unrelated cord blood transplant (UCBT) in adults with haematologic malignancies [abstract]. Blood. 2000;96:587a) (Eurocord)	Goldberg et al (Goldberg SL, Chedid S, Jennis AA, Preti RA. Unrelated cord blood transplantation in adults: a single institution experience [abstract]. Blood. 2000;96:208a)	Cornetta et al (Cornetta K, Laughlin M, Carter S, et al. Umbilical cord blood transplantation in adults: results of a prospective, multi-institutional, NHBLI sponsored trial [abstract]. Blood. 2002;100:42a)  (COBLT)	Ooi et alref	Ooi et alref13	Long et alref14
number of patients		68	22	108	19	34	18	13	57 (19 of the 57 patients were included in the study published by Laughlin et al.)
median age (range, years)		31.4 (17.6–58.1)	29 (18–46)	26 (15–53)	48 (20–59)	34.5 (18.2–55)	43 (21–52)	40 (20–51)	31 (18–58)
median weight (range, kg)		69.2 (40.9–115.5)	70 (41–85)	60 (35–110)	69 (52–126)	NA	55.2 (NA)	51 (43–68)	70 (46–110)
diseases (n)	hematological malignancies	54	21	96	18	32	18	13	50
	bone marrow failure syndrome/MDS	13	1	12	1	2			6
number of HLA loci disparity (n)	0	2	1	6	4	1	0	0	2
	1	18	13	38	8	10	4	4	8
	>= 2	48	8	64	7	23	14	9	47
preparative regimen (n)		TBI-based/ATG  (n = 51) Bu-based/ATG  (n = 14)  Others/ATG (n = 4)	Thio/Bu/Cy/ATG  (n = 21)  Thio/Flu/ATG  (n = 1)	NA	Cy/TBI/ATG  (n = 13)  Mel/TBI/ATG  (n = 4)  Bu/Cy (n = 2)	Cy/TBI  (n = 27)  Bu/Mel  (n = 7)	TBI/Ara-C/Cy  (n = 17)  TBI/Ara-C/Flu  (n = 1)	Cy/TBI/Ara-C  (n = 13)	Cy or Mel with  TBI or BU/  ATG n = 41  thoraco-abdominal radiation (TAI)/Cy/ATG (n = 1)
GVHD prophylaxis		CSA/Pred (n = NA)  CSA (n = NA)	CSA/Pred (n = 22)	CSA/Pred (n = 77)  Others (n = 31)	CSA/Pred (n = 13)  FK506/Pred (n = 6)	NA  CSA/MTX (n = 16)	CSA (n = 2)	CSA/MTX (n = 13)	CSA (n = 1)  CSA/Pred (n = 56)
median time to engraftment (range, days)	ANC > 500/µL	27 (13–59)	22 (13–52)	32 (13–60)	28 (NA)	28.5 (13–55)	23 (16–41)	22.5 (19–35)	26 (12–55)
	Platelet > 20,000/µL	58 (35–142)	69 (49–153)	129 (26–176)	56 (NA)	NA	49 (31–263) (platelet > 50,000/µL)	49 (30–164) (platelet > 50,000/µL)	84 (35–167)
probability of myeloid engraftment		90% by 42 days	100% at 60 days	81% by day 60	75% by day 60	72% at day 60	94%	92%	80% at day 50
GVHD (n) (probability, %)	acute grade II–IV	33 (60%)	16 (NA)	44 (38)	NA	11 (38%)	10 (55%)	9 (9 of 12 evaluable patients had acute acute graft-versus-host disease)	17 (41%)
	acute grade III–IV	11 (20%)	7 (NA)	27 (NA)	NA	6 (21%)	1 (6%)	-	9 (22%)
	chronic/patients at risk	12/33	9/10	15/58	NA	NA	14/18	8/11	8/25
median cell dose (range)	N.C. infused (x 107/kg)	1.6 (0.6–4)	1.71 (1.01–4.96)	1.71(0.2–6)	1.8 (0.4–5.3)	1.73 (1.11–3.75	2.51 (1.16–5.29)	2.43 (2.09–4.06)	1.5 (0.54–2.78)
	CD34+ cells infused (x 105/kg)	1.2 (0.2–16.7)	0.79 (0.27–2.60)	NA	NA	NA	NA	NA	1.37 (0.02–12.45)
therapy-related mortality (%)		50% at 100 day	43% at 100 day	54% at 100 day	NA	NA	6%	0%	56%
survival (%)		NA	NA	27% at 1 year	20% for good risk  and 21% for poor risk  at 1 year	30% at 180 days	76% at 2 years	76.2% at 2 years	19% at 3 years
event-free survival (%)		26% at 40 months (EFS is better in patients receiving graft with CD34+ cells >1.2 x 105/kg)	53% at 1year (patients under the age of 30 had significantly better survival)	21% at 1 year (100 day TRM is lower in patients with disease in chronic phase or remission, N.C.dose infused  2.0 x 107/kg and transplant performed after January 1998)	NA (10 patients received ex vivo expanded UCBT)	NA (the high mortality and relapse reflects the poor risk patients enrolled)	77% at 2 years (all de novo AML)	NA (all except 1 patients received UCB transplant as an upfront treatment and all patients received > 2 x 107 N.C. per weight, perhaps due to smaller size of patients (median weight 51 kg))	15% at 3 years (CD34+ cell dose infused correlated with rate of platelet recovery and age > 31 years was significant predictor of poorer event-free survival)

The median age varied considerably from a low of 29 to a high of 48 years with a range of 15 to 58 years. Age is an important factor in the outcome, with the younger patients generally doing better than the older patients. The median weight for these studies also varied from a low of 51 to 70 kg. As the cell dose of the UCB is given per recipient weight, the patients with lower body weights would have a higher chance of receiving a higher cell dose per cord blood unit. The range of mononuclear cells/kg recipient weight ranged from 1.5 to 2.43 x 10⁷/kg. The total numbers of CD34⁺ cells has been correlated with both engraftment and the speed of engraftment. The diseases for which these patients were transplanted consisted primarily of hematological malignancies. The differences in outcome can be partially attributed to the proportion with more favorable disease status at transplantation. For example, most of the data from Sanz et al^ref or Ooi et al^ref included recipients with chronic myeloid leukemia (CML) in first chronic phase or acute leukemia in first remission or de novoacute myeloid leukemia or untreated myelodysplastic syndrome. In contrast, the less favorable survival in the other studies reflects the poor-risk subjects enrolled into the study. The probability of myeloid engraftment ranged from 72% to 100% by day +60; however, some patients were censored to this event if they died prior to the set date for the definition of engraftment. The median times to engraftment of both neutrophils and platelets varied as well with approximately 1 month to reach 500 neutrophils/mL and 2–4 months to reach platelet counts > 20,000/µL. Again, the pace of recovery is likely to reflect the UCB stem cell dose as well as the amount of prior therapies a patient could have received. As compared to the pediatric patients, the incidence of severe grade III-IV acute GVHD was low (approximately 20%) given that the majority of the patients received HLA mismatched unrelated grafts. Of note, the HLA matching in general was done serologically for HLA-A and-B and molecularly for HLA-DR. Thus it is likely that the mismatching would have been greater than reported if molecular matching had been performed. Therapy-related mortality (TRM) was high, reaching approximately 50% in most series. The overall survival again varied considerably, reflecting the risk factors outlined above. Comparative studies of UCB transplantation versus unrelated bone marrow have been performed from two registry datasets. These two studies have demonstrated that if there is a cord blood unit that is at least a 4/6 HLA match with sufficient cells, the outcomes are similar following UCB transplantation to matched or one antigen mismatched unrelated donor bone marrow grafts^ref (Laughlin M, Rubinstein P, Stevens C, et al. Comparison of unrelated cord blood and unrelated bone marrow transplants for leukemia in adults: a collaborative study of the International Bone Marrow Transplant Registry and the New York Blood Center. Blood. 2003;103:857). Thus, UCB contains sufficient numbers of hematopoietic stem cells to achieve engraftment in adult patients with lower than anticipated risk of severe acute GVHD, even when HLA-disparate grafts are infused. The use of UCB allows allografting to be offered to a greater proportion of patients, many of whom do not have a matched sibling or unrelated donor to allow allogeneic therapy as the only chance to cure the underlying disease. The results thus far suggest that UCBT can result in long-term disease-free survival in many of these patients. Similar to the pediatric series, clinical experience in the adult patients has also documented the importance of graft cell dose in determining engraftment and survival. The critical "threshold dose," below which engraftment and survival become significantly inferior, remains to be defined in a larger study with a longer follow-up. Based on current results, it appears that a UCB graft that contains at least 2 x 10⁷ nucleated cells/kg and 1.7 x 10⁵ CD34⁺ cells/kg is acceptable for adult recipients^{ref1, ref2, ref3}. It is hoped that the advantage of a lower GVHD without any apparent increase in relapse will offset the adverse impact of reduced cell dose on survival. As in the pediatric setting, TRM remains the main obstacle for success in adult recipients, although several investigators have demonstrated that the TRM is not different from that seen with matched unrelated donors. With the profound influence of UCB cell dose (both nucleated cell dose and CD34⁺ cell dose) on engraftment, survival and probably TRM in the adult setting, future research should also focus on increasing the cell dose of the UCB graft. Possible methods to increase cell dose include ex vivo expansion, infusion of multiple cord blood units or use of possible additional cells such as mesenchymal stem cells or improving HLA matching.
• nonmyeloablative regimens : the nonmyeloablative stem cell transplantation (NST) regimen was proposed initially based on the rationale that the therapeutic benefit of an allogeneic transplantation is partially related to the crucial immune-mediated graft-versus-malignancy effect. The concept of graft-versus-malignancy as the pivotal therapeutic component of allogeneic transplantation is supported by the observation from clinical studies that :
• patients with acute and chronic GVHD have a reduced risk of relapse
• patients with syngeneic BMT and after T cell–depleted allotransplant have a higher incidence of relapse compared with other allogeneic donors
• patients with a relapsed malignancy after allogeneic transplantation can be re-induced into complete remission without any chemotherapy by donor lymphocyte infusion (DLI).
NST, with its reduced-intensity preparative regimen, makes allogeneic transplantation applicable to patients with relative contraindications to myeloablative regimens given its lower rate of TRM with fewer infections and less GVHD. However, the preparative regimen does not contribute significantly to the antimalignancy effect; thus, the risk of disease progression after transplantation remains higher compared with the myeloablative approach. NST using UCB provides an opportunity for immunotherapy for older patients, sicker patients and also patients without suitable donors who are not eligible for this potentially curative approach. However, there is increased concern about graft rejection using this approach given that there are, on average, 2 logs fewer cells infused than would be considered for a standard matched sibling or unrelated donor transplant. The clinical outcome of 2 patients with malignant lymphoma using this novel approach was first reported by investigators at Duke University^{ref1, ref2}. In that study, 2 patients with relapsed lymphoma who had no matched siblings, partially matched family members, or matched unrelated donors successfully underwent nonmyeloablative conditioning therapy followed by infusion of 4 and 6 matched, unrelated donor UCB cells, respectively, at the nucleated cell dose of 2.9 and 6.5 x 10⁷/kg, respectively. The conditioning regimens consisted of fludarabine 30mg/m² and cyclophosphamide 500 mg/m² daily for 4 days with antithymocyte globulin 30 mg/kg per day for 3 days. Cyclosporine and prednisolone were given for acute GVHD prophylaxis. Both patients had 100% donor engraftment by the third month of transplant and remained in remission 6 and 12 months following transplantation. Extension of these data to 11 other patients confirms the low morbidity and the potential for robust engraftment using this approach. The favorable outcome demonstrates the feasibility of the mismatched unrelated UCB cells, even with the NST regimens. Summary of results of nonmyeloablative unrelated umbilical cord blood transplantation in adult patients :

investigator	Barker et alref	McSweeney PA, Bearman SI, Jones RB, et al. Nonmyeloablative hematopoietic cell transplant using cord blood [abstract]. Blood 2001;98:666a	Chao et alref
number of patients	43	5	13
age, years: median (range)	49.5 (22–65)	64.5 (25–78)	49 (19–62)
diagnosis	HM	HD; AML; CLL; NHL	NHL; MDS; AML; melanoma; ALL
preparative regimen	F + Bu + TB I (200cGy (n = 21)  F + C + TBI (200cGy) (n = 22)	F + TBI (200cGy)	F + C + ATG (n = 10)  F + C + ATG + TBI (200cGy) (n = 3)
GvHD prophylaxis	CsA + MMF	CsA + MMF	CsA + Pred (n = 8) CsA + MMF (n = 5)
cell dose
nucleated cell (x 107/kg): median (range)	2.6 (1.6–3.8)  3.2 (1.1–5.1)	1.1 (1.75–1.3)	2.07 (1.07–5.53)
CD34 (x 106/kg): median (range)	3.7 (1.1–8.1)  4.3 (1.1–10.3)	NA (0.01–0.04)	1.3 (0.5–9.6)
CD3 (x 106/kg): median (range)	0.06 (0.02–0.15)  0.05 (0.02–0.12)	NA (1.4–3.3)	4.6 (2.02–22.82)
engraftment (n)	C.I. = 76%  C.I. = 94%	2	12
median days to ANC  > 500/µL (range)	26 (12–30)  9.5 (5–28)	NA	12 (6–24)
median days to platelet  > 20,000/µL (range)		NA	14 (6–61)
grade II–IV aGVHD / cGVHD (n)	C.I. for aGVHD = 44%  C.I. for cGVHD = 21%	1/NA	1/1
outcome	O.S. : 39% at 1 year  DFS.: 31% at 1 year	Alive: n = 3  PR: n = 1	O.S.: 43% at 1 year  DFS: 43% at 1 year

The largest experience with transplantation using a reduced-intensity regimen was evaluated by investigators from the University of Minnesota on a cohort of high-risk patients with hematological malignancies^ref. In their study, unrelated UCB graft with a median nucleated cell dose of 3.7 x 10⁷ per kg (range, 1.6–6.0 x 10⁷/kg) was infused into 43 patients (median age 49.5 years; range, 22–65 years), after receiving 2 types of conditioning regimens: fludarabine 200 mg/m², total body irradiation (TBI) 200 cGy and busulfan 8 mg/kg (Flu/Bu/TBI) for the initial 21 subjects; fludarabine 200 mg/m², TBI 200 cGy and cyclophosphamide 50 mg/kg (Flu/Cy/TBI) for the subsequent 22 subjects. All patients received GVHD prophylaxis with cyclosporine and mycophenolate mofetil. The median time to neutrophil recovery of more than 0.5 x 109/L was 26 days (range, 12–30 days) for the Flu/Bu/TBI recipients, but only 9.5 days (range, 5–28 days) for the Flu/Cy/TBI recipients. The cumulative incidence of engraftment for Flu/Bu/TBI and Flu/Cy/TBI recipients was 76% and 94%, respectively. Despite the use of 1–2 HLA-antigen mismatched graft in 93% of the recipients, the cumulative incidence of grade II–IV GVHD and grade III–IV GVHD for the entire cohort of patients were 44% and 9%, respectively. The disease-free survival of these high-risk subjects was also favorable: 24% at 1 year for Flu/Bu/TBI recipients and 41% at 1 year for Flu/Cy/TBI recipients. A similar approach and data have also been reported by investigators in University of Colorado Health Sciences Center (McSweeney PA, Bearman SI, Jones RB, et al. Nonmyeloablative hematopoietic cell transplant using cord blood [abstract]. Blood 2001;98:666a). Immune recovery in 5 recipients of UCB transplant following the NST regimen were compared to recovery in adult recipients of UCB following a myeloablative regimen by investigators at Duke University^ref. Recipients of NST regimens had a more rapid and robust recovery of myeloid and lymphoid cells. The T cell repertoire in UCB recipients treated with the NST regimen was markedly more diverse and robust compared with the repertoire in those receiving the myeloablative regimen at similar time points. T cell receptor excision circles (TRECs), which are generated within the thymus and identify new thymic emigrants and those that have not divided, were detected 12 months after transplantation in the NST recipients. This compared favorably to the delayed detection of TRECs at 18–24 months among recipients of myeloablative regimens. Thus, in adults receiving an NST preparatory regimen, the quantitative and qualitative recovery of T cells occurs through rapid peripheral expansion. The ability of patients receiving NST transplantation to recover within a few months suggests that the peripheral "niches" in which T cells can proliferate are preserved in these patients compared to those receiving myeloablative regimens. Moreover, the presence of TREC-positive cells within 1 year suggests that thymic recovery is likewise accelerated in NST compared with recipients of myeloablative regimens. The favorable results of T cell recovery following the NST suggest that it may be possible to have an excellent outcome with an unrelated mismatched UCB transplantation in adult patients. Patients have a rapid recovery of T cells with a complex diversity. The primary difference between the recipients of ablative and NST regimens was the extent of physiologic damage caused by the preparatory regimen. When the damage is relatively mild, as in nonmyeloablative regimens, the donor T cells are able to expand effectively in the periphery, and the development of new T cells through the thymus is also accelerated compared to the rate of development in those receiving ablative regimens. Alternatively, the lower incidence of GVHD in NST may also play an important role in the preservation of the peripheral and central niches for T cell development.
Value of pretransplant screening for colonization of Pseudomonas aeruginosa in reduced-intensity umbilical cord blood transplantation for adult patients^ref.
Future directions for UCB transplantation in adults : while the use of NST signifies an advancement in the field of transplantation and immunobiolology, several unresolved questions remain:
• what is the optimal UCB cell dose required for adult patients and can we increase it?
• what is the optimal NST conditioning regimen and GVHD prophylaxis?
• is the incidence of GVHD similar in ablative and NST regimens?
• does the incidence of infection differ in ablative versus NST regimens and can one improve immune recovery?
• what is the difference in overall efficacy between myeloablative and NST UCBT?
UCB is a viable alternative to bone marrow and peripheral blood as a source of stem cells capable of hematopoietic reconstitution for adults, when related or unrelated marrow donor is not available. UCBT following NST preparative regimen is an exciting new approach that provides an option for patients who are otherwise excluded from conventional hematopoietic stem cell transplantation, including elderly or medically infirm patients with no matched sibling donor. Preliminary results have shown that such an approach can be associated with timely engraftment with full donor chimerism. Comparison between myeloablative and NST approaches will be needed before this therapy can be considered for younger patients eligible for myeloablative transplant. At the moment, the use of NST UCBT cannot be encouraged outside of a clinical trials or selected patients. The future challenge will be to develop strategies to optimize the chance of early and durable engraftment, as well as to minimize the risk of GVHD and transplant-related death.
Expansion of UCBC progenitors and stem cells : the close correlation between preclinical assays and in vivo clinical biology that is observed with bone marrow stem cells and with hematopoietic growth factors such as Epo, G-CSF and GM-CSF has been very reassuring and has accelerated the development and improvement of clinical hematology practice. Clinical experience with UCB transplants, in contrast, has proved much more paradoxical and problematic. Early laboratory measurements showed that the density of both CD34⁺ cells and clonogenic progenitor cells was extremely high in UCB^ref. Similarly, the proliferative capacity of progenitor cell differentiation in laboratory assays and of UCB stem cells transplanted into immunodeficient mice was measurably higher than for progenitors and stem cells from bone marrow (BM) or peripheral blood (PB)^{ref1, ref2}. But when comparable numbers of UCB progenitors (1–5 x 10⁶/kg) and stem cells are transplanted as are delivered to patients with standard BM or PB grafts, engraftment is far slower, and graft failure is more likely^ref. Given that opportunistic infection and organ failure rise exponentially with time to engraftment, and that overall long-term survival correlates closely with platelet engraftment, this delay has proved to be a major barrier to adoption of what would otherwise be an overwhelmingly attractive stem cell source. One of the striking lessons of BM and peripheral stem cell (PSC) transplant is that time to engraftment correlates inversely with progenitor and stem cell dose, with most graphed comparisons showing hyperbolic curves with asymptotic limits at 7–8 days for neutrophil engraftment and 10–12 days for platelet engraftments, both achieved with PSC stem and progenitor cell doses of > 5 x 10⁶ CD34⁺ cells per kg. Providing lower doses of early hematopoietic cells results in increasingly longer nadirs, but providing higher doses does not further accelerate hematopoietic recovery. Based on these well-documented observations, the question has been raised whether one could significantly shorten UCB transplant nadirs by providing increased numbers of stem and progenitor cells to patients by expanding the numbers of hematopoietic stem cells (HSCs) and progenitor cells, in the laboratory, prior to transplant. This approach should succeed if the delay in engraftment is due to a paucity of HSC and progenitor numbers, but would fail if the delay were due to an uncharacterized-but-insurmountable qualitative defect in UCB stem and progenitor cells. Attempts to characterize UCB expansion protocols have relied directly on the assays used to measure the cell populations that are targets for expansion. Over the past 20 years, these assays have become more sophisticated, and clinical investigators have progressively adjusted their clinical trial designs based on more recent, better assays. Pluripotent hematopoietic stem cells are very rare, representing only 1 cell in 2–10 x 10⁶ BM cells, perhaps slightly more frequent in UCB. There is no phenotypic assay, to date, that directly identifies and measures these rare cells. Rather, all efforts to measure HSCs rely on functional assays. The easiest and most popular assays have been colony-forming cell (CFC) assays, which measure cells that are partially differentiated between HSCs and mature blood cells. These cells, which correlate closely but inexactly with CD34⁺ cell numbers, produce easily detectable blood cell colonies in 2 weeks in the laboratory. But careful cell fractionation studies have shown that the intermediate-maturity cells detected in these assays contribute only very slightly, and temporarily, to hematopoietic recovery after transplantation, since they don’t produce enough cells that survive long enough after transplantation; it is only the immature pluripotent stem cell population that contributes to sustained engraftment^{ref1, ref2}. So, although CD34⁺ cell numbers in an unmanipulated graft may correlate with clinical time to engraftment after transplantation, that is likely because the ratio of intermediate progenitor cells to very primitive stem cells is fairly constant between clinical samples, and therefore CD34⁺ cell numbers indirectly reflect primitive stem cell numbers transplanted. This assumption, however, may not be true following many ex vivo expansion procedures, in which progenitors may be greatly expanded but primitive stem cells may not^ref. Better functional assays for primitive stem cells, developed in the late 1980s and 1990s, include the long-term culture initiating cell (LTCIC), cobblestone area forming cells (CAFC), and best of all the human to immunodeficient NOD/SCID mouse (NOD/SRC) repopulating cell assay^ref. In evaluating UCB expansion protocols, it is important to determine what populations were measured and specifically whether LTCIC, CAFC or NOD/SRC were identified as being expanded.
• UCB expansion ex vivo: cytokine cocktails : when UCB progenitors were first detected, investigators noted that the progenitor cells formed colonies in agar more rapidly than did BM or PSC progenitors, and that a higher fraction of UCB colonies could be replated to form secondary colonies. These data on UCB progenitors, together with their much higher frequency in the UCB mononuclear cell fraction, suggested that they were more proliferative than their adult counterparts. In the early 1990s Moore and Hoskins applied the techniques of PB CD34⁺ cell isolation followed by culture in high concentrations of multiple hematopoietic growth factors first pioneered by Haylock et al to UCB, with prima facie very promising results. After removal of more mature CD34^– cells, clonogenic progenitors could be expanded 50- to 100-fold, while slightly more primitive  cells could be multiplied 10–20X, all in 2 weeks^ref. Subsequently, many investigators confirmed that UCB progenitor populations could be readily expanded, and some showed that LTCIC could also be expanded 10X, particularly if combinations of low-dose cytokines were combined with perfusion conditions that simultaneously removed the secreted products of maturing blood cells and supplied continuous fluxes of cytokines^ref. Of note, however, no assays for primitive NOD/SRC repopulating cells were measured in these studies, so it is not known to what extent, if any, true HSC were expanded in these protocols. When Lewis et al assayed UCB NOD/SRC repopulating cells in parallel with CFC and LTCIC during ex vivo cytokine expansion cultures, they found that while CFC were increased 20–25X, LTCIC were only increased 40%, and NOD/SCR were maintained but not increased at all^ref. True UCB NOD/SRC stem cell expansion may be achievable by very judicious combinations of HSC purification and cytokine selection. SDF-1 treatment increases the engraftment of UCB HSCs, but whether this effect reflects quantitative increases in HSCs or a qualitative improvement in their behavior is not certain^ref. This may be the basis of the reported enhancement of UCB engraftment achieved by supplementing UCB grafts with mesenchymal stem cells. UCB HSCs are selectively responsive to IL-6 stimulation^ref, and recent studies suggest that combining UCB CD34⁺ cell selection with IL-6/soluble IL-6R/SCF/Flt-3l/Tpo stimulation induces NOD/SRC expansion 4–5X. Overall, it appears that UCB stem cell expansion may be possible with ex vivo cytokine cocktails, but conditions that can generate such HSC expansions, as opposed to the expansion of more mature progenitor populations, may have not yet been used in clinical trial settings.
• stem cell expansion: Hox gene products : individual cytokines are known to trigger many intracellular signal transduction pathways, which presumably activate multiple gene transcription pathways. Thus, in retrospect, combinations of cytokines might well be expected to have multiple effects on HSCs that might not permit UCB stem cell expansion. An alternative, more direct, approach to expanding stem cells might focus on the specific genetic pathways known or believed to support primitive HSC proliferation. Over the past decade several such pathways and transcription factors have been proposed, including Notch-1 receptor activation, Wnt/LEF-1 pathway induction and telomerase. One of the most promising intracellular targets for HSC expansion are Homeobox (Hox) gene products. These transcription factors, which were first identified as master switch transcriptional regulators of early development, were discovered a decade ago to be expressed in the most primitive HSCs. When cloned and overexpressed in mouse HSCs, several of these Hox genes induce HSC proliferation, in addition to more or less disruption in differentiation. One of these Hox genes, HoxB4, has proven to be the most interesting to date. HoxB4 is highly expressed in primitive HSCs, and its expression declines with differentiation. Overexpression of HoxB4 mRNA by retroviral infection results in HSCs that expand over 100X if transplanted in very low numbers (analogous to the clinical HSC scenario)^ref. Of even more interest, recent experiments suggest that the same effect can be achieved by treating purified HSCs with a modified form of soluble HoxB4 protein (TAT-HoxB4), which increases intracellular HoxB4 protein levels for several hours only^ref. While this approach has thus far only been applied to murine HSCs, and not to human BM or UCB HSC expansion, the application to human UCB expansion could be very direct. More recently, Zhu et al have discovered that the protein NF-Y is a normal transcriptional activator for multiple Hox genes, as well as telomerase, Notch-1 and LEF-1^ref. When overexpressed in murine HSCs by as little as twofold, NF-Ya increases HSC numbers by 20X following transplantation. Application of TAT-NF-Y protein transduction to UCB HSC expansion thus offers the attractive possibility of activating multiple HSC expansion pathways simultaneously. The absolute and relative efficiencies of these protein treatments for expanding human UCB HSCs will need to be evaluated directly in NOD/SCID mouse xenotransplants, and then in appropriate Phase I clinical trials. Similar approaches using other soluble, reversible, biochemical stimulators of stem cell transcription programs, including perhaps copper chelation, have also been proposed and are under preclinical and clinical trial.
• clinical experience with ex vivo expanded HSCs : clinical trials of ex vivo culture UCB cells have begun to be reported, but their interpretation should be tempered by the knowledge that the conditions of culture used to date have not been shown to support USC amplification, only CFU expansion. At least three trials evaluating the combination of unmanipulated with ex vivo expanded UCB grafts have been published to date; in all three studies expanded cells were added on day 10–12 to supplement unmanipulated cells. One study involved the use of a static culture system in defined media while the other two used a perfusion culture expansion system. Although no toxicities were seen in these studies, no significant acceleration of neutrophil or platelet engraftment was seen^{ref1, ref2, ref3}. Neutrophil engraftment to ANC > 500 was seen on day 24–33, and platelet engraftment to > 50,000 at approximately day 90. These results, while disappointing, highlight the importance of building clinical trial design based upon the most precise and appropriate preclinical data. The cytokine cocktails employed in the three published studies included some but not all of the cytokines required to achieve UCB HSC expansion in vitro, as measured by our best NOD/SRC assays: One trial used SCF and a Tpo analogue, but not Flt3-l, and the other 2 used Flt3-l, but not Tpo or SCF, and neither included IL-6 or soluble IL-6R. In addition, a lesser but possibly contributing variable was that in all three trials the ex vivo expanded cells were added 10–12 days later, thus mitigating any accelerating effect they might have otherwise have achieved. It must be pointed out that these trial design limitations were known to the clinical investigators, but the lack of availability of clinical grade cytokines, and possibly also HSC selection devices, prevented the optimal cocktails and culture conditions from being employed. These difficulties would be surmounted if simpler expansion conditions, requiring few added reagents and/or devices while still supporting HSC expansion, could be devised and employed.What if UCB HSC Numbers Are Not Rate Limiting? Exploring the Null Hypothesis Another possibility that must be considered is that increasing UCB numbers will not, by itself, accelerate engraftment. Perhaps there is a qualitative "immaturity" defect that prevents rapid engraftment. If so, then is there a way to circumvent this defect: providing a maturation effect through some combination of in vivo cytokine treatment, or perhaps ex vivo cell activation targeted at maturation? Viewed through this perspective, one can envision manipulations of the non-HSC components of the UCB graft, including the mature hematopoietic cells, the immune cells, antigen-presenting cells, and mesenchymal stromal cell components of the graft. Interactions between HSC and non-HSCs could form the basis for potentially improved engraftment in mixed graft settings as well^ref. Each of these manipulations could be evaluated by comparing engraftment in NOD/SCID mice, prior to clinical transplantation trials. In summary, experience with hematopoietic cell expansion to date supports the fundamental principle of experimental hematology, that the best clinical results follow from the most careful, comprehensive and creative preclinical trials in the proper in vitro and in vivo models. For the future, this experience suggests concentrating on ex vivo manipulations that can be shown to directly increase HSC numbers following NOD/SCID engraftment and/or accelerate the pace of engraftment in these same models.
Immune reconstitution after UCBSCT : one of the events necessary for the success of HSCT, regardless of the source of stem cells, is the development of a functional immune system from donor-derived cells. The production of adequate granulocytes, platelets, and red blood cells usually occurs rapidly after HSCT. In contrast, the ability to produce lymphocytes, especially T lymphocytes, is delayed. As a result, serious infection in the first 1–2 years after HSCT occurs in about 50% of uncomplicated transplants from histocompatible sibling donors, and up to 80–90% of recipients of matched unrelated donor (MUD) marrow transplants or histocompatible recipients who developed GVHD^ref. Infections attributable to poor lymphocyte function include viral pathogens such as the herpes group (CMV, HSV, VZV, HHV-6 and HHV-8), respiratory viruses (RSV, parainfluenza viruses), adenovirus, and enteroviruses (Coxsackie and ECHO viruses), as well as EBV-associated lymphoproliferative disorders. Other infections that may result from inadequate cellular immunity include fungal infections such as Pneumocystis carinii, Candida, and Cryptococcus. Increased susceptibility to Aspergillus infection may reflect defects in innate immunity. Susceptibility to some bacterial infections, notably those caused by encapsulated organisms (Staphylococcus pneumoniae, Haemophilus influenzae), is determined by the inability to produce antipolysaccharide antibodies. The emphasis of this review will be the relationship between stem cell source and the development of T lymphocyte functions required for protection of the HSCT recipient from infection. In particular, the unique aspects of UCBT, compared to either BMT or mobilized peripheral blood stem cell transplantation (PBSCT), will be described. Factors influencing the development of adequate immune function after HSCT include those related to the donor progenitor cell populations, pre-formed lymphocytes in the donor product, the host microenvironment, pharmacological interventions, and antigenic exposure. All clinically relevant stem cell sources include both progenitor cells and preformed lymphocytes. Prethymic progenitors may include HSC, common lymphoid progenitors (CLP), committed T progenitors (CTP), whose T lymphoid progeny depend on the host thymus for their development^ref. 2 pathways for generation of T lymphocytes after hematopoietic stem cell transplantation (HSCT) :

Shown at top is the adoptive transfer of post-thymic T cells which then undergo expansion in the host, resulting in a limited repertoire of mature T lymphocytes that can rapidly confer antigen-specific responses, including graft-versus-host disease (GVHD). Below is shown the pathway for differentiation of pre-thymic progenitors by migration into the recipient thymus, resulting in the slow generation of a diverse repertoire that is host-tolerant.
• development of thymic progenitors : upon entry to the thymus, progenitor cells undergo expansion; differentiation events including V(D)J recombination to generate functionally rearranged TCR genes; and both positive and negative selection events that cull potential T lymphocytes that would either be unresponsive to antigenic peptides presented by self-major histocompatibility complex (MHC) antigens or overly responsive to either MHC or self antigens. The ultimate fate of negatively selected cells is apoptosis. Since most thymocytes are fated to die, maintenance of thymic output depends on continual expansion and differentiation of the immature prothymocytes, which lack expression of the TCR invariant CD3 complex, and the co-receptors CD4 and CD8 ("triple negative," TN). The major signals for expansion of immature prothymocytes are 2 cytokines, IL-7 and c-kit ligand (KL), which are produced by thymic epithelial cells (TEC). Murine studies have demonstrated that pre-HSCT radiotherapy and chemotherapy kill TEC, resulting in defective intrathymic IL-7 production. Early studies demonstrated that the thymic microenvironment is a target of GVHD, and more recent studies have shown that TEC organization and function is abnormal in experimental GVHD models. In non-HSCT studies, aging also results in TEC damage and decreased intrathymic IL-7 production. Thus, thymic function may be adversely affected by several phenomena commonly associated with clinical HSCT-cytotoxic therapies given pre-HSCT, GVHD, and recipient aging. Clinical studies have demonstrated that decreased capacity for production of new T lymphocytes is associated with GVHD and increasing recipient age^{ref1, ref2}. The effects of pre-transplant conditioning on thymopoiesis has been more difficult to assess because of the complexity of the problem of dose intensity as well as the effects of previous therapy for malignancies. Any assessment of the effect of stem cell source, e.g., UCB, on immune reconstitution must take into account the function of the thymic microenvironment. The biology of prethymic progenitors is also highly relevant to the outcome of HSCT. The lack of pre-existing TCR rearrangements means that donor-derived prethymic progenitors are unlikely to cause GVHD, as long as the recipient thymus maintains the ability to select out undesirable T cells. However, the development of an immune repertoire from prethymic progenitors may be delayed by ontogenic factors such as commitment to the T lymphoid lineage and the time normally required for development of large numbers of diverse T cells. Between 7 to 9 weeks of gestation, hematopoietically derived cells enter the thymic anlage, which forms from mesenchyme and endoderm of the III-IV pharyngeal pouches. Alloreactive T cells are present by week 14–15 and antigen-specific T lymphocytes appear later in the second trimester. Recapitulation of fetal ontogeny provides a framework for understanding the development of lymphoid cells after HSCT. The generation of T lymphocytes post-HSCT requires events similar to those observed in normal immune development, e.g., lymphoid commitment, thymic migration and entry, and thymic differentiation. It is likely that the time period required for such development is similar to the time normally required for prenatal T cell development. For example, in patients with severe combined immune deficiency (SCID) receiving CD34⁺ haploidentical HSCT, the appearance of functional mature T lymphocytes in the peripheral blood is 90–120 days after transplant, a time period similar to that required for normal prenatal immune ontogeny.
• post-thymic T lymphocytes : in contrast to prethymic progenitors, post-thymic T cells have already undergone maturation and have antigenic and functional specificities that were conferred by the donor’s thymus. Donor-derived post-thymic T cells express a fixed TCR and thereby are programmed to respond to a set of antigenic determinants. Although adoptive transfer of mature T lymphocytes had long been thought to be a relatively static process, recent studies have indicated that mature T lymphocyte populations are dynamic and regulated by interactions with other T lymphocytes as well as the host microenvironment. Mature T lymphocytes may undergo activation if they encounter a stimulating antigen and the proper set of co-stimulatory signals. Activation of T lymphocytes leads to acquisition of functional properties such as cytolytic capacity or cytokine secretion. Activated T cells may proliferate and mature into memory cells, undergo apoptosis (activation-induced cell death [AICD]), or be anergized. Expansion of activated T lymphocytes is dependent on production of IL-2 by helper T lymphocytes and expression of high affinity IL-2R by the responsive helper and cytotoxic cells. Recent studies have demonstrated that naive T lymphocytes in a lymphopenic environment can undergo proliferation while maintaining a mainly naive phenotype^ref. Such homeostatic proliferation differs from activation-induced proliferation in which naive T cells recognize foreign antigens and proliferate in response to IL-2. In homeostatic proliferation, the stimuli recognized by the TCR are the same self antigens that normally induce positive selection in the thymus. Like thymopoiesis, homeostatic proliferation also requires IL-7, although the source is probably extrathymic, not TEC. Homeostatic proliferation has also been described for memory T lymphocytes with evidence that IL-15 is critical for the expansion of these cells. The appearance of T lymphocytes after HSCT is conventionally seen as evidence of de novo generation of T lymphocytes from progenitor cells. However, as previously noted, transplantation of stem cell products that have been extensively depleted of mature T lymphocytes in order to prevent GVHD results in significant delays in immune reconstitution. Therefore, it is likely that much of what has been attributed to T cell generation in the first few months after transplant is the result of adoptive transfer of post-thymic cells. Such cells may undergo homeostatic proliferation in the lymphopenic environment of the host, or IL-2 driven proliferation if activated by exposure to alloantigens or nominal antigens. Analyses of the length of time required for de novo generation of T lymphocytes after HSCT are best performed in patients receiving highly purified progenitors are depleted of all post-thymic T lymphocytes.
• UCBC HSC : HSC derived from UCBC differ from those of adult marrow or PBSCT in several ways. The most notable difference is quantitative—the cell dose in UCBT is significantly less than the HSC dose in adult sources such as PBSC or marrow. While low cell dose has been associated with failure of hematopoietic engraftment, HSC dose may also influence immune reconstitution. In a murine congenic transplant model, increasing the dose of phenotypic HSC administered resulted in greater thymic cellularity post-transplant, as well as greater numbers of mature T lymphocytes derived from the transplanted cells^ref. Thus, one of the limiting factors for T lymphocyte reconstitution after HSCT, besides microenvironmental defects, may be the number of progenitor cells entering the thymus and contributing to thymopoiesis. Limited numbers of HSC in UCBC compared to marrow or PBSC products may delay or decrease thymic regeneration. There are also qualitative differences between UCBC and other HSC sources. Comparisons of CD34⁺ CD38^– phenotypic HSC derived from UCBC versus adult marrow have shown that UCBC contain a higher frequency of LTCICs and a higher percentage of cycling cells^ref. UCBC-derived HSC also had greater cloning efficiency and generative capacity. Thus, the HSC in UCBC may be both more primitive and more capable of regenerating hematopoiesis in the recipient. The contrasting biological properties may represent intrinsic or environmental differences in the UCBC and adult marrow HSC. The most likely intrinsic difference may be the age of the HSC—a few months for UCBC versus decades for adult HSC. There have been some studies of the aging process in hematopoietic progenitors, but there is little direct evidence that HSC from adult donors have decreased capacity to contribute to lymphocyte development compared to other progenitors. Properties of umbilical cord blood (UCB) cell populations relevant to immune responsiveness :

hematopoietic stem cells	dendritic cells	T lymphocytes
decreased number increased cell cycling increased generative capacity more immature?	decreased immune activity decreased co-stimulation decreased IL-12 production	decreased number naive phenotype resistance to activation decreased cytokine expression skewing to Th2 responses (decreased IL-2, INF-g production)

The placenta as unique microenvironment that influences UCBT : besides intrinsic differences, HSC in UCBC have had a different set of microenvironmental exposures compared to those of adult marrow or PBSC. All HSC sources are influenced by the microenvironment from which they are derived. An example of differences between sources are some of the observed changes in HSC cell cycle status, gene expression and adhesive and invasive properties induced by mobilization procedures used to generate PBSC, e.g., G-CSF. The placenta is a complex organ that regulates maternal-fetal interactions. Many cytokines that can influence lymphohematopoietic development, e.g., G-CSF, c-kit ligand (SCF), GM-CSF, IL-15, and others, are produced by the placenta. Production of G-CSF by the placenta may be especially relevant to UCBT. G-CSF is produced both by the maternal decidua and the fetal chorionic villi8 and enters the fetal circulation by a process that does not require a functional G-CSF receptor. G-CSF from the mother probably does not enter the fetal circulation as administration of recombinant human G-CSF (rhG-CSF) to pregnant macaques did not result in detectable rhG-CSF in the fetuses. The function of placental G-CSF production is unknown; however, it may serve as an immunoregulator that protects the mother and fetus from each other’s allogeneic immune systems. G-CSF inhibits the ability of placental mononuclear cells to mediate cytotoxicity against allogeneic targets including choriocarcinoma cells. The production of G-CSF and other cytokines capable of HSC mobilization, e.g., SCF, is likely to explain the high percentage of HSC in UCBC and their rapid disappearance from the neonate’s circulation after birth. In contrast to mobilization induced by a short period of monotherapy with G-CSF before PBSCT, the HSC in UCBC have been exposed continually to a complex mixture of cytokines that likely affect their behavior, e.g., induce a high rate of cell cycling^ref, or alter the homing and invasive properties needed for entry of progenitors into the thymus. Placental G-CSF production affects umbilical cord blood cells (UCBC). G-CSF produced in the placenta may affect both fetal HSC and dendritic cells. Placental G-CSF may induce mobilization of the fetal HSC, resulting in significant numbers of circulating HSC among UCBC, which declines rapidly after delivery. G-CSF may also suppress the immunological potency of fetal dendritic cells by interfering with IL-12 production and co-stimulatory activity. As a result, the immune responsiveness of fetal T lymphocytes is attenuated and skewed away from T_h1 responses.

UCBC and lymphoid progenitors : little is known about the numbers and functional properties of circulating lymphoid progenitors present in UCBC compared to other sources. Commitment to lymphoid differentiation by donor HSC could be a rate-limiting step in the ability to repopulate the recipient immune system after HSCT. At present, there is some controversy regarding the nature of intermediate lymphoid progenitors. The murine common lymphoid progenitor (CLP) has been described as a phenotypic population in the marrow that can contribute to T, NK, and B lymphopoiesis, but it is not clear if CLP generate all thymocytes. There is some evidence that early thymic progenitors (ETP) in the thymus can arise in a CLP-independent manner. At present, CLP are the only lymphoid progenitor that are relevant to transplantation; besides the logistical problem of transplanting the intrathymic ETP population, it is unclear if such cells could home and engraft after transplantation. Murine CLP can be transplanted into irradiated hosts and contribute to post-transplant lymphopoiesis. Co-transplantation of CLP with HSC is able to more rapidly restore immune function than transplantation of HSC alone, and can result in increased protection of mice from experimental challenge with murine CMV^ref. Although human cells with CLP-like features can be found abundantly in UCBC, rigorous comparison of the numbers and functional properties of CLP in UCBC versus marrow or PBSC still need to be performed.
• UCBC post-thymic T cells : studies over the last 20 years have demonstrated differences between neonatal T lymphocytes and those of adults. The major notable difference between the T lymphocytes in UCBC and those derived from adult marrow or mobilized PBSC is the maturational status. Because the fetus is exposed to few foreign antigens, the T lymphocytes in UCBC are almost exclusively naive. Naive T lymphocytes express a phenotype that is identifiable as CD45RA⁺ CD45R0^– and CD62L⁺. As individuals age, there is an increase in the frequency of T cells that have differentiated into a memory phenotype, CD45RA^– CD45R0⁺ and CD62L^–/low as a result of antigenic exposure. Memory cells are more readily activated by antigen than naive T cells and may be able to respond to weaker co-stimulatory signals through the B7-CD28 pathway. The predominant naive phenotype of T lymphocytes in UCBC may contribute to the reduced alloreactivity observed after UCBT. Investigations of neonatal T cell function have been driven by interest in the increased susceptibility to severe viral infections in neonates. For example, newborns infected with HSV have higher rates of dissemination (sepsis, meningoencephalitis) than adults with primary infection, and increased severity of some viral infections such as enteroviruses is observed until at least several months of age. Investigations of neonatal helper T lymphocytes have demonstrated decreased production of T_h1 cytokines needed for cytolytic and anti-viral responses, compared to adult T lymphocytes^ref. Specifically, the activation of the IFN-g gene is markedly reduced in neonatal T lymphocytes, because of decreased induction of transcription factors, e.g., NF-AT, which are required for activation of the IFN- gene after TCR stimulation. A central question is whether such differences are intrinsic to UCBC versus adult T lymphocytes. Interpretation of the differences has been confused by comparison of signaling properties of UCBC T lymphocytes to unfractionated adult T lymphocytes. Since adult T lymphocytes are comprised of both naive and memory T cells, while UCBC are almost exclusively naive T cells, ascribed differences between them may simply reflect differences between the stringent activation requirements of naive helper T lymphocytes and the more easily activated memory cells. Only comparisons between UCBC and purified naive T lymphocytes from adult blood can be used to evaluate whether UCBC T cells have unique properties different from adult peripheral blood naive T lymphocytes. Regardless of the reason, T lymphocytes from UCBC appear to be less capable of mediating T_h1 cytotoxic responses than those derived from adult sources. The decreased capacity for T_h1 responses has both positive and negative implications for HSCT. The decreased absolute numbers and decreased responsiveness of UCBC T lymphocytes contribute to the decreased alloreactivity and greater tolerance for MHC disparity observed in clinical UBCBT. However, the decreased responsiveness probably means that there is also less adoptive immunity derived from UCBC T lymphocytes. There are fewer T cells overall, and the number of memory cells capable of mediating recall responses to antigen is extremely limited. Any adoptive immunity must be derived from the naive T lymphocytes present in the UCBC product. As discussed above, naive T lymphocytes are difficult to activate, rendering them less capable of responses to pathogens. These properties of the mature T lymphocytes in UCBC would predict that antiviral and fungal immunity may be decreased after UCBT than after BMT or PBSCT.
• UCBC dendritic cells : besides intrinsic differences in T lymphocytes, there may be differences in antigen presenting cells (APC) derived from UCBC versus adult sources. Analyses of dendritic cells (DC) in UCBC have shown that both so-called myeloid and plasmacytoid DC are present, although there may be a decreased proportion of myeloid DC compared to adult peripheral blood. The myeloid dendritic cells are thought to promote T_h1 responses by producing IL-12. Studies of the DC derived from UCBC have indicated that there may be intrinsic defects in production of IL-12 as well as other functions of DC. Recent data on DC generation after HSCT suggests that G-CSF administration significantly decreases the ability to produce IL-12^ref. The decrease in IL-12 production after post-HSCT G-CSF administration may be relevant to UCBT. As discussed above, cells in the fetal circulation are probably constantly exposed to placental G-CSF. Furthermore, many clinical UCBT protocols have used post-transplant G-CSF administration to accelerate the development of granulocytes. The routine administration of G-CSF, combined with intrinsic defects in DC function, may decrease the generation of T_h1 responses after UCBT. The expected consequences of such an effect would be decreased capacity to control opportunistic viral and fungal infections.
Anticipated and empiric properties of UCBT : based on the progenitor and immunological properties of UCBC, certain predictions can be made regarding the expected immune reconstitution after UCBT. Compared to the detailed information available about the recovery of marrow function and the decreased risks of severe GVHD, there are few systematic data on lymphoid recovery, and even less on such properties as antigenic repertoire and effector function. As discussed above, immune function after HSCT can be derived from either the adoptive transfer of preformed mature T lymphocytes in the stem cell product or from the development in the host of donor-derived lymphocytes from the donor HSC or other prethymic populations, e.g., CLP^ref. The adoptively transferred T cells would be expected to represent most of the lymphocytes seen in the first few months after transplant, but are more likely to be both impermanent and narrow in repertoire. Adoptively transferred T cells after UCBT are largely naive cells which will be less responsive to specific antigen than the memory cells in adult sources and probably less able to mediate T_h1 effector functions than marrow or PBSC. The HSC and pre-thymic progenitors are expected to produce a long-lasting, broad immune repertoire but would be slow to develop. The increased generative capacity of UCBC HSC might predict more rapid engraftment kinetics, but would be expected to be offset by the decreased dose of transplanted HSC in the average UCBT. Analyses of UCBT recipients have demonstrated slow de novo generation of T lymphocytes in the first year after transplant; however, by 2 years post-UCBT recipients have evidence of both a broad repertoire and thymic function^{ref1, ref2, ref3}. It is unclear if there are differences in the kinetics of post-transplant lymphopoiesis in UCBT versus BMT. More rapid recovery in children versus adults and nonmyeloablative than myeloablative conditioning suggests that damage to the thymic microenvironment as a result of aging or cytotoxic therapies may be important determinants of post-HSCT immune reconstitution^ref. Although several studies have demonstrated the importance of UCBC CD34⁺ cell dose for likelihood of hematopoietic engraftment and engraftment kinetics, analyses of the engraftment kinetics of lymphoid populations are more limited. In a study from Case Western University, unrelated donor UCBT was compared to the matched unrelated donor marrow transplantation^ref. UCBT led to decreased rate of appearance of lymphoid populations in the first 3 months after transplantation. In the subsequent year, the absolute lymphocyte count of the UCBT recipients exceeded that of the MUD recipients. Indeed, the mean ALC in the UCBT group was normal by day 200 post-transplant but remained abnormally low in the MUD recipients during the entire first year. Since the T lymphocytes seen in the first 100 days post-transplant are likely to be derived from adoptively transferred mature T cells, the decreased lymphocyte count in the first 3 months after UCBT would be expected as a result of lower amount of such cells in UCBC products. The increased numbers of lymphocytes observed later could reflect greater capacity of the UCBC progenitors to produce lymphocytes de novo. However, this is uncertain: an alternative explanation could be that the increased GVHD observed in MUD recipients led to more severe lymphopenia than that seen after UCBT. The differences in UCBC T cells from adult sources would be expected to translate into UCBT recipients having less GVHD but also less protection from viral infections such as CMV or EBV-LPD. Overall, the risk of infectious death in studies of UCBT may be higher than that of transplant from other sources, but large, multi-institutional, randomized studies have not been done, and there is a bias toward use of UCBT in sicker patients who cannot await a MUD BMT^{ref1, ref2, ref3, ref4, ref5}. In a non-randomized study of children with ALL, the incidence of viral infections was greater after UCBT than after unrelated BMT^ref. Complicating the analysis of actual infectious risk for some infections like herpes viruses is the difference in potential source of virus between UCBT and either BMT or PBSCT. CMV- or EBV-infected B cells can be derived from either the donor or recipient in BMT or PBSCT. In UCBT, CMV- or EBV-infected cells would be expected to exclusively arise from the recipient. Assuming that CMV-negative blood products are administered to CMV-negative recipients, then only CMV-positive recipients of UCBT are at significant risk of CMV disease, while CMV-negative BMT or PBSCT recipients could be infected through their seropositive donor. Therefore, comparisons of herpes virus infections after UCBT versus BMT or PBSCT need to analyze at risk populations, not the total number of recipients. For several infections, notably CMV and HHV-6, there is evidence that at-risk recipients may have greater rate of progression to symptomatic infection, with decreased evidence of viral control, although this is not a universal finding^{ref1, ref2, ref3}. In the comparison of UCBT with MUD transplants at Case Western University, the overall risk of infection, including bacterial infection, correlated with the degree of lymphopenia^ref. The risk of early bacterial infection in UCBT recipients may reflect differences in duration of neutropenia, not just lymphocyte numbers or function^ref. The available information about immune reconstitution after clinical UCBT is quite limited. In order to better understand both the kinetics and quality of immune reconstitution after UCBT, analyses of specific lymphoid populations, immunological repertoire, mitogen and antigen-driven proliferation and function, and responses to test neo-antigens, e.g., keyhole limpet hemocyanin, are needed. Ideally, such studies would be multi-institutional but use similar degrees of matching, CD34⁺ cell dose, pretransplant conditioning, supportive care and post-transplant immune suppression to control for the variables of GVHD, conditioning related microenvironmental damage and progenitor cell dose that likely have an impact on immune reconstitution. At this time, the lack of direct comparisons of immune reconstitution hinders the ability to make decisions regarding the optimal source of HSC for transplantation. In a patient with an active viral infection, e.g., CMV or a history of Epstein-Barr virus lymphoproliferative disease (EBV-LPD), unrelated marrow from an immune donor may be better than UCBT. However, potential HSCT recipients may have limited choices regarding potential HSC sources, and frequently the ability to rapidly perform transplant may trump any immunological disadvantages of UCBT.
Strategies to overcome the problems of immune reconstitution after UCBT : a paradox of UCBT is that some of the associated immunological problems are causally related to the properties that make UCBT attractive in the first place. For example, the decreased alloreactivity that has been clinically observed in UCBT reflects the quantitative and functional abnormalities that make adoptive transfer of antiviral immunity so unlikely immediately after UCBT. Efforts to improve immune reconstitution after any form of HSCT have been directed at two somewhat different questions—accelerating the rate of development of functional immunity, especially that required to control certain early infections, e.g., CMV; or altering the long-term qualitative and functional defects such as hypogammaglobulinemia that contribute to morbidity and mortality over the first decade after transplant. The first goal has been more pressing because of the high impact of opportunistic infection on transplant-related mortality. Strategies in BMT or PBSCT designed to increase the adoptive transfer of antigen-specific T cells directed against important pathogens such as CMV or EBV have used co-transplantation of cloned T cells that have been expanded in vitro. The application of such strategies to UCBT would require either cloned cells that are derived from the host or a third party, or techniques for in vitro priming of the naive T cells in the UCBC graft. Nonspecific activation of the T cells in the UCBC product would likely increase the risk of GVHD. Besides the manipulation of the preformed T cells in a graft, other strategies that have been proposed for accelerating immune reconstitution have been aimed at increasing the rate at which prethymic cells contribute to the mature T lymphocyte compartment, either by increasing the number of transplanted lymphoid progenitors or by accelerating their development in vivo. One potential strategy that has been demonstrated in murine models is the co-transplantation of committed progenitors, such as CLP^ref. Administration of 3000 CLP in addition to 500 HSC in lethally irradiated mice provided significant protection from experimental infection with murine CMV, compared to HSC alone. Impressively, small numbers of CLP provide levels of protection from MCMV that are comparable to that of millions of post-thymic cells. The technical difficulty for UCBT would be obtaining sufficient CLP to affect reconstitution, e.g., via ex vivo expansion. The use of ligands for the Notch receptors that regulate both proliferation and commitment to lymphoid lineages might be useful for in vitro generation of prethymic progenitors that could be used to augment those present in the UCBC product^ref. Based on the data of Chen et al, expansion techniques to increase the number of transplanted HSC might be expected to increase thymic output^ref. Other strategies have focused on ways to increase the in vivo development of T lymphocytes in the recipient after transplantation. These studies have been driven by the hypothesis that the thymic microenvironment of HSCT recipients lacks sufficient capacity to efficiently produce new T lymphocytes from prethymic progenitors. Our group has emphasized that defects in TEC numbers or function caused by aging, cytotoxic therapies, or GVHD lead to a relative lack of intrathymic IL-7 needed for thymopoiesis. Systemic administration of IL-7 in murine models can dramatically improve thymic output after transplantation, but the effects have not been reproducibly observed in larger animal models and IL-7 has additional effects on mature T cells that could limit its usefulness in allogeneic HSCT. Besides promoting the development of thymocytes, IL-7 treatment also increases the expansion of mature T lymphocytes by promoting their proliferation and survival. These effects have led to increased GVHD in some preclinical experiments but have not been observed by others. Experimental usage of IL-7 in clinical allogeneic transplants should probably be initially restricted to T cell–depleted grafts, not UCBT, until more is known about both efficacy and promotion of GVHD. Another strategy to address the problem of microenvironmental damage has been to decrease the toxicity of the conditioning regimen. Besides decreasing the intensity of conditioning, another approach is the administration of a biological response modifier, recombinant keratinocyte growth factor (KGF). KGF is a mesenchymally derived member of the fibroblastic growth factor family which interacts with an epithelial-specific receptor. Administration of KGF in both preclinical experimental models and clinical trials has demonstrated decreased regimen-related toxicities such as oral mucositis, but also less GVHD. KGF protects TEC in murine transplant and aging models, thereby increasing thymopoietic capacity and improving both the immediate rate of lymphocyte recovery and medium-term (3–4 months) immune reconstitution. The clinical relevance of protection of murine thymopoiesis by KGF needs to be determined.
Immune reconstitution after UCBT is promoted by the increased generative capacity of UCBC HSC, and the decreased GVHD, but is hampered by fewer HSC, decreased adoptively transferred antigen-specific T cells, and functional defects in the T cells. Clinical studies to understand the relative importance of each of these biological properties to outcome are needed. At this time, it is difficult to use relative differences in immune reconstitution between HSC sources as a basis for choosing the mode of HSCT, although BMT might be preferential to UCBT for a recipient with an active viral infection. Some but not all strategies aimed at improving immune reconstitution after HSCT in general may be relevant to addressing the immunological and infectious problems observed after UCBT. Currently available assays of hematopoietic progenitor and stem cells in umbilical cord blood :

assay	ease and rapidity for clinical application	correlation with stable engraftment
colony assays	+/–	indirect, unmanipulated grafts only
CD34+	+++	indirect, unmanipulated grafts only
long-term culture-initiating cell (LTCIC)	-	possibly direct
human to immundeficient NOD/SCID mouse repopulating cell assay (NOD/SRC)	-	likely direct

Ex vivo approaches to progenitor and stem cell expansion :

	evidence for HSC expansion	application to clinical trials
CD34+ selected cells, high-dose cytokines	+	limited trials, without best combinations due to cytokine availability. No nadir reduction
low-dose cytokines, continuous perfusion	?	in trials, without best cytokine combinations due to cytokine availability. No nadir reduction.
direct transcriptional activation of HSC cycling: HOXB4, telomerase, NF-Y	+++	future

One of the criticisms of UCBT, compared with other unrelated sources of hematopoietic stem cells, has been that donor anonymity precludes augmenting the GVL effect by infusions of donor-derived lymphocytes^{ref1, ref2, ref3}. The ability to generate B-ALL–specific T cells from UCB by enforcing expression of a chimeric immunoreceptor provides a potential solution to this limitation. Furthermore, the setting of UCBT may be particularly well suited to adoptive immunotherapy, since the slow pace of hematopoietic recovery provides a period of relative lymphopenia in which adoptively transferred T cells might expand using homeostatic mechanisms inherent to recovering the lymphocyte pool, and the relatively low incidence of severe GVHD means that recipients will be less likely to be taking immunosuppressive medications that might impair the function and persistence of the adoptively transferred T cells.
9 patients with haematological malignancies (5 with AML, 1 with ALL, 1 with HL and 2 with NHLs) received transplants of cryopreserved cord blood after conditioning therapy with high-dose cyclophosphamide, total body irradiation and antithymocyte globulin. Cord units contained a median 2.6 x 10⁷ nucleated cells/kg recipient bodyweight and were matched for 4 (7 cases) or 5 (2 cases) major histocompatibility complex class 1 and 2 antigens. Patients were given post-transplant immunosuppression with cycosporin and methylprednisolone. Neutrophil recovery to 0.5 x 10⁹/L was seen by median day 30 after transplant in all seven patients who survived more than 1 month post-transplant. Platelet recovery to 50 x 10⁹/L occurred by median day 81 in five evaluable patients. aGVHD grades II-IV was seen in 4 of 7 evaluable patients and limited cGVHD was seen in 4 of 5. Infection was the most common complication. 4 patients died before day 100 of infection (methicillin-resistant Staphylococcus aureus septicaemia, RSV pneumonia), GVHD and MOF, and intracranial bleeding. 5 patients survived 7-69 months post-transplant, without evidence of relapse of the underlying malignancy^ref
Web resources : LifebankUSA - Placenta and Cord banking
• placental stem cell. Unlike other sites where blood stem cells are found during embryonic development, such as the liver, the stem cells in the placenta can increase in number without giving rise to mature, specialized cells.
• fetal liver : experimental

• in vivo expansion :
• recombinant cytokines
• gene transfer of transcription factors : HOXB4 homeoprotein protein is of particular interests as it promotes the expansion of mouse HSCs without inducing the development of leukemia. Direct delivery of the HOXB4 homeoprotein by passive translocation through cell membranes (alone or as recombinant HIV Tat-HOXB4 protein eliminates any deleterious effects that might be associated with stable HOXB4 gene transfer
• in vitro expansion :
• a 3D, tantalum-coated porous biomaterial (Cytomatrix) supports the maintenance and expansion of human BM pluripotent HPCs (with multi-lineage engraftment capability) in the absence of cytokines and animal-derived serum. The number of CD45⁺ and CD34⁺ cells both increase 3-fold. Trends demonstrate an increase in the frequency of CD34⁺C38^- cells, and an increase in both CD34⁺C33⁺ cells and CD34⁺C61⁺ cells. No expansion of T or B lymphocytes is observed^ref

• cytofluorimetry
• standardization on the lyse-no-wash and single platform (SP) method reduces variation of results^ref

• systemic capillary leak syndrome (engraftment syndrome) during neutrophil recovery following HSCT^ref : a constellation of symptoms and signs including fever, erythrodermatous skin rash, and noncardiogenic pulmonary edema often occur. While described most often following autologous HSCT, a similar clinical syndrome has been observed followed allogeneic HSCT. Distinction from graft-versus-host disease in the allogeneic setting however, has been difficult. Recent experience with non-myeloablative conditioning for HSCT, however, reveals that an engraftment syndrome independent of GVHD may occur. In some cases, this engraftment syndrome may be a manifestation of a host-versus-graft reaction (graft rejection). While cellular and cytokine interactions are believed to be responsible for these clinical findings, a distinct effector cell population and cytokine profile have not been defined. Engraftment syndromes are likely associated with an increased transplant-related mortality, mostly from pulmonary and associated multi-organ failure. Corticosteroid therapy is often dramatically effective for engraftment syndrome, particularly for the treatment of the pulmonary manifestations. A proposal for a more uniform definition of engraftment syndrome has been developed in order to allow for a reproducible method of reporting of this complication and for evaluating prophylactic and therapeutic strategies^ref

• intravenous
• intramarrow : peripheral blood granulocyte marking peaked at 2 to 3 weeks after transplantation and decreased thereafter before stabilizing. In all animals, marking levels of IM-injected cells (GFP) were lower than those of IV-injected cells (YFP) early after transplantation. However, in two of the four monkeys, GFP marking steadily increased after 2 months resulting in higher marking levels from IM-injected cells. In one animal, this trend sustained up to the last follow-up at 1 year after transplantation, with marking levels of 63.4% and 9.7% from IM- and IV-injected cells, respectively. Transplantation of both IM- and IV-injected CD34⁺ cells resulted in polyclonal multilineage engraftment^ref

• syngeneic HSCT
• syngeneic BMT
• syngeneic PBSCT
Cutaneous and gastrointestinal graft-versus-host disease (GVHD) are described after syngeneic hematopoietic cell transplantation (HCT), but the occurrence of syngeneic GVHD (sGVHD) remains controversial and seems to defy the concept that the host must appear "foreign" to the graft^{ref1, ref2, ref3, ref4, ref5, ref6, ref7} (Billingham R. The biology of graft-versus-host reactions. The Harvey Lecture Series, 1966-1967. Vol 62. Orlando, FL: Academic Press; 1968: 21-78). Although a number of mechanisms could potentially explain this observation, recent studies reveal that women harbor fetal cells decades after pregnancy, suggesting that HLA-incompatible fetal cells could be infused from parous donors^ref. Either aGVHD in a twin transplant is a direct consequence of conditioning or that pretransplant conditioning is a prerequisite for developing features resembling aGVHD : a subject with Philadelphia chromosome-positive CML received 2 syngeneic transplants of blood cells. In the first transplant of 2.6 x 10⁸ mononuclear cells/kg, no pretransplant conditioning was given; in the second transplant of 4.9 x 10⁸ mononuclear cells/kg, pretransplant conditioning therapy consisted of chemotherapy and TBI. Although no symptoms were seen after the first transplant, the second was followed by fever, diarrhea, rash and liver function test abnormalities coincident with engraftment. Symptoms resolved spontaneously. The patient was not on any medication and had not received any transfusions^ref. A hypothesis that the definition of 'self' (in the immunological sense) should be broadened to include both genetically determined molecules (e.g. HLA) and the microbial flora that colonize an individual^ref
• autologous HSCT (ASCT)
• autologous PBSCT (90-95%) : 4-8 ^. 10⁶ CD34⁺ cells/kg is the minimal dose to ensure a safe engraftment = 8 x 10⁴ CFU-GM
• autologous BMT (ABMT) (for bad mobilizers only) : 2 ^. 10⁸ CD34⁺ cells/kg is the minimal dose to ensure a safe engraftment
Contaminations :
• microbiological contamination of manipulated blood products, including hematopoietic progenitors obtained from peripheral blood, is an infrequent but persistent problem in transplant units. The relevance of such contamination in causing patient infection has been reported as insignificant, but the effect on the post-transplant course has not been well documented. We studied the incidence of bacterial contamination in autologous peripheral blood progenitor cell transplants in two of the bench processing steps, as well as the repercussions in the post-transplant course affecting incidence of infections, transfusion requirements and time to engraftment. A total of 365 aphereses performed on 152 patients were cryopreserved in 617 bags. In 31 of these bags (5.0%), bacterial cultures were positive for coagulase-negative Staphylococcus (31.1%), S. epidermidis (21.9%), Corynebacterium sp. (6.3%), S. warneri (6.3%), Stenotrophomonas maltophilia (6.3%), Streptococcus sp. (9.4%), Viridans group Streptococcus (3.1%) and > 1 bacteria (Coagulase-negative Staphylococcus plus Corynebacterium) (15.6%). Half of the bags were contaminated at the time of freezing and the others at the time of thawing. The 31 contaminated bags were infused into 17 patients. In 5 of these the same contaminating bacteria was found. No difference between the 2 groups of patients (contaminated and non-contaminated) was found on the day the fever started, length of fever, blood transfusion requirements and engraftment, but length of hospitalization was significantly greater in patients receiving contaminated transplants^ref.
• neoplastic cells : purging :
• positive selection :
• anti-CD34 antibodies
• immunomagnetic CD34⁺ cell selection (ICS) is a widely employed technology in autotransplant and allotransplant settings. When an haploidentical transplant is performed, a high dose of purified CD34⁺ cells together with efficient T and B cell depletion are required to minimize the risks of GVHD and EBV-related lymphoma. To ameliorate the performances of the CliniMACS (Miltenyi Biotec) ICS device, 73 ICS performed following the manufacturer's recommended platelet depletion were compared versus 48 performed after adjunctive centrifugations to increase platelet depletion of the leukapheresis (LKF) product. A total of 121 ICS (from single or fractioned LKF products) were carried out on 93 LKF collected from 47 related healthy donors. A statistical significance in terms of CD34⁺ cell recovery (81.8% vs. 71.2%) was found in favor of the modified ICS procedure (p=0.0049) with a comparable stem cell purity and viability. The modification of the standard manufacturer's technique for increasing platelet depletion can further improve the recovery of stem cells with no influence on T and B cell depletion. These results demonstrate the negative influence exerted on CD34⁺ cell recovery by LKF platelet contamination^ref
• immunoadsorption on avidin-biotin columns
• panning techniques (obsolete)
• FACS
• anti-CD133 antibodies
• negative selection :
• pharmacological methods
• cyclophosphamide derivatives (mafosfamide and 4-hydroxyperoxycyclophosphamide)
• etoposide
• methylprednisolone
• vincristine
• alkyllysophospholipids
• dibutylphthalate
• eilatine
• photoactive substances (merocyanine, hematoporphyrin derivatives)
• immunological methods :
• monoclonal antibodies and complement-mediated lysis
• in vivo
• in vitro
• immunotoxin
• immunomagnetic beads
• biological methods
• in vitro culture
• IL-2, IL-3, IL-6
• antisense oligonucleotides
• TNF-a
• combinations :
• drug combinations
• drug and mAb combinations
• in contrast to leukaemia-initiating cells, HSCs were therefore unable to maintain themselves without Pten. These effects were mostly mediated by mTOR as they were inhibited by rapamycin. Rapamycin not only depleted leukaemia-initiating cells but also restored normal HSC function. Mechanistic differences between normal stem cells and cancer stem cells can thus be targeted to deplete cancer stem cells without damaging normal stem cells^ref.
Indications :
• diseases with bone marrow involvement : following results in animal models^{ref1, ref2} and clinical observations in patients with malignancies and concomitant MS^{ref1, ref2, ref3}, HSCT was first proposed for the treatment of autoimmune diseases^{ref1, ref2}. Debulking of autoreactive clones followed by restoring of self-tolerance during the immunologic recovery has been postulated^ref :
• AML
• CML
• ALL
• CLL
• lymphomas
• autoimmune diseases :  in this age of cost-effectiveness, it will be difficult to arrange third-party reimbursement for a hematopoietic stem cell transplant that may lead to medical charges of between $100,000 and $200,000. However, the cost of standard medical care for patients with SADS is not trivial. Dialysis for an SLE patient with renal failure costs $40,000 per year, while the medical resources required to care for a patient with progressive multiple sclerosis may exceed $35,000 per year. Unique BMT regimen-related toxicities may occur, including intracranial hemorrhage in the SLE or rheumatoid arthritis patient who has vasculitis; acute neurologic decompensation in patients with multiple sclerosis, especially if the conditioning regimen contains neurotoxic agents that cross a compromised blood-brain barrier; respiratory failure in patients with myasthenia gravis; and increased renal or pulmonary toxicity in patients with scleroderma and parenchymal fibrosis. Scleroderma-associated gastrointestinal dysmotility and bacterial overgrowth may also lead to greater fungal and bacterial infections^ref
• rheumatoid arthritis : 1 patient relapsed after 5 weeks^ref, another^ref
• juvenile chronic arthritis : 5^ref
• systemic lupus erythematosus^ref
• multiple sclerosis : autologous HSCT was first adopted for the therapy of MS in the late 1990s^{ref1, ref2, ref3}. Data on the MS cases treated with autologous HSCT have been analyzed and published as single-center studies^{ref1, ref2, ref3}, multicenter trials^{ref1, ref2}, and as a collective European Group for Blood and Marrow Transplantation (EBMT) retrospective study^ref. It is significant that 74% of 85 MS patients reviewed are progression free at 3 years from transplantation^ref. However, due to the different clinical forms of MS treated as well as the multiplicity of the employed immunoablative regimens, these results should be interpreted with caution. A phase II trial showed that HSCT is able to induce a prolonged clinical stabilization in severe progressive MS patients, resulting in both sustained treatment-free periods and quality of life improvement^ref. From baseline to 1 month after treatment, brain atrophy in 9 patients was 10 times faster than before treatment. A patient with non-CNS lymphoma showed comparable acute brain atrophy after analogous therapy. These observations suggest that brain atrophy after immunoablation may not be due entirely to the resolution of edema but may be related to chemotoxicity^ref
• systemic sclerosis or CREST syndrome : 1 case relapsed after 2 months^ref
• Hashimoto's thyroiditis : 1 patient with associated myasthenia gravis relapsed after 6 months^ref
• myasthenia gravis : 1 patient with associated Hashimoto's thyroiditis relapsed after 6 months^ref, another^ref
• systemic vasculitis : 1 patient^ref
• Sjogren's syndrome : 1 patient^ref
• Behcet's disease : 1 patient^ref
• Crohn's disease : 1 patient^ref
• diseases without bone marrow involvement : ^ref
• lymphomas
• solid chemo- and radiosensitive tumors
• CHF : intracoronary infusion of G-CSF mobilised PBSC improves left ventricular systolic function but causes in-stent restenosis after coronary stenting in myocardial infarction (MAGIC cell randomised clinical trial^ref). Some researchers have argued that any human trials are premature because animal experiments have not yet shown exactly how the stem cells repair the heart. They may generate new heart muscle, encourage new blood vessels to grow or secrete molecules that boost tissue survival. Ultimately, blood stem cell injections might be unnecessary if doctors can persuade stem cells dwelling within the heart to start dividing and make new tissue. There is evidence in laboratory animals that an adult stem cell can differentiate into a cell that normally belongs to a different lineage — bone marrow stem cells into hepatocytes, for example. But such reports of a pluripotent stem cell that can transdifferentiate^ref have been challenged^{ref1, ref2}. The fact that a single, rigorously defined hematopoietic stem cell from an adult mouse can reconstitute the entire hematopoietic system of a lethally irradiated mouse but has no capacity to differentiate into other tissues of the animal does not, however, rule out the existence of a pluripotent stem cell^ref. Such a cell may not look like a hematopoietic stem cell, and it could lurk in the marrow, waiting to make an appearance only under special circumstances. For these reasons, experiments in animals suggesting that the transfer of cells derived from bone marrow (BMC) could dramatically improve cardiac function after infarction through regeneration of the myocardium^ref1 or neovascularization^ref2 generated tremendous excitement. In addition, they stimulated clinical studies suggesting that this approach is feasible, safe, and potentially effective in humans^{ref1, ref2}.3,4

trial or investigator group	setting	design	no. of cells administered in treatment group	results
Bone Marrow Transfer to Enhance ST-Elevation Infarct Regeneration (BOOST)ref1, ref2	PCI after AMI	randomized trial; 30 patients received BMC, 30 received no infusion. LVEF assessed by MRI	approximately 2.5 x 109 unfractionated BMC	at 6 mo : LVEF 6% greater in BMC group than in control group at 18 mo : no significant difference in LVEF between the 2 groups, suggesting that the main effect was an acceleration of recovery
Janssens et alref	PCI after AMI	randomized, double-blind trial. 33 patients received BMC. 34 received placebo infusions. LVEF assessed by MRI	approximately 3x108 Ficoll-separated BMC	at 4 mo : no significant difference in overall LVEF; decreased infarct size and better regional function in BMC group
Transplantation of Progenitor Cells and Recovery of LV Function in Patients with Chronic Ischemic Hearth Disease (TOPCARE-CHD)ref	chronic left ventricular dysfunction	randomized, crossover trial. In the second phase, 24 patients received circulating progenitor cells (CPC), 28 patients received BMC, 23 received no infusions. LVEF assessed by left ventricular angiography	approximately 2x108 Ficoll-separated BMC or approximately 2x107 Ficoll-separated , cultured CPC	at 3 mo : greater increase in LVEF (2.9 percentage points : significant, but clinically uncertain) in BMC group than in CPC group or control group
Autologous Stem-Cell Transplantation in Acute Myocardial Infarction (ASTAMI)ref	PCI after AMI	randomized trial .47 patients received BMC; 50 received no infusion. LVEF assessed by SPECT, echocardiography and MRI	approximately 7x107 Ficoll-separated BMC	at 6 mo : no significant difference in LVEF between the 2 groups
Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI)ref	PCI after AMI	multicenter, randomized, double-blind and fully controlled trial. 101 patients received BMC; 98 received placebo infusion. LVEF assessed by left ventricular angiography	approximately 2.4x108 Ficoll-separated BMC	at 4 mo : greater absolute increase in LVEF in BMC group than in placebo group (5.5% vs. 3.0% : significant, but clinically uncertain). Subgroup analysis suggested that the benefit was greatest in patients with the worst LVEF at baseline.  at 1 yr : reduction in combined adverse clinical events in BMC group as compared with placebo group

It may be challenging to achieve significant improvements in LVEF in small cohorts of patients who have relatively preserved ventricular function and who are already receiving state-of-the-art therapy. Even some early trials of reperfusion in patients with acute myocardial infarction demonstrated either no improvement in LVEF^ref10 or a modest improvement^ref.11 Ultimately, the validation of cardiac cell therapy will require demonstration of benefit with regard to clinical outcomes — as was the case with reperfusion. Studies performed to date have not been designed or powered to evaluate clinical outcomes. Nevertheless, it is encouraging that the REPAIR-AMI investigators found the rate of adverse clinical events to be significantly lower at 1 year among patients receiving BMC than among those receiving placebo. Given the relatively small number of events, this result will require replication in larger cohorts. However, it reinforces the message that BMC infusion is not only feasible but also safe, and it raises the possibility that clinical benefits may exceed the modest improvement seen in ventricular function. Data on ventricular function at 1 year are not available.
. The study was powered to have an 80% chance of detecting a change of 5 percentage points in LVEF; thus, a smaller effect could have been missed. However, the change closest to achieving significance — the change in LVEF as measured by magnetic resonance imaging (P=0.054) — actually favors the control group, arguing against this explanation. Technical differences in the characteristics or handling of the infused BMC might explain the different outcomes. Janssens et al. also did not detect an improvement in global ventricular function at 4 months in the BMC group as compared with the control group, although infarct size was reduced and regional wall motion was improved in the BMC group.8 The identification of features of BMC preparations and of patients that are predictive of a favorable response should help to resolve these discrepancies and to focus future trials.
. The groups received the other type of cell in the next phase of the trial, but the result was independent of the order in which the cells were given, suggesting that the BMC effect is somewhat specific. Which quantitative or qualitative differences in the cell populations account for their different effects is currently unknown. Although the benefit observed after BMC infusion was modest (an increase in LVEF by 2.9 percentage points), it is remarkable that any benefit was seen in these patients, who were studied on average more than 6 years after infarction and who were already receiving optimal medical care. The TOPCARE-CHD trial suggests that BMC can have effects beyond simple acceleration of healing after infarction. Whether repeated infusions would yield additive benefits and whether these benefits would persist will be important questions for future trials. Although the prospect of regeneration of cardiac tissue provided an initial stimulus for cell-based therapies,1 subsequent work in animals has questioned the ability of BMC to effectively generate cardiomyocytes,12,13 and clinical studies have suggested that only 1.3 to 2.6% of infused BMC are retained in the heart^ref.14 Functional benefits may also be mediated through paracrine secretion of growth factors or cytokines, which could indirectly promote survival of cardiomyocytes, mobilization of endogenous progenitor cells, or neovascularization. Do these distinctions matter? As pointed out by others,15 patients benefited from many established therapies — including aspirin — before we understood the underlying mechanisms. There is no doubt that the ultimate success or failure of cell therapy will rest on its ability to show clinical efficacy rather than on the imputed mechanism. However, the heterogeneous cell populations used make BMC infusion fundamentally different from most medical treatments. This complexity may help explain why apparently similar protocols can yield disparate results.5,7 Identifying which — if any — of the cellular constituents is necessary for beneficial effects, and whether these effects are mediated directly by the transplanted cells or indirectly through involvement of other cells, would enable targeted delivery of essential components and is likely to be a critical step in the full realization of the potential of this therapeutic approach. Even aspirin might not be as effective if it were still being delivered as willow bark. As articulated in the consensus statement of the task force of the European Society of Cardiology, the clinical need, feasibility, and safety of the treatment,15 as well as the need to resolve discrepant results, mandate additional clinical trials. However, as illustrated by recent randomized trials,4,5,6,7,8,9 we should proceed in a manner that maximizes both the information gained and the safety of patients. Patients should be treated with cells only as part of randomized, controlled trials and only after they understand that neither the efficacy nor the long-term risks of this approach are established. Future trials should be powered to examine clinical end points and patients should be followed over the long term and for both beneficial and adverse effects. Simultaneously, we must continue to support basic and translational research that can help guide clinical investigation. The enrollment of patients with a poor prognosis (i.e., large infarcts, poor left ventricular function) makes sense. They have the greatest need for therapeutic approaches and thus have the most favorable risk–benefit ratio. Demonstration of incremental benefit, as compared with conventional therapy, is easier in these populations, and subgroup analyses suggest that they are the most likely to benefit.5 The enrollment of patients with heart failure who use left ventricular assist devices as a bridge to transplantation would also provide a unique opportunity to examine cellular and molecular mechanisms through analyses of cardiac tissue acquired both before cell infusion (at implantation) and after (at transplantation). Recent randomized studies of cell therapy for heart disease4,5,6,7,8,9 represent a milestone in this rapidly developing field while serving as a cogent reminder that many important clinical and fundamental questions have yet to be addressed. We should guard against both premature declarations of victory and premature abandonment of a promising therapeutic strategy. The ultimate success of this strategy is likely to depend on continued and effective coordination of rigorous basic and clinical investigations.

These results are not unexpected. In a mouse model of myocardial infarction, infused bone marrow stem cells differentiated only into blood cells, not cardiac myocytes, and they failed to contribute to myocardial regeneration^ref. If there was no transdifferentiation in the studies of myocardial infarction, though, there is another possibility: the injected bone marrow cells may not settle in the heart for long, but while there, they could produce cytokines or other diffusible molecules that enhance myocardial regeneration. The heart does produce cytokines, and cytokines are involved in the pathogenesis of coronary artery disease, but whether cytokines can explain the small effects of bone marrow cells on ventricular function is a matter of conjecture.
Web resources :

• VesCell
• t2cure
• BM mononuclear and side population (SP) cells generated progeny in some compartments of the lung (mean 45%, range 4-70% : primarily monocytes and macrophages, but also fibroblasts and type I, but not type II, alveolar cells and rare cells in the bronchial epithelium) and liver (mean 4%, range 0.4-8.3% : Kupffer cells, inflammatory cells and small clusters of hepatocytes, but not bile duct cells), but only in 7-9.5 Gy TBI recipient mice. Stem cells in BM can contribute to repair of tissue injury in some compartments, but not to the same extent in the lung and liver^ref.
• in elderly patients with NHL. In 1994-2004 altogether 88 NHL patients > 60 years old received ASCT in six Finnish transplant centres. There were 57 male and 31 female patients with a median age of 63 years (range 60-70 years); 17 patients were>65 years. The histology included diffuse large B cell (n = 29), mantle cell (n = 27), follicular (n = 15), peripheral T cell (n = 12) and other (n = 5). Disease status at ASCT was I complete remission/partial remission (CR/PR) in 53 patients, II CR/PR in 30 patients and other in five patients. The conditioning regimens included BEAC (n = 49), BEAM (n = 34), TBI-CY (n = 4) and other (n = 1). 84 patients received PB grafts. The medians to reach neutrophils > 0.5 and platelets > 20 were 10 and 14 days, respectively. The early treatment-related mortality (TRM) (<100 days) was 11%. With a median follow-up of 21 months for all patients, 45 patients (51%) are alive. A relapse or progression after ASCT has been observed in 32 patients (36%). ASCT is feasible in selected elderly patients with NHL, but the early TRM seems to be higher than in younger patients^ref
Hematopoietic regeneration after HCT is associated with increased proliferation and differentiation of primitive progenitors. Increased proliferative stress on stem cells bearing genotoxic damage could contribute to the pathogenesis of t-MDS/AML. Extended follow-up of a larger number of patients is required to confirm whether alterations in progenitor and telomere recovery predict for increased risk of t-MDS/AML^ref.
• tandem autologous transplantation : used for ...
• multiple myeloma
• primary progressive or refractory Hodgkin's disease (HD) or aggressive non-Hodgkin's lymphoma (NHL) : tandem HDCT followed by autologous stem cell transplantation (ASCT) offers a chance of cure in these poor prognostic patients, but is associated with risks^ref
Contraindications : creatinine clearance < 40 mL/min because of concerns regarding excessive toxicity of HDT and pharmacokinetics of melphalan in patients with renal failure, despite renal failure has no impact on the quality of stem cell collection and does not effect engraftment
Conditioning regimens :
• busulphan-melphalan
• idarubicin-intensified busulphan-melphalan (ida-bu-mel)^ref
• busulphan (Bu), cyclophosphamide (Cy), and etoposide (E) that was originally developed for allogeneic HSCT. 49 patients with intermediate- and high-risk aggressive NHL underwent autologous HSCT using the regimen : 18 patients treated before 1999 received Cy 2.5 g/m² on days -3 to -2 and E 1800 mg/m² on day -3 after oral (PO) administration of Bu 1 mg/kg every 6 hours x 4 days for a total of 16 doses beginning on day -7. After April 1999, 31 patients similar in all pretransplantation risk assessments received the same regimen except that intravenous (IV) Bu was substituted for PO Bu and pharmacokinetic-directed (PKD) dosing was attempted to achieve an AUC time curve of 1000-1500 mmol/min for each dose. Nonrelapse mortality was 28% for PO Bu patients versus 3% for the IV PKD group (P = .01, chi-square test). Actuarial 5-year overall survivals were 28% for patients who received the PO Bu regimen and 58% for patients who received the IV Bu regimen (P = .010, log-rank test), and PFSs were 17% and 50%, respectively (P = .008, log-rank test). After substitution of PKD IV Bu in the BuCyE regimen, we observed lower nonrelapse mortality with increased overall and progression-free survivals in patients with intermediate- and high-risk aggressive non-Hodgkin lymphoma who underwent autoHSCT^ref
• BEAM
• standard-dose ibritumomab tiuxetan followed by BEAM high-dose chemotherapy and autologous HSCT : 23 patients, median age 55 years (range, 35-66) with chemo-refractory lymphoma, either primary refractory (n = 11) or in refractory relapse (n = 12). Rituximab 250 mg/m² followed by ibritumomab tiuxetan 0.4 mCi/kg were given on day -14 and high-dose BEAM chemotherapy started on day -6. All patients engrafted. 21 patients are evaluable for response; 11 achieved CR, 9 achieved PR, 5 of whom converted to CR with additional radiation therapy (overall CR rate 76%). With a median follow-up of 17 months (range, 4-27) 16 patients are alive and 12 are progression-free. The estimated 2-year OS and PFS are 67% (95% CI, 46-87%) and 52% (95% CI, 31-72%), respectively. The day-100 rate of treatment-related mortality was 9% (95% CI, 2-33%) and the 2-year cumulative incidence of relapse was 31% (95% CI, 17-57%). Extensive prior therapy (>3 lines), high LDH and IPI score at ASCT, bulky disease, and progression during last chemotherapy were risk factors for reduced survival. Ibritumomab tiuxetan-BEAM and ASCT is relatively safe and may improve outcome in patients with refractory lymphoma. Although excess nonrelapse mortality can not be ruled out, relapse rate was relatively low, resulting in improved outcome. Standard-risk patients with chemo-sensitive disease may also benefit from ibritumomab tiuxetan-BEAM and ASCT^ref
• BVAC
• CBV
• cyclophosphamide 60 mg/kg i.v. for 2 days + total body irradiation (TBI) (12 Gy in 4 fractions over 4 days)
Reconstitution :
• CD3⁺ T-cell repopulation typically occurs by 2 to 4 months. Low CD4/CD8 ratios, related to both low numbers of CD4⁺ and elevated CD8⁺ with low levels of naive CD4⁺ CD45RA cells may exist during the first 6 months but return to baseline by 1 year. T-cell function, as measured by mitogen studies and mixed lymphocyte reactions, returns to baseline by 1 year^ref. Even the introduction of CD34⁺ selection techniques to reduce potential tumor contamination does not greatly influence long-term immunologic reconstitution after PBSC infusions, with no significant differences in the kinetics of CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺ cells^{ref1, ref2}.
• CD19⁺ B-cell numbers return to baseline within 3 months, but in vivo function measured by Ig levels typically requires > 6 months after both selected and unselected infusions. Preliminary studies have suggested delayed immune reconstitution with the combination of rituximab and CD34-selected transplantation. Among recipients of CD34-selected, B-cell antibody-purged HSC infusions receiving posttransplantation rituximab, decreased IgG levels were noted in 10 of 20 patients at 6 months and 6 of 11 at 1 year. T-cell recovery was also impaired with CD4 counts less than 200/µL observed in 12 of 20 patients at 6 months and in 4 of 11 at 1 year (Horwitz SM, Breslin S, Negrin RS, et al. Adjuvant rituximab after autologous peripheral blood stem cell transplant results in delayed immune reconstitution with increase in infectious complications [abstract]. Blood. 2000;96:707a). A second study of CD34-selected autographs with pretransplantation rituximab noted 5 of 13 patients with T_h < 200/µL at 6 months, and 8 of 8 CMV-seropositive patients developing reactivation CMV-DNA in blood at least once after transplantation (Flohr T, Hess GR, Kreiter S, Meyer RG, Huber C, Derigs G. Immune reconstitution after autologous CD34-positive selected peripheral blood stem cell transplantation combined with rituximab for refractory B-cell non-Hodgkin's lymphoma [abstract]. Blood. 2000;96:709a). Cases of PML and CMV disease suggest that the addition of peritransplantation rituximab, which results in delayed T-cell reconstitution after autografting, may have serious consequences for late clinical infectious disease^ref. B-cell engraftment was 100- to 200-fold less efficient in RAG2-deficient recipients that harbored host pro-B cells^ref : why the presence of recipient pro-B cells compromises donor B-cell engraftment in the murine model? One possibility is that limiting amounts of IL-7 might exist in a "B lymphopoietic niche," adequate to support proliferation of host pro-B cells but inadequate to support expansion of donor pro-B cells as well. In a similar vein, and as discussed elsewhere^ref, an important nutrient such as arginine could be rate limiting. Donor pro-B cells may also have reduced expression of the IL-7 receptor making them unable to compete, reminiscent of the situation with reduced IL-7 receptor expression on T lymphocytes^ref. In the event that the murine study holds relevance for human allogeneic HSCT, what strategy could be used to promote donor B-cell engraftment? It is noteworthy that SCID patients with residual pro-B cells who undergo HSCT in the absence of myeloablation often retain host B-lineage cells many years after transplantation^ref. Depletion of residual host B-lineage cells before transplantation with, for example, anti-CD19 antibodies (potentially conjugated to drugs or radionuclides) might facilitate donor B-cell engraftment. Administration of IL-7 after transplantation might also overcome the putative competitive disadvantage of donor B-lineage cells and facilitate their engraftment, given evidence that IL-7 can transduce a replicative signal to human pro-B cells^ref.
• dendritic cell (DC) content has been identified as a predictor of relapse and EFS after autologous HSCT^ref. In all patients DC type 1 (DC1) and DC type 2 (DC2) were already significantly lower than in normal individuals before conditioning therapy (DC1/microl 3.1 +/- 1.0, DC2/microl 3.0 +/- 1.1). On day 0 and day +7 the mean DC1 and DC2 numbers were very low in both groups. Patients who received unmanipulated marrow or peripheral blood stem cells reached pre-conditioning levels of DC1 and DC2 cells on day +20. In patients receiving selected CD34 cells, DC increased slowly and pre-transplant counts were observed only on day +60. Nearly 'normal' levels of DC1 and DC2 could be observed in the first group from day +180, and were maintained thereafter; in CD34(+) selected patients DC1 and DC2 counts remained lower than normal. Our data emphasise that circulating antigen presenting cells (APC) recover quickly. It remains to be determined if DC frequency in PB reflects their tissue function^ref. Both DC1 and DC2 counts were already significantly lower than in normal individuals before conditioning therapy. Pre-conditioning levels of DC1 were reached in unmanipulated patients at day 30 and became normal at 6 months. In selected patients, DC1 pre-transplant level was observed at day 60 and was maintained thereafter. DC2 recovery showed a similar trend. In unselected patients, DC2 count increased to pre-conditioning level at haematological recovery and was normal after 1 year. In selected transplants, DC2 increased more slowly than DC1 in the same patients: pre-transplant level was detected at day 90 but was still significantly lower than normal 1 year after transplant. The incidence of infection was similar in both groups^ref.
• serum immunoglobulin levels : adjuvant immunotherapy with rituximab may reduce relapse rates for high-risk B-cell NHL following high-dose chemotherapy with autologous HSCT. However, the potential adverse effects of rituximab on immune reconstitution following SCT are not fully characterized. A retrospective analysis of Ig levels and peripheral blood neutrophil counts was performed in 11 patients who received adjuvant rituximab following autologous HSCT for B-cell NHL. Results were compared to a contemporaneous group of 24 patients who received an identical conditioning regimen and autologous HSCT for lymphoma, but no adjuvant rituximab. Adjuvant rituximab was associated with a significantly increased incidence of hypogammaglobulinaemia between 12 and 24 months post-SCT, but not neutropenia. Despite suppression of Igs, there were no late or atypical infective complications attributable to rituximab^ref. A total of 14 patients with high-risk CD20⁺ lymphoma underwent an autologous PBHSCT (APBSCT). After a hematological recovery, rituximab was given weekly for up to 4 doses as an adjuvant therapy. After a median follow up of 33.5 months, 6 patients (group A) who had hypogammaglobulinemia, while the 8 other patients (group B) had normal serum immunoglobulin levels. A phenotypical analysis revealed that group A patients had already achieved B-cell recovery. However, a severe delay in the recovery of CD27⁺ memory B cells, especially in the IgD^-/CD27⁺ switched populations in group A, was found but CD27^- naive B-cells reverted to a normal range in both groups. Consistent with this, RT-PCR studies with peripheral blood mononuclear cells revealed that most patients in group A lacked > 2 classes of isotype transcripts^ref
Autologous bone-marrow transplantation (ABMT) is feasible in heavily pretreated patients who do not mobilize peripheral blood progenitor cells (PBPC), suggesting that BM progenitor-cells are not as sensitive to chemotherapy as are PBPC. However, information regarding the impact of previous chemotherapy on the performance of BM grafts is scanty. 40 consecutive lymphoma patients treated with the BEAM regimen and ABMT at a single institution were retrospectively invetigated. Pre-transplant chemotherapy has little or no influence on progenitor-cell yield and short-term engraftment after ABMT.In contrast, cumulative doxorubicin doses have an independent influence on long-term engraftment. In heavily pretreated lymphoma patients in whom poor PBPC mobilization is expected, BMT may represent an attractive option^ref.
Complications :
• mucositis : amifostine protects against mucositis and other toxicities in patients with advanced, refractory, or recurrent hematologic malignancies undergoing HDT and TBI. 35 patients (20 with NHL, 12 with HL, and 3 with AML) who underwent autologous stem cell transplantation were conditioned with TBI 2 Gy twice daily on days -8 through -6; cyclophosphamide 6 g/m², etoposide 1.8 g/m², and carboplatin 1 g/m² on days -5 through -3; and amifostine 500 mg/m² on days -8 through -2. Prior institutional experience in patients treated without amifostine was used as a historical comparison (no-amifostine group). Severe mucositis occurred in 14 (40%) of 35 patients in the amifostine group, compared with 33 (94%) of 35 in the no-amifostine group (P < .0001). TPN was used by 4 (11%) of 35 amifostine-treated patients and 34 (97%) of 35 no-amifostine patients (P < .0001). The median duration of narcotic use decreased from 15.5 days with no amifostine to 11 days with amifostine (P = .002). Granulocyte and platelet engraftment times were similar. Prospective trials with innovative designs and clearly defined stopping rules are warranted to confirm whether amifostine reduces the toxicities of a myelosuppressive conditioning regimen before autologous stem cell transplantation without compromising therapeutic response^ref.
• reactivation of latent viruses :
• HHV-1 / HSV-1
• HHV-3 / VZV
• HHV-4 / EBV
• HHV-5 / CMV : although viral reactivation occurs with similar frequencies (approximately 40-50%) after allogeneic and autologous HSCT, serious clinical disease occurs in only 2% to 8% of autologous HSCT recipients^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10, ref11, ref12, ref13, ref14, ref15}. Clinical disease after HSCT typically involves a pattern of early (first 3 months) pneumonitis and enteritis rather than retinitis that is more common among patients with HIV disease and severe T-cell deficiencies^ref.
• CMV pneumonia developed in 2% of 795 patients. The frequency was higher among patients who were seropositive than those who were seronegative (3.3% vs 0%, P = 0.008). Among seropositive patients, the frequency was higher among patients with hematological malignancies than patients with solid tumors (5.0 % vs1.0%, P = 0.019). 11 cases occurred <30 days, and 5 cases occurred >100 days post transplant. The overall CMV pneumonia-related mortality rate was 31%. 7 (78%) of 9 patients treated with ganciclovir and IVIG prior to respiratory failure survived; neither of two patients treated after respiratory failure survived. 4 of 5 (80%) untreated patients survived. In conclusion, CMV is a not infrequent cause of pneumonia among autologous BMT recipients. Risk factors include CMV seropositivity and an underlying hematological malignancy. A favorable response hinges on the prompt initiation of therapy. The survival of 25% of the patients without antiviral therapy suggests that the isolation of CMV from a BAL specimen occasionally reflects oropharyngeal contamination or that CMV pneumonia may sometimes be self-limited in more immunocompetent autologous BMT recipients^ref. The lack of cases attributable to primary infections is consistent with previous studies demonstrating the prophylactic efficacy of administering only CMV seronegative or filtered leukocyte-depleted blood products to seronegative BMT recipients^{ref1, ref2, ref3, ref4}. The diagnostic value of the detection of CMV in BAL, which is high in allogeneic BMT recipients with pneumonia and low in patients with acquired immunodeficiency syndrome, has not been determined in autologous BMT recipients^{ref1, ref2}
• HHV-8 / KHSV^ref
• HBV and HCV
• JCV
• community respiratory virus : during 2 winters, a virus was isolated from 37 (36%) of 102 patients and 30 (26%) of 115 patients, respectively.
• respiratory syncytial virus (RSV) (49%)
• influenza virus (18%)
• picornaviruses (18%)
• parainfluenza virus (9%)
• adenovirus (6%)
58% of these infections were complicated by pneumonia, with an associated mortality of 51%. The pneumonias that complicated RSV infection were almost exclusively viral in origin and were associated with a mortality of 100% if not treated promptly with antiviral agents. In contrast, many of the pneumonias that complicated the other viral infections, such as influenza, appeared to be either self-limited viral pneumonias or secondary bacterial or fungal pneumonias^ref
• spontaneous cortisol secretion is maintained in children after autologous BMT irrespective of whether cranial or total body irradiation has been given or not^ref
• recipients of autologous HSCT were no more likely than siblings to report diabetes mellitus or systemic arterial hypertension^ref
• late (>100 d) non-relapse mortality (NRM)^ref :
• another malignancy was the most common cause of late NRM (35%)
• secondary haematological malignancy (17%)
• infectious causes (32%)
Prognostic factors :
• obesity : TRM was similar among the normal, overweight, and obese groups; the underweight group had a higher risk of TRM (relative risk [RR], 2.46; 95% confidence interval [CI], 1.59-3.82; P<0.0001) compared with the normal-BMI group. No differences in relapse were noted. Overall mortality was higher in the underweight group (RR, 1.48; 95% CI, 1.17-1.88; P=.001) and lower in the overweight (RR, 0.87; 95% CI, 0.79-0.96; P=.004) and obese (RR, 0.76; 95% CI, 0.67-0.86; P<.0001) groups compared with the normal-BMI group. In light of our inability to find differences in survival among overweight, obese, and normal-weight patients, obesity alone should not be viewed as a contraindication to proceeding with auto-HCT for lymphoma when it is otherwise indicated^ref
Hospital in the home (HITH) management following autologous HSCT for patients with multiple myeloma or lymphoma is both safe and feasible with adequate infrastructure support and rigorous patient selection^ref
• allogeneic HSCT : 2-4 ^. 10⁸ CD34⁺ cells/kg is the minimal dose to ensure a safe engraftment. If RBC incompatibility exists, RBCs can be removed by chemical lysis of gradient centrifugation. See HLA and HLA typing. The competence of the immune system of the developing fetus to act as a barrier to in utero hematopoietic cell transplantation (IUHCT) has been a source of debate. Until now, comparisons of allogeneic and congenic engraftment have been inconclusive due to methodologic limitations resulting in minimal and inefficient engraftment. In this study, we transplanted E14 fetal mice with either allogeneic or congenic BM using a new intravascular technique that allows definitive administration of much higher doses of donor cells. Our results demonstrate that 100% of surviving recipients demonstrate engraftment at 1 week of age, but that 70% of allogeneic recipients lose engraftment by 1 month of age, and 80% ultimately fail to sustain long-term chimerism. In contrast, all congenic recipients maintain stable, long-term, multilineage chimerism. These results strongly support an immune barrier to allogeneic engraftment after IUHCT^ref

Sources :
• allogeneic PBSCT : PBSCT provides 10 times more T cells than BMT, but G-CSF converts granulocytes into low-density granulocytes (LDG), which undergo apoptosis after 48 h in culture, able to inhibit T-cell function by H₂O₂ production => shifts donor T cells to T_h2 cytokine secretion (IL-4 and IL-10) and downregulates T_h1 (IL-2 and IFN-g) cytokine production => estimated numbers of T_h1 and T_h2 cells infused in BMT and PBSCT do not differ significantly^ref => the incidence and severity of acute GvHD is similar among recipients of both types of transplants. Nephrotic syndrome^ref. More rapid engraftment
• G-CSF donor-marrow priming : G-CSF at 3 to 4 micro/kg per day for 7 days prior to the marrow harvest accelerates both neutrophil and platelet engraftment and is associated with a very low incidence of grades II to IV acute GVHD in CML patients after HLA-matched sibling marrow transplantation^ref
• allogeneic BMT : usually performed in patients under 50 years of age and having a HLA identical sibling
• related donor
• HLA-matched related donor (MRD) : In 1959, Thiomas and his colleagues reported that a patient with end-stage leukemia who was treated with total-body irradiation, followed by infusion of her identical twin's marrow, had a 3-month remission^ref. Transplantation of bone marrow from an HLA-matched child to his immunodeficient sibling was successful because the recipient could not reject the allograft^ref. In the 1970s, Thomas and colleagues cured some patients who had end-stage leukemia by using marrow from their HLA-identical siblings after ablating the host marrow with total-body irradiation combined with cyclophosphamide^ref. Transplantation during the first remission of the leukemia was successful in > 50% the patients^ref. The occurrence of GVHD reduced the incidence of leukemic relapse^ref, which suggested that donor lymphocytes can eradicate tumor cells that survive preparative regimens.
• prognostic significance of NOD2/CARD15 variants in HLA-identical sibling HSCT : effect on long-term outcome is confirmed in 2 independent cohorts and may be modulated by the type of gastrointestinal decontamination^ref
• HLA-partially matched related donor (PMRD) : multivariate analysis showed that incompatibility for HLA-A alleles and incompatibility for HLA-C alleles were independent risk factors for severe aGVHD (HLA-A, P=0.006; HLA-C, P=0.001). Mismatching of HLA-A, but not of HLA-C, alleles was an independent risk factor for death (P<0.001). Matching of HLA-C alleles was a significant risk factor for relapse of leukemia (P=0.035). HLA-B disparity was a significant risk factor for both GVHD and death in the univariate analysis, but not in multivariate analysis. Disparities in class II HLA alleles of the DRB1, DQA1, DQB1, DPA1, and DPB1 loci were not identified as significant risk factors for acute GVHD or death in the multivariate analysis^ref. Mismatches at HLA-A, -B, -C, and -DRB1 each had similar adverse effects on mortality. Only HLA-A mismatches demonstrated significant adverse effects on GVHD. These adverse effects on outcome were more evident in transplants with low-resolution versus only high-resolution mismatches. Mismatches for HLA-DQ or -DP did not significantly affect outcome. When high-resolution mismatches at HLA-A, -B, -C, and -DRB1 were considered together, adverse effects on survival and GVHD were observed. We therefore conclude that matching for HLA-C should be incorporated into algorithms for unrelated donor selection. High-resolution mismatches at HLA-A, -B, -C, and -DRB1 adversely affect outcome, but less so than low-resolution mismatches^ref.
• haploidentical (or semi-allogeneic or parental) BMT : transplantation of HSCs from mismatched related donors makes a potential donor available for every child in need of stem cell transplantation. G-CSF primed bone marrow (G-BM) and peripheral blood stem cells (PBSC) without in vitro T-cell depletion (TCD) has been proposed : 171 patients, including 86 in high-risk group, underwent transplantation from haploidentical family donors. All patients achieved sustained, full donor chimerism. 111 patients were alive in remission at a median of 682 (253-1502) days. The cumulative incidence of grade III-IV acute GVHD was 23% and that of extensive chronic GVHD, 47%; these were not influenced by HLA disparity. Patients younger than 15 years had less grade III-IV acute GVHD than older patients (P=0.044). The 2-year probability of relapse was 12% for standard-risk disease and 39% for high-risk disease. The 2-year probability of leukemia-free survival (LFS) was 68% for standard-risk patients and 42% for high-risk patients (P=0.0009). Grade III-IV acute GVHD was associated with better LFS (P=0.0017). The results require confirmation and show that G-BM combined with PBSC from haploidentical family donors, without in vitro TCD, may be used as a good source of stem cells for allo-HSCT^ref. 3 different graft manipulation methods were compared in patients with leukemias and lymphomas: positive selection of stem cells with either CD34 (n = 39) or CD133-coated magnetic microbeads (n = 14) and a new strategy which depletes T- and B-cells through the use of CD3- and CD19-coated microbeads (n = 11). Median purity of stem cells was comparable after CD34⁺-selection and CD133⁺-selection, whereas stem cells were only slightly enriched after CD3⁺/CD19⁺-depletion (97.5 %, 93.4 % and 1.02 %). Indirect depletion of T-cells by positive selection resulted in 1 x 10⁴ (median) residual CD3⁺-cells/kg (0.7-3 x 10⁴). Patients with CD3/CD19-depleted grafts received 3.2 x 10⁴ (median) (0.7-16 x 10⁴) residual T-cells/kg. Those grafts also comprised NK-cells (median number: 86 x 10⁶/kg), dendritic cells and monocytes/granulocytes. Primary engraftment of the stem cell products was comparable after CD34- and CD133-selection (85 and 72 %). In the CD3/CD19 group, 91 % had a primary engraftment. After reconditioning, all patients (64/64) were finally engrafted. Patients with CD34-selected or CD133-selected grafts had similar incidences of a GvHD II-IV (3 and 7 %), whereas a GvHD was slightly increased in patients receiving CD3/CD19-depleted cells (27%). Reconstitution of CD3⁺ T-cells was faster in the CD3/CD19 group than in the CD34 or CD133 group. These preliminary results indicate, that CD3/CD19-selected grafts may be advantageous regarding engraftment and immunoreconstitution. Since effector cell with potential antileukemic activity are cotransfused, such grafts may be suited in particular for patients with insufficient remission^ref. Myeloablative TBI followed by haploidentical BMT in mice results in a gradual reduction of splenic CD3, CD4 and CD8 cells until day 45 post-BMT. Concomitantly, there was an increase in monocytes and CD4⁺/CD8⁺ (double positive) cells, accompanied by a persistent elevation in the percentage of B lymphocytes. The total thymic and splenic T cell populations were reduced until day +30. The cellular reduction correlated with the poor proliferative response of the thymic and splenic cells. A decrease occurred in IL-2 mRNA expression in thymic cells during days 15-20 post-transplant, corresponding with the low level of IL-2 secretion in the spleen and thymus of the transplanted mice. In conclusion, following semi-allogeneic BMT, there was an overall immune down-regulation in the cells, gene and protein levels. Reduced immunological responsiveness following BMT reinforces the need for improving the immune dysfunction by immunotherapy post-BMT^ref
• RIC haploidentical HSCT : fludarabine, cyclophosphamide, and alemtuzumab were used as the preparatory regimen. Additional GVHD prophylaxis included mycophenolate with or without cyclosporine. Patients with persistence of disease had a donor lymphocyte boost planned. Toxicities, engraftment, response, survival, and immune recovery are reported. 49 patients with hematologic malignancies or marrow failure and no other available donors were enrolled. 94% of patients had successful engraftment, and 8% had secondary graft failure. The TRM rate was 10.2%, and 8% of patients had severe GVHD. Encouraging evidence of quantitative lymphocyte recovery through expansion of transplanted T cells was noted by 3 to 6 months. Seventy-five percent of patients attained a CR, and 1-year OS rate was 31% (95% CI, 18% to 44%). A standard-risk group of 19 patients with aplasia or in remission at transplantation demonstrated a 63% 1-year OS rate (95% CI, 38% to 80%) and 2.9-year median overall survival time (95% CI, 6.2 to 48 months). Nonmyeloablative therapy using haploidentical family member donors is feasible because the main obstacles of GVHD and graft rejection are manageable, allowing readily available stem-cell donors to be found for nearly all patients. Further qualitative and quantitative improvement in immune recovery is needed to address the high rate of relapse and risk of severe infections^ref
KIR epitope mismatch : Although KIR epitope incompatibility and direct pretransplant functional analysis indicated the risk of NK cell-mediated graft rejection in, rejection occurrs extremely rarely, indicating the strong conditioning regimen had ablated NK cell ability to reject the graft.
• recipient should not have
• The speed of the NK cell recovery suggested these donor-versus-recipient alloreactive NK clones had differentiated from the engrafted stem cells. In theory, differentiating NK cells could have been selected by KIR epitopes on donor hematopoietic cells, and on host stromal cells^ref.21 However, the present data indicate the host MHC failed to select a compatible NK cell repertoire. Therefore, as shown in mice^ref,24 hematopoietic cells, rather than stromal cells, appear to play a major role in the selection of the NK cell repertoire. The mechanisms involved in the disappearance of the alloreactive clones require further studies to establish whether they are deleted from the repertoire or whether they survive in a state of anergy as in MHC class I-deficient mice^ref.25 The absence of GVHD despite the donor-versus-recipient NK cell alloreactivity is unexpected. The same biological phenomenon, although in the opposite sense, is found in the hybrid resistance mouse model^ref,26 where the host F1 mouse NK cells reject parental bone marrow cells but tolerate parental skin and organ grafts. Both the transient failure of host tissues to educate the donor NK cell repertoire and the lack of GVHD may be two facets of the same phenomenon, ie, NK cell sensitivity to class I polymorphism is restricted to hematopoietic cells. Tissues may lack the ligands which bind to and/or activate NK cells. In addressing the GVL potential of donor-versus-recipient NK cell alloreactivity we found that 100% myeloid leukemias, but only a minority of lymphoblastic leukemias, were killed by alloreactive NK clones
Natural killer (NK) cells achieve tolerance through inhibitory receptors for major histocompatibility complex (MHC) antigens^ref. In humans, the 2 main classes of inhibitory MHC receptors are (1) the killer cell immunoglobulin-like receptors (KIRs) with distinct specificity for supertypic epitopes shared by groups of HLA class I antigens^ref, and (2) the NKG2A receptor, which binds to HLA-E and therefore has broad specificity^ref. Full understanding of the KIR binding patterns is essential for predicting NK-cell alloreactivity in HLA-mismatched^{ref1, ref2}, and possibly also HLA-identical^ref HSCT. There is consensus that KIR2DL1 binds to HLA-C antigens expressing lysine at position 80; KIR2DL2 and KIR2DL3 are engaged by HLA-C antigens with asparagine at position 80; KIR3DL1 binds to HLA-B antigens with the Bw4 serologic specificity derived from leucine/arginine at positions 82/83. Initial data on KIR3DL1 indicated binding of Bw4 epitopes on both HLA-B and HLA-A^ref, whereas a later study using antibody blocking experiments suggested that only HLA-B antigens with Bw4 specificity acted as ligands for KIR3DL1^ref. Consequently, HLA-A antigens with Bw4 specificity have been ignored when assessing KIR ligand mismatches in hematopoietic transplantation^ref. The HLA-A*2301, A*2402, and A*3201 but not A*2501 are ligands for KIR3DL1 and should be taken into consideration when assessing the potential for NK alloreactivity in hematopoietic stem cell transplantation^ref.


• HLA-matched unrelated donor (MUD) / voluntary unrelated donor (VUD)
• age < 10 years : congenital anomalies
• age < 25 years : severe bone marrow aplasia
• age < 30 years : acute leukemias even in second CR
• age < 40 : CML, MDS, MM
• unrelated CB transplantation (UCBT) : unrelated HLA partially matched UCBT in adults is associated with delayed neutrophil and T- and B-lymphocyte recovery, T-cell subsets recovery, platelet recovery, higher rates of bacteraemia, deficiencies of CB mononuclear cells (MNC) in producing cytokines (including G-CSF, GM-CSF, M-CSF, IL-12, and IL-15) at early time points after transplantation compared with MUD grafting. While phenotypically similar, CB and APB DC have differential potency in allogeneic MLR, which may account for the difference in GvHD and infection incidence and severity between UCBT and allogeneic stem cell transplantation^ref. The "naive" nature of UCB lymphocytes may explain the lower incidence and severity of GvHD encountered in UCBT compared to the allogeneic transplant setting. Furthermore, UCB is rich in primitive CD16^-56⁺⁺NK cells, which possess significant proliferative and cytotoxic capacities and can be expanded using IL-12 or IL-15, so as to mount a substantial GvL effect. The major disadvantage of UCB is the low yield of HSCs, resulting in higher graft failure rates and slower time to engraftment compared to bone marrow transplantation. A rational approach thus involves ex vivo expansion of UCB-derived HSCs^ref.
After allogeneic transplantation MSC from the donor can engraft in bone marrow. Moreover, since the stem cells were obtained from peripheral blood, it can be concluded that MSC circulate among mobilized peripheral blood stem cells and can engraft in bone marrow after allogeneic transplantation^ref.
Conditioning regimens : the choice of conditioning regimens is based on two key factors [3]. First, the agents should preferably have some effect against the tumor. Second, one or more of the agents should provide sufficient immunosuppression to prevent rejection of the donor cells by the recipient immune system
• myeloablative or conventional stem cell transplantation (CST) : mortality = 10%
• cyclophosphamide^ref
• cyclophosphamide (100-120 mg/kg for 2-4 days)/total body irradiation (TBI) (CY/TBI) 5-10 Gy has been the standard preparation since the 1980s. In one study, higher-than-standard doses of TBI reduced the rate of relapse but did not improve survival, because transplantation-related mortality increased^ref.
• busulphan/cyclophosphamide (BU/CY)
• cyclophosphamide, BCNU / carmustine, etoposide (CBV)
• carmustine, etoposide, cytarabine, melphalan (BEAM)
• melphalan 140 mg/m² + etoposide 60 mg/kg + total body irradiation (TBI) 500 cGy
• total body irradiation (TBI) 12 Gy/etoposide/cyclophosphamide
• selective radiation of leukemias involves the delivery of radiolabeled monoclonal antibodies against antigens on marrow cells^ref
• newer methods of selective radiation promise increased specificity^ref
• busulphan alone, 4 mg/kg/day for 2, 3, or 4 consecutive days. No additional pre- or post-transplant immunosuppressive agents were used in order to exploit the capacity of donor lymphocytes to induce GVT effects. Conditioning was well tolerated, trilineage engraftment was documented in all patients and none exhibited immune-mediated rejection. Time to recovery of ANC >0.5x10⁹/L and 1.0x10⁹/L was 12 - 38 (median 15) days and 12 - 41 (median 15) days, respectively. The time to platelet recovery >or=20 and >or=50x10⁹/L ranged from 0 to 26 (median 11) days, and from 0 to 83 (median 14) days, respectively. Acute GVHD (<or=grade I) occurred in 13/15 patients. 3 patients benefited from long-term survival^ref
Blood group incompatibility does not appear to affect the overall outcome in patients undergoing myeloablative conditioning before allogeneic BMT. Older age has been linked to increased TRM from GvHD, but depletion of T cells from stem cell grafts seems to protect from complications of GvHD particularly in older patients. Myeloablative conditioning is well tolerated up to the age of 59 in patients receiving TCD grafts^ref.
• reduced-intensity conditioning (RIC) HSCT (RIST / RI-HSCT) / minitransplant / mini-allotransplant / nonmyeloablative stem cell transplantation (NMSCT): submyeloablative, immunosuppressive conditioning regimen without bone marrow destruction, which reduces early transplant-related mortality (TRM) in older patients^ref
• fludarabine-based RIC regimens :
• fludarabine (30 mg/m²/day on days -6, -5, and -4), cyclophosphamide 60 mg/m² on days -6, -5, and -4
• fludarabine (30 mg/m²/day on days -6, -5, and -4), cyclophosphamide 60 mg/m² on days -6, -5, and -4, rituximab 375 mg/m² on day -6 and 1,000 mg/m² on days +1, +8, and +15^ref
• fludarabine + low-dose busulfan + antithymocyte globulin (ATG). In 37 heavily pretreated patients >/=55 years, aGVHD grade III-IV and cGVHD developed in 15.6% and 44.4% of cases, respectively. With a median follow-up period of 22 (range 3-113) months, the 1-year OS and DFS were 55% and 53%, respectively, while the overall non-relapse mortality was 35%^ref.
• fludarabine (30 mg/m²/d x 6 days) or cladribine (0.11 mg/kg/d x 6) + busulfan (BU, 4 mg/kg/d orally x 2), with or without antithymocyte globulin (ATG)
• fludarabine (30 mg/m²/d x 4 days) + melphalan 140 mg/m² as a single dose or
• fludarabine 25 mg/m² for 5 days + melphalan 90 mg/m² daily for 2 days.
• fludarabine 200 mg/m², busulfan 8 mg/kg, and total body irradiation 200 cGy
• fludarabine, BCNU / carmustine and melphalan (FBM)^ref reduced intensity conditioning results in reduced, if any, stromal damage as compared to standard myeloablative treatment. The novel, donor-derived, hematopoiesis in FBM patients after allogeneic transplantation is supported and maintained by a host-derived BM stromal microenvironment^ref
• fludarabine 30 mg/m² on days -8, -7, -6, -5, -4 (total dose 150 mg/m²) as 1 hr infusion, and thiotepa 5 mg/kg on days -7, -6, -5 (total dose 15 mg/kg) 2 h after the end of fludarabine as a 2 h infusion. For GVHD prophylaxis all patients receive CYA 2.5 mg/kg every 12 h starting at day 3 and alemtuzumab 20 mg/d absolute on days -2, -1 (total dose 40 mg)^ref.
• cyclophosphamide-based RIC regimens :
• cyclophosphamide 100 mg/kg i.v. on days -5 and -4; thiotepa 10 mg/kg
• cyclophosphamide + thiotepa + melphalan
• cyclophosphamide 60 mg/kg i.v. on days -5 and -4; 15 mg/kg equine antithymocyte globulin (ATG) IV on days -1, +1, and +3; and thymic irradiation (700 cGy) on day -1^ref
• low-dose total body irradiation
• cyclophosphamide, thymic irradiation, and in vivo T-cell depletion
• 10 doses of total lymphoid irradiation (80 cGy each) + antithymocyte globulin, followed by an infusion of HLA-matched peripheral-blood mononuclear cells from related or unrelated donors who received G-CSF. Of the 37 HSCT recipients, only 2 had acute GVHD after hematopoietic-cell transplantation. Potent antitumor effects in patients with lymphoid malignant diseases were shown by the change from partial to complete remission. In the transplant recipients who underwent conditioning with total lymphoid irradiation and antithymocyte globulin, the fraction of donor CD4⁺ T cells that produced IL-4 after in vitro stimulation increased by a factor of 5, and the proliferative response to alloantigens in vitro was reduced, as compared with normal control subjects and control subjects who underwent conditioning with a single dose of TBI (200 cGy)^ref. With a period of observation now ranging from 492 to 1339 days after transplantation, only 1 of the 37 patients (<3 percent) died from complications related to either acute or chronic GVHD — a number we consider to be impressively low, especially given the fact that almost 40 percent of the patients received grafts from unrelated donors. 6 patients in our study had graft loss within 75 to 200 days after transplantation. However, in four of these patients, the graft loss was associated with tumor progression or relapse of disease in the bone marrow. Thus, only 2 of the 37 patients (5 percent) had non–relapse-related graft loss — a fraction that is consistent with other nonmyeloablative host-conditioning regimens^ref.
• pentostatin/extracorporeal photopheresis (ECP)/total body irradiation have been shown to be well tolerated and associated with early full donor engraftment with a predominance of donor DC2 cells and a low incidence of aGVHD^ref
• busulfan alone, 4 mg/kg/day for 2, 3, or 4 consecutive days : it is the most commonly used myeloablative alkylating agent, but is considered a poor anti-lymphocyte agent. Engraftment of allogeneic stem cells depends not only on adequate immunosuppression but also on successful hematopoietic competition : residual lymphocytes of host origin may play a beneficial role in preventing GVHD. No additional pre- or post-transplant immunosuppressive agents were used in order to exploit the capacity of donor lymphocytes to induce graft-versus-malignancy (GVM) effects. Conditioning was well tolerated, trilineage engraftment was documented in all patients and none exhibited immune-mediated rejection. Time to recovery of absolute neutrophil count >0.5x10⁹/L and 1.0x10⁹/L was 12 - 38 (median 15) days and 12 - 41 (median 15) days, respectively. The time to platelet recovery >or=20 and >or=50x10⁹/L ranged from 0 to 26 (median 11) days, and from 0 to 83 (median 14) days, respectively. Acute GVHD (<or=grade I) occurred in 13/15 patients. Three patients benefited from long-term survival. Using busulfan alone for the preparation of patients for SCT may be sufficient for engraftment, in very high-risk heavily pre-treated patients^ref.
• radioimmunotherapy provides an attractive, minimally toxic modality to safely prevent early progression of B-cell lymphomas and induce remissions without incurring the risks of traditional intensive therapy. This approach provides a time window during which a robust GvL effect may be established before tumor progression, thereby providing more effective long-term disease control. The rationale for incorporation of radioimmunotherapy into RIC allogeneic HSCT regimens for NHL is discussed, as are current study designs, preliminary results, and future direction^ref
RIC compared to standard conditioning preserves the in vitro growth capacity of bone marrow stroma, which remains of host origin^ref. It is used to exploit graft-versus-tumor reaction in myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), and leukemias and lymphomas.
The safety and efficacy of RIC allo-HSCT using either unrelated umbilical cord blood (UCB) donors or matched-sibling donors (MSDs) was assessed in 21 adults at high risk with advanced HL (UCB, n = 9; MSD, n = 12). Both groups were comparable except for younger age in the UCB cohort (median, 28 vs 42 years; P = .02). Neutrophil recovery occurred earlier in the MSD group (median, 7 vs 10 days; P = .02). All patients had sustained donor engraftment by day 60. Cumulative incidence of acute severe graft-versus-host-disease (33% vs 33%; P = .99), chronic graft-versus-host-disease (11% vs 33%; P = .24), and 100-day treatment-related mortality (11% vs 17%; P = .80) were comparable. With median follow-up periods of 17 and 24 months, the 2-year progression-free survival rates were 25% (95% confidence interval [95% CI], 0%-55%) for UCB and 20% (95% CI, 0%-44%) for MSD allo-SCT (P = .67). These results suggest comparable outcomes for RIC allo-SCT using UCB or MSD in adults at high risk with advanced Hodgkin lymphoma^ref.
Impact of graft composition : the influence of cell subsets [CD34, CD3, CD4, CD8, CD14, CD20, natural killer (NK; CD3/CD56⁺), NKT (CD3⁺/CD56⁺), DC1, and DC2 cells] of G-CSF mobilized peripheral blood stem cells (PBSC) on early T-cell chimaerism and later clinical outcomes was studied in 125 patients with haematological malignancies who received HLA-matched related grafts after non-myeloablative conditioning. Conditioning consisted of 2 Gy TBI alone (n = 28), or 2 Gy TBI preceded by either 90 mg/m² fludarabine (n = 62) or planned autologous HSCT (n = 35). Post-transplant immunosuppression included mycophenolate mofetil and ciclosporin. Multivariate analysis showed that higher numbers of grafted NK cells predicted higher early T-cell chimaerism (P = 0.03), while higher numbers of B cells were associated with better clinical outcomes and a higher risk for chronic graft-versus-host disease (P = 0.05). Higher numbers of CD14⁺ cells were associated with worse overall survival (P = 0.03), while higher numbers of CD34⁺ cells showed better survival (P = 0.03). The addition of fludarabine or autologous HCT predicted higher early T-cell chimaerism (P = 0.001), while advanced donor age predicted lower chimaerism (P ? 0.02). Patients with aggressive diseases were at higher risk for relapse/disease progression, and shorter progression-free and overall survival (P < 0.01). These results suggest that the dosing of certain cellular subsets of PBSC products can influence important outcomes post-HCT after non-myeloablative conditioning. G-CSF mobilized PBSC have been widely used in allogeneic HSCT^{ref1, ref2, ref3, ref4, ref5, ref6} (Vigorito et al, 1998; Ringden et al, 1999; Blaise et al, 2000; Champlin et al, 2000; Heldal et al, 2000; Bensinger et al, 2001). The numbers of T cells, CD34⁺ progenitor cells and monocytes, along with many other cell subsets, have been higher in PBSC grafts compared with marrow grafts^{ref1, ref2, ref3, ref4, ref5} (Dreger et al, 1994; Korbling et al, 1995; Ottinger et al, 1996; Theilgaard-Monch et al, 1999; Storek et al, 2001). Several studies have suggested that the cellular composition of PBSC grafts affected engraftment kinetics and clinical outcomes after allogeneic HSCT. For instance, T-cell depletion of PBSC grafts resulted in higher relapse rates after myeloablative HSCT compared with unmanipulated grafts^{ref1, ref2} (Barge et al, 2001; Chakraverty et al, 2001), and graft rejection was recently reported with T-cell depleted PBSC grafts after non-myeloablative conditioning^ref (Kreiter et al, 2001). Furthermore, higher doses of CD34⁺ cells within PBSC grafts were shown to increase the risk of chronic GVHD after conventional and reduced-intensity conditioning for HLA-identical sibling HSCT^{ref1, ref2} (Zaucha et al, 2001; Perez-Simon et al, 2003). In addition, improved overall disease-free survivals have been reported with PBSC compared with marrow after both myeloablative and non-myeloablative HCT in patients with advanced disease^ref (Bensinger et al, 2001; Maris et al, 2003). Another study noted that higher numbers of CD14⁺ cells (monocytes) with decreased expression of co-stimulatory molecules within the PBSC grafts might impair the proliferation rate of donor T cells^ref (Tanaka et al, 1998). This was cited as a possible reason for similar rates of acute GVHD after PBSC compared with marrow grafts despite the approximately 10-fold higher numbers of T cells given with PBSC products. Both T cells and NK cells from HCT donors have been implicated in the GVT effects after allogeneic HSCT^{ref1, ref2, ref3} (Truitt & Atasoylu, 1991; Glass et al, 1996; Giebel et al, 2003). The GVT effect of T cells has been indirectly confirmed by the results of T-cell depletion trials^{ref1, ref2, ref3} (Link, 1999; Barge et al, 2001; Chakraverty et al, 2001). Preclinical studies in the canine HCT model showed that grafts created their own marrow space via subclinical GVH reactions, thereby ensuring stable engraftment^ref (Storb et al, 1999). Data from clinical trials in patients showed that a low degree of donor T-cell chimaerism on day +28 after non-myeloablative HCT was associated with an increased risk of rejection and that high donor T-cell chimaerism was beneficial in mediating GVT effects^{ref1, ref2, ref3, ref4} (Antin et al, 2001; McSweeney et al, 2001; Maris et al, 2003; Niederwieser et al, 2003). The level of donor T-cell engraftment (chimaerism) is currently being used as a surrogate marker for donor engraftment after non-myeloablative HCT^{ref1, ref2, ref3} (Childs et al, 1999; Antin et al, 2001; McSweeney et al, 2001). Given these observations and the known heterogeneity of cellular subsets among leukapheresis collections from healthy donors after G-CSF mobilization (Chatta et al, 1994), we investigated whether the cellular composition of PBSC grafts from HLA-identical related donors was important for engraftment, occurrence of GVHD, progression-free and overall survival after non-myeloablative HCT. We retrospectively analysed data from 125 patients with various haematological malignancies undergoing HCT from HLA-matched related donors after non-myeloablative conditioning and studied associations between infused cell doses, early T-cell chimaerism levels and clinical outcomes. This study confirms the substantial variation in PBSC cellular subset composition as reported in previous studies of conventional myeloablative allogeneic HCT recipients^{ref1, ref2} (Dreger et al, 1994; Ottinger et al, 1996). Positive correlations occurred between almost all cellular subtypes within the PBSC product. Results from this study are the first published indication in the truly non-myeloablative setting that infused quantities of some cellular subsets of PBSC products from HLA-matched siblings are associated with important post-HCT outcomes of engraftment (T-cell chimaerism) and survival. The cellular components that were associated with higher donor T-cell chimaerism on day +28 were TNC and CD56⁺ cell numbers. Given the observation that TNC numbers were highly correlated with CD3⁺ cell numbers in this study, the relationship of TNC on donor T-cell chimaerism possibly reflected the known role of T cells in establishing donor engraftment after myeloablative and non-myeloablative HCT^{ref1, ref2, ref3} (Champlin, 1993; Drobyski, 2000; Kreiter et al, 2001). NK and NKT cells analysed separately were marginally associated with T-cell chimaerism; however, the association was stronger when the two values were combined. CD56 has been used as a surrogate marker to describe NK cells, and it was known that NK-cell alloreactivity could influence engraftment and relapse rates after HLA-mismatched myeloablative HCT^{ref1, ref2, ref3}(Bornhauser et al, 1999; Ruggeri et al, 1999, 2001). This alloreactivity was thought to be the result of the missing expression of HLA-class I epitopes on haematopoietic cells (Ciccone et al, 1992). Whether donor NK cells can exert anti-host activity after HLA-identical related donor HCT is presently unknown^ref (Dupont & Hsu, 2004). Interestingly, a recent report showed that patients who rejected their unrelated grafts after a non-myeloablative conditioning regimen had significantly lower donor NK-cell chimaerism compared with patients who did not experience rejection^ref (Bornhauser et al, 2001). However, it was unclear if, and to what level, these patients were HLA-matched in this report, and no data were given about the actual number of donor cells transplanted. We suspected that certain cellular subsets in PBSC grafts would be associated with better progression-free and overall survival after non-myeloablative HCT, as this approach relies mainly on graft anti-tumour responses mediated by donor cells^{ref1, ref2} (Barrett & Malkovska, 1996; Appelbaum, 2001). In fact, only B cells and CD34₊ cells were associated with improved outcomes. Specifically, higher doses of B cells appeared to be associated with a greater occurrence of chronic GVHD, better PFS, and improved OS, while the effect of higher numbers of CD34⁺ progenitor cells was associated only with better overall survival. The finding that higher B-cell numbers were associated with an increase in chronic GVHD in the current study was unexpected, two previous reports had shown that high numbers of CD34+ cells were associated with an increased risk of chronic GVHD after both myeloablative and RIC HSCT^{ref1, ref2} (Zaucha et al, 2001; Perez-Simon et al, 2003). The association of B cells with chronic GVHD is also the probable explanation for the positive effect of B cells on PFS and OS found in this study, possibly through GVT effects. In addition, humoral responses of B cells against malignant host cells, which have been described for potential CML-associated antigens^{ref1, ref2} (Ling et al, 1998; Wu et al, 2000), might have been responsible for the anti-tumour responses. However, it seems most likely that the donor CD20+ cells could have functioned as antigen-presenting cells^{ref1, ref2} (Chesnut & Grey, 1986; Pernis & Weber, 1989), thereby facilitating the donor T cells to exert more GVT and GVHD effects. Higher numbers of CD14⁺ cells (monocytes) were associated with worse overall survival in our analysis. As CD14⁺ cells in PBSC might decrease allogeneic T-cell responses^ref (Mielcarek et al, 1997), one might speculate that GVT effects may have been diminished in patients who received a higher CD14⁺ cell dose in the PBSC graft. As expected, patients categorized before HCT as having aggressive malignant disease had a higher risk for disease relapse/progression and worse progression-free and overall survival. These results confirmed the prognostic value of this clinical categorization, similar to the high dose conventional HCT setting; patients classified as having aggressive diseases had worse outcomes after non-myeloablative conditioning as well. Another finding consistent with previous studies was that patients who had received 2 Gy TBI only as conditioning showed lower donor T-cell chimaerism on day 28 compared with patients who had received additional fludarabine or a combination of autologous and allogeneic transplantation^ref (McSweeney et al, 2001). As fludarabine is known to be immunosuppressive and to enhance the effect of radiation^{ref1, ref2} (Gregoire et al, 1999; Van Den Neste et al, 2001), it is likely that additional suppression of host immune-competent cells caused by fludarabine enabled the achievement of more rapid donor T-cell chimaerism. Our data were consistent with previous studies showing that donor T-cell chimaerism can be used as a surrogate marker for engraftment^{ref1, ref2, ref3, ref4} (Childs et al, 1999; Antin et al, 2001; McSweeney et al, 2001; Niederwieser et al, 2003), although there were too few rejections in this set to confirm this hypothesis. In summary, the doses of CD34⁺ cells, B cells and monocytes in the HLA-matched related PBSC graft influenced outcomes after non-myeloablative HCT and variations in conditioning regimens, disease status, and age of donors and recipients were important. Evaluations in patients receiving PBSC from alternative donors (e.g. HLA-matched unrelated or HLA-mismatched donor recipient pairs) are needed to support and further exploit these findings. Furthermore, prospective clinical studies with larger numbers of patients will be required. If these results are confirmed, then clinical studies evaluating graft engineering for selective cellular enrichment or depletion may be important in improving clinical outcomes after non-myeloablative conditioning for allogeneic HCT.
Immunosuppression to avoid GvHD :
• FK506since day -2 0.03 mg/kg i.v. continuous infusion (maintaining morning levels 5-15 ng/ml and checking dose adjustements after 72 hrs. Reduce since day +60 and discontinue at day +90 if MRD is present, otherwise continue until month 6) + methotrexate 5 mg/m² at days +1, +3, and +6
• cyclosporine and methotrexate^ref
• increased mycophenolic acid (MPA), the active metabolite of MMF, concentrations steady state (Css) predict higher degrees of donor T-cell chimerism after unrelated donor nonmyeloablative HCT and suggest that targeting MPA Css's > 2.5 mg/mL could prevent graft rejection^ref.
Patients with ABO mismatch have problems with engraftment, including graft rejection, pure RBC aplasia, and mixed-lineage chimerism. RBC transfusions are significantly higher in the ABO-mismatched recipients. GVHD may play a role in engraftment, possibly by facilitating the disappearance of native ABO antibodies via graft-versus-plasma cell effect. Donor red blood cell (RBC) chimerism (initial detection of donor RBCs in peripheral blood) was markedly delayed following NST versus myeloablative SCT (median, 114 versus 40 days; P <.0001) and strongly correlated with decreasing host antidonor isohemagglutinin levels. Antidonor isohemagglutinins declined to clinically insignificant levels more slowly following NST than myeloablative SCT (median, 83 versus 44 days; P =.03). Donor RBC chimerism was delayed more than 100 days in 9 of 14 (64%) and PRCA occurred in 4 of 14 (29%) patients following NST, while neither event occurred in 12 patients following myeloablative SCT. Conversion to full donor myeloid chimerism following NST occurred significantly sooner in cases with, compared with cases without, PRCA (30 versus 98 days; P =.008). Cyclosporine withdrawal appeared to induce graft-mediated immune effects against recipient isohemagglutinin-producing cells, resulting in decreased antidonor isohemagglutinin levels and resolution of PRCA following NST^ref. Following exposure to RhD antigen, anti-D develops in up to 20% of RhD-negative patients on chemotherapy, but seldom in the recipients of HSCT or solid-organ transplants. Data on anti-D formation come from HC transplants using myeloablative conditioning; no data are available for the non-myeloablative HSCT transplants. The 2 types of transplant have a distinct isohaemagglutinin disappearance rate and different kinetics of post-transplant red-cell engraftment. The objective of the study was to analyse anti-D formation in patients receiving non-myeloablative transplants from RhD-incompatible donors. 16 patients were analysed: 9 RhD^- recipients of RhD⁺ HSCs; and 7 RhD⁺ recipients of a graft from a RhD^- donor. Patients were sequentially tested for irregular antibodies, as well as donor/recipient chimerism by cytogenetics and analysis of DNA variable-number tandem repeats. Despite having received 7-499 ml of D⁺ red cells, none of the RhD^- recipients developed anti-D. The median follow-up was 202 days. By contrast, anti-D was identified in 1 of 7 RhD⁺ recipients of an RhD^- graft. Non-myeloablative conditioning containing fludarabine and/or Campath 1H, with CsA given post-transplant, effectively prevents anti-D formation in RhD^- recipients of a RhD⁺ graft. However, anti-D developed in an RhD⁺ recipient of an RhD^- graft, who was also exposed to RhD⁺ blood products before and after the transplant. Transfusion of RhD⁺ products should be avoided in such patients^ref. A total of 89 consecutive adult patients with hematological diseases including 49 ABO-identical, 20 major, 15 minor, and 5 bidirectional ABO-incompatible transplants were enrolled from 4 medical centers in Korea. No significant difference in engraftment times, graft failure, or transfusion requirements between groups was noted. A clinical diagnosis of severe immune hemolysis or pure red cell aplasia was not made for any patient after transplantation. The incidence of acute or chronic GVHD did not statistically differ between groups. With a median follow-up duration of 13 months (range, 0.5-61 months), the 3-year overall survival estimates for the ABO-identical, major/bidirectional, and minor group were 44.6.0+/-9.0, 43.1+/-11.6, and 43.8+/-13.5%, respectively (P=0.8652), while the 3-year disease-free survival estimates were 33.8+/-7.6, 39.9+/-11.4, and 45.7+/-13.1%, respectively (P=0.8546). We observed that time to neutrophil, platelet, and red blood cell engraftment, transfusion requirements, incidence of acute or chronic GVHD, relapse, and survival were not influenced by ABO incompatibility after allogeneic PBSCT from HLA-matched sibling donors^ref. A total of 158 patients with major or bidirectionally mismatched donors were identified. After major mismatched HPCT, patients with anti-A directed against the donor blood group required RBC transfusion support for a median of 109 days (range, 0-324 days) compared to 21 days (range, 2-98 days) for patients with anti-B directed against donor blood group (log-rank test, p = 0.0001). Other risk factors associated with prolonged RBC transfusion support in univariate analysis were age (p = 0.024), cytomegalovirus infection (p = 0.016), hemolytic anemia (p = 0.027), and chronic bleeding disorders (p = 0.038). The independent influence of donor blood group and recipient age were confirmed in a multivariate analysis^ref.
Immune reconstitution : G-CSF 5 mg/kg/day since day 0 until ANC > 10⁹/ml
Chimerism :
• full donor chimerism : donor CD3⁺ T cells in bone marrow > 95%
• mixed hematopoietic chimerism : donor CD3⁺ T cells in bone marrow = 1-95%
• graft rejection : lack of donor cells in bone marrow at day +56 after donor cells were > 50% at day +28
• graft failure : lack of donor cells in bone marrow at day +56 after donor cells were 1-50% at day +28
• primary graft failure
• delayed or secondary graft failure :

Aetiology :
• hemophagocytic syndrome (HPS)^ref : a 40-year-old man who presented with acute onset of hemophagocytic syndrome (HPS) after allogeneic BMT for AML. On day 8 after alloBMT, the patient suddenly manifested high-grade fever, transfusion-resistant severe anemia, and thrombocytopenia. Neither VOLD nor TMA was documented. The level of ferritin in serum was elevated to 1192 ng/mL. A BMA test on day 16 showed a markedly increased number of activated macrophages showing massive hemophagocytosis. Serum levels of IFN-g, sIL-2R, IL-6, TNF-a, and M-CSF were elevated. From these findings, we determined his transfusion-resistant cytopenias to be attributable to HPS. No viruses (including CMV, EBV, HHV-6, parvovirus B19, and adenovirus B11) were detected in serum or urine by polymerase chain reaction amplification. In addition to the administration of M-CSF, hypercytokinemia during the early phase post-alloBMT might have contributed to the onset of HPS in this patient. Methylprednisolone pulse therapy was very effective for the treatment of the HPS. This case reveals that HPS could develop after alloBMT, even when engraftment of hematopoietic cells is not confirmed^ref. A female patient with B-cell NHL with graft failure due to HPS 12 days after autologous PBSCT. Autologous PBSCT was carried out during unconfirmed/uncertain complete remission according to the Cotswolds classification after 6 cycles of biweekly (cyclophosphamide, doxorubicin, vincristine, and prednisolone) therapy and 3 courses of salvage chemotherapy including etoposide. The patient developed a high fever on day 2 post-PBSCT. Her white blood cell count rose to 0.9 x 10(9)/L on day 10 post-PBSCT, but then began to decrease. A bone marrow aspirate on day 12 post-PBSCT revealed an increase in the number of benign histiocytes with hemophagocytosis, and the patient was diagnosed with HPS. Although high-dose methylprednisolone therapy was continued, her white blood cell count further decreased to 0.3 x 10⁹/L, and the patient died of MOF on day 29 post-PBSCT. A CT scan did not identify recurrent NHL, and necropsy specimens from the BM, liver, and kidney revealed no neoplastic infiltration. Bone marrow necropsy showed marked hypocellularity with active histiocytic hemophagocytosis. HPS may have been induced by infection with MRSA rather than by lymphoma-associated HPS^ref. A 7-year-old girl with AML with multilineage dysplasia received unrelated cord blood transplantation but developed HPS after sepsis with MRSA before engraftment. BMA on day 20 revealed a markedly increased number of activated macrophages showing hemophagocytosis. The presence of donor-type chimera in the bone marrow was confirmed at that time. Immunosuppressive and antibacterial treatment were therefore quickly started. Although her condition gradually improved, the patient suffered graft failure due to HPS. She received PBSCT from her HLA 2-loci-mismatched mother on day 54 and continued in CR 12 months after the second transplantation. The results in this case suggested that because of fetomaternal microchimerism it may be useful to select an HLA-haploidentical mother as a backup donor for stem cell transplantation^ref.
• cytomegalovirus (CMV)is the pathogen most often associated with this complication.’
• HHV-6^ref : the primary target cells of HHV-6 appear to be CM’ T lymphocytes and macrophage^{ref1, ref2} although a number of other cell types have been reported to be infectable as well^ref. Over the past several years, the potential for HHV-6 to serve as a pathogen in BMT patients has begun to be explored. Reactivation of HHV-6 can be frequently detected in the blood of BMT patients after transplant^{ref1, ref2, ref3, ref4}. However, few clinical correlations have been made except for concurrence of HHV-6 viremia and graft-versus-host disease (GVHD)^ref and rash^ref. Other studies of HHV-6 infection of tissues from BMT patients have indicted the virus as causing interstitial pneumonitis^{ref1, ref2} and fatal encephalitis^ref. Other work has suggested a role for HHV-6 in the suppression of BM function in the posttransplant period in BMT patients^ref. In that study, 4 of 5 allogeneic BMT patients with severe, posttransplant idiopathic BM suppression (IBMS) were found to have active HHV-6 infection of their BMs. Further support of the hypothetical role of HHV-6 as a cause for BM suppression was obtained by showing the ability of the virus to suppress the proliferation and differentiation of BM cells in vitro^ref. Also, other in vitro studies have shown that exposure of BM precursors to HHV-6 inhibited their abilities to respond to growth factors such as GM-CSF and IL-3^ref
• parvovirus B₁₉^ref : long-term parvovirus B₁₉ viraemia associated with PRCA after allogeneic HSCT^ref
• HGV infection may significantly influence the engraftment in patients after HSCT, particularly if coinfection with HCV occurs^ref
• parainfluenza virus type I (PIV-I)^ref : parainfluenza virus URI is primarily a disease of young children, although adults can also be infected and transmit the disease^ref. While PIV is a recognized cause of respiratory disease and mortality in HCT recipients^{ref1, ref2, ref3}, it has not been associated with secondary marrow failure in this patient population. However, there have been reports of allograft rejection associated with PIV (albeit PIV-III) infection in recipients of kidney^ref and liver^ref transplants.
• adenovirus type 11 : 4 weeks following autologous BMT for Wilms' tumor, a patient developed fever, hepatomegaly, coagulation disorders and pancytopenia. Bone marrow studies showed progressively increased hemophagocytosis of normal hematopoietic progenitors by histiocytes resulting in aplasia. Adenovirus type 11 was consistently isolated from urine and stool cultures, and one of the marrow aspirates. At autopsy, adenovirus was isolated from the lungs, liver, heart, intestine and spleen. These findings are consistent with the previously described virus-associated hemophagocytic syndrome, which have not been associated with BMT^ref
• anti-MHC class II antibodies are made late after transplantation and contribute to late graft failure. This has been demonstrated for renal and lung transplantation, but has not yet been confirmed for other organs. Clearer definition of non-HLA antibodies has been achieved, such as the autoantigen vimentin and MICA. Experimental studies using minor mismatched strain combinations confirm that non-HLA antibodies bind to donor endothelial cells; these antibodies seem to cause apoptosis but not complement-mediated lysis^ref.
Therapy :
• SCF rescues^ref a canine model of anti-MHC class II mAb-induced delayed autologous HSCT failure^{ref1, ref2}
• sequential IL-3 and GM-CSF administration, following an ineffective 5-day trial with G-CSF, after allogeneic^ref or autologous^ref HSCT
Therapy :
• GM-CSF :
• after autologous HSCT :
• 12 patients with recurrent NHL or HL having an ANC < 150 x 10⁶/l on day 30 after autologous HSCT in an open-label, nonrandomized study. These patients were compared to 21 similar historical control patients who were not treated with colony stimulating factor. Overall, the patients treated with granulocyte-macrophage colony stimulating factor had a mean absolute granulocyte count of 704 x 10⁶/l on day 44 after stem cell infusion compared to a mean absolute granulocyte count of 408 x 10⁶/l in historical controls (p = 0.008). The number of documented bacterial and fungal infections occurring after day 30 (9 vs 0, p = 0.01) was significantly reduced in the study group. The toxicity attributed to the granulocyte-macrophage colony stimulating factor was minimal with only one patient experiencing chills. Recombinant human granulocyte-macrophage colony stimulating factor appears to be effective for the treatment of delayed engraftment following high-dose therapy and autologous hematopoietic transplantation for lymphoid malignancies, with most patients having accelerated granulocytic recovery and a reduced incidence of infections^ref
• 26 patients with hematologic malignancies (20) or solid tumors (6) were treated with non-glycosylated rhGM-CSF (E. coli) for delayed hematopoietic recovery (granulocytes < 0.1 x 10⁹/l on day 21 or < 0.5 x 10⁹/l on day 28) after autologous marrow or PBSCT. Median pretreatment granulocytes were 0.1 x 10⁹/l (range 0-0.4 x 10⁹/l). Treatment with rhGM-CSF was initiated at 60-250 mg/m² subcutaneously daily with dose escalation every 7 days if there was no response. Within 14 days, 21 (84%) of the 25 evaluable patients achieved granulocytes > 0.5 x 10⁹/l and 17 (68%) had granulocytes > 1.0 x 10⁹/l. For those who responded within 14 days, granulocytes were > 0.5 x 10⁹/l at a median of 3 days (range 1-13) and > 1.0 x 10⁹/l at 6 days (range 2-12). 16 of the 23 patients receiving an initial rhGM-CSF dose of 60-125 mg/m² achieved granulocytes > 1.0 x 10⁹/l. 3 patients discontinued use of rhGM-CSF because of toxicity, and 4 patients never recovered despite use of rhGM-CSF doses as high as 1000 mg/m². Graft failure-related mortality was 16% at 4 months after transplantation. These results demonstrate that relatively low doses of non-glycosylated rhGM-CSF administered subcutaneously daily can be used to promote granulocyte recovery in patients with delayed engraftment after autologous transplantation. No beneficial effects were seen on red cell or platelet recovery^ref.
• in 37 patients with marrow graft failure after allogeneic (n = 15), autologous (n = 21), or syngeneic (n = 1) HSCT, rhGM-CSF was administered by 2-hour infusion at doses between 60 and 1,000 mg/m²/d for 14 or 21 days. At doses < 500 mg/m², rhGM-CSF was well-tolerated and did not exacerbate GvHD in allogeneic HSCT recipients. No patient with myelogenous leukemia relapsed while receiving rhGM-CSF. 21 patients reached an ANC >= 0.5 x 10⁹/L within 2 weeks of starting therapy while 16 did not. None of 7 patients who received chemically purged autologous marrow grafts responded to rhGM-CSF. The survival rates of GM-CSF-treated patients were significantly better than those of a historical control group^ref. In 9 patients with delayed engraftment or graft failure after autologous (n = 6) or allogeneic (n = 3) BMT, 6 patients were given a dose of 10 mg/kg/d over 30 min; three patients received lower doses of 3-5 mg/kg/d. 7 patients lived > 3 days after commencing GM-CSF and could be evaluated for their response. 6 of them had a marked rise in neutrophil counts; there was no effect on platelet and reticulocyte counts. 2 patients died within the first 3 days after starting GM-CSF, although both seemed to have some response to GM-CSF (increasing blood neutrophils in one, and increasing macrophages in the bone marrow on autopsy in the other). Side effects most likely attributable to GM-CSF administration were mild and included diarrhea and abdominal pain, low grade fever and mild rash. Severity of GVHD was not enhanced in the recipients of allogeneic marrow^ref.
• in 25 patients, rhGM-CSF was administered i.v. at a dose of 5 or 10 mg/kg. 14 patients (7 allogeneic BMT, 7 autologous BMT) were treated for primary bone marrow failure (no granulocyte recovery after BMT), and 11 cases (all allo-BMT) received rhGM-CSF for secondary bone marrow failure (ANC < 0.5 x 10⁹/L after a previously sustained granulocyte recovery). 2 allo-BMT and 3 ABMT patients with primary BM failure achieved a granulocyte response to rhGM-CSF. In contrast, 9 patients with primary graft failure did not respond to rhGM-CSF (4 ABMT, 3 HLA-identical T-depleted BMT, 1 minor mismatch BMT, 1 unrelated BMT). 10 of 11 allo-BMT patients treated for secondary bone marrow failure attained an ANC > 0.5 x 10⁹/L, but most became severely neutropenic again at a median time of 4 weeks. The possible cause triggering graft failure (GVHD, CMV infection) remained unsolved in most of these cases. Actuarial probability of survival of the entire series was 16 +/- 9% at 15 months. The severity of graft failure and the presence of other concomitant complications in most of our patients may justify these poor results. In conclusion, rhGM-CSF had less efficacy in patients with primary bone marrow failure than in those with secondary bone marrow failure. In the latter setting, measures addressed to correct the initial cause of graft failure are mandatory^ref.
• a prospective, randomized trial comparing GM-CSF (250 mg/m²/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 mg/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a WBC count > or = 100/ml by day +21 or > or = 300/ml by day +28, no ANC > or = 200/ml by day +28, or secondary sustained neutropenia after initial engraftment. 47 patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM-CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/mL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to RBC transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the 2 treatment groups (GM-CSF: median, 30 days; GM-CSF+G-CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the 2 treatment groups (GM-CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT^ref
• a second allogeneic HSCT from a different donor :
• 12 patients with graft failure (5 primary, 7 secondary) had a second transplant, 5 from their original donor and 7 from a different donor. Their median age was 9 years. 2 patients died before day 10 of regimen-related toxicity. 9 of 10 evaluable patients engrafted in a median of 17 days. Secondary graft failure was seen in 1 patient. TRM was significant. 6 of 9 developed aGHVD, there were 5 severe infections and 5 patients developed Bearman grade 3 or 4 extramedullary toxicity. Overall, 5 patients survive at a median of 38 months after the second BMT and 2 are in continuous CR. Second transplants from unrelated donors for graft failure can result in prolonged survival^ref.
• Between March 1984 and December 1999, a total of 43 second related allogeneic BMT procedures after myeloablative conditioning were carried out in our institution, 37 following allogeneic, and 6 following autologous BMT. 31 patients were males (72%). At 1st BMT (BMT1), median age was 11.5 years (range, 0.16-45 years). BMT1 was carried out for the diagnosis of AML in 13 patients (30%), SAA in 9 (21%), ALL in 6 (14%), CML in 6 (14%), immunodeficiency in 3 (7%), NHL in 2, beta-thal in 2, HD in 1, Red cell aplasia in 1. HLA matching status for allogeneic BMT1 was full match in 33, 1 antigen mismatch in 2 and haplo identical in 2 patients. Median age at the 2nd BMT (BMT2) was 14 years (range, 0.41-46.7 years). Indications for BMT2 were recurrent hematologic neoplasm in 23 patients (53%), primary graft failure in 12 (28%) and late graft failure in 8 (19%). Median time from BMT1 to recurrence of hematologic neoplasm or late graft failure was 10 months (range, 2.5- 88 months). Median BMT1 to BMT2 interval was 13 months (range, 1-107 months). For BMT2, the same donor was used in 29 patients, while 14 patients had alternate related donor (12 full match, 1-one Ag mismatch, 1 haplo identical). A different conditioning regimen was used in the majority of the patients (39, 91%). Radiation containing conditioning regimen were used mostly for patients previously conditioned with chemotherapy only for BMT1 and chemotherapy conditioning +/- ATG for those who received radiation containing conditioning at BMT1. Bone marrow was the stem cell source for all patients at BMT2 and all except 3 autologous peripheral stem cell transplantation patient at BMT1. Significant organ toxicity leading to procedure related death in 13 patients (30%) was observed after BMT2. At a median follow up of 36 months after BMT2, 22 patients (51%) are alive (20 free of disease, 2 with recurrent disease) with median OS of 47.5 (SD +/- 9) months. Univariate analysis of relevant clinical factors identified the following variables as the only statistically significant favorable prognostic factors for overall survival: BMT1-BMT2 interval of > or = 6 months (P=0.0007) and age at BMT2 < or = 10 years (P=0.041). The nature of underlying disease (neoplastic or non-neoplastic) was not statistically significant (P=0.23). There was no statistically significant difference in survival outcome of BMT2 using same donor vs. alternate related donor (P=0.51). Due to the relatively limited sample size, multivariate analysis was not attempted. This single institution study suggests that second allogeneic BMT after myeloblative conditioning has an acceptable treatment related morbidity/mortality and favorable outcome if performed at age < or = 10 years and with an interval of > or = 6 months after the first BMT. Additionally same donor can successfully be used for the second transplant with similar survival outcome to alternate donor^ref.
• 32 thalassemic patients with a median age of 7.7 years (range 3.4-26 years) were given a second HLA-identical related marrow transplant (BMT2) for graft failure. 4 patients were in class 1 and 28 patients in classes 2 and 3. 21 patients had full thalassemia recurrence (first group) and 11 patients had aplastic marrows (second group) either with or without residual donor marrow cells after the first BMT (BMT1). As conditioning regimen for BMT2 all but 5 patients received BUCY or CY in association with TLI and/or ALG, whereas 9 patients received a new preparative regimen with hydroxyurea, azathioprine, fludarabine before conditioning with BUCY. 21 of 31 evaluable patients (67.7%) had initial, and 16 (51.6%) had sustained engraftment. 10 patients (32.3%) failed to engraft. OS and EFS for the entire group of patients were 49% and 33%, respectively, with a median follow-up of 4 years (range 0.6-14 years) for surviving patients. EFS was higher in the second group of patients compared with the first group (41% vs 29%). The second group of patients appeared to have less graft failure compared with the first group (30% vs 63%; P = 0.1). TRM was 28%. A linear stepwise regression analysis revealed that occurrence of graft failure within 60 days after BMT1 (P = 0.04) and absence of residual donor marrow cells (P = 0.009) predicted for graft failure following BMT2, whereas the occurrence of graft failure after 60 days (P = 0.03) had a positive influence on survival following BMT2. The incidence of grade >/=2 acute GVHD was low (14%). 8 of 9 patients who received the new preparative regimen are alive, 4 without thalassemia^ref
• a boost dose of HSCs from the original donor :
• with additional conditioning :
• successful non-T-cell-depleted HLA-haploidentical HSCT with RIC from a second child for late graft failure after the first HLA-haploidentical HSCT for MDS/overt leukemia based on feto-maternal microchimerism^ref
• profound marrow aplasia occurring 13 years after sibling allogeneic BMT for CML in CP1. Although the patient remained a complete donor chimera, thereby suggesting that an unselected infusion of donor peripheral blood stem cells (PBSC) or bone marrow might be indicated, the newly acquired aplasia was thought to be immune in aetiology and some immunosuppression was therefore considered appropriate. Rapid haematological recovery was achieved after the infusion of unselected PBSC from the original donor following conditioning with ATG^ref
• a 53-year-old woman with AML M2 (normal karyotype) in first remission underwent the nonmyeloablative allogeneic PBSCT from her HLA-identical brother, with conditioning consisting of fludarabine and low dose TBI (2Gy). Karyotype analysis of bone marrow on day 28 after the recovery of the hematopoiesis showed 46 XY (20/20). However, pancytopenia progressed from day 130 and the patient became transfusion dependent. Because of the hypoplastic bone marrow and the high ratio (81%) of recipient cells among the peripheral T-cells, she was diagnosed as the late graft failure. Cyclophosphamide was added to the conditioning and the second transplant was performed using the same donor's cryopreserved stem cells. Hematopoiesis recovered and the complete chimerism in T-cells was confirmed on day 28. Although the transplant dose of the CD34 and CD3 positive cells was the same between the two transplantation, the patient suffering from the late graft failure obtained the stable engraftment after the second transplant with more immunosuppressive conditioning regimen^ref
• without additional conditioning :
• between March 1983 and August 1999, 20 of 788 (2.5%) patients receiving allogeneic BMT were treated with an additional boost dose of donor cells. The reasons for the use of the boost treatment were primary graft failure (early rejection; n = 7), secondary graft failure including late rejection (n = 10), refractory PRCA caused by the remaining recipient cells producing anti-erythrocyte antibodies (n = 2), and donor lymphocyte infusion induced pancytopenia (n = 1). The patients were aged from 17 to 48 years (median age 31 years). The underlying diseases of the patients were sAA in 12 patients, AML in 3, ALL in 3, and CML in 2. The donors were human leukocyte antigen-identical siblings in 18 cases, 1 mismatched related donor, and 1 unrelated donor. The cell source was bone marrow in 6 cases and peripheral blood progenitor cells in 14. The median interval between BMT and the boost treatment was 7 weeks (range 1-124). No conditioning regimen was given prior to the boost treatment for 11 patients, while 4 received total nodal irradiation (TNI) + ATG, 3 ATG alone, and 2 TNI plus steroid. The median infused booster mononuclear cell dose was 2.55 x 10⁸/kg (range 0.28-37.0). 15 (75%) patients achieved a hematological recovery. After the boost treatment, 6 of 20 (30%) patients developed aGVHD > or = grade II, 3 of whom had had prior GVHD. 5 (31.3%) of the evaluable 16 patients developed cGVHD. The GVHDs were easily controlled using immunosuppressive agents except in the case of 1 patient. 5 patients died after the boost treatment; 2 within 30 days, 2 within 60 days, and 1 after 32 months. The causes of death were: 3 engraftment failures, 1 late rejection, and 1 infection following GVHD. With a median follow-up of 31.5 months (range 6-92), the Kaplan-Meier method estimated that the OS rate 1 and 3 years after the boost treatment was 80 and 71%, respectively. The survival of patients with primary graft failure was determined to be significantly lower compared to that of patients with secondary graft failure, using the log rank test (p = 0.0176). Disease category, stem cell source, conditioning prior to a boost treatment, and year of boost treatment did not have an influence on survival. The reinfusion of donor HSCs is frequently successful in achieving engraftment with rare occurrence of fatal GVHD. Furthermore, relatively good long-term survival was demonstrated^ref.
• 20 allogeneic BMT patients were treated with an additional dose of donor cells (boost dose) for graft failure (n = 7), partial graft failure (n = 11) or extensive hemolysis caused by remaining recipient cells producing anti-erythrocyte antibodies (n = 2). Donors were in 12 cases HLA-identical siblings, 3 mismatched related donors and 5 unrelated donors. Cell source was in 13 cases bone marrow and in 7 peripheral blood progenitor cells. Median time from BMT to booster dose was 3.4 months (range 0.7-59.3). Median infused cell dose was 2.4 x 10⁸/kg patient (range 0.5-19.0). As GVHD prophylaxis most patients were already receiving different combinations of cyclosporine, prednisolone and methotrexate. No preparative treatment was given prior to boost in 16 patients; 4 received ATG. After boost, 11 patients developed aGVHD, 6 grade I, 4 grade II and 1 grade III. Except for 1 patient, aGVHD after boost was less, or the same grade as after BMT. 6 patients developed chronic GVHD, 3 limited and 3 extensive. 5 patients died within 30 days of the boost. 9 of 15 (60%) evaluable patients became transfusion independent within 30 days and 3 more within 60 days. Causes of death were: infections 6 (IP 4, pneumonia 2), relapse 3; and GVHD 3. 3 out of 5 patients transplanted with unrelated marrow suffered from severe immunological reactions and died 2-3 months after the boost dose. Patient survival 1 and 3 years after boost was 55% and 43%, respectively. Among patients with hematological malignancies, LFS at 3 years was 41%^ref.
• of 54 patients with poor graft function (PGF), 20 patients received no further donor cell infusion (group A), 14 received a boost of unmanipulated marrow or blood cells from the original donor, without further conditioning (group B), and 20 received donor cells after CD34 selection without conditioning (group C). The 3 groups were comparable for disease phase, patients' age, donor type, primary or secondary PGF, full donor chimerism and duration of PGF. Trilineage recovery was seen in 40%, 36% and 75% of the patients in, respectively, groups A, B and C (p=0.02). In multivariate Cox analysis trilineage recovery was more frequent in patients with secondary PGF (RR of complete recovery 2.82, p=0.01) and in patients receiving CD34⁺-selected cells (RR of complete recovery 3.0; p=0.007). There was no effect of donor type on hematologic recovery. The rate of NRM was 55%, 64%, 20% in groups A, B and C, respectively (p=0.06) and was highly correlated with trilineage recovery (RR 0.36, p<0.0001). PGF was the primary cause of death in 30%, 21% and 10% of the patients in the 3 groups, GVHD in 5%, 36%, and 10%. In patients with poor graft function (a) a boost of CD34⁺-selected peripheral blood donor cells is associated with a high chance of trilineage recovery and a low risk of acute GVHD; (b) a boost of unmanipulated donor cells does not seem to offer a survival advantage over no infusion of cells; and (c) NRM is lower when using peripheral blood cells for the boost^ref.
• 7 (28%) of 25 AA patients who received BMT from HLA-identical sibling donors developed late graft failure at a median of 7 months (range, 2.0-9.3 months) after transplantation. The patients with graft failure were treated with PBSC collected from the original donor after mobilization with G-CSF. The median boost dose of PBMCs was 3.1 x 10⁸/kg (range, 1.4-11.9 x 10⁸/kg). Median times to reach an ANC > 0.5 x 10⁹/L and a platelet count > 50 x 10⁹/L were 7 days (range, 4-14 days) and 9 days (range, 3-41 days), respectively. There was sustained graft function in 6 of 7 patients, with a median follow-up duration of 3.3 yr (range, 1.0-6.2 yr). Grade-I aGVHD occurred in 2 patients, while extensive cGVHD developed in 3 patients^ref.
• between March 1983 and August 1999, 20 of 788 (2.5%) patients receiving allogeneic BMT were treated with an additional boost dose of donor cells. The reasons for the use of the boost treatment were primary graft failure (early rejection; n = 7), secondary graft failure including late rejection (n = 10), refractory PRCA caused by the remaining recipient cells producing anti-erythrocyte antibodies (n = 2), and donor lymphocyte infusion induced pancytopenia (n = 1). The patients were aged from 17 to 48 years (median age 31 years). The underlying diseases of the patients were sAA in 12 patients, AML in 3, ALL in 3, and CML in 2. The donors were HLA-identical siblings in 18 cases, 1 mismatched related donor, and 1 unrelated donor. The cell source was BM in 6 cases and PB progenitor cells in 14. The median interval between BMT and the boost treatment was 7 weeks (range 1-124). No conditioning regimen was given prior to the boost treatment for 11 patients, while 4 received TNI + ATG, 3 ATG alone, and 2 TNI plus steroid. The median infused booster mononuclear cell dose was 2.55 x 10⁸/kg (range 0.28-37.0). 15 (75%) patients achieved a hematological recovery. After the boost treatment, 6 of 20 (30%) patients developed aGVHD > or = grade II, 3 of whom had had prior GVHD. 5 (31.3%) of the evaluable 16 patients developed cGVHD. The GVHDs were easily controlled using immunosuppressive agents except in the case of 1 patient. 5 patients died after the boost treatment; 2 within 30 days, 2 within 60 days, and 1 after 32 months. The causes of death were: 3 engraftment failures, 1 late rejection, and 1 infection following GVHD. With a median follow-up of 31.5 months (range 6-92), the Kaplan-Meier method estimated that the OS rate 1 and 3 years after the boost treatment was 80 and 71%, respectively. The survival of patients with primary graft failure was determined to be significantly lower compared to that of patients with secondary graft failure, using the log rank test (p = 0.0176). Disease category, stem cell source, conditioning prior to a boost treatment, and year of boost treatment did not have an influence on survival^ref
• a patient with type I Gaucher disease was treated by allogeneic BMT from HLA-matched sibling donor. However, the treatment resulted in late graft failure and the patient developed severe bone involvement. Fifty months after the first BMT, the patient was treated by allogeneic PBSC transplantation without preconditioning. G-CSF was used to mobilize PBSC. CsA) was administered for the prophylaxis of GVHD. Full donor-derived hematopoiesis was obtained, and clinical symptoms including severe bone involvement improved completely with increased glucocerebrosidase activity. It was shown that an engraftment could be obtained without intensive preconditioning when a recipient receives an rhG-CSF-mobilized PBSCs infusion as a secondary transplant. Another important finding of this study is the complete reversal of severe bone involvement by the supply of abundant glucocerebrosidase from high proliferating PBSC graft^ref
Complications :
• infections : in 62 patients undergoing NMSCT with low-dose TBI +/- fludarabine and postgrafting CsA and MMF, the proportion of patients with any infection was 77%, but the majority of infectious events occurred beyond day 30. Donor other than sibling, older age, early disease and male gender were significant risk factors. The incidence of bacteremia was 55% at 1 year and the number of bacteremic episodes was 0.9 per patient (0.08 before day 30). The risk of bacteremia increased with older age and the use of a donor other than an HLA-identical sibling, but not with neutropenia. The incidence of infections other than bacteremia correlated with the use of corticosteroids. The risk of CMV infection increased with high-risk CMV serology, and risk of CMV disease with high-risk CMV serology, older age, first transplantation and a diagnosis of lymphoma^ref
• deep vein thrombosis (DVT) might be a significant problem associated with RIST in patients with solid tumours and anticoagulation therapy might be needed.
• monoclonal antibodies in conditioning :
• alemtuzumab : TCD (reduced GvHD, increased graft rejection (20%), relapse (expecially CML, not related to lymphomas), and delayed immune recovery)
• Campath-1G in vivo + Campath-1M in vitro reduce both rejection and GvHD
• Campath-1H 50-100 mg days -8 to -4 or -6 to -2 (dosable until day + 10, so with greater anti-rejection activity) or -3 to -1 (dosable until day +40, so with greater anti-GvHD activity). Still dosable after 2 months, T-cell recovery after 1 year
• Campath in the bag : (CD52 binding sites per lymphocyte = 5x 10⁵) x (lymphocytes per transplant = 3 x 10¹⁰) x (molecular weight of IgG = 1.5 x 10⁵) x 10³ / NA (= 6 x 10²³) = 3.75 mg per bag
• ATG^ref : reduces cGvHD to 4% => indicated in PBSCT^ref.
Indications :
• malignancies :
• leukemias and lymphomas : allo-SCT for relapsed or refractory aggressive histology (DLBCL, PTCL, transformed B cell) NHL results in long-term EFS and OS of 40-50%. Patients with chemorefractory disease can have a durable remission post-transplant. TRM was 25% at 1 year. Grade III-IV aGVHD was the only significant variable affecting OS and EFS, and had a negative impact. cGVHD did not influence survival. Lymphoma relapse <12 months after initial therapy predicted for increased risk of relapse post-transplant^ref
• myeloproliferative disorders (MPD)
• selected advanced solid tumors^ref (see situation in Europe^ref and USA^ref), including nonmyeloablative conditioning regimens^{ref1, ref2, ref3}
• metastatic breast cancer^{ref1, ref2}, including nonmyeloablative conditioning regimen^ref
• renal cell carcinoma, including nonmyeloablative conditioning regimen^ref
• colorectal carcinoma, including nonmyeloablative conditioning regimen^ref
Among patients who have relapsed after allogeneic HSCT, an immunologically based GVT effect may be obtained :
• simply by transfusing T cells obtained by leukopheresis of the original bone marrow donor (donor leukocyte infusion (DLI)) : this technique has been used to obtain complete remissions in relapsed acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), multiple myeloma, non-Hodgkin's lymphoma, myelodysplastic syndrome (MDS), and chronic myeloid leukemia (CML)
• combination of nonmyeloablative BMT followed by immunotherapy with DLI.
• tumor vaccines in conjunction with autologous transplantation offer a promising method for eliminating tumor.
Patients undergoing autologous HSCT may have bone marrow and peripheral blood that has been contaminated with tumor (Minimal residual cancer detection in hematopoietic stem cell products and its prognostic significance in patients with breast cancer, lymphoma, or multiple myeloma by Thomas J. Moss, MD), which places them at a higher risk of relapse :
• purging bone marrow with either 4-hydroperoxycyclophosphamide, mafosfamide or with mAb have proven feasible although inducing prolonged myelosuppression. Selection of CD34⁺ progenitors appears to be a promising alternative with a 2.5-4.5 log depletion of plasma cells.
• PBSCT have possibly lower tumor contamination than BMT, despite sensitive immunofluorescence studies or PCR based techniques have demostrated that virtually al PBPC harvests are contaminated with malignant cells. Several pilot studies have confirmed the feasibility of autologous transplants with CD34⁺ selected PBPC in MM
Prior exposure to chemotherapy, specially to alkylating agents, significantly impact stem cell collection and hematopoietic recovery : thus PBSC should be collected prior to ASCT.
• nonmalignant conditions : RI-HSCT provides a good alternative to myeloablative HSCT for children with non-malignant disorders, except for haemoglobinopathies, in which engraftment is poor : even patients with MUD-HSCT have adequate engraftment with acceptable toxicities^ref.
• paroxysmal nocturnal hemoglobinuria
• congenital immune deficiencies
• Glanzman thromboasthenia
• thalassemia
• Diamond-Blackfan anemia (DBA)
• sickle cell anemia
• osteopetrosis
• inborn errors of metabolism (IEM) :
• childhood cerebral form of X-linked adrenoleukodystrophy (X-ALD) is a rapidly progressive demyelinating condition affecting the cerebral white matter, which rapidly leads to total disability and death. The only known curative treatment for this condition is allogeneic HSCT. Procedure-related toxicity is assumed to be the cause of death of patients with X-ALD. 3 cases of ALD successfully transplanted with the use of non-myeloablative fludarabine based conditioning are described. Patients showed smooth peri-HSCT course with fast and stable engraftment. In the 3- to 5 yr follow-up period, patients showed no deterioration in their clinical and neurological condition. Levels of very long chain fatty acids were very variable and had a tendency to decrease in at least 1 of the 3 patients. In another patient, an improvement of MRI changes was found. Non-myeloablative HSCT should be considered as an early treatment for X-ALD^ref
• aplastic anemia
• Fanconi anemia : RIC has been suggested as a desirable therapeutic modality for the treatment of patients with malignant and nonmalignant indications, but it seems particularly attractive for patients with Fanconi anemia due to their increased sensitivity to chemoradiotherapy. Between November 1996 and September 2003, 7 patients (1 male and 6 female; age range, 3-31 years; median age, 9.5) were conditioned with a fludarabine-based protocol for HSCT without radiation. In vivo T-cell depletion was accomplished with ATG or Campath-1H (alemtuzumab). GvHD prophylaxis consisted of low-dose cyclosporine alone. 8 transplantations were carried out for 7 patients using bone marrow, peripheral blood, and/or cord blood as sources of stem cells. All patients received transplants from HLA-A, -B, -C, and -DR matched donors, 5 from family members and 2 from MUDs. 1 patient did not engraft her first matched unrelated donor and underwent a second transplantation from another MUD, after which she engrafted well. All 7 patients are alive and well, fully reconstituted with donor cells, and with 100% performance status. In conclusion, fludarabine-based preparative protocols are well tolerated, facilitate rapid engraftment with minimal toxicity, and should be considered an essential component of choice for patients with Fanconi anemia^ref
• myeloablation resulting from anti-proliferative chemotherapy or radiotherapy (sometimes to increase the MTD)
• autoimmune diseases : graft versus autoimmunity effects
• rheumatoid arthritis : of 9 allogeneic bone marrow transplants, 4 patients have done well for many years while one relapsed after 2 years
• myasthenia gravis
• insulin dependent diabetes mellitus
• spinal cord inury (SCI) : Hwang Mi-Soon, 37-yo, received UBSCT in injured spinal cord and stood up her wheelchair after 20 yrs of paralysis caused by an accident. Stem cells transplanted after SCI mostly generate astrocytes, which can promote aberrant sprouting of sensory neurons, leading to allodynia. Making cells produce more oligodendrocytes reduces allodynia and improves functional recovery^ref.
Contraindications :
• HHV-5 / CMV serostatus^ref : prior to the arrival of effective antiviral therapy in the early 1990s, CMV disease was the leading infectious cause of death among CMV-seropositive recipients of HSCT. The initial goal of prevention strategies was to eliminate CMV disease, which presents as often-fatal pneumonia despite combination treatment with ganciclovir and immunoglobulin. Thus, both prophylaxis and preemptive therapy strategies have been extensively studied and prevention protocols based on these strategies are now used in transplant centers around the world. As a result, CMV disease during the first 3 months after HSCT has been reduced from 20-30% to < 5% in most studies^ref. Despite these accomplishments, several lines of investigation indicate that OS remains inextricably linked to the pretransplant CMV serostatus of the patient. Thus, the overriding goal of antiviral supportive care—to reduce the TRM for the CMV-seropositive patient to the level of a seronegative patient—has remained elusive. Schema demonstrating the impact of pretransplant CMV infection on overall survival after allogeneic HSCT. Despite the near-complete elimination of early CMV disease with current strategies, a survival disadvantage persists for high-risk, CMV-seropositive patients (R+) compared with D–/R– patients :


Additional studies^{ref1, ref2, ref3} have also examined the impact of donor serostatus among seropositive recipients; these studies have come to conflicting conclusions. The issue of donor CMV serostatus is important, as it may be relevant for donor selection for both seronegative and seropositive transplant recipients. The CMV serostatus of the donor, however, must be considered as one selection factor among many that are important for transplantation outcome including HLA compatibility, donor age, and sex matching^ref.
• CMV serostatus of the recipient : despite the significant decline in the incidence of CMV disease among CMV seropositive HSCT recipients early after transplantation, several recent studies have indicated that a substantial mortality disadvantage still exists for the CMV-seropositive patient. The studies differ with regard to sample size, time of follow-up, endpoints, underlying disease, and graft manipulation (ie, T-cell depletion); the multitude of factors known to be associated with mortality raises the issue of residual confounding in many of these studies. Large studies of CMV serostatus and mortality in more homogeneous populations, however, demonstrate similar findings^{ref1, ref2} :

reference, by first author	no. patients	underlying disease	T-cell-depleted donors, %	unrelated donors, %	results among CMV-seropositive recipients compared with CMV-seronegative recipients with a seronegative donor
Broersref (The seropositive group includes some seronegative recipients with a seropositive donor)	115	Mixed	95	0	24% absolute decline in OS (P = .01)
McGlaveref	1423	CML	23	100	20% relative decline in DFS (P = .002)
Cornelissenref (The seropositive group includes some seronegative recipients with a seropositive donor)	127	ALL	26	100	38% relative decline in DFS (P = .05)
Craddockref (the difference in OS is between seropositive recipients and seronegative recipients (some of whom have seropositive donors); the difference between seropositive recipients and D—/R— patients is 14% at 5 years)	106	CML	100	100	22% absolute decline in OS (P = .006)
Krogerref	125	Mixed	100	100	41% absolute decline in OS (P < .001)
Castro-Malaspinaref	510	MDS	24	100	46% relative decline in DFS (P = .001)
Nicholsref	1750	Mixed	0	57	26% relative decline in OS (P = .03)
Kollmanref	6978	Mixed	25	100	7% absolute decline in OS (P < .001)
Meijerref	48	Mixed	100	100	41% absolute rise in TRM (P < .001)
Doneyref	182	ALL	0	52	99% relative rise in TRM (P = .01)

Analysis of published studies suggests that specific subgroups of CMV-seropositive HSCT recipients appear to be at particularly high risk for TRM in the current era. The use of T-cell depletion (either via graft manipulation ex vivo or via the use of anti–T-cell antibodies in vivo) appears to be one such factor. Indeed, most of the studies performed to date have focused on these patients. A small study addressed the particular method of TCD, suggesting that the dose of antithymocyte globulin (ATG) is an important factor in the observed effect on survival^ref. Larger studies are needed to confirm this hypothesis. CMV-seropositive recipients of transplants from unrelated donors may also be at higher risk for mortality; this association was demonstrated in a study by McGlave et al^ref (where CMV seronegativity was still associated with a 20% lower hazard for mortality after controlling for graft manipulation) and the studies by Nichols et al^ref and Meijer et al^ref. A recently completed subanalysis of the study by Nichols et al indicates that the survival disadvantage associated with CMV seropositivity in T-cell–replete patients is restricted to recipients of HLA-mismatched or unrelated transplants^ref :

donor/recipient CMV serostatus	mortality for matched related donors (hazard ratio, 95% CI)	mortality for mismatched related or unrelated donors (hazard ratio, 95% CI)
D-/R-	1.0 (reference)	1.0 (reference)
D+/R-	0.98 (0.70-1.38)	1.36 (1.06-1.74)
D-/R+	0.90 (0.65-1.24)	1.29 (1.04-1.60)
D+/R+	1.07 (0.81-1.42)	1.26 (1.01-1.58)

Shown are results of multivariable models, which controlled for patient/donor age, underlying disease and disease risk, conditioning regimen, cell source, GVHD prophylaxis, cell dose, and year of transplantation. Recipients of HLA-mismatched transplants from relatives and unrelated donors had similar outcomes (data not shown). For matched related donors, N = 749; for mismatched related or unrelated donors, N = 1001
The mechanism for increased mortality among CMV-seropositive patients is often not presented, though TRM rather than relapse accounts for the majority of the findings. Some indirect effects (eg, nonviral infections) may be difficult to separate from consequences of drug toxicity (eg, ganciclovir-associated neutropenia). Both Broers et al^ref and Cornelissen et al^ref documented a higher incidence of aGVHD in CMV-seropositive recipients, which resulted in a high incidence of opportunistic infections and respiratory insufficiency. Craddock et al^ref found no difference in GVHD, but more infectious death in the CMV-seropositive group. Meijer also failed to find a difference in aGVHD according to serostatus, but a significant increase in infectious mortality was detected among seropositive recipients of unrelated donor transplants^ref. The putative immunomodulatory effects of CMV infection have long been recognized in the setting of solid organ transplantation and HIV/AIDS, and have recently been reviewed^ref. Possible reasons for association of CMV with poor outcome in high-risk patients :

• CMV serostatus of the donor : selection of an appropriate HSCT donor usually begins with a search for an HLA-matched sibling. If one is not available, the use of stem cells from an HLA-matched unrelated donor is pursued. Outcome after unrelated donor transplantation has been associated with similar outcomes for some underlying diseases^{ref1, ref2}. Searches of donor registries may identify multiple suitable matches; in such instances, donor selection may be based on other factors, including CMV serostatus. It is known that many transplant physicians prefer CMV-seronegative donors^ref. In theory, however, the selection of donor according to CMV serostatus may depend on the serostatus of the HSCT recipient. For the CMV-seronegative recipient, it may be preferable to utilize CMV-seronegative donors in order to reduce the possibility of primary CMV infection associated with a seropositive allograft^ref. In contrast, the use of stem cells from a seropositive donor may be preferred in the seropositive recipient due to the potential for active transferred immunity, a phenomenon shown to be operative in the case of HSV^ref. As detailed below, however, the data to support these hypotheses have been conflicting. In a landmark study that examined the effect of donor factors on transplant outcomes among almost 7000 patients receiving unrelated marrow transplants in the National Marrow Donor Program (NMDP) Registry from 1987 to1999, Kollman et al^ref showed that donor age and HLA mismatch (but not donor CMV serostatus) were the primary drivers of TR. Although CMV seropositivity in the recipient was clearly associated with poorer outcome, the survival curves for donor-positive versus donor-negative were virtually superimposed for both the CMV⁺ and -negative recipient^ref. In contrast, Ljungman et al^ref conducted a similar analysis of over 7000 CMV-seropositive patients in the EBMTR who received transplants over > 2 decades, and found somewhat different results. No significant effect of donor serostatus could be discerned among the almost 6000 patients who received transplants from HLA-identical siblings, but transplant-related mortality and overall mortality were higher among 542 CMV-seropositive recipients of stem cells from seronegative unrelated donors when compared with the 566 seropositive recipients of stem cells from seropositive unrelated donors in both univariate and multivariable models^ref. Donor serostatus did not affect the risk for acute or chronic GVHD in that study, but there was a trend toward a higher rate of death associated with infections among patients who received grafts from CMV-seronegative donors (28% vs 23%, P = .07) :


Recently published studies of donor CMV seropositivity's impact on mortality :

author	N	setting	% of patients with T-cell depletion	% of transplants from unrelated donors	effect on mortality
					positive recipient	negative recipient
Kollman et alref	6878	mixed	25	100	none	none
Nichols et alref	1001 (also included recipients of HLA-mismatched transplants from related donors)	mixed	0	100	none	increased
Ljungman et alref	1108	mixed	27	100	decreased	NA

Reconciling these results is difficult, but of significant clinical importance. Given the option of an equally matched CMV-seronegative or -seropositive donor for a seropositive recipient, Ljungman et al^ref suggest that selecting the seropositive unrelated donor for the seropositive patient decreases the hazard for overall mortality by up to 35%. Interestingly, donor serostatus appeared to alter the hazard of death in some diseases (eg, chronic myeloid leukemia [CML]) but not others (eg, acute leukemia); this may be due to the competing risks of relapse and noninfectious mortality, both of which are higher in patients with acute leukemia. Indeed, Ljungman and colleagues^ref suggest that by adjusting for disease state (rather than examining disease groups separately), the NMDP study may have missed this interaction between CMV serostatus and disease. Other subgroups of interest (the influence of T-cell depletion or mismatched transplants, for example) did not appear to have been specifically analyzed in the context of donor serostatus in the NMDP study. Residual confounding may also explain some of the results, given that increasing donor age (the primary predictor of poor outcome in that study) was associated with CMV seropositivity. Finally, by including a significant number of patients from the pre–ganciclovir era (20%), the NMDP study may have also attenuated the true relationship between donor serostatus and outcome. Results were adjusted for time periods in both studies^{ref1, ref2}, however, it is possible that important differences in posttransplant prevention strategies and supportive care were not accounted for. The study by Nichols et al^ref (which was performed exclusively in the era of effective prophylaxis and preemptive therapy) did not find a difference in survival between seropositive and seronegative donors for the seropositive transplant recipient, though all patients in that series received T-cell–replete transplants.
For the CMV-seronegative recipient of allogeneic HSCT, it has been demonstrated that the receipt of stem cells from a seropositive donor (D+/R–) is associated with a significant increase in the hazard for overall mortality when compared with transplants from a seronegative donor, findings that have been offered as further evidence of the immunomodulatory properties of primary CMV in the HSCT setting^ref. Notably, this difference has been particularly apparent in unrelated donor transplants. Cwynarski et al^ref also showed that seronegative children receiving stem cells from seropositive donors had decreased overall survival when compared with D–/R– patients. Kollman et al^ref, however, did not confirm these findings in the setting of unrelated transplantation. Given the risk of primary infection due to the seropositive allograft, however, the prevailing practice of selecting a seronegative donor for the seronegative recipient seems prudent.
Recipient CMV serostatus continues to be an important variable for overall survival after transplantation in the era of antiviral preemptive therapy and prophylaxis. This survival disadvantage appears largely restricted to highly immunosuppressed patients, such as recipients of T-cell–depleted grafts or grafts from HLA-mismatched or unrelated donors. Mechanistically, the increased mortality in seropositive recipients appears to be due to both direct and indirect effects of CMV as well as drug toxicities. The impact of donor seropositivity, however, remains controversial, particularly for seropositive recipients.
These results raise several questions that are in urgent need of investigation in order to optimize supportive care for the HSCT recipient.
• 1. What groups of CMV-seropositive and D+/R– patients are at highest risk for mortality in the current era? Studies to date suggest that the survival disadvantage is limited to high-risk patients, yet these patients are not very well characterized. T-cell depletion and HLA-mismatched or unrelated donor status appear to be important variables for poor survival, but the impact of more sensitive HLA matching strategies^{ref1, ref2, ref3, ref4}, posttransplant antiviral prevention strategies, and the impact of underlying disease remain to be investigated. The method and degree of T-cell depletion may also be an important variable^ref. To more accurately define groups of patients who might benefit from improved prevention strategies, analysis of large cohorts of patients who received transplants under current supportive care protocols (including preemptive or prophylactic anticytomegalovirus strategies and effective antifungal prophylaxis) are needed. The impact of high-resolution HLA matching strategies on CMV-related mortality is also likely to be important, and should be a focus of investigation. Also, the effect of serostatus with use of nonmyeloablative as well as reduced-toxicity conditioning regimens^{ref1, ref2} as well as socioeconomic status or ethnicity as confounding factors^{ref1, ref2} should be studied.
• 2. What is the mechanism of excess mortality among high-risk, CMV-seropositive patients? Although the studies cited above show that seropositivity in the recipient is associated with increased mortality, less accurate and mostly retrospective information is available on the causes of death. Knowledge of the causes of death in seropositive recipients (relative to D–/R– patients) may provide important leads regarding operative mechanisms, and may also offer opportunities for intervention.
• 3. What strategies are available to improve outcomes in high-risk recipients? If survival among D–/R– patients is used as the gold standard, the current literature indicates that preemptive therapy (the predominant strategy for allogeneic transplant recipients^{ref1, ref2}) is less effective for high-risk recipients of HSCT. Thus, improved prevention strategies are needed and the issue of preemptive therapy versus prophylaxis should be revisited. By suppressing reactivation, prophylaxis may also decrease the indirect immunomodulatory effects of viral replication. However, significant disadvantages are ascribed to this strategy. Ganciclovir given at engraftment historically caused prolonged neutropenia and was associated with a higher risk of invasive bacterial and fungal infections^{ref1, ref2, ref3}. A delayed recovery of CMV-specific immune responses compared with patients who receive culture-based preemptive therapy has also been described^ref, which may lead to an increased risk of late-onset CMV disease (Boeckh M, Riddell SR, Cunningham T, Myerson D, Flowers M, Bowden RA. Increased incidence of late CMV disease in allogeneic marrow transplant recipients after ganciclovir prophylaxis is due to a lack of CMV-specific T cell responses. Blood. 1996;88(suppl 1): 302a). An effect of ganciclovir prophylaxis on recovery of CMV-specific immunity was not seen when compared with antigenemia-based preemptive strategies^ref. Thus, new anti-CMV drugs with an improved toxicity profile are needed to provide effective and well-tolerated prophylaxis in high-risk patients. Although drug development is being pursued^{ref1, ref2} (Kern ER, Bidanset DJ, Quenelle DC, et al. Enhanced efficacy of orally active cidofovir prodrugs against experimental cytomegalovirus infections (abs, V-1289). 43rd Annual Interscience Conference on Antimicrobial Agents and Chemotherapy. Chicago, IL; 2003: 501), these new compounds are early in development and may not be readily available for several years. Immunologic strategies including adoptive immunotherapy and vaccine strategies are another option as they may overcome issues of drug toxicity by hastening immune reconstitution^{ref1, ref2}. There have been major laboratory improvements in T-cell therapy, which may make this therapy more readily available in the near future^{ref1, ref2, ref3, ref4, ref5, ref6}. The use of cytokines such as IL-7 may also prove useful^ref. One potential obstacle for widespread use of immune-based strategies may be the use of systemic steroids^ref. Clinical trials are needed to evaluate these strategies. A look at presently available drugs may also be useful. A review of recently conducted studies indicates that the sequelae of ganciclovir-induced neutropenia (ie, bacterial and fungal infections) may not be as common as they once were with current management of neutropenia (via the use of growth factors and/or early discontinuation of ganciclovir in the setting of toxicity and/or alternative use of foscarnet) and better antifungal agents. In 2 randomized trials the incidence of severe nonviral infections was not increased between the ganciclovir and the control arms^{ref1, ref2}. Thus, it is possible that ganciclovir or valganciclovir prophylaxis may be of benefit in subgroups of highly immunosuppressed transplant recipients, where even preemptive strategies based on highly sensitive PCR–based surveillance often fail^ref. With improvements in supportive care (ie, early use of growth factors), use of alternative agents with different toxicity profiles (eg, foscarnet) in cases of neutropenia, improved diagnostics for fungal infections, and better agents for antifungal prophylaxis and treatment, it may be possible to design an effective and well-tolerated long-term antiviral prophylaxis strategy aimed at both the direct and indirect effects of CMV. These intensified strategies should be studied in a randomized fashion. Valacyclovir has not shown a strong antiviral effect in high-risk patients^ref and is thus unlikely to be useful as a single agent in this setting. Whether it could be part of a strategy could be tested in randomized trials. Finally, the available data suggest that CMV seropositivity is primarily associated with poor outcome in patients receiving T-cell–depleted HSCT, yet appears to be relatively benign among T-cell–replete transplants from related donors. This raises the question if alternatives to T-cell depletion should be pursued for the CMV-seropositive recipient with a family donor until improved prevention strategies are developed. Although evidence in support of this position stems from retrospective analyses rather from randomized trials, recognition and open debate of this issue are warranted. Recipients of TCD grafts, however, may be particularly good candidates for adoptive transfer studies due to their lower risk of GVHD and thus lower utilization of steroid therapy. At the present time, however, no data exist on the efficacy of this approach in affecting outcome.
• 4. What priority should donor CMV serostatus assume in the process of donor selection? As summarized above, the selection of a CMV-seronegative donor for the seronegative HSCT recipient seems prudent^{ref1, ref2}. For seropositive recipients, however, the impact of donor CMV status remains controversial^{ref1, ref2}. The European study had fewer numbers of "high-risk" transplant recipients, and was not able to entirely control for donor age; importantly, both studies also included patients from the era prior to effective CMV therapy, which may confound their results, and did not include high-resolution HLA typing. Future studies should focus on cohorts exclusively from the current era, and should include other important variables such as antiviral prevention strategy and degree of HLA match using contemporary techniques.
• performance status > 2
Pathogenesis :
Supportive therapies :
• BMT and PBSC patients treated with G-CSF had a faster engraftment of absolute neutrophils greater than 0.5 x 10(9)/L (P <.01), but platelet engraftment ( > 50 x 10⁹/L) was slower (P <.001). In the BMT patients, aGVHD grades II to IV was 50% +/- 5% (+/- 95% CI) in the G-CSF group versus 39% +/- 3% in the controls (relative risk [RR], 1.33; P =.007, in the multivariate analysis). The incidence of chronic GVHD was also increased (RR, 1.29; P =.03). G-CSF was associated with an increase in transplantation-related mortality (TRM; RR, 1.73; P =.00016) and had no effect on relapse but reduced survival (RR, 0.59; P <.0001) and leukemia-free survival rates (LFS; RR, 0.64; P =.0003). No such effects of G-CSF were seen in patients receiving PBSC^ref.
• enteral nutrition is a safe and effective method for feeding allo-SCT patients. A randomized trial is warranted to confirm its advantage on early patient outcome^ref.
Reconstitution : engraftment within 2 weeks
• ANC > 500/ml at week 3
• slower for PLT and RBC
• long periods of post-transplant immune deficiency (4 months to 3 years). Peripheral T-cell reconstitution is particularly slow : in mice 3 populations of donor T cells are found in allogenic hosts after transplantation : fast proliferating alloreactive cells (that downregulate IL-7R, respond to host antigens and cause GvHD), slow proliferating non-alloreactive cells and non-proliferating non-alloreactive cells. IL-7 administration could improve the immunocompetence of patients after allogeneic HSCT driving the clonal expansion of non-alloreactive T-cells without inducing GVHD^ref. Allograft dendritic cell (DC) content has been identified as a predictor of relapse and event-free survival after allogeneic HSCT. The immune recovery of 66 patients undergoing allogeneic HSCT with either conventional or non-myeloablative conditioning regimen was studied. Infections post-transplant were enumerated and quantitative immunoglobuilins (IgG, IgA, IgM) and lymphocyte sub-sets 3, 6 and 12 months post-transplant were measured. A significant difference was found in the immunologic recovery of non-myeloablative and conventional ASCT in the patient population. The T-helper cell reconstitution was significantly faster after NMA than conventional transplantation and the recovery of B cells was faster after conventional transplantation. Regarding immunoglobulin levels, a faster recovery of IgM levels after NMA-ASCT and a delayed recovery of IgA levels was observed in both groups. These were accompanied by a significant difference in the frequency and severity of infectious episodes^ref.
Prognosis :
• plasma cholinesterase : on day +7 after HSCT, the median level for surviving patients was 2598 IU/l vs 2309 IU/l for patients who subsequently died (P = 0.0002), on day +21 CHE levels were respectively 3348 vs 2528 IU/l (P < 0.00001), on day +50, 3575 vs 2358 IU/l (P < 0.00001) and on day +100 4193 vs 2729 IU/l (P < 0.00001). CHE levels on day +50 strongly correlated with aGVHD (3803 vs 3070 vs 1933 IU/l for patients with GVHD grade 0-I, II, and III-IV, respectively (P < 0.00001) and relapse (3569 for patients relapsing vs 3115 IU/l for patients not relapsing, P = 0.0006)^ref
• CRP : a rise in the first week^refis a surrogate for acute and/or chronic GvHD which themselves are powerful predictors of relapse^ref. The background of interindividual differences in CRP level may be related to polymorphisms in CRP gene^{ref1, ref2} and genetic variations in components governing the inflammatory response. IL-1 and IL-6 are the central regulators of the acute phase proteins. Several variations in the IL-1 gene cluster and the IL-6 gene correlate with the expression of these interleukins, and they have also been associated with a number of inflammatory diseases^ref. More recently, it was shown that the serum IL-6 levels may be an early predictor of transplant-related complications and might trigger both pro- and anti-inflammatory cytokine release^ref
• eosinophilia : OS rates improved in those with eosinophilia compared to those without eosinophilia (88.7 vs 43.0%, P=0.0034). In particular, in patients with malignant diseases, those with eosinophilia showed a higher event-free survival (81.1 vs 44.6%, P=0.0025) and a lower relapse rate (16.0 vs 43.0%, P=0.0287) than those without eosinophilia, independently from preconditioning regimen and immunosuppressive therapy. However, no statistical difference in serum IL-5 was observed between SCT patients with and without eosinophilia, although IL-5 levels at each sampling point were slightly (but not significantly) higher in patients with eosinophilia than those without. Instead, high serum levels of IL-12 and low serum levels of IL-10 were observed in SCT patients with eosinophilia^ref. Eosinophilia during SCT might be an indicator of high IL-12/low IL-10 levels; nevertheless, the mechanism underlying these cytokine patterns during eosinophilia remains unknown. In previous studies, some authors demonstrated that combined IL-12 and GM-CSF, which contributes to the production and activation of eosinophils, provided antitumor immunity^{ref1, ref2}. 20% of patients develop eosinophilia (AEC > 500/mL) after day 100 : a rise in the AEC may herald the development of cGVHD in adults^ref, but not in children^ref. McNeel et al^ref reported 2 cases of hypereosinophilia as an early sign of aGVHD after allogeneic HSCT. Patients were treated with glucocorticoids, resulting in resolution of both eosinophilia and GVHD symptoms. Daneshpouy et al^ref examined the relationship between eosinophilia and digestive GVHD, and found that the eosinophil density in the digestive tissue correlated with GVHD severity. Therefore, although not yet confirmed, these authors have concluded that eosinophilia indicates a potential development of GVHD^{ref1, ref2}.
• serum sTNFRI level is more likely to reflect the degree of inflammatory activation induced by pretransplant conditioning^ref
• G-CSF increases serum levels of sIL-2R^{ref1, ref2, ref3}
• serum sIL-2R and sFas levels were significantly raised in allograft, autograft (allo and auto) and non-graft groups compared to the normal controls (HC), but there was no statistical difference between the three patient groups. TNF-a was raised in the auto and allo groups and the non-graft patients compared to the HC group (median 4.37 pg/ml), but only reached significance in the allo group (median 6.02?pg/ml; p=0.008) when this was compared with the non-graft patients. There was no significant difference in TGF-ß levels between any of the groups. The median serum VEGF levels were decreased in allo and auto patients compared to HC, (31 and 62 pg/ml versus 90 pg/ml, respectively), with a significant difference in the auto group (p=0.007). VEGF levels were significantly lower in the auto versus the allo group (p=0.008) and also in the auto versus the non-graft group (median 104 pg/ml; p=0.011). When the allo group was divided into patients who developed GvHD and those who did not, serum VEGF levels were significantly higher in those with GvHD (p=0.028)^ref.
• during the pretransplant conditioning, full donor chimera (DC) patients had higher levels of TNF-a and lower levels of TGF-b and IL-10, compared with mixed chimera (MC) patients. During the first week after HSCT, lower levels of TGF-b and IL-10 and higher levels of soluble Fas (sFas) were found in DC patients compared with MC patients. During the second and third weeks after HSCT, increased levels of TNF-a, IFN-g and sFas were found among DC patients compared with MC patients. Patients who developed moderate-to-severe acute GVHD had higher levels of TNF-a, IFN-g, IL-10 and sFas at 2 weeks post-HSCT than in those with less GVHD. Patients homozygous for the TNFd microsatellite alleles 3 or 4 had significantly higher TNF-a levels during conditioning and more often developed acute GVHD grades II-IV^ref.
Complications : procalcitonin (PCT) differentiates between infection and other transplant-related complications. Exclusive aGVHD did not increase CRP (4.4 mg/dL vs. 5.7 mg/dL; n.s.), IL-6 (93 ng/mL vs. 153 ng/mL; n.s.) and PCT (0.8 ng/dL vs. 0.8 ng/dL; n.s.). CRP (6.1 mg/dL vs. 3.1 mg/dL; P < 0.001) and IL-6 (295 ng/mL vs. 122 ng/mL; P = 0.001) were decreased during steroid therapy, but not PCT (2.3 ng/dL vs. 2.0 ng/dL; n.s.)^ref

• early complications :
• acute renal failure : during the first 2 weeks after HSCT, it develops in up to 75% of patients receiving myeloablative regimens and approximately 33% of these patients require hemodialysis^ref. The mortality rate among patients with acute renal failure is 2-7 times that among patients without this complication^ref.  From 2 weeks to 3 months after transplantation, a spectrum of renal insults occurs secondary to aGVHD. These insults range from prerenal azotemia to a TTP-like microangiopathy that does not respond to pheresis and is accompanied by high mortality rates (50-60%)^ref. By 3 to 6 months after transplantation, the GFR decreases in 65-100% of patients^ref. In some patients with cGVHD, the nephrotic syndrome, primarily membranous glomerulonephropathy, develops^ref. A combination of radiation-induced nephritis and thrombotic microangiopathy may also occur^ref. These frequencies pertain only to allogeneic HSCT with myeloablative regimens; but even in that setting, the stated need for hemodialysis is higher than that reported in most studies^{ref1, ref2}. Renal failure often occurs in association with the sinusoidal obstruction syndrome, as stated in my review article, and it may be secondary to that condition or to other complications, particularly sepsis
• graft rejection :
• HLA-matched related donor : 2%
• MUD : 12%
• 7% in HLA-matched
• 9% in 1-HLA mismatched
• 21% in 2-HLA mismatched
Anti-HLA antibody-mediated rejection (AMR)^ref : patients who have become sensitized to allogeneic HLA antigens through pregnancies, blood transfusions, failed transplants, or other means represent a challenge when requiring a transplant or platelet transfusion. Currently, approximately 30% of the patients on the waiting list have evidence of sensitization, and only 6.5% of highly sensitized patients (those with a panel-reactive antibody level above 80%) receive a transplant each year^ref.
Risk factors :
• female sex
• high parity
• high number of blood transfusions (not depleted, not irradiated)
• failed previous transplant
• G-CSF-mobilized peripheral blood HSCT compared with bone marrow transplantation^ref
Laboratory examinations : sensitization is reported as the percent panel reactive antibody (PRA), which is the percentage of potential donors' cells tested that were killed by the patient's serum (so they are not necessarily donor specific antibody (DSA)). The PRA also gives an estimate of the likelihood of finding a suitable donor. Computerized analyses of lymphocytotoxicity reaction patterns on well-characterized cell panels identify the specific HLA-antigens against which antibodies are directed, and "safe" antigens, which are not recognized. Patients who have autoimmune diseases, are on particular medications, or have infections may develop false positive reactions that are not caused by anti-HLA antibodies. Flow bead tests provide results separately for class I and class II antibodies and since they are based on purified HLA antigens, they avoid many of the false positive reactions. Flow bead testing can also be used to identify HLA specificities even with low antibody titers. Results are reported as the percent PRA for cytotoxicity tests, whether or not reactivity is DTT sensitive, and the anti-HLA specificities that were identified. Luminex and flow cytometry test reports indicate percent PRA for class I and class II antigens and antibody specificity. Single recombinant HLA class I and class II antigens coupled to microparticles are used to determine the HLA specificity of broadly reactive sera or sera with undefined specificity. Anti-HLA antibody testing provides an accurate assessment of an individuals sensitization status and identification of the HLA antigens specifically targeted by those antibodies. The tests used to identify anti-HLA antibodies are based upon assessing reactions of antibodies with well-characterized panels of individual lymphocytes (using complement-dependent lymphocytotoxicity), and with purified HLA antigens coupled to specialized microparticles (luminex, flow cytometry).
• complement-dependent  lymphocytotoxicity is used to identify patients at risk for antibody-mediated damage to grafted organs or tissue. This test can also be performed after transplantation.
• Luminex is used before and after transplantation to monitor patients for presence or absence of anti-HLA class I and class II antibodies or to monitor the appearance or disappearance of antibodies over time. Positive sera are subsequently tested by luminex class I and class II identification assays to determine the HLA specificity of the antibodies.
• fluorescence cytometric cross-match (FCXM) is used before and after transplantation to assess the specificity of anti-HLA antibodies that are not normally detected by less sensitive methods such as cytotoxicity. Single HLA antigen beads are used to determine specific HLA antibodies in high PRA containing sera. Values of median fluorescence (MdFl) < 0.5 are regarded as negative FCXM
• MICA antibody test detects antibodies against a panel of MICA single antigens. The test detects antibodies directed against the most common MICA alleles and allows high throughput antibody screening and identification using luminex technology. The test can be performed before or after transplantation
Therapy :
• plasmapheresis plus low-dose (100 mg per kilogram of body weight per dose) intravenous immune globulin protocol^ref1,ref2. Blood was drawn from the patient’s Hickman catheter, and plasma was separated by centrifugation (Spectra®, Cobe, Unterschleißheim, Germany). The plasma was pumped over either of two protein A-Sepharose columns (Immunosorba®, Fresenius HemoCare, St. Wendel, Germany) using a plasma flow monitor (Citem 10®, Fresenius HemoCare). Sodium heparin and 0.13 M sodium citrate achieved anticoagulation of the extracorporeal circuit. Immunoadsorption was started on day -8 and repeated on days -7, -6, -5, -1, and 0. Three plasma volumes (PV) were processed on each occasion.
• immunoadsorption onto staphylococcal protein A for IgG^ref
• high-dose (2 g per kilogram per dose)  intravenous immune globulin protocol ^ref
• rituximab significantly reduces both Class I and Class II PRA in lung^ref and renal^{ref1, ref2} translant recipients
• graft-versus-host disease (GvHD) (> 70%)
• acute GvHD (30-70%) : aGVHD (grade II or higher) developed in 20 to 65% of the patients in single-center or multicenter trials^{ref1, ref2, ref3, ref4, ref5, ref6}. Death due to this complication accounts for approximately 50% of the deaths that are not due to a relapse of the neoplasm^{ref1, ref2, ref3}
• chronic GvHD (30%) : the incidence of cGVHD in 584 individuals who had undergone allogeneic HSCT between 1976 and 1999 and had survived > 2.5 years was 54%, of whom 46% reported active cGVHD. In multivariable analyses, subjects with active cGVHD were more likely to report adverse general health, mental health, functional impairments, activity limitation, and pain compared to those with no history of cGVHD. However, health status did not differ between those with resolved cGVHD and those who never had cGVHD^ref
• investigators from the Dana-Farber recently reported a Phase II trial in which sirolimus / rapamycin was substituted for methotrexate (MTX) in a tacrolimus-based GVHD prophylaxis regimen for PBSC HSCT utilizing a fully myeloablative conditioning regimen^ref. Engraftment was prompt and only 3/30 (10%) patients developed acute GVHD (all grade II) compared to the expected incidence of 35-40% acute GVHD in sibling-donor HSCT with tacrolimus/MTX. TRM was only 6% at 1 year compared to 25-30% in most traditional allogeneic studies. Chronic GVHD developed in 40% of the study patients. Toxicities included significant hyperlipidemia, thrombotic microangiopathy and a 10% incidence of veno-occlusive disease primarily in patients with recent exposure to gemtuzumab ozogamicin
• other studies of the substitution of MMF for MTX in GVHD prophylaxis regimens have demonstrated decreased regimen-related toxicities of severe mucositis and interstitial pneumonias, as well as more rapid engraftment compared to a MTX-containing regimen (14-16 days versus 19-22 days for neutrophil engraftment)^{ref1, ref2}.
• fatal immune-mediated pancytopenia and a TRALI-like syndrome associated with high titers of recipient-type antibodies against donor-derived peripheral blood cells after allogeneic HSCT following dose reduced conditioning^ref
• infections :
• invasive fungal infections (IFIs) (3%; 6.9% in MUD alloHSCT, 4.5% in MRD alloHSCT, 0.9% in autoHSCT; 3-mo OS after IFI : 38%) :
• Candida glabrata
• Aspergillus fumigatus. Amphotericin B, itraconazole, and caspofungin show negligible levels in CSF or brain tissue^{ref1, ref2, ref3} (Heykants J, Michiels M, Meuldermans W, et al. The pharmacokinetics of itraconazole in animals and man: an overview. In: Fromtling RA, ed. Recent Trends in the Discovery, Development and Evaluation of Antifungal Agents. Barcelona, Spain: J.R. Prous Science Publishers; 1987: 223-249). Voriconazole, a novel triazole, penetrates into the CNS in humans with a CSF-to-plasma ratio of 0.22 to 1. In addition, high voriconazole brain tissue levels (11.8 µg/g and 58.5 µg/g) were found at autopsy in 2 patients with pulmonary aspergillosis, indicating that voriconazole is enriched in brain tissue^ref. In a large randomized trial, initial therapy with voriconazole has shown superior efficacy compared to that with amphotericin B in all forms of invasive aspergillosis^ref. Moreover, voriconazole has been used successfully in a few patients with CNS aspergillosis^{ref1, ref2, ref3, ref4}. 81 patients who were treated with voriconazole for definite (n = 48) or probable (n = 33) CNS aspergillosis. Complete and partial responses were recorded in 35% of patients and varied by the underlying disease group: hematologic malignancies (54%), other underlying conditions (50%), chronic immunosuppression (45%), solid organ transplantation (36%), and hematopoietic stem cell transplantation (16%). 31% of patients survived CNS aspergillosis for a median observation time of 390 days. There were 31 patients who underwent neurosurgical procedures, including craniotomy/abscess resection (n = 14), abscess drainage (n = 12), ventricular shunt (n = 4), and Ommaya-reservoir (n = 1). Multifactorial analysis revealed that neurosurgery was associated with improved survival (P = .02). Patients who underwent hematopoietic stem cell transplantation had a poorer survival (P = .02), but 7 (22%) of 32 survived for a median of 203 days. Voriconazole treatment together with neurosurgical management, whenever feasible, is currently the best approach to treat patients with CNS aspergillosis^ref. However, our ability to more easily reduce or reverse the underlying immunosuppression in these patients is another factor that may have played a significant role in the improved outcome of CNS aspergillosis during recent years. Long-term survivors in Schwartz et al's series were mostly patients with low or chronic underlying immunosuppression, and patients with hematopoietic stem cell transplantation had an inferior outcome compared with all other patients. Experimental studies as well as case series strongly suggest that underlying immunosuppression is one of the most powerful predictive factors in invasive aspergillosis^ref. According to the Infectious Diseases Society of America (IDSA) guidelines, the ultimate response of invasive aspergillosis to antifungal therapy is largely related to host factors, such as the resolution of neutropenia and the return of neutrophil function, lessening immunosuppression and the return of graft function from a bone marrow or organ transplant^ref. The emerging repertoire of novel immunosuppressive agents allows more specific tailoring on the immune system^ref, while the growing use of adjuvant therapies such as growth factors in neutropenic patients can augment antifungal therapy. Invasive aspergillosis (IA), a complication with high mortality rates, especially in disseminated IA with cerebral involvement, is difficult to diagnose. Biopsy of cerebral lesions is often not feasible, and culture of Aspergillus spp. from cerebrospinal fluid (CSF) is frequently negative. New molecular methods have emerged for diagnosing IA. So far, there are only few reports of Aspergillus DNA detection in CSF. After modifying the DNA extraction protocol, we detected Aspergillus DNA in CSF samples by a previously described nested PCR assay. In 6 patients with hematologic malignancy and cerebral aspergillosis, CSF samples were investigated for Aspergillus DNA. IA was classified according to the EORTC/MSG 2002 criteria. 2 patients each had proven, probable, and possible IA. 35 CSF samples were investigated for Aspergillus DNA by nested PCR. Samples with positive results in the nested PCR assay were quantified by LightCycler PCR assay. 14 CSF samples showed positive results in the nested PCR assay. Of these, 6 samples gave positive results in real-time PCR. The range of CFU per ml was 2,154 to 63,100,000. The highest number of CFU per ml was found in a CSF sample of a patient with ALL and probable cerebral aspergillosis. Detection of AspergillusDNA in CSF samples is thus possible and has the potential to improve diagnosis of cerebral aspergillosis. Further prospective studies with larger numbers of patients must be performed to evaluate the clinical significance of Aspergillus PCR with CSF samples^ref. It is well established that various types of immunosuppression (eg, neutropenia, corticosteroid therapy, and GvHD after HSCT) increase the risk for invasive fungal infections^{ref1, ref2, ref3}. However, it is less clear that modulation of immunosuppression always impacts positively on the outcome. Clinicopathologic data clearly indicate that the use of higher corticosteroid doses and OKT3 is associated with a higher mortality rate in nonneutropenic patients with filamentous fungal infections after allogeneic hematopoietic stem cell transplantation^ref. Interestingly, the predominant histopathologic finding in these patients was acellular necrosis, suggesting that corticosteroids severely impair leukocyte trafficking. Although regaining neutrophil function is commonly regarded as an important factor for a successful outcome, recovery from neutropenia was not associated with an improved survival in a recent study evaluating 87 patients with invasive aspergillosis^ref. Furthermore, fatal pulmonary complications in patients with mold infections have been reported repeatedly upon recovery from neutropenia^{ref1, ref2}. This is supported by experimental data demonstrating that the neutrophil response is a major cause of tissue damage^ref

Risk factors :
• polymorphisms in genes
• recipient : IL-10^{ref1, ref2}, TNFR2 VNTR^ref, TLR1, and TLR6^ref
• donor : TLR4 haplotype S4^ref
• Aspergillus ustus
• Cryptococcus neoformans var. neoformans
• Fusarium spp.
• zygomycosis
• Pneumocystis carinii
 No serious adverse events were associated with rIFN-g_1b immune enhancement in patients with systemic opportunistic infections who received donor GTX therapy^ref. A 9-yr-old boy received the highest cumulative dose so far reported of liposomal amphotericin B after an allogeneic HSCT for adrenoleucodystrophy, after a conditioning regimen with busulfan, thiothepa and cyclophosphamide. Rabbit antithymoglobulin, cyclosporin and prednisone were used as prophylaxis against GVHD. Post-transplant EBV-related lymphoma was diagnosed on day +68 and was treated with DLIs. The patient developed a severe form of GVHD, and a progressive worsening of his neurological status because of progression of his underlying disease. Death from septic shock occurred 23 months after BMT. During prolonged hospitalization, 19,750 mg of liposomal amphotericin B, about 1000 mg/kg, were given for prophylactic or empirical therapeutic purposes without significant nephrotoxicity. This case suggests that liposomal amphotericin B is safe and well-tolerated even if is administered for long periods and a cumulative dose 5-fold greater than the nephrotoxic threshold of amphotericin B deoxycholate is achieved^ref
• reactivation of latent viruses :
• HHV-1 / HSV-1(70-80%) : recipients of partially matched related donor transplantation using stringently T- and B-cell–depleted stem cell products experience uniform reactivation of latent HSV, despite acyclovir prophylaxis, and high levels of drug resistance. All 7 cases in which the recipient and/or donor were HSV positive demonstrated post-transplant HSV reactivation, and in 5 cases acyclovir-resistant viral isolates were identified. Importantly, despite antiviral drug therapy, herpetic lesions only healed following normalization of T-cell counts^ref, which is further evidence for the important role of cellular immunity in the control of post-transplant viral infections.
• HHV-3 / VZV (50-60%)
• HHV-4 / EBV (60%)
• HHV-5 / CMV (70%) : Historically, during the period of immune reconstitution following HSCT, up to 80% of CMV-seropositive allogeneic recipients developed CMV reactivation, 50% had viremia (similar to autologous HSCT) and 40% developed CMV disease^ref. These rates have declined in recent years, but CMV nonetheless still constitutes an important and potentially lethal post-transplant opportunistic pathogen^ref, facilitated by T-cell depletion (TCD) of the donor component, recipient conditioning prior to HSCT and Steroid therapy for GvHD^ref. A comparative study noted an increased odds ratio of 17.0 of CMV disease among recipients of CD34-selected peripheral blood stem cells (7 of 31 patients) versus recipients of unselected peripheral blood stem cell support (10 of 237 patients)^ref. Clinical disease after HSCT typically involves a pattern of early (first 3 months) pneumonitis and enteritis rather than retinitis that is more common among patients with HIV disease and severe T-cell deficiencies^ref. A murine model of a liver-adapted B-cell lymphoma has previously shown a survival benefit and tumor growth inhibition by nonlethal subcutaneous infection with murine CMV. Virus replication proved to be required for antitumoral immunity, since inactivated virions or the highly attenuated enhancerless mutant mCMV-DMIEenh did not impact the lymphoma in the liver. Surprisingly, the dissemination-deficient mutant mCMV-DM36 inhibited tumor growth, even though this virus fails to infect the liver. On the other hand, various strains of HSV consistently failed to control the lymphoma, even though they infect the liver. A quantitative analysis of the tumor growth kinetics identified a transient tumor remission by apoptosis as the antitumoral effector mechanism. Tumor cell colonies with cells surviving the CMV-induced "apoptotic crisis" lead to tumor relapse even in the presence of full-blown tissue infection. Serial transfer of surviving tumor cells did not indicate a selection of apoptosis-resistant genetic variants. NK cell activity of CD49b-expressing cells failed to control the lymphoma upon adoptive transfer. An innate antitumoral mechanism is triggered by CMV infection and involves an apoptotic signal effective at a distant site of tumor growth^ref. While donor CMV seropositivity had no effect on OS, having a CMV-seropositive donor was associated with a significantly lower risk for relapse (20% vs. 52%, P = 0.02). The beneficial effect of a CMV-seropositive donor, however, was offset by a higher TRM (42% vs. 19% in patients with a CMV-seronegative donor, P = 0.2). Donor CMV serostatus had no effect on the incidence of CMV infection/disease, and acute or chronic GvHD. Overall, 27 patients died during the observation period. Causes of death in patients with a CMV-seropositive donor were primarily transplant-related (11 vs. 4 in patients with CMV-seronegative donors), whereas leukemic relapse was the leading cause of death in patients with a CMV-seronegative donor (8 vs. 4 in patients with a CMV-seropositive donor, P = 0.038). Multivariate Cox regression analysis showed risk category by the underlying disease and donor CMV seropositivity to be significant predictors of leukemic relapse following RIC HSCT. These preliminary results suggest a possible role of donor CMV serostatus on the risk of relapse following RIC allogeneic HSCT^ref. Ganciclovir, foscarnet or cidofovir are frequently used for anti-CMV prophylaxis or to treat diagnosed CMV infections in immunosuppressed HSCT recipients. These drugs can block CMV replication, reduce viral load, and decrease but not eliminate CMV disease^ref. However, because HSCT recipients with low levels of anti-CMV CTLs respond relatively poorly to ganciclovir therapy^ref, antiviral drugs are not a definitive solution to CMV infection in immunosuppressed transplant recipients. Furthermore, prolonged antiviral drug therapy can worsen immunosuppression leading to extended neutropenia, further delay in recovery of anti-CMV CTLs, and outgrowth of resistant viral mutants^ref. In one study, even limited use of ganciclovir caused severe neutropenia in 33% (10 of 30) of treated patients, leading to increased mortality from other opportunistic pathogens including Aspergillus fumigatus, Streptococcus pneumoniae, and Pneumocystis carinii^ref. Up to 15% of bone marrow transplant (BMT) recipients develop late CMV disease after discontinuation of ganciclovir prophylaxis, and the mortality of late-onset disease is in the range of 50 to 70%^ref. These studies underscore the drawbacks of using anti-CMV drug therapy as the definitive approach to prevent viral disease in HSCT recipients, and the importance of developing alternative antiviral therapies, such as antiviral vaccination, that directly address the underlying problem of prolonged immunosuppression. There is also evidence that CMV induces or increases the severity of GVHD following allogeneic HSCT^ref. Although the mechanism(s) is unclear, it may involve heterologous immunity^ref. Heterologous immunity can best be understood in the context of a normal antiviral immune response, which typically involves activation of naïve antiviral CD8⁺ T cells that subsequently differentiate into CTLs that then kill virus-infected cells^ref. This is not, however, a high fidelity process since virus infection activates a broad range of CTLs with diverse TCR specificities. In fact, some of the activated CTLs have much higher affinities for alternative targets such as alloantigens^ref. The resulting heterologous immunity reshapes the immune profile of the host, modifying subsequent responses to authentic alloantigens such as might be encountered after transplantation. The model virus lymphochoriomeningitis virus (LCMV), a potent inducer of heterologous immunity, breaks transplantation tolerance and promotes rejection of allogeneic HSCTs including bone marrow allografts^{ref1, ref2}. While CMV has not yet been shown to induce heterologous immune responses, its ability to activate alloreactive CTLs and induce or increase the severity of GvHD has not been well studied in detail in HSCT recipients. Ongoing GvHD can accentuate CMV disease through mechanisms unrelated to steroid therapy^ref. Using a mouse model, higher levels of murine CMV (MCMV) DNA were found in the lungs of mice that received allogeneic donor lymphocytes and developed GvHD than in those that did not^ref. The relationship between CMV and GvHD is composed of feedback loops that can amplify both CMV disease and GvHD following allogeneic HSCT. CMV is central to these pathways. Anti-CMV vaccination may be an effective alternative strategy to rapidly reconstitute anti-CMV immunity, which in turn should interdict the downstream effects of CMV disease (and antiviral drug therapy) on immunosuppression, GvHD, and other opportunistic infections, thereby reducing morbidity and mortality after allogeneic HSCT.

Although early studies suggested a beneficial effect of acyclovir^{ref1, ref2}, it has weak in vitro activity against CMV as compared with ganciclovir. Two randomized controlled trials assessed the impact of ganciclovir prophylaxis when administered to all allogeneic HSCT recipients to day 100 post transplant^{ref1, ref2}. In these trials, CMV disease was strikingly reduced, but no survival benefit could be demonstrated. Neutropenia was a significant side-effect in the ganciclovir-treated group, with a concomitant higher risk of fungal infection^ref. It has also been suggested that ganciclovir prophylaxis may delay the development of effective CMV-specific immune responses, and thus may lead to more late CMV disease^ref. More recently, interest has focused on the possibility of pre-emptive therapy with ganciclovir based on screening for early CMV with a sensitive diagnostic test^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10, ref11} (Zaia JA, Schmidt GM Chao NJ et al. Use of preemptive ganciclovir based solely on asymptomatic pulmonary cytomegalovirus infection in marrow transplant recipients - long-term follow-up. Blood 1994; 84: (10 Suppl. 1) 249a). Tests used as triggers for pre-emptive therapy have included CMV-DNA by PCR^{ref1, ref2, ref3, ref4}, the pp65 antigenemia assay^{ref1, ref2, ref3, ref4, ref5, ref6}, CMV-DNA by hybrid capture assay (Digene), CMV shell vial or tissue culture^{ref1, ref2, ref3, ref4}, and bronchoalveolar lavage^ref (Zaia JA, Schmidt GM Chao NJ et al. Use of preemptive ganciclovir based solely on asymptomatic pulmonary cytomegalovirus infection in marrow transplant recipients - long-term follow-up. Blood 1994; 84: (10 Suppl. 1) 249a). Advocates of universal ganciclovir prophylaxis cite the benefits of a low rate of CMV pneumonitis^{ref1, ref2, ref3, ref4}. Advocates of pre-emptive strategies cite less neutropenia, less cost, and possibly less fungal infection as reasons to utilize pre-emptive therapy. Some clinicians have advocated intermittent (eg 3 times weekly) ganciclovir prophylaxis rather than daily prophylaxis as a way to avoid the problem of neutropenia, but concerns have been raised as to whether this strategy is adequate in high-risk recipients^ref. Several centers have devised hybrid strategies^ref in which patients judged to be at higher risk for CMV^ref receive universal ganciclovir prophylaxis while those judged to be at lower risk are followed with monitoring and pre-emptive therapy (Zaia JA, Schmidt GM Chao NJ et al. Use of preemptive ganciclovir based solely on asymptomatic pulmonary cytomegalovirus infection in marrow transplant recipients - long-term follow-up. Blood 1994; 84: (10 Suppl. 1) 249a). Few studies have directly compared prophylaxis and pre-emptive therapy. Boeckh et al^ref performed a randomized comparison, and found that pre-emptive therapy based on antigenemia resulted in more CMV disease by day 100, but universal prophylaxis was associated with more early invasive fungal infections and more late CMV disease, and there was no difference in survival^ref. This group has subsequently published a modification of the pp65 antigenemia-based strategy, in which ganciclovir is initiated for antigenemia at any level, instead of high-grade antigenemia, and is continued to day 100 after transplant^ref. This strategy resulted in a lower incidence of CMV disease, comparable to that with ganciclovir prophylaxis, and also a low incidence of fungal infections. Compared with the previous antigenemia-based strategy, a somewhat higher incidence of late CMV disease was noted, consistent with a possible delay in reconstitution of immune responses to CMV in patients treated with ganciclovir early after transplant^ref. Thus, this modified pp65 antigenemia-based strategy appears to combine the advantages of both prophylactic and pre-emptive strategies seen in the previous randomized trial, with a low incidence of both CMV disease and invasive fungal infections^ref. Recent recognition of the importance of reconstitution of the anti-CMV cellular immune response^{ref1, ref2} has led to the proposal of innovative strategies based on adoptive immunotherapy^ref
• HHV-6 (48%) : DNA levels ranged from 200 to 200,000 copies/mL of plasma, and HHV-6 DNAemia was observed significantly more frequently after CBT than after BMT/PBSCT (92.3% vs 30.3%; P < .001). Multivariate analyses identified CBT (vs BMT/PBSCT), HLA mismatches between recipient and donor, and low anti-HHV-6 IgG titer before transplantation as the only risk factors for developing HHV-6 DNAemia^ref. HHV-6B was detectable at least once in 47.2% (34/72) of HSCT recipients on the median day 21 (range, 7-84 days); HHV-6A reactivation occurred in only 1 recipient (1.4%). Detectable HHV-6B reactivation was associated with increased probability of skin rash by day 30 after HSCT (hazard ratio [HR], 3.68; 95% confidence interval [CI], 1.24-10.92; P = .019), cytomegalovirus (CMV) antigenemia (HR, 2.35; 95%CI, 1.32-4.19; P = .004), and hemorrhagic cystitis (HC) (HR, 2.59; 95%CI, 0.96-6.98; P = .061) by day 100 after HSCT. Neutrophil and platelet engraftment, mortality for 100 days after HSCT were not affected by HHV-6B reactivation^ref. It may cause myelitis^ref and encephalitis^{ref1, ref2, ref3}
• HHV-8 / KHSV^ref (in RIC^ref)
• HBV and HCV
• JCV (6.7%)
• BKV (55%) : hemorrhagic cystitis (HC) is a well-known complication of HSCT. Its overall incidence has been reported to vary from 7-16^ref-68%. The spectrum of clinical presentation varies from asymptomatic microhematuria to life-threatening bleeding. Sodium hyaluronate is a glycosaminoglycan present on the bladder mucosa, which serves as an important protective substance against uroepithelial damage. Preparations of this component have been shown to be effective in the treatment of interstitial cystitis. 5 out of the 7 patients included in a study achieved CR, while 1 patient had only PR. Sodium hyaluronate administration was not associated with any local or systemic adverse effects^ref. BK-viruria was more common in patients with hemorrhagic cystitis than in those without (p<0.01), and hemorrhagic cystitis was less common in patients with related donors than in those with unrelated donors (p=0.02). Finally, hemorrhagic cystitis and BK-viruria were less common in patients receiving RIC, rather than full conditioning (p<0.01 and p<0.01, respectively)^ref.
• late complications :
• malignant^ref.4 : secondary malignant diseases are of particular clinical concern as more patients survive the early phase after transplantation and remain free of their original disease^{ref1, ref2}.
• secondary cancers : 17 cases of squamous cell carcinoma owing to oral GVHD in patients undergoing allogeneic HSCT^ref
• 10 cases of porokeratosis of Mibelli have been reported following HSCT. The diagnosis of porokeratosis was made between 1 and 13 years post-transplantation. The underlying malignancy in four of the five cases was leukemia, while the fifth patient had NHL. Porokeratosis developed during remission in the four leukemia patients, whereas, in the fifth patient, it occurred during a relapse of lymphoma. As cutaneous carcinomas have been reported in association with porokeratosis, careful surveillance for porokeratosis in bone marrow transplant recipients is warranted^ref
Experiments in the 1960s and 1970s in murine models suggested, furthermore, that a graft-versus-host reaction after allogeneic spleen cell transplantation could transform from an immunologic to a neoplastic disorder, ie, the development of lymphoma^ref. Finally, marrow transplant studies in rhesus monkeys and dogs in the 1970s and 1980s showed a significant increase in the incidence of malignancies relative to controls in radiation chimeras, ie, in animals irradiated with lethal doses of total body irradiation (TBI) and infused with autologous or allogeneic marrow cells (reviewed in Deeg et al^ref, Kolb et al^ref (Broerse JJ, Hollander CF, Van Zwieten MJ: Tumor induction in Rhesus monkeys after total body irradiation with X-rays and fission neutrons. Int J Radiat Biol 40:671, 1981). Thus, it should not be surprising that new malignancies occur in patients after hematopoietic stem cell transplantation, in which one or several of these risk factors are present. New malignancies after marrow or blood HSCT
• posttransplant lymphoproliferative disorders (PTLD) and lymphomas: the majority of cases of PTLD after hematopoietic stem cell transplantation have been observed in allogeneic (rather than autologous) recipients^ref. Most of these PTLD are best classified as B-cell PTLD rather than non-Hodgkin's lymphoma^{ref1, ref2, ref3, ref4, ref5} ((suppl 1) Socie G, Henry-Amar M, Devergie A, Esperou-Bourdeau H, Ribaud P, Traineau R, Gluckman E: Malignant diseases after allogeneic bone marrow transplantation: An updated overview (review). Nouv Rev Fr Hematol 36:S75, 1994; O'Reilly RJ, Lacerda JF, Lucas KG, Rosenfield NS, Small TN, Papadopoulos EB: Adoptive cell therapy with donor lymphocytes for EBV-associated lymphomas developing after allogeneic marrow transplants, in De Vita TD, Hellman S, Rosenberg SA (eds): Important Advances in Oncology 1996. Philadelphia, PA, Lippincott-Raven, 1996, p 149). In addition, some T-cell PTLD have been reported. Thirdly, lymphomas with clinical and biological characteristics typical for non-Hodgkin's lymphoma or Hodgkin's disease as seen in nontransplanted patients have occurred after stem cell transplantation. Although lymphoproliferative disorders do not represent a frequent posttransplant complication, important insights have been gained into the pathophysiology and considerable progress has been made in regards to treatment.
• B-cell PTLD :
• incidence.   B-cell PTLD are clinically and morphologically heterogeneous; usually they are associated with T-cell dysfunction and the presence of EBV. B-cell PTLD have been observed with almost any organ transplant^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. Cohen^ref recently reviewed 100 well-documented cases, including 32 in marrow transplant recipients. Additional cases have been described since then, bringing the total number of PTLD after hematopoietic transplants to about 70 to 100^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8} (Simon M, Bartram CR, Friedrich W, Arnold R, Schmeiser T, Hampl W, Muller-Hermelink HK, Heymer B: Fatal B-cell lymphoproliferative syndrome in allogeneic marrow graft recipients. A clinical, immunobiological and pathological study. Virchows Arch 60:307, 1991 ) In Cohen's review^ref, the mean interval from transplantation to the development of B-cell PTLD was 5 months, with most being diagnosed within 3 months. It appears that patients transplanted for congenital immunodeficiencies are at a particularly high risk for PTLD, presumably due to the underlying immunodeficiency and T-cell depletion of the donor graft generally used for these diseases. Because the diagnostic criteria may differ from study to study (eg, a nonlethal infectious mononucleosis-like syndrome may resolve spontaneously, whereas acute-onset extensive disease may be diagnosed only at autopsy), the true incidence of B-cell PTLD after hematopoietic stem cell transplantation is difficult to determine. In a large single-center survey (1,400 allografted patients), the cumulative incidence of B-cell PTLD reached a plateau of 1.6% by 4 years after transplantation; other published data range from 0.6% to 10%^ref.
• clinical features.   The most frequent presentation of PTLD is with fever and lymphadenopathy. Intra-abdominal lymphadenopathy, splenomegaly, or hepatomegaly may cause symptoms such as abdominal pain, vomiting, or diarrhea. Extrahematopoietic organ involvement, including lungs, kidneys, and the central nervous system (CNS), is frequent. CNS involvement is of particular concern, because it has been associated with a dismal prognosis. The differential diagnosis in a symptomatic patient should include PTLD a priori in high-risk situations such as in recipients of T-cell-depleted or HLA-nonidentical transplants. Early diagnosis has become important since powerful therapeutic instruments have been developed. Early diagnosis can be established and the effect of therapy can now be monitored by semiquantitative polymerase chain reaction (PCR) of the EBV DNA (see pathogenesis and treatment).
• pathology.   B-cell PTLD occurring after allogeneic hematopoietic stem cell transplantation are almost always of donor origin and associated with EBV-genomic DNA integration. Biopsies show monomorphic or polymorphic, DLBCL. However, whereas the morphology of B-cell PTLD occurring after solid organ transplantation has been described extensively, few studies have examined in detail the histologic features of PTLD in HSCT recipients^{ref1, ref2, ref3, ref4} (Simon M, Bartram CR, Friedrich W, Arnold R, Schmeiser T, Hampl W, Muller-Hermelink HK, Heymer B: Fatal B-cell lymphoproliferative syndrome in allogeneic marrow graft recipients. A clinical, immunobiological and pathological study. Virchows Arch 60:307, 1991 ). Those reports show that, whereas some of these PTLD are histopathologically similar to the polymorphic PTLD described in solid organ transplant recipients, as many as half of the cases after HSCT show aggressive features of immunoblastic lymphoma (O'Reilly RJ, Lacerda JF, Lucas KG, Rosenfield NS, Small TN, Papadopoulos EB: Adoptive cell therapy with donor lymphocytes for EBV-associated lymphomas developing after allogeneic marrow transplants, in De Vita TD, Hellman S, Rosenberg SA (eds): Important Advances in Oncology 1996. Philadelphia, PA, Lippincott-Raven, 1996, p 149). Also, in contrast to PTLD after organ transplantation, most B-cell PTLD occurring after HSCT are oligoclonal or monoclonal, as determined by analysis of Ig gene rearrangements and fused termini of episomal EBV DNA^{ref1, ref2, ref3, ref4} although some discrepancies between these 2 methods (tumors appearing monoclonal on the basis of EBV genomic analysis and polyclonal by analysis of Ig gene rearrangement) have been observed^{ref1, ref2, ref3}. PTLD express the full array of latent EBV antigens, including EBNA-1, -2, -3, -4, -5, and -6 and LMP1^{ref1, ref2, ref3, ref4}. Karyotypic analyses have identified nonconsistent cytogenetic abnormalities, more frequently in monoclonal lesions of more aggressive histology. However, with the exception of 2 cases of B-cell PTLD developing in heart transplant recipients^ref, the characteristic translocation of Burkitt's lymphoma has not been observed in lymphoproliferative disorders developing after marrow (or solid organ) HSCT. In a recent report of 10 cases of PTLD in marrow transplant recipients, Orazi et al^ref attempted to correlate morphology with clonality (based on Ig chain gene rearrangement and immunochemistry), proliferative activity as measured by immunostaining for the proliferating cell nuclear antigen (PCNA), and presence of p53 overexpression. The cases included 7 polymorphic B-cell lymphomas and 3 immunoblastic lymphomas. IgH chain gene rearrangement analysis showed B-cell clonality in three of seven polymorphic lymphomas and in all three immunoblastic lymphomas. The EBV genome, the expression of the EBV latent membrane protein, or both were found in all 10 cases. High proliferative activity as assessed by the expression of the PCNA antigen was found in all cases, and 5 specimens were p53⁺.
• risk factors.   B-cell PTLD were the first posttransplant malignancies for which risk factors were identified^{ref1, ref2, ref3, ref4}. In 1989, Witherspoon et al54 showed in multivariate analysis that treatment of aGVHD with either antithymocyte globulin or monoclonal anti-CD3 antibody, total body irradiation, T-cell depletion of donor marrow, and HLA nonidentity between donor and recipient were risk factors for PTLD. A more recent survey by Bhatia et al^ref showed the following factors to be associated with an increased risk of B-cell PTLD: T-cell depletion of the graft (relative risk [RR] = 11.9), HLA mismatch (RR = 8.9), use of antithymocyte globulin for acute GVHD prophylaxis (RR = 5.9) or in the preparative regimen (RR = 3.1), and primary immune deficiency disease (RR = 2.5). The cumulative risk of developing a B-cell PTLD in patients with primary immune deficiency who received a T-cell-depleted HLA-mismatched transplant was 64.8% ± 17.7% at 4 years, compared with 0.9% ± 0.2% (P < .001) in patients who received an HLA-matched transplant with no in vitro manipulation of the graft. The role of HLA-mismatching in the pathogenesis of B-cell PTLD is not clear but may consist in chronic antigenic stimulation or delayed immune reconstitution. In unrelated transplants, the National Marrow Donor Program (NMDP) reported an incidence of PTLD of 2% overall, 5% in patients receiving a T-cell depleted marrow, and 1% for those receiving a T-replete graft.55 However, available data suggest that the risk is not uniform but depends on the method of T-cell depletion and the type of additional immunosuppression used in the posttransplantation period. Although in patients transplanted with marrow depleted of T cells with specific monoclonal antibodies the incidence of EBV-positive PTLD ranged from 11% to 25%, the incidence was less than 1% with techniques removing both T and B lymphocytes (eg, soybean agglutinin or Campath-1), possibly reflecting the 2 to 3 log reduction in B lymphocytes associated with these procedures^ref1 (O'Reilly RJ, Lacerda JF, Lucas KG, Rosenfield NS, Small TN, Papadopoulos EB: Adoptive cell therapy with donor lymphocytes for EBV-associated lymphomas developing after allogeneic marrow transplants, in De Vita TD, Hellman S, Rosenberg SA (eds): Important Advances in Oncology 1996. Philadelphia, PA, Lippincott-Raven, 1996, p 149). However, when additional posttransplant immunosuppression with steroids and antithymocyte globulin was administered after HLA-matched or mismatched related transplants or transplants from unrelated donors using soybean agglutination/E-rosetting for T-cell depletion, the incidence of PTLD increased to 6% to 18%. Finally, even in the absence of in vitro T-cell depletion, the use of intensive in vivo immunosuppressive prophylaxis or therapy of GVHD, especially with anti-T-cell agents such as OKT3 antibody or antithymocyte globulin, is associated with the development of B-cell PTLD^{ref1, ref2}
• Pathogenesis.   B-cell PTLD are thought to develop because of depressed EBV-specific cellular immunity and the inherent transforming capacities of EBV. EBV is a ubiquitous herpes virus that infects 95% of individuals by adulthood. The virus persists as a latent infection in certain epithelial cells, where reactivation and replication may occur intermittently, and in B lymphocytes^ref. EBV type A and type B have been defined on the basis of sequence divergence in the EBNA-2 gene. In a recent series of 27 solid organ transplant recipients who developed PTLD, type A EBV was present in 24 of 27 cases (89%) by PCR amplification of EBNA-2 and EBNA-3c regions. In addition, there was polymorphism at the EBER locus documenting the presence of 4 different type A EBV strains. None of the 27 cases harbored type B EBV^ref. Whether the same applies to marrow transplant recipients remains to be determined. Among the 80 to 100 EBV-encoded proteins, the latent membrane protein 1 (LMP-1) plays an essential role in B-cell immortalization. LMP-1 has recently been shown to induce the expression of bcl-2, which inhibits programmed death of the infected cells. LMP-1 is also considered an oncogene because of its ability to transform rodent fibroblasts. Deletions near the 3 end of the LMP-1 gene, in a region that affects the half-life of the LMP-1 protein, have been reported in some EBV-related lymphoproliferative disorders^{ref1, ref2}; B-cell PTLD after marrow or stem cell transplantation have not been analyzed yet. Infection of B cells by EBV also induces high levels of IL-1, IL-5, IL-6, IL-10, CD23, and TNF. The cellular IL-10 and the EBV-induced BCRF1, a homolog of IL-10, act as autocrine growth factors, stimulating the proliferation of EBV-transformed B cells and inhibiting their susceptibility to apoptosis. Much of the initial work investigating anti-EBV cellular responses was performed in patients with acute infectious mononucleosis (O'Reilly RJ, Lacerda JF, Lucas KG, Rosenfield NS, Small TN, Papadopoulos EB: Adoptive cell therapy with donor lymphocytes for EBV-associated lymphomas developing after allogeneic marrow transplants, in De Vita TD, Hellman S, Rosenberg SA (eds): Important Advances in Oncology 1996. Philadelphia, PA, Lippincott-Raven, 1996, p 149). Early in the course of the disease, natural killer cells and cytotoxic and suppressor T cells reactive against EBV emerge. Using standard assays of cell-mediated cytolysis, Crawford et al^ref found that, in recipients of unmodified marrow, 7 of 10 patients studied had defective killing of autologous targets at 3 months posttransplant, but all were normal by 6 months. In a recent study, investigators at Memorial Sloan-Kettering Cancer Center explored whether deficiencies of EBV-specific cellular immunity contribute to EBV-PTLD susceptibility^{ref1, ref2}. They performed limiting dilution analysis to quantify anti-EBV specific cytotoxic T-lymphocyte precursor (CTLp) frequencies in 26 recipients of unmodified or T-cell-depleted grafts from EBV-seropositive donors. At 3 months, only 5 of the 26 patients had EBV CTLp frequencies in the normal range of seropositive controls, whereas at 6 months, 9 of 13 patients were within the normal range. This time interval of low CTLp frequency corresponds to the period in which B-cell PTLD are observed. The same investigators showed that EBV-specific cytotoxic T lymphocytes home preferentially to and induce selective regression of autologous EBV-induced B-cell lymphoproliferative lesions in xenografted SCID mice^ref. These studies have led to clinical trials on the role of EBV-specific T lymphocytes in controlling EBV-induced B-cell proliferation. Rather definitive proof has been provided by the St Jude group using adoptive transfer of gene-modified EBV-specific T lymphocytes^ref. Preliminary clinical results^ref showed that adoptive transfer of EBV-specific cytotoxic T lymphocytes offered effective therapy for B-cell PTLD. The investigators showed long-term persistence of gene-marked EBV-specific cytotoxic T lymphocytes in vivo. These cells not only restored cellular immunity against EBV, but also provided a population of CTLps that responded to in vivo or ex vivo challenge with the virus for as long as 18 months.
• prophylaxis and treatment.   Because various recognized risk factors such as initial diagnosis (primary immune deficiency syndrome) or type of donor (HLA-nonidentical) cannot be changed and others (eg, GVHD prophylaxis) are considered an integral part of the overall treatment regimen, it has been proposed to use early identification of EBV-associated PTLD as an indication for therapy rather than apply true prophylaxis. The St Jude group used both the outgrowth of transformed B lymphocytes ex vivo and detection of EBV DNA by a PCR method as tools to detect EBV-associated lymphoproliferation before clinical disease developed^ref. A semiquantitative PCR assay is used to assist in the detection of EBV DNA in peripheral blood and in monitoring the effect of therapy^{ref1, ref2, ref3, ref4}. Complete regression of B-cell PTLD has been reported in 40% of patients after reduction or discontinuation of immunosuppressive therapy, particularly in renal transplant recipients^ref. Immunosuppression is intrinsic to marrow transplantation, and discontinuation of immunosuppression is likely to result in flares of GVHD and a further delay in recovery of T-cell-mediated immunity. EBV-transformed B cells contain a circular viral DNA that is not susceptible to inhibition by thymidine kinase (TK) inhibitors. Nevertheless, anecdotal reports suggest tumor regression with either acyclovir and ganciclovir therapy (reviewed in Benkerrou et al^ref and Sullivan et al^ref). Chemotherapy and irradiation have been useful in selected cases, and in a recent series of cardiac transplant recipients, among 19 consecutive patients with PTLD, 6 of 8 treated with aggressive chemotherapy are surviving in complete remission, at a median follow-up of 38 months^ref. Surgical resection has proven effective when the PTLD was limited to single sites in solid organ transplant recipients^ref. More recently, three approaches have shown promise in the treatment of B-cell PTLD in marrow transplant recipients:  interferon, B-cell-specific monoclonal antibodies, and cellular therapy. A combination of  interferon and intravenous Ig was first reported in 1988 by the Minneapolis group to be effective in B-cell PTLD. Remissions were maintained in several patients^ref. In a recent update, 3 of 7 patients receiving  interferon achieved a complete remission (Gross and Filipovich, personal communication, July 1997). 2 anti-B-cell antibodies (anti-CD21 and anti-CD24) were used by Alain Fischer's group and by one of the authors (G.S.) in a multicenter trial^ref (Blanche S, Le Deist F, Veber F, Lenoir G, Fischer AM, Brochier J, Boucheix C, Delaage M, Griscelli C, Fischer A: Treatment of severe Epstein-Barr virus-induced polyclonal B-lymphocyte proliferation by anti-B-cell monoclonal antibodies. Two cases after HLA-mismatched bone marrow transplantation. Ann Intern Med 108:199, 1988). Among 19 marrow transplant recipients, 10 had a complete remission and 6 survived at a median follow-up of 20 months.72 The survivors in this series all were patients with oligoclonal disease. Studies in a SCID mouse model (Durandy A, Brousse N, Rozenberg F, de Saint Basile G, Fischer AM, Fischer A: Control of human B cell tumor growth in severe combined immunodeficiency mice by monoclonal anti-B cell antibodies. J Clin Invest 90:945, 1992) show that, after initial remission, with such an approach 30% to 50% of mice relapsed within 30 to 70 days, providing a very strong indication that persistence of residual B cells can provoke a second tumor in the absence of efficient cytotoxic T cells. Currently, the anti-CD21 and CD24 antibodies used in these studies are not available for clinical use (Alain Fischer, personal communication, July 1997). Based on in vitro data showing an antitumor effect of anti-IL-6 antibody in neutralizing the IL-6-dependent proliferative loop^{ref1, ref2}, the same investigators are now testing this antibody in patients with PTLD (Alain Fischer, personal communication, July 1997). In 1994, Papadopoulos et al^ref first reported therapeutic efficacy of the infusion of donor leukocytes in five patients who developed a B-cell PTLD after T-cell-depleted allogeneic marrow transplantation. Unirradiated donor leukocytes were infused at doses calculated to provide 1.0 × 10⁶ CD3⁺ T cells/kg of body weight. All 5 patients had complete pathologic or clinical responses. 3 of the 5 patients developed chronic GVHD and 2 died of respiratory failure with no evidence of PTLD at autopsy. Subsequently, Rooney et al^ref reported on the use of gene-marked EBV-specific T lymphocytes to control or prevent B-cell PTLD in 10 patients. Three of the patients had shown signs of EBV reactivation, with or without overt lymphoproliferation, and 7 received T-cell infusions as prophylaxis. In the 3 patients with EBV reactivation, EBV DNA levels that had increased 1,000-fold or more returned to control levels within 3 to 4 weeks of immunotherapy. In a recent update, the Sloan-Kettering team reported data on 15 patients with eradication of B-cell PTLD in 14; GVHD occurred in 6 among the 12 evaluable patients ((abstr, suppl 1) Papadopoulos EB, Small T, Ladanyi M, Boulad F, Castro-Malaspina H, Childs B, Kernan N, Mackinnon S, Szabolcs P, Young J, O'Reilly RJ: Current results of donor leukocyte infusions for treatment of Epstein-Barr virus associated lymphoproliferative disorders following related and unrelated T cell depleted bone marrow transplant. Blood 88:681a, 1996). The St Jude team described the prophylactic use of EBV-specific T-cell clones in 25 high-risk patients, none of which developed PTLD. Among 6 patients who either refused CTL therapy or were ineligible for treatment, 2 developed lymphomas that were successfully treated with CTL ((abstr, suppl 1) Heslop HE, Smith CA, Ng C, Loftin SK, Sixbey J, Krance RA, Brenner MK, Rooney CM: Efficacy of adoptively transferred virus specific cytotoxic T lymphocytes for prophylaxis and treatment of EBV lymphoma. Blood 88:681a, 1996). Bordignon's group most recently reported on the use of HSV-TK gene transfer in donor lymphocytes infused to control B-cell PTLD in two patients. One of these patients subsequently developed GVHD that was successfully treated with ganciclovir by way of activating the HSV-TK suicide gene^ref. Thus, promising approaches have been developed for the treatment of B-cell PTLD in high-risk marrow^ref and solid organ transplant recipients^ref. However, the numbers of patients treated are still limited. Also, the use of cellular therapy may induce GVHD if non-EBV-specific CTL are used and still requires high-level biotechnology laboratories to provide either EBV-specific CTL clones or HSV-TK-transduced T lymphocytes.
• T-cell lymphoproliferative disorders : besides the well-defined B-cell PTLD, an entity of T-cell proliferative disorders without EBV association has been reported both after solid organ and marrow transplantation. After solid organ transplantation these disorders have occurred predominantly at extranodal sites and were monoclonal^{ref1, ref2}. After marrow transplantation, only three such cases have been reported^ref; 2 occurred late after transplant and may be included in the late-onset lymphoma category. None of the cases was associated with HTLV1, HIV, or HHV6 infection.
• late-onset lymphoma : some 20 cases of late occurring lymphomas have been reported in the literature^{ref1, ref2, ref3, ref4, ref5, ref6} (Socie G: Secondary malignancies. Curr Opin Hematol 3:468, 1996; Meignin V, Devergie A, Brice P, Brison O, Ribaud P, Cojean I, Gluckman E, Socie G, Janin A: Late occurrence of Hodgkin's disease after allogeneic bone marrow transplantation for chronic myelogenous leukemia. Transplantation (in press)). At least 2 have been linked to EBV infection (just as early onset PTLD) and three were associated with T-cell depletion of the graft. These cases presented like ordinary non-Hodgkin's lymphoma with lymph node enlargement with or without generalized symptoms; one of these patients has been reported to be disease-free after chemotherapy. At least two of the late lymphomas were Hodgkin's disease. At Hôpital Saint Louis in Paris, such a late occurrence of EBV-related Hodgkin's disease in donor cells was observed in a patient transplanted 8 years before for chronic myelogenous leukemia (Meignin V, Devergie A, Brice P, Brison O, Ribaud P, Cojean I, Gluckman E, Socie G, Janin A: Late occurrence of Hodgkin's disease after allogeneic bone marrow transplantation for chronic myelogenous leukemia. Transplantation (in press)). Although more work is needed, ongoing studies seem to support the notion that these late-occurring lymphomas represent an entity distinct from the early occurring B-cell PTLD (R. Curtis, personal communication, November 1997).
• MDS and acute leukemia :
• after allogeneic HSCT : in the early 1970s, Fialkow et al^ref and Thomas et al^ref reported on 2 patients with ALL receiving TBI and transplanted with marrow from an HLA-identical sibling donor, who within 2 to 4 months experienced what appeared to be a relapse of their original disease. However, further studies using cytogenetic analysis showed that the leukemic cells were donor-derived. Both donors continued to be healthy. Several similar cases, including patients with AML, ALL, and CML, were subsequently reported from other institutions (reviewed in Deeg et al^ref). Conditioning regimens in those patients consisted of chemotherapy only or chemotherapy plus TBI, and the diagnosis of recurrent leukemia in donor cells was made 6 months to more than 3 years after transplantation. Boyd et al^ref estimated that as many as 3% to 5% of leukemia recurrences may in fact be new leukemias in donor cells. However, no molecular tools were used in that study. The mechanism that would lead to leukemia in previously healthy transplanted cells was not clear. Several hypotheses have been proposed. Donor cells may have been transformed by antigenic stimulation through host tissue^{ref1, ref2, ref3}, as observed in murine models of marrow transplantation^ref. However, if this was the case, one would expect a higher frequency of this event. Alternatively, the recipient lymphohematopoietic environment in which the original leukemia had developed might trigger a similar development in donor cells^ref. Furthermore, fusion of normal cells with leukemic cells still residing in the recipient or transfection of an etiologic agent (virus/oncogene) might have transformed donor cells^{ref1, ref2, ref3}. Although these possibilities are conjectural, the clinical observations are of interest in the context of leukemogenesis in general. More recent studies have used refined molecular biology tools (eg, variable number tandem repeat [VNTR] analysis) to determine the origin (host v donor) of normal or abnormal cells in patients posttransplant. As determined by microsatellite analysis, disease reappearance in donor-derived cells is infrequent^ref. A rare case of transplantation of leukemia from the donor into the patient has been reported^ref. MDS, of some concern in autologous HSCT recipients, has occurred extremely infrequently after allogeneic HSCT (even in patients with Fanconi anemia in whom MDS develops frequently if not transplanted with normal cells). This observation provides indirect support for the notion that MDS after autologous HSCT is related to pretransplant factors rather than the transplant itself.
• donor cell leukemia (DCL), a rare although well-recognized disease entity following hematopoietic cell transplantation (HCT), occurs as the result of oncogenic transformation of apparently normal donor hematopoietic cells in the transplant recipient. DCL was first described in 1971,1 and, since then, descriptions of more than 50 cases have been published.2–6 DCL is less common after HCT than leukemic recurrence or relapse of the patient's original malignancy. In addition to de novo leukemia, there are cases documenting the occult transfer of malignant cells from donors during HCT.7 Notably, transfer of malignant cells can also occur with solid-organ transplantation.8–10 Defining the true incidence of DCL is difficult because of vagaries in its recognition and in reporting; therefore, the majority of evidence in the literature is anecdotal. In 1982 Boyd et al11 reported an incidence as high as 5%, and a recent large survey by the European group for Blood and Marrow Transplantation (EBMT) reported 14 cases from 10 489 transplantations performed during a 21-year period.12 The diagnosis of DCL is dependant on the ability to accurately identify the donor origin of the leukemic cells. With steady improvements in the molecular analysis of donor-host chimerism, the stage is set for improved and more accurate detection. A variety of methods to diagnose DCL have been used, including karyotype analysis, chimerism analyses, and, more recently, short tandem repeat analyses and variable number of tandem repeats, but in some of the earlier cases the diagnosis was based on differences in morphology alone.3 DCL can occur within 4 months after HCT, and the median time reported is 17 months after HCT. Therefore, it is scientifically interesting and potentially informative to speculate on the target cell for leukemogenesis among the apparently healthy donor cells, as well as what occurs during the earliest steps in what may be a unique neoplastic process. Posttransplant lymphoproliferative disorder (PTLD), a more extensively studied disease, may provide clues to the pathogenesis of DCL.13–15 PTLDs have a reported cumulative incidence of 1% to 2% after HCT and most occur within the first 6 months after HCT. The majority of PTLDs after HCT are donor in origin, although host-derived PTLDs after HCT are described. The recipient or donor origin of the PTLD after HCT appears to depend on the status of engraftment.16 In contrast, PTLDs following solid-organ transplantation are predominantly host derived.17 Epstein-Barr virus plays a pivotal role and is almost always associated with PTLD. Other risk factors include ex vivo T-cell depletion of the graft, in vivo T-cell therapy during transplantation, and histocompatibility between donor and host. Host-derived PTLDs suggest impaired tumor surveillance secondary to T-cell dysfunction after HCT allows an uncontrolled proliferation of transformed B cells. Although immunoregulatory dysfunction likely has a role to play in donor-derived PTLDs and DCL, additional mechanisms are also speculated. In DCL, the host environment in which the original malignancy developed might trigger a similar oncogenic process in donor cells favored by the immunocompromised status after transplantation. The correlation of engraftment status and origin of the PTLD suggests that either the recipient or donor cell population that is present can become transformed. This would support the theory that the environment in which the cell is placed has a role in its fate. Alternatively, chronic antigenic stimulation by minor histocompatibility differences between the donor cells and the host may trigger leukemogenesis in a setting of impaired tumor surveillance.1,18 Perturbations within the host bone marrow microenvironment following multiple rounds of chemotherapy may contribute.19 In some cases, as described later, premature aging of the donor cells and the associated chromosomal instability, as a consequence of the replicative stress ensuing from the effort to repopulate the recipient's marrow after transplantation, may also play a role.20,21 Certainly, combinations of these processes, or alternative mechanisms, are also feasible contributions to the development of lymphoid and myeloid malignancies after HCT.22,23

Cellular changes after HCT : following HCT, there are vigorous proliferative demands on the transplanted cells. A rapid proliferative rate often correlates with a higher likelihood of replication error or mutation. A preleukemic mutation may occur, and, with ensuing immunosuppression and impaired immune surveillance, this may more readily result in leukemia. Even unselected umbilical cord blood (UCB) units (a cell source becoming more common for HCT) have been shown to harbor preleukemic clones.24 These clones may be associated with an increased risk of leukemia when combined with the proliferative stress after HCT, although this remains to be formally proven.25 Studies of telomere length in HSCs from donors and recipients of bone marrow (BM) and peripheral blood (PB) HC transplants show an accelerated rate of shortening equivalent to approximately 15 years of aging after transplantation, compared with age-matched controls.20 Shortened telomeres are associated with premature replicative senescence and genomic instability, factors that could contribute to development of DCL. It is unlikely that telomere shortening after HCT is sufficient by itself to cause hematologic dysfunction, although it may be additive or synergistic to other changes in the bone marrow environment.26 There has been no consistent correlation between age of the donor or recipient, nor the type and extent of pre-HCT therapy, with after-HCT changes in telomere length.27 Interestingly, mononuclear cells of HC transplant UCB recipients have significantly longer telomeres compared with allogeneic PB HC transplant recipients, and it has been hypothesized that UCB transplants may provide patients with replicative "reserve" potential and a delay in the onset of hematologic disorders.28 Also the telomere dynamics of mesenchymal stem cells were shown to be unaffected by HCT, suggesting a distinct role of the microenvironment as a homeostatic regulator.29
Stem cell source for development of DCL : differences in the ontogenic source of stem cells may also account for some of the variation in the incidence and timing of DCL. Until 2005, reports of DCL were exclusively in allogeneic bone marrow HC transplant recipients; however, there have now been at least 4 cases reported in PB HC transplant recipients.7,12 The first report in an UCB transplant recipient was in 200530 and was quickly followed by 2 additional cases reports.31,32 The predominance of DCL in allogeneic BM HCTs compared with both PB and UCB may simply be a result of the longer historical use of this cell source. However, there are functional differences between the different ontogenic sources.33 Repopulation capacity differs between these cell sources, and the response of transplanted stem cells to replicative stresses may differ accordingly. These differences may reflect the frequency of HSCs in the cell source, although to date there has been a lack of correlation between the number of infused cells and changes in telomere length. UCB HC transplant recipients receive a lower total cell dose compared with other cell sources; the biologic differences in stem cell repopulation may have an impact on the replicative stresses on engrafting stem cells. These differences may be particularly relevant to UCB as a stem cell source, with the incidence of preleukemic mutations reported.24,25 However, on the basis of the information from the EBMT survey with prolonged follow-up of donors,12 donors from whom DCL develops are not typically destined to develop leukemia themselves. Future analysis of DCL, its clonal origin, and stem cell source may yield more information.
Leukemic and cancer stem cells : leukemic stem cells (LSCs) were first described in 1994.34 The studies of cancer stem cells have since been extended to other tissue types, including breast cancer,35 brain tumors,36 prostate cancer,37 melanoma,38 and lung cancer.39 The recognition that malignancies may develop from a population of "cancer stem cells" has caused both scientists and clinicians to consider the important implications such a cell population and developmental process could have on cancer pathogenesis and therapeutics. The characterization of cancer stem cells, in particular LSCs, has led to several proposed models of leukemogenesis.40 The paradigm of cancer stem cells considers leukemia a hierarchical disease process whose growth is sustained by a rare population of LSCs.41 Two factors suggest the stem cell as the target: (1) self-renewal machinery is already active and (2) quiescence that allows greater opportunity for the accumulation of mutations. LSCs maintain the capacity to self-renew and give rise to malignant progeny with extensive proliferative potential. The precise cell of origin and the mechanism of transformation is unknown in the majority of cases. It is speculated that the transformation involves at least a 2-step process, one mutation blocking differentiation and another event conferring its progeny with a proliferative advantage. Myeloid leukemia has been shown to originate from a primitive hematopoietic cell.42,43 Other LSCs may result from dedifferentiation of more committed progenitors that reacquire the ability to self-renew prior to accumulating transforming mutations.44 In BCR-ABL–associated leukemias, the transformation occurs at the stem cell and progenitor cell level depending on the phenotype and fusion transcript isoform.45 Several other exceptions exist, including PML-RAR,46 MLL-ENL,47 and MOZ-TIF2,48 whereby leukemia is promoted by fusion oncogenes, and self-renewal characteristics are imposed on more committed progenitors in the hematopoietic hierarchy. These models suggest an intrinsic cellular mechanism for leukemogenesis. However, it is evident that the fate of normal HSCs and LSCs is also determined by cell-independent mechanisms, including regulatory cues from their microenvironment.49,50
Donor cell leukemia and stem cells : the shared intrinsic and extrinsic mechanisms governing HSCs and LSCs may shed light on the unique pathogenesis of DCL whereby transplanted "apparently healthy" donor cells become leukemogenic over a short time. In most cases, specific mechanisms that result in the development of DCL are unknown. No consistent pattern of cytogenetic abnormality is apparent among the reported cases of DCL; almost half are associated with a normal karyotype. The remainder includes a heterogeneous mix of complex karyotypes, deletion, and fusion oncogenes.
We speculate that DCL is an illustration that leukemia development is not entirely cell autonomous. The important influence of the microenvironment must be considered. Studies of cancer stem cells have never made external factors irrelevant to tumorigenesis, but these aspects are often overlooked. Clearly distinct cell niches, including the endothelium and vasculature and other cell types, have an impact on tumor cell development.51 A better understanding of DCL may shift the emphasis from the stem cell toward the external homeostatic influence of the bone marrow microenvironment on the HSC. If the niche becomes dysregulated, the homeostatic balance may be disturbed, leading to changes in hematopoietic developmental pathways. These critical regulatory processes, both positive and negative, that govern HSC development are undoubtedly strained and potentially more prone to abnormalities at times of replicative stress, such as those that exist when donor HSCs repopulate the recipient marrow after HCT. The seminal "seed and soil hypothesis" of cancer development was published by Paget in 1889 and can be used as a model for DCL.52,53 Paget was struck by the discrepancy between the relative blood supply and frequency of cancer metastasis in certain organs. He proposed that the "seed" (cancer cell) will only grow if it falls on congenial "soil," and he used this hypothesis to explain the nonrandom pattern of cancer metastasis to visceral organs and bones. Some parallels can be drawn with the original seed and soil hypothesis and the relationship between the newly engrafted HSCs (seeds) and the complex bone marrow niche (soil) in which they arrive after HCT. Optimal development of HSCs occurs when the seed and the soil are in equilibrium. The soil plays an essential role in the maintenance and development of the seed. It regulates the quiescence, self-renewal, proliferation, and differentiation of the stem cells.50,54,55 Niche structures have the potential to determine stem cell fate, and the niche can become damaged after HCT. Important insight for this process comes from other developmental models, such as development of Drosophila germ cells in which nonstem cells can acquire either a more proliferative phenotype or frankly revert to a less-differentiated stem cell-like state when transplanted into certain permissive microenvironments.56,57 This finding suggests that the niche may actively impose stem cell characteristics on more differentiated cells. Other studies have also shown that a vacant viable niche can engage other cell types and cause a phenotypic change by providing "stem cell-like" features to more mature cells.54 Following HCT, HSCs engraft into a complex stromal environment composed of endosteal bone, osteoblasts, endothelial-lined sinuses, blood vessels, adipocytes, insoluble extracellular matrix proteins, and soluble peptides (chemokines and cytokines)58 (Figure 1A). All the molecular events that govern HSC decisions and its associated stroma are not known. In addition, after HCT, the effects of endogenous and pharmacologic cytokines on the stromal equilibrium are not well understood. Physical interaction between the HSC and trabecular bone via N-cadherin is known to help anchor the HSC at this interface.59,60 Osteoblasts under the influence of bone morphogenic signaling and parathyroid hormone (PTH) or PTH-related protein receptor also represent a regulatory element for the HSC.59,61 Osteoclasts may have a role in the localization and regulation of HSCs within the niche.62 Notch receptors have a role in HSC self-renewal,63 and interactions between angiopoietin-1 on bone and Tie-2 on stem cells seem to maintain quiescence.60 Disturbances in the cross talk between these cytokine and cellular stimuli and the HSC could contribute to leukemogenesis.
   Figure 1. Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells reside in the bone marrow in tissue composed of trabecular bone adjacent to endothelial lined sinuses and surrounded by various stromal cells that include osteoblasts (OB), osteoclasts (OC), chrondrocytes (CC), adipocytes (Ad), and fibroblasts (FB). This tissue also contains a complex environment of extracellular matrix proteins (EMP) and soluble cytokines. The interaction and homeostasis of these stromal components with the HSCs may determine its normal or neoplastic fate after hematopoietic cell transplantation (HCT). Figure 1A illustrates the normal HSC niche in which donor HSCs undergo self-renewal and differentiation to donor progenitor cells (PCs). Figure 1B illustrates the altered environment after HCT in which changes in the balance of extrinsic signals because of chemotherapy and radiation therapy may influence normal donor HSC development toward donor cell leukemia (DCL). These changes after HCT include injured stroma, leaky endothelium, impaired tumor surveillance, and a dysregulated extracellular matrix. Illustration by Kenneth Probst.
This normally supportive bone marrow environment is markedly changed by chemotherapy and radiation therapy that precedes HCT (Figure 1B). Patients are exposed to these agents both as a part of the treatment for the underlying malignancy and as a part of the conditioning prior to HCT. Ionizing radiation exposure during total body irradiation has direct effects on irradiated cells and the microenvironment.64 Radiation-induced injury can also contribute to secondary cell damage in the microenvironment as a consequence of an inflammatory-type response.65 Transplantation of growth factor–dependant cells into syngeneic mice produces leukemia from donor cells in higher numbers and at a faster rate in irradiated compared with nonirradiated recipients.66 These findings may reflect dysregulation of the stroma and alteration of the growth and phenotypic characteristics of stem cells.67 Chemotherapeutic drugs such as cyclophosphamide also cause endothelial injury and allow cells, proteins, and cytokines to move between the vascular space and the bone marrow niche.68
Almost all cases of DCL are acute myeloid leukemia. The original diseases included chronic myeloid leukemia and acute myeloid and lymphoblastic leukemias2,4,5,12,32; there have been 3 case reports with aplastic anemia and single cases with several other diseases (chronic lymphocytic leukemia, Langerhans histiocytosis, thalassasemia, renal cell carcinoma, and adult T-cell lymphoma). The stem cell niche likely has a role in de novo leukemogenesis and DCL; it appears to impact early hematopoietic development, as mature cellular malignancies are rarely seen. The association of DCL with myeloid disease suggests that the equilibrium between lymphoid and myeloid differentiation may be altered after HCT. Prolonged follow-up, as already mentioned, has shown that donors from whom DCL develops are not destined to develop leukemia.12 Until recently, in most cases described the donors were siblings; however, there are recent reports of cases in unrelated donors, including UCB donors30,31 which brings a new set of issues. The question of whether to inform an unrelated donor about the development of leukemia from his or her cells in a recipient, and the potential consequences for the donor, represents a significant ethical and medical challenge. DCL is a unique disease entity, although there is undoubtedly some mechanistic overlap between the development of DCL and other forms of leukemia. In the pathogenesis of DCL, it is important to consider that its cause is multifactorial in nature. Factors intrinsic to the cell and external signaling cues from the niche determine a normal versus neoplastic fate for the transplanted donor cells. Continued research to characterize DCL will help to understand better the dynamic equilibrium between both normal and leukemic stem cells and the hematopoietic microenvironment
• after autologous HSCT^ref : high-dose chemotherapy and autologous stem cell transplantation are used with increasing frequency in the treatment of NHL, HL, breast cancer, and other indications. Recent randomized trials have shown that this approach is more effective than conventional chemotherapy in patients with chemotherapy-sensitive relapse^ref and in some patients with high-risk NHL^{ref1, ref2}. Secondary MDS and AML have been observed after conventional chemotherapy and to a lesser extent radiotherapy for HL and NHL^{ref1, ref2, ref3, ref4, ref5}. Alkylating agents, epipodophylotoxins, combined modality therapy, and splenectomy have been implicated as risk factors^ref. Clearly, therefore, this complication does occur in patients who have not been transplanted, and a thorough evaluation of all transplant candidates, particularly in regard to cytogenetic abnormalties, before autologous transplantation is mandatory^ref. Nevertheless, beginning in 1993, several studies reported the development of secondary MDS and AML in patients with Hodgkin's disease and non-Hodgkin's lymphoma who had undergone autologous transplantation at a frequency that appeared unusually high (reviewed in Socié (Socie G: Secondary malignancies. Curr Opin Hematol 3:468, 1996), Blume^ref, Kumar^ref, Taylor et al^ref, Rohatiner^ref, and Stone^ref). Marolleau et al^ref first reported 3 cases of AML among 168 patients treated with autologous HSCT for advanced lymphomas (median follow-up, 3 years). In 1994, the University of Nebraska team^ref reported its experience in a case-control study. Twelve cases of MDS/AML occurred in 511 patients after autologous transplants for HL (n = 249) or NHL (n = 262). The cumulative incidence at 5 years was estimated to be 4% (11% and 12% for the 2 groups, respectively, among patients alive at 5 years). Age > 40 years at the time of transplant and the use of TBI were risk factors. Among 262 patients receiving autologous HSCT for NHL at the Dana Farber Cancer Center, 12 developed MDS/AML for a 6-year cumulative incidence of 18% ± 9%^ref. Pretreatment variables predictive (in univariate analysis) for the development of MDS included prolonged interval between initial treatment and HSCT, duration of exposure to chemotherapy (alkylating agents), and use of radiotherapy, especially pelvic irradiation. The Minneapolis team^ref reported on 206 patients with either HL (n = 68) or NHL (n = 138) who showed a 5-year cumulative incidence of MDS of 14.5% ± 11.6%. Recipients of peripheral blood transplants had an apparent higher risk than marrow transplant recipients (31% ± 33% v 10.5% ± 12%, respectively; P = .0035). In these 3 series combined, the elapsed time between transplant and diagnosis of MDS/AML ranged from 30 to 103 months. In a study at City of Hope Medical Center, clonal chromosomal abnormalities were detected in 10 of 275 patients after autologous transplant for HL or NHL^{ref1, ref2}. The diagnosis was made 1.8 to 6.5 years after chemotherapy and 0.5 to 3.1 years after HSCT, respectively. In nine patients the abnormalities involved chromosome 5, 7, 11q23, 21q22, or combinations thereof. 5 patients had morphologic evidence of MDS or AML. The cumulative probability of developing clonal chromosomal abnormalities reached 9% ± 4.7% at 3 years after transplantation.  The Minneapolis team recently updated their results^ref. Among 258 patients receiving autologous transplants for HL or NHL, 10 developed MDS/AML, for a cumulative probability of 13.5% ± 4.8% at 6 years. In multivariate analysis, the use of peripheral blood stem cells (RR = 5.8) and age over 35 years at transplant (RR = 3.5) were associated with an increased risk of MDS/AML. A French study of 467 patients also observed a higher incidence of MDS after peripheral blood than after marrow stem cell transplantation ((abstr, suppl 1) Andre M, Henry-Amar M, Blaise D, Colombat P, Fleury J, Milpied N, Cahn JY, Pico JL, Bastion Y, Kuentz M, Biron P, Ferme C, Gisselbrecht C: Incidence of second cancers (SC) and causes of death after autologous stem cell transplantation (ASCT) for Hodgkin's disease (HD). Blood 86:460a, 1995). MDS/AML have also been reported after HSCT for breast cancer^ref. Although studies are less extensive than in patients with HL or NHL, there is evidence that in particular after accelerated dose adjuvant therapy, the incidence of MDS may be high. These observations are of interest and raise several questions. Is MDS/AML after transplantation related to pretransplant chemoradiotherapy administered as primary or salvage therapy? Among 188 patients who underwent transplant for multiple myeloma at the University of Arkansas^ref, 71 were enrolled in a total therapy program and received no more than one course of standard chemotherapy (median, 7.6 months of treatment), whereas 117 patients had received more prolonged treatment courses before transplantation (median, 24 months). 7 patients developed MDS, all in the group of patients who had received prolonged treatment, leading the investigators to conclude that pretransplant therapy was the major risk factor for MDS after autologous transplantation. A closely related question is whether MDS/AML arises from the infused marrow (or peripheral blood) stem cells or from residual cells in the patient. If the disease develops from reinfused stem cells, then it is unlikely that TBI administered in preparation for transplant is a risk factorunless we postulate that TBI modifies the microenvironment in a way that enhances the risk of leukemogenesis. However, if the development of MDS/AML is related to the transplant procedure, we need to ask the following questions. Is it the procedure itself or, eg, the status of immunoincompetence following the transplant that contributes to the development of MDS? Do peripheral blood stem cells modify the milieu in a way different from marrow? Investigations into the function of growth factor mobilized peripheral blood stem cells show indeed cellular function (T cells and monocytes) and cytokine patterns different from marrow^ref. In fact, the term disordered engraftment has been proposed to describe the hematopoiesis in these patients^ref
• solid tumors : observations in animal models suggested that posttransplant (or postirradiation) solid tumors occurred with considerable delay, ranging from 7.5 to 15 years (median, 11.5 years) in x-irradiated and 4 to 15 years (median, 8 years) in rhesus monkeys irradiated with fission neutrons (Broerse JJ, Hollander CF, Van Zwieten MJ: Tumor induction in Rhesus monkeys after total body irradiation with X-rays and fission neutrons. Int J Radiat Biol 40:671, 1981 ). The time interval in g-irradiated dogs was 1.6 to 10.5 years (median, 8 years)^ref. Extrapolation to humans with a longer expected life span would suggest that solid tumors might develop a decade or more after transplantation. This appears to be born out by the actual data (Deeg HJ: Delayed complications after bone marrow transplantation, in Forman SJ, Blume KG, Thomas ED (eds): Bone Marrow Transplantation. Boston, MA, Blackwell Scientific, 1994, p 538 )^{ref1, ref2}
• solid tumors after allogeneic HSCT : initial reports, generally on small numbers of patients who had undergone allogeneic (or syngeneic) marrow transplantation, documented the development of some adenocarcinomas of the rectum, brain tumors (glioblastomas) particularly in patients who had also received cranial irradiation (1,800 to 2,400 cGy) before transplantation, squamous cell carcinomas of the skin, and cancers of the oropharyngeal mucosa^{ref1, ref2}. In contrast to PTLD, which generally were diagnosed within 2 to 4 months of transplantation, these solid tumors were observed at 1 to 5 years (Deeg HJ: Delayed complications after bone marrow transplantation, in Forman SJ, Blume KG, Thomas ED (eds): Bone Marrow Transplantation. Boston, MA, Blackwell Scientific, 1994, p 538 )^{ref1, ref2}. In the first larger series, analyzing results in 2,145 patients transplanted from 1970 through 1987 in Seattle, Witherspoon et al54 found 35 new malignancies; 13 of these were solid tumors, including glioblastoma, melanoma, squamous cell carcinoma, adenocarcinoma, hepatoma, and basal cell carcinoma. These tumors were diagnosed between 2.5 months and 14 years (median, 4.6 years) after transplantation. Although TBI was a significant risk factor when all malignancies were considered, only the use of antithymocyte globulin as an immunosuppressive agent was identified as a significant risk factor for solid tumors. Subsequent analysis of the results in patients with aplastic anemia transplanted in Seattle and at Hôpital Saint Louis in Paris, as well as reports from other European centers, showed that irradiation, in particular total lymphoid or thoraco-abdominal irradiation (as compared with conditioning regimens that did not involve irradiation), was a significant risk factor for the development of solid tumors^ref. A combined analysis of results in 700 patients with aplastic anemia transplanted at the Fred Hutchinson Cancer Research Center or Hôpital Saint Louis suggested that, in addition to irradiation (RR [RR] = 3.9), treatment of chronic GVHD with azathioprine (RR = 7.5) and older age (RR = 1.1) increased the risk of a posttransplant malignancy^ref. Not surprisingly, the highest incidence of malignancy was observed in patients in whom the etiology of marrow failure was Fanconi anemia (Kaplan-Meier estimate at 15 years, ~40%). However, it is of note that no hematologic malignancies (MDS, etc) were observed in either idiopathic or Fanconi-associated aplastic anemia, an indication that the transplanted (allogeneic) stem cells were able to develop and differentiate normally in the patient's marrow microenvironment. Bhatia et al^ref summarized observations in patients transplanted in Minneapolis. Among 2,150 patients, 15 developed a solid tumor (8 in 1,400 allogeneic and 7 in 750 autologous transplant recipients)^ref for a cumulative probability of 5.6% at 13 years. Irradiation was the major risk factor (RR = 6; P = .008). Kolb et al ((abstr, suppl 1) Kolb HJ, Duell T, Socié G, Van Lint MT, Carreras E, Tichelli A, Ljungman P, Jacobsen N, Apperley JF, Hertenstein B, Weiss M, Nekolla E, Goldstone AH: New malignancies in patients surviving more than 5 years after marrow transplantation. Blood 86:460a, 1995) determined the incidence of posttransplant malignancies in 1,211 patients who had survived at least 5 years after transplantation at 45 European centers. Forty-seven patients developed malignancies, including squamous cell carcinoma, breast cancer, glioblastoma, lymphoma, and others. In comparison to normal controls, the incidence rates were increased significantly for malignancies of the oral cavity, skin, esophagus, uterine cervix, and brain. In univariate analysis, donor age, chronic GVHD, and treatment of GVHD with cyclosporine, thalidomide, azathioprine, or methotrexate, and the number of agents used were found to be significant. In multivariate analysis using a Cox model, donor age above 30 years and chronic GVHD were significant risk factors, but the use of irradiation for conditioning was not. In a large collaborative study, Curtis et al^ref analyzed results in 19,220 patients (97.2% allogeneic and 2.8% syngeneic HSCT recipients) transplanted between 1964 and 1992 at 235 centers. There were 80 solid tumors for an observed/expected (O/E) ratio of 2.7 (P < .001). In patients surviving at least 10 years after HSCT, the risk was increased eightfold. The cumulative incidence of tumors was 2.2% at 10 years and 6.7% at 15 years. The risk was increased significantly for melanoma (O/E = 5.0), cancers of the oral cavity (11.1), liver (7.5), CNS (7.6), thyroid (6.6), bone (13.4), and connective tissue (8.0). The risk was highest for the youngest patients and declined with age (P for trend, <.001). Most striking was the link of squamous cell carcinoma with chronic GVHD and male gender. The underlying diagnosis was important insofar as the risk of solid tumors was higher for patients with acute leukemia and lower in patients with lymphoma or aplastic anemia. The risk associated with TBI decreased if irradiation was administered with a fractionation regimen, but increased with the total cumulative dose administered. This analysis strongly suggests that reduced doses of TBI, the omission of limited field irradiation, and the prevention of GVHD, in particular chronic GVHD, should reduce the risk of posttransplant solid tumors. Most frequent solid tumor malignancies and significant variables^ref :

Tumor	RR by variable
	high-dose TBI	limited field irradiation	GVHD		T-cell depletion	HLA nonidentity	male gender
			acute (II-IV)	chronic
squamous cell carcinomaref
Buccal cavity (n = 14)	3.0	136 (P < 0.01)	1.7	6 (P < 0.01)ref	0	0	9.7 (P < 0.01)
Skin (n = 8)	0.2	0	2.1	22 (P < 0.01)	0	0	infinity  (P < 0.01)
thyroid CA (n = 7) ref	5.8	0	1.9	0	4.9	0	0.4
bone or connective tissue (n = 8)	0.6	0	0.4	0.6	0	2.2	1.2
brain or other CNS (n = 10)	4.3	0	0.4	0	0	0.6	2.5
melanoma, skin (n = 9)	8.2	0	0.6	0.4	4.5	0	0.4



• solid tumors after autologous HSCT : although studies to date have focused on allogeneic transplant recipients, there is evidence for an increased incidence of new malignancies in autologous patients as well. A French study analyzed results in patients with Hodgkin's disease, 467 of whom had received an autologous stem cell transplant and 3,855 had been treated with conventional therapy ((abstr, suppl 1) Andre M, Henry-Amar M, Blaise D, Colombat P, Fleury J, Milpied N, Cahn JY, Pico JL, Bastion Y, Kuentz M, Biron P, Ferme C, Gisselbrecht C: Incidence of second cancers (SC) and causes of death after autologous stem cell transplantation (ASCT) for Hodgkin's disease (HD). Blood 86:460a, 1995). Among the transplanted patients, 18 developed a new malignancy for an the incidence of 8.9% at 5 years. The incidence was particularly high in patients above the age of 35 years and in patients who had received peripheral blood (rather than marrow) stem cells. Whereas the incidences of MDS were similar in transplanted and nontransplanted patients, transplanted patients were at a higher risk of solid tumors (P = .039). As noted before, a recent analysis of results in patients transplanted in Minneapolis also showed seven solid tumors in 750 autologous transplant recipients. Unpublished Seattle data show 6 solid tumors among 684 autologous transplant recipients conditioned with a radiation-containing or chemotherapy-only regimen (R.P. Witherspoon, personal communication, November 1997). Further observations in autologous transplant recipients will be of great interest because etiologic factors, such as chronic alloantigenic stimulation and GVHD, can basically be excluded.
Pathogenesis of solid posttransplant tumors : much less is known about the pathogenesis of solid tumors than of PTLDs. However, the interaction of various factors appears to apply to these malignancies as well. Using a PCR technique, Socié et al (unpublished observations) found evidence for involvement of human papilloma virus (HPV) 13, 15, or 16 in 3 of 8 tumors examined; HHV8 was detected in one tumor. In addition, the pattern of p53 expression suggested mutations of this gene in all eight tumors studied. Mutations might be induced by cytotoxic therapy, and suppressed immunity would interfere with a normal surveillance. Clearly, considerable work is needed for a better understanding of those questions.
Therapy of solid tumors after HSCT has followed the standards used in nontransplant patients. Experimental studies suggest that selective immunostimulation and measures aimed at scavenging free radicals may be beneficial in preventing tumor development.
Statistical considerations in evaluating the risk of new malignancies after HSCT : the development of new malignancies has long been recognized as a potential complication of cytotoxic therapy, either with chemotherapeutic agents or irradiation. An increased incidence has been observed, eg, in patients treated for Hodgkin's disease, acute leukemia, or solid tumors in childhood. Intensive cytotoxic conditioning therapy is also used in preparation for stem cell transplantation to eradicate the underlying disease. Furthermore, in allogeneic transplants, the conditioning regimen provides immunosuppression, thereby assuring sustained engraftment of donor-derived cells. As a result, transplantation is followed by a period of severe immunodeficiency that is further enhanced, in allogeneic transplants, by immunosuppressive agents administered for prophylaxis or therapy of GVHD. These and other factors (including the primary disease and treatment administered pretransplant) contribute to the development of second malignancies after HSCT. The data reviewed here raise questions about the best approach to estimate risks and to provide information for physicians and patients about the excess risk of second malignancy after stem cell transplantation. The most commonly used method is the standardized incidence ratio (SIR), ie, the ratio of observed (O) incidence of malignancies in the patient cohort compared with the expected (E) incidence of these malignancies in the general population of the same age and gender. High SIR, or RRs, in cohorts of young patients must be viewed within the context of the frequency of events in the comparable general population at similar ages. For example, few cases of a new acute leukemia in a cohort lead to a high SIR because of the rarity of this disease in the general population, whereas a substantial number of second breast cancers is needed before the O/E ratio becomes significant because of the relative frequency of this tumor type within the general population. Another commonly used method is actuarial risk estimates (using Kaplan-Meier method, eg). These actuarial estimates often lead to alarming figures once the interval after treatment exceeds 5 to 10 years, due to the fact that data for most of the study population are censored at follow-up intervals shorter than those at which second malignancies are recognized. As a result, this method magnifies the percentage of change caused by any event. This problem should be kept in mind when comparing actuarial estimates provided by two different studies (ie, 5% and 15% actuarial incidences of second malignancies at 10 years might not be different, due to large confidence interval limits). These methodologic aspects have been reviewed in a recent editorial on second cancers after Hodgkin's disease in childhood treated with conventional chemoradiotherapy^ref. Finally, in the context of HSCT, one has to ask whether the general population is the best reference group. Because other conventional (standard chemotherapy) treatment is administered for some (if not all) diseases that are also treated with transplantation, it would be important to compare the risk of second malignancy (and survival!) in patients receiving transplants versus conventional therapy rather than transplanted patients versus the healthy population.

• nonmalignant late effects are heterogeneous, and although often not life threatening they significantly impair the quality of life of long-term survivors^ref. The main aims of this review by the Late Effects Working Party of the European Study Group for Blood and Marrow Transplantation (EBMT) are to present physicians with an overview of these nonmalignant late complications and provide some recommendations regarding their prevention and early treatment. The major risk factors for nonmalignant complications after SCT are chronic graft-versus-host disease (cGVHD) and/or its treatment and the use of irradiation in the pretransplantation conditioning. Interrelationship between total body irradiation, chronic graft-versus-host disease in the genesis of late complications after allogeneic stem-cell transplantation.
• chronic GVHD, and its associated immune-deficiency state, is the prime cause of transplant-related mortality late after marrow grafting and contributes directly or indirectly to most nonmalignant complications. Because cGVHD has recently been reviewed in Blood^ref, only the main points relating to nonmalignant complications will be highlighted here. Following HLA-identical marrow transplantation, the 5-year probability of transplant-related mortality following discharge is > 20% in patients with and around 5% in patients without chronic GVHD^ref. Despite the advent of new treatment modalities, the incidence of cGVHD is being sustained by changes in clinical SCT practice as follows: (1) the expanded use of matched unrelated as well as mismatched related donors^{ref1, ref2, ref3} (2) the increasing use of SCT in older patients, (3) the increasing use of donor lymphocyte infusions to treat relapsed disease or to achieve full donor chimerism after nonmyeloablative transplantation, and (4) current evidence that suggests patients receiving allogeneic peripheral blood stem cell transplants have an equally high or a higher incidence of chronic GVHD compared with patients receiving marrow grafts. Long-term follow-up data from Seattle indicated that, although the cumulative incidence of chronic GVHD at 3 years was similar in patients whose stem cells came from marrow versus peripheral blood, cGVHD after peripheral blood SCT was more protracted and less responsive to treatment than cGVHD after marrow SCT^ref. Whereas the prophylactic use of multiagent immunosuppression has reduced the incidence and severity of acute GVHD, the incidence of chronic GVHD remains unchanged. The incidence of chronic GVHD after sibling-matched related, unrelated, and peripheral blood stem cell transplantation lies between 27-50%, 42-72%, and 54-57%, respectively^ref. Factors that increase the development of chronic GVHD include increased donor and recipient age, prior acute GVHD, use of alloimmune female donors, type of GVHD prophylaxis, and history of recipient herpes virus infection. There are several grading schemes that predict survival of patients with chronic GVHD. In these grading schemes, poor prognostic variables include lichenoid skin changes, extensive skin involvement (> 50% body surface area), elevated bilirubin, progressive onset, thrombocytopenia, and prior steroid refractory/dependent acute GVHD (reviewed in Lee et al^ref). Immune reconstitution has a pivotal role in the long-term issue of allogeneic SCT. Several studies have characterized the immune reconstitution in the few months following allogeneic SCT, but data on long-term immune reconstitution involving large numbers of patients are scarce^{ref1, ref2, ref3, ref4}. A large number of variables related to patient or transplant characteristics, such as age of recipients, stem cell engineering, type and duration of their disease, or conditioning regimen, may influence the recovery of immunity after SCT. Other posttransplantation variables, and in particular chronic GVHD and the consequent administration of immunosuppressive drugs, also have an effect. Chronic GVHD is the major factor affecting immune reconstitution of B cells and CD4 and CD8 T cells. Donor source (marrow versus peripheral blood), unrelated versus sibling transplant, and the degree of HLA compatibility between donor and recipient also affect the pace of immune reconstitution. Low B-cell count, inverted CD4/CD8 ratio, and a decreased IgA level are all risk factors associated with late infections. The role of slow B-cell reconstitution on the susceptibility to infections occurring in the years following SCT has been pointed out in 2 recent studies^{ref1, ref2}. Factors contributing to the immune deficiency post-HSCT are summarized in Figure 2. Recent immunologic methods such as repertoire analysis or the identification of recent thymic emigrants are currently being used as tools to evaluate immune reconstitution following SCT. Using these new tools, it has recently been suggested^ref that the immune system could regain normal functioning in the long term post-SCT. However, because most of the patients in the latter study were treated for severe aplastic anemia and did not have cGVHD^ref, it still remains to be proven whether leukemic survivors who have undergone TBI-conditioned SCT and developed cGVHD are also able to recover normal immune function 15 to 20 years post-SCT.

Oral beclomethasone dipropionate (BDP) control gastrointestinal GvHD (anorexia, vomiting, and diarrhea). Patients were randomized to prednisone for 10 days and either oral BDP 8 mg/day (N=62) or placebo (N=67) tablets for 50 days. At Study Day-10, prednisone was rapidly tapered while continuing study drug. On an intent-to-treat basis, the risk of GVHD-treatment failure was reduced for the BDP group at Study Day-50 (hazard ratio 0.63, 95% CI 0.35-1.13) and at 30 days follow-up (HR 0.55, 95% CI 0.32, 0.93). Among patients eligible for prednisone taper at Study Day-10, the risk of GVHD-treatment failure was significantly reduced at both Study Days-50 and -80 (HR 0.39 and 0.38, respectively). By day-200 post-transplant, 5 patients randomized to BDP had died, compared to 16 deaths on placebo, a 67% reduction in the hazard of mortality (HR 0.33, p=0.03). In 47 recipients of unrelated and HLA-mismatched stem cells, mortality at transplant day-200 was reduced by 91% in the BDP group, compared to placebo (HR 0.09, p=0.02). The survival benefit was durable to 1-year post-randomization. Oral BDP prevents relapses of gastrointestinal GVHD following tapering of prednisone; survival is statistically significantly better among patients receiving BDP^ref
Factors contributing to late immune deficiency and late infection following allogeneic stem cell transplantation.
 
• late bacterial and viral infections have been extensively reviewed, and guidelines for preventing and treating these opportunistic infections after SCT are proposed in a document published under the hospice of the Centers for Disease Control and Prevention (CDC), the Infectious Disease Society of America, and the American Society of Blood and Marrow Transplantation^ref. Susceptibility to encapsulated bacteria (Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis) has been well documented, especially in patients with current or previous chronic GVHD. Late (> 2 years) fungal or cytomegalovirus (CMV) infections are rare and almost invariably occur in patients with ongoing immune suppression for GVHD. Varicella-zoster, in contrast, is extremely frequent even in patients without GVHD, but it usually occurs within several months of SCT after acyclovir prophylaxis has been discontinued. Finally, of the parasitic infections, late Pneumocystis carinii pneumonia (PCP) and Toxoplasma gondii infections are more common in patients receiving active treatment for cGVHD. Because PCP prophylaxis with trimethoprim-sulfamethoxazole is highly active, this regimen should be given to all patients receiving treatment for cGVHD and/or those with CD4⁺ cells < 0.2 × 10⁹/L. Probably, PCP prophylaxis should be continued for several weeks after the cessation of immunosuppressive therapy given the long-lasting T-cell defects characteristic of patients who have developed cGVHD.
• late ocular effects :
• ocular complications of the posterior segment : these complications can be divided into^ref :
• microvascular retinopathy occurs mainly after TBI-conditioned allogeneic SCT in patients receiving cyclosporine as GVHD prophylaxis. Visual acuity is decreased in most patients but recovers when cyclosporine is withdrawn. Atypical retinal microvasculopathy, without cotton-wool spots, has also been described^ref. Radiation may provoke an occlusive microangiopathy but only if the radiation dose exceeds 35 Gy. Thus, TBI alone cannot explain ischemic retinopathy in patients who have received a transplant, and additional factors, such as cyclosporine, may lower the threshold for radiation retinopathy^ref. Ischemic retinopathy has been reported in patients conditioned with busulfan and cyclophosphamide without irradiation^ref, or in patients receiving Campath-1G for GVHD prophylaxis^ref. In most cases, retinal lesions resolve with withdrawal or reduction of immunosuppressive therapy, and resolution has even been described in a case associated with complete blindness^ref. Although ischemic retinopathy is still an enigma, these data suggest that the development of ischemic eye lesions is a multifactorial process leading to capillary damage of the eye fundus. These endothelial changes might not be restricted to the eye but may reflect a generalized process within the microvasculature.
• optic disk edema
• hemorrhagic complications
• infectious retinitis : fungal infections typically occur within 120 days of HSCT, whereas herpes zoster, CMV, and toxoplasmic retinitis occur later. Ischemic retinopathy with cotton-wool spots and optic-disk edema has been described in 10% of patients following HSCT^{ref1, ref2, ref3}
• ocular complications of the anterior segment : the 2 most common late complications affecting the anterior segment are cataract formation and keratoconjunctivitis sicca syndrome. Cataract formation, particularly posterior subcapsular cataracts, has long been recognized in recipients of HSCT as one of the most frequent late complications of TBI^{ref1, ref2}. After single-dose TBI almost all patients develop cataracts within 3 to 4 years, and most if not all need surgical repair. Although the probability of developing cataracts after fractionated TBI lies around 30% at 3 years, the incidence may reach more than 80% in 6 to 10 years post-SCT^{ref1, ref2}. In a multivariate analysis, the use of TBI, fractionation of its dose, and the use of steroid treatment for longer than 3 months were associated with a significant risk of cataract development^ref. The effect of the dose rate of irradiation on the subsequent development of cataracts is also established^{ref1, ref2, ref3}. In the largest series evaluating a cohort of 1064 patients^ref, factors independently associated with an increased risk of cataracts were older age (> 23 years), higher dose rate (> 0.04 Gy/minute), allogeneic HSCT, and steroid administration. Finally, in prospective studies comparing cataract incidence rate and risk factors it has been shown that patients who receive cyclophosphamide and TBI (Cy/TBI) have a higher incidence of cataract formation than those treated with busulfan and cyclophosphamide (BuCy)^ref. Although the total dose of TBI is the most important factor for cataract formation, the incidence, severity, and time course of cataract formation differ depending on the number of fractions, dose rate of irradiation, the age of the patient, and the use of steroids. The only treatment for cataract is to surgically remove the opacified lens from the eye to restore transparency of the visual axis. Today, cataract surgery is a low-risk procedure and improves visual acuity in 95% of eyes that have no other pathology. Results of surgical repair in patients who have received a transplant are not yet available.
• cataract after HSCT : cataract formation after TBI. (A) Cataract formation occurs earlier after single dose than after fractionated dose total body irradiation (reprinted from Tichelli et al^ref with permission). (B) Fractionated TBI is associated with a significant, dose-dependent risk of cataract formation (reprinted from Benyunes et al^ref with permission, nonexclusive world English rights only). Keratoconjunctivitis sicca syndrome is usually part of a more general syndrome with xerostomia, vaginitis, and dryness of the skin^ref. All these manifestations are closely related to cGVHD^ref that may lead in its most extensive forms to a Sjögren-like syndrome^ref. The ocular manifestations include reduced tear flow, keratoconjunctivitis sicca, sterile conjunctivitis, corneal epithelial defects, and corneal ulceration^{ref1, ref2, ref3, ref4, ref5}. The incidence of late-onset keratoconjunctivitis sicca syndrome may reach 20% in 15 years after SCT^{ref1, ref2, ref3} but reaches nearly 40% in patients with cGVHD, compared with < 10% in those without GVHD. Risk factors for late-onset keratoconjunctivitis include cGVHD, female sex, age older than 20 years, single-dose TBI, and the use of methotrexate for GVHD prophylaxis^ref. Treatment is based on the management of cGVHD with repeated use of topical lubricants. Topical corticosteroids may improve symptoms but can cause sight-threatening complications if used inappropriately in herpes simplex virus or bacterial keratitis. Topical retinoic acid may also be used^ref
• pulmonary late effects : significant late toxicity involving both the airways and lung parenchyma affects > 15-40% of patients after HSCT. Most of the studies have been performed in adult patients, and results are still conflicting because of varying selection and evaluation criteria, limited sample size, and short follow-up. Moreover, clinical syndromes are not well defined or definable because of overlapping mechanisms and/or because they represent a continuum rather than a distinct disorder. Sensitivity to cytotoxic agents and irradiation, infections, and immune-mediated lung injury associated with GVHD are the most prominent factors that contribute to late respiratory complications^ref. Impaired growth of both lung and chest can be an additional factor in children.
• restrictive lung disease : abnormal pulmonary function tests (PFTs) with loss of lung volume and diffusing capacity may exist prior to SCT as a result of the intensification of front-line treatment^ref. Although the relevance of pretransplantation PFTs is still debated, some studies indicate that PFTs can be predictive of complications and outcome after SCT and should, therefore, be included within the pretransplantation investigation program^{ref1, ref2, ref3}. Restrictive lung disease is frequently observed 3 to 6 months after SCT in patients conditioned with TBI and/or receiving an allogeneic SCT, but in most cases it is not symptomatic. Restrictive disease is often stable and may recover, partially or completely, within 2 years^{ref1, ref2, ref3}. However, some patients do develop severe late restrictive defects and may eventually die of respiratory failure^ref
• chronic obstructive lung disease with reduced forced expiratory volume in 1 second/forced vital capacity (FEV₁/FVC) and FEV₁ can be detected in up to 20% of long-term survivors after SCT^{ref1, ref2, ref3}. Its pathogenesis is not yet well understood. It has been mainly associated with cGVHD, but other potential risk factors, including TBI, hypogammaglobulinemia, GVHD prophylaxis with methotrexate, and infections, have been described. Although direct immune-mediated damage by donor T lymphocytes and cytokines is classically the main mechanism, airflow obstruction can also be due to indirect consequences of cGVHD, for example aspiration secondary to esophageal GVHD, sicca syndrome, abnormal mucociliary transport^ref, and recurrent infections. Mortality is high among these patients, particularly in those with an earlier onset and rapid decline of FEV₁. Symptoms consist of nonproductive cough, wheezing, and dyspnea; chest radiography is normal in most cases. HRCT scanning may reveal nonspecific abnormalities^ref. Symptomatic relief can be obtained in some patients with bronchodilators; however, in most cases obstructive abnormalities are not improved by this treatment. Patients with low IgG and IgA levels should receive immunoglobulins to prevent infections, which may further damage the airways. Immunosuppressive therapy may be of benefit, but typically improvements occur in < 50% of cases probably because the damage has already become irreversible or because other pathogenetic factors persist. Asymptomatic patients with abnormal PFTs should be closely monitored for the development of respiratory symptoms; an early recognition of airflow obstruction allows the initiation of treatment at a potentially reversible stage.
• obliterative bronchiolitis (OB), the best characterized obstructive syndrome, has been reported in 2% to 14% of allogeneic HSCT recipients and carries a mortality rate of 50%^{ref1, ref2, ref3, ref4, ref5}. OB is strongly associated with cGVHD and low levels of immunoglobulins. GVHD is probably responsible for the initial epithelial injury to the small airways^ref with further damage caused by repeated infections. Initial symptoms often resemble those of recurrent upper respiratory tract infections, and then persistent cough, wheezing, inspiratory rales, and dyspnea appear. PFTs gradually deteriorate with severe and nonreversible obstructive abnormalities. Chest radiographs and CT scanning may reveal hyperinflation with or without infiltrates and vascular attenuation; however, radiologic findings do not correlate with lung function changes probably because of the patchy nature of the disease^ref. Bronchoscopy with transbronchial biopsy can help to rule out infection and may reveal obliteration of bronchioles with granulation tissue, mononuclear cell infiltration, or fibrosis. It is not clear to what extent combined immunosuppressive treatment can be effective in the treatment of this disease, which typically does not respond to treatment with steroids. Azathioprine and mycophenolate may lead to improved symptoms in some cases. Prophylaxis and prompt treatment of infections are the most important elements of clinical management and may help to alter the clinical course of a disease whose pace can vary from slow progression to rapidly fatal respiratory failure. Single or double lung transplantation has been suggested for patients with advanced disease^ref, although the transplanted lung may also be a target for immune-mediated damage.
• late liver complications : unraveling the cause of liver dysfunction can pose difficulties, first, because several causes of liver disease may coexist and, second, because patterns of viral serology may be atypical. Furthermore, in addition to the most important hepatotropic viruses, other agents, like herpesviruses (including CMV), adenoviruses, and Epstein-Barr virus, may be implicated, sometimes leading to life-threatening fulminant hepatitis ( Locasciulli A, Nava S, Sparano P, Testa M. Infections with hepatotropic viruses in children treated with allogeneic bone marrow transplantation. Bone Marrow Transplant. 1998;21:S75-S77). Useful tools for differential diagnosis are timing posttransplantation, type of clinical and biochemical deterioration, previous evidence of liver complications, including veno-occlusive disease (VOD), acute or chronic GVHD, and infection. Liver biopsy is often difficult to interpret, but histologic examination can be helpful in discriminating between an acute exacerbation of viral hepatitis and an episode of GVHD.
• HBV and HCV infections^ref may be nonsymptomatic or may progress to fulminant hepatitis or evolve to chronic active hepatitis and cirrhosis. Today, the risk of acquiring HBV and HCV infection from blood transfusion is greatly reduced. A recent prospective study of the EBMT, which included patients transfused in the "post-screening" era, showed that the prevalence of serum HBsAg- and HCV-RNA-positive HSCT patients was 3.1% and 6.0%, respectively. The prevalence of "de novo" infection in patients receiving HBsAg and HCV-RNA-negative HSCT was 2.0% and 7.4%, respectively; the prevalence of donors positive for HBsAg and HCV-RNA, was found to be 2.6% and 3.6%, respectively^ref. Chronic viral hepatitis still remains an important clinical problem in this setting^{ref1, ref2}. Patients infected with HBV generally show mild to moderate liver disease on long-term follow-up, but cirrhosis as a result of chronic hepatitis B has not been reported to date. Chronic hepatitis C is often asymptomatic with fluctuating transaminases levels and no signs or symptoms of uncompensated liver disease at least during the first decade following HSCT^{ref1, ref2, ref3}. Progression to cirrhosis or advanced liver disease in patients surviving > 10 years does occur, however^ref (G.S., unpublished observations, 2003). These observations indicate that, although the outcome of chronic hepatitis was thought to be benign, it may represent an important clinical problem in very long-term survivors. Careful tapering of immunosuppressive therapy posttransplantation, with monitoring of biochemical parameters and viral load, is crucial to prevent and allow early treatment of hepatitis reactivation. Increasing HBV viral load may need treatment with lamivudine^ref. However, prolonged therapy with this drug is followed by the emergence of HBV DNA polymerase mutants. As the selection of these mutants is a function of time, the indication for long-term therapy should be reviewed. The combination of lamivudine and anti-HBs immunoglobulins may also be of value. In patients with active chronic HCV, hepatitis treatment with interferon, with or without nucleotide analogues, is indicated^ref
• iron overload : on the basis of serum ferritin levels, the diagnosis of iron overload can be made in up to 88% of long-term survivors of HSCT^ref. Prolonged dyserythropoiesis and increased iron absorption both contribute to the accumulation of iron. Liver biopsies performed early after HSCT show siderosis in most patients^ref. Autopsies performed in patients dying early after HSCT show iron accumulation in a range equivalent to that of patients suffering from hereditary hemochromatosis^ref. Iron deposition has also been demonstrated in others tissues, such as the myocardium or bone marrow^{ref1, ref2}. Iron overload may be associated with a number of clinical consequences, but these consequences have not been extensively evaluated in patients posttransplantation. A clear correlation exists between iron deposition and persistent hepatic dysfunction, probably as a consequence of intracellular iron accumulation and the toxic effect of free radicals. In heavily transfused patients, such as those with thalassemia, iron can contribute to hepatic fibrosis, cirrhosis, and hepatocellular carcinoma as well as to cardiac dysfunction^{ref1, ref2}. Hepatic iron overload may also worsen the natural course of chronic hepatitis, in particular hepatitis C, and the response to antiviral therapy^ref. Finally, iron overload increases the risk of opportunistic infections in immunocompromised patients, mucormycosis being the infection most usually observed in HSCT^ref. Although iron overload spontaneously decreases in the years following HSCT^ref, the true effect of iron overload on post-HSCT complications such as diabetes mellitus, impotence, hypogonadism, or growth retardation has not yet been established. All survivors (even if asymptomatic) should, therefore, be assessed for iron overload by measuring serum ferritin. Iron overload should be treated by means of phlebotomy and/or chelation therapy, especially when iron overload coexists with chronic viral hepatitis. Phlebotomy has the advantage over chelation of better compliance, fewer side effects, and lower costs. The use of rhuEPO may facilitate this strategy in patients who have low hemoglobin levels^{ref1, ref2}
• late complications of bones and joints :
• avascular necrosis of bone (AVN) : the published incidence of AVN varies from 4% to more than 10% in the largest series^{ref1, ref2, ref3, ref4, ref5}. The mean time from transplantation to AVN is 18 months (range, 4-132 months), and pain is usually the first sign. Early diagnosis can rarely be made using standard radiography alone, and magnetic resonance imaging is the investigation of choice. The hip is the affected site in > 80% of cases, with bilateral involvement occurring in > 60% cases. Other locations described include the knee (10% of patient with AVN), the wrist, and ankle. Symptomatic relief of pain and orthopedic measures to decrease the pressure on the affected joints are of value, but most adult patients with advanced damage will require surgery. The probability of total hip replacement following a diagnosis of AVN is approximately 80% at 5 years (Bizot P, Witvoet J, Sedel L. Avascular necrosis of the femoral head after allogenic bone-marrow transplantationa retrospective study of 27 consecutive THAs with a minimal two-year follow-up. J Bone Joint Surg Br. 1996;78B:878-883)^ref. Although short-term results of joint surgery are excellent in the majority (> 85%) of cases, it is clear that long-term follow-up of the protheses are needed in young patients who have a long life expectancy. Studies evaluating risk factors for AVN have clearly identified steroids (both total dose and duration) as the strongest risk factor. Thus, unnecessary long-term low-dose steroids for nonactive chronic GVHD should be avoided. The second major risk factor for AVN is TBI, the highest risks being associated with receipt of single doses > 10 Gy or > 12 Gy in fractionated doses^ref. Finally, some underlying conditions may predispose a patient to develop AVN after SCT, in particular patients receiving transplants for severe aplastic anemia^ref or acute lymphoblastic leukemia.

Avascular necrosis of the hip. Standard radiography versus MRI as a tool for the diagnosis of avascular necrosis. These panels illustrate that standard radiography could be strictly normal at the first clinical sign while MRI is already highly abnormal. The top panel shows the normal appearance of the hip using standard radiography. The middle and bottom panels show 2 different MRI sequences showing that, although the femoral head is still spherical, the adjacent bone is already affected with necrosis, with even cartilage modification.
• osteoporosis : HSCT can induce bone loss and osteoporosis via the toxic effects of TBI, chemotherapy, and hypogonadism (reviewed in Weilbaecher^ref and Schimmer et al^ref). Osteopenia and osteoporosis are both characterized by a reduced bone mass and increased susceptibility to bone fracture. These conditions are further distinguished by the degree of reduction in bone mass and can be quantified by T and Z scores on dual photon densitometry. The Z score is similar to the T score but uses mean bone mass from an age- and sex-matched control population as a reference value. The relative risk of fracture doubles for every standard deviation below the control adult peak bone mass. The incidence and clinical course of bone mineral density (BMD) abnormalities following hematopoietic SCT have been studied in 2 large series^{ref1, ref2}. In both, the cumulative dose and number of days of glucocorticoid therapy and the number of days of cyclosporine or tacrolimus therapy showed significant associations with loss of BMD. Nontraumatic fractures occurred in 10% of patients. These results indicate that loss of BMD after allogeneic HSCT is common and is accelerated by the length of immunosuppressive therapy and cumulative dose of glucocorticoid. With the use of WHO criteria, nearly 50% of the patients have low bone density, a third have osteopenia, and roughly 10% have osteoporosis, 12 to 18 months posttransplantation. The true incidence and morbidity rate of osteoporosis in very long-term SCT survivors is currently unknown. Preventative measures of osteoporosis must include sex-hormone replacement in patients with gonadal failure; the efficacy of new treatments for osteoporosis in long-term survivors of HSCT requires evaluation
• dental late effects : both TBI-based regimens and those without irradiation can result in severe damage to the enamel organ and developing teeth. These defects may be prolonged or permanent^{ref1, ref2}. After TBI in children, underdevelopment of the mandible and anomalies in the mandibular joint may also occur^ref. In children, long-term clinical and radiologic follow-ups reveal hypoplasia and microdontia of the crowns of erupted permanent teeth and thinning and tapering of the roots of erupted permanent molars or incisors^{ref1, ref2, ref3, ref4}. Caries are found more frequently in patients who have received a transplant compared with age-matched healthy children. The defects in dental elements post-HSCT may occur at any age of tooth development, and only the severity seems to depend on age at HSCT. Recommendations to minimize this adverse effect aim to preserve the enamel layer and prevent, by active oral hygiene, dental plaque, periodontal and oral mucosal infections, and xerostomia, all of which contribute to the development of caries. Specialist dental consultation before HSCT and yearly posttransplantation examinations should be requested to register any specific dental problems, to provide treatment, and to give instruction on oral and dental care. In the long-term, 3 key elements to reduce dental complications are brushing teeth, application of fluoride, and the use of antiseptic mouthwashes. Brushing teeth should be done twice daily, with a soft brush and fluorinated toothpaste.
• endocrine function after HSCT
• thyroid dysfunction has long been recognized as one of the most frequent late complications of HSCT^ref. The most common thyroid abnormalities can be classified into 3 distinct patterns: subclinical-compensated hypothyroidism, overt hypothyroidism, and autoimmune thyroid disease. Subclinical-compensated hypothyroidism. 7-15.5% of patients will develop subclinical hypothyroidism (slightly high serum TSH and normal fT4 levels) in the year post-SCT^{ref1, ref2, ref3}. It is not yet clear if patients who develop subclinical hypothyroidism should be treated with L-thyroxin because most of these cases are mild, compensated, and may resolve spontaneously^{ref1, ref2, ref3}. Furthermore, treatment with L-thyroxin might induce early osteoporosis, especially if given to those women after HSCT with gonadal failure. It is also not clear if a relationship exists between high TSH levels and carcinogenesis^ref. One possible approach is to monitor TSH and free-T4 levels twice yearly and to consider L-thyroxin treatment only if TSH concentration remains high or is increasing^ref.
• overt hypothyroidism. The great majority of cases of overt hypothyroidism following HSCT are due to direct damage to the thyroid gland. Secondary hypothyroidism, caused by pituitary damage, is rare following SCT. Hypothyroidism is usually diagnosed after a median period of 50 months posttransplantation. The frequency of hypothyroidism requiring L-thyroxin replacement therapy is highly variable^{ref1, ref2},109,110 depending to a large extent on the type of pretransplantation conditioning applied as follows: nearly 90% in patients who have received 10 Gy single-dose TBI,110 14% to 15% of patients following fractionated TBI^ref,103 and smaller numbers after conditioning with BuCy^{ref1, ref2, ref3, ref4}.32,109-111 Treatment with L-thyroxin is indicated in all cases of frank hypothyroidism (elevated TSH with low free-T4 blood levels). Thyroid hormone levels should be measured 4 to 6 weeks after commencement of replacement therapy, and dosage should be tailored thereafter to the individual patient and adjusted according to thyroid function evaluation every 6 months. Elderly patients should have an electrocardiogram (ECG) prior to commencing treatment to exclude associated ischemic heart disease and/or arrhythmias.
• autoimmune thyroid disease. Autoimmune thyroiditis, presumably transferred via donor cells, has been reported^ref.112 This same autoimmune mechanism can also lead to hyperthyroidism if the donor is affected with Grave disease^ref.113
• growth : linear growth is an intricate process that may be affected by several systems, including genetic (ie, midparental height), nutritional, hormonal, and psychological factors. Intensive anticancer therapy during childhood may influence all or some of these factors resulting in decreased growth. Children who undergo HSCT form a heterogeneous group because of the different treatment protocols used. In addition, posttransplantation factors such as GVHD and its treatment, especially the use of long-term steroids, may induce growth failure in childhood^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}.104,114-119 Final height achievement has been reported in some studies^{ref1, ref2, ref3, ref4, ref5, ref6}.115-117,120-122 Decreased growth has been described in patients who underwent HSCT during childhood. The mean loss of height (as estimated by the standard deviation score [SDS]) is estimated to be approximately 1 height SDS (equivalent to 6 cm) compared with both the mean height at the time of HSCT and mean genetic height^{ref1, ref2, ref3, ref4, ref5, ref6}.115-117,120,121,123 Nevertheless, nearly 80% of the children will achieve adult height values above the 3rd percentile (or 2 height SDS) for the healthy general population^ref.116 Growth deficiency is more pronounced in children who receive transplants at a younger age (< 10 years) and in those who have received irradiation. In contrast, children who are conditioned with non-TBI regimens, such as cyclophosphamide or busulfan-cyclophosphamide, usually grow normally. Patients who have been exposed to cranial radiotherapy (CRT) prior to conditioning with TBI show a greater decline in growth^ref.124 The role of growth hormone (GH) deficiency as a cause of growth failure and its substitution in children after HSCT is still controversial. Although some reports show some benefit of GH treatment in children after HSCT^{ref1, ref2},125,126 others have failed to document GH deficiency^{ref1, ref2, ref3}.106,121,122 Furthermore, some studies have failed to demonstrate any correlation between growth rate and the GH secretion level after pharmacologic provocative tests^{ref1, ref2}.116,127 In a study involving children who had survived more than 5 years after HSCT for severe aplastic anemia (SAA)^ref,117 the decrease in growth observed in a population of 11 children who had received thoracoabdominal irradiation in their pretransplantation conditioning was significantly higher than seen in a group of 27 children who had received cyclophosphamide only. The decrease in growth found in the irradiated group cannot be explained by impaired secretion of growth hormone because thoracoabdominal irradiation spares the hypothalamus and pituitary gland. The reduction in growth observed in the irradiated group may, however, be explained by the direct effect of irradiation on the gonads, the thyroid gland, and/or the bone epiphyses.
• puberty and gonadal failure : gonadal failure (both testicular and ovarian) is a common long-term consequence of the chemotherapy given prior to HSCT and of the pretransplantation conditioning. The major cause of gonadal damage leading to hypergonadotropic-hypogonadism is irradiation^{ref1, ref2}. Similar damage can also be caused by busulfan. Among patients who have received a transplant, it is uncommon to find either a condition of hypogonadotropic-hypogonadism or precocious puberty because of irradiation damage to the hypothalamic-pituitary area^{ref1, ref2}.
• In males, the testicular germinal epithelium (Sertoli cells) where spermatogenesis occurs is more vulnerable to radiation and chemotherapy than the testicular Leydig cell component, which is involved in testosterone secretion. Therefore, testosterone levels are usually normal even when spermatogenesis is reduced or absent. The serum follicle-stimulating hormone (FSH) is typically elevated, whereas luteinizing hormone (LH) levels may remain in the normal range. The great majority of patients will not, therefore, require testosterone replacement to ensure sexual activity, libido, erection, and ejaculation. Furthermore, boys transplanted during childhood will usually spontaneously start and complete puberty. Patients transplanted before puberty, however, might achieve a reduced testicular volume as a result of damage to the germinal epithelium^ref. Sex hormone replacement therapy (SHRT) with testosterone derivatives in males is indicated in patients with severe uncompensated hypogonadism.
• In females, the ovaries are more vulnerable to irradiation and chemotherapy than the testes, and hypergonadotropic-hypogonadism is almost the rule. Busulfan is one of the most gonadotoxic agents, whereas cyclophosphamide is usually associated with only minor effects on gonadal function.
Prepubertal patients conditioned with cyclophosphamide alone for SAA usually have normal puberty^ref. The age at transplantation is of major importance because the younger the age, the better will be the chances for gonadal recovery. In fact, ovarian failure in adult women is usually irreversible, whereas in prepubertal girls, although uncommon, there is still a greater possibility for a subsequent spontaneous recovery and achievement of spontaneous menarche^{ref1, ref2, ref3, ref4}. Fractionation of the irradiation reduces the effect on the ovaries^{ref1, ref2}; girls treated with 12 Gy fractionated TBI are 5 times more likely to have a spontaneous recovery to normal ovarian function than girls receiving single-dose TBI. With the increasing utilization of BuCy in pediatric pretransplantation conditioning, it becomes clear that most girls of any age at transplantation develop ovarian failure^{ref1, ref2}
Most females will need SHRT, both for the induction of puberty in girls who have had SCT prior to the menarche and for maintaining menstrual cycles and bone turnover/mineralization in adult women. In prepubertal girls who do not undergo puberty spontaneously post-HSCT, estrogen treatment should be started at the age of 12 to 13 years to promote breast and uterine development and the pubertal growth spurt. The dose of estrogen treatment will need to be gradually increased, and a combination of cyclical estrogen-progesterone treatment introduced after 1 to 2 years to initiate menstruation and to reduce the risk of future osteoporosis. SHRT can be interrupted once every 2 to 3 years, for a period of 6 months, to evaluate possible spontaneous recovery of ovarian activity, which occurs in a minority of women. Because of the high incidence of gonadal dysfunction and early menopause in patients after SCT, an annual clinical and biologic gynecologic assessment is recommended.
• Fertility following stem cell transplantation : despite the potential gonadotoxicity of pretransplantation conditioning (see "Puberty and gonadal failure"), gonadal recovery and pregnancies following HSCT are well described. The precise incidence of fertility following HSCT is hard to establish. Unpublished data from the EBMT Late Effects Working Party (LEWP) pregnancy database relating to incidence of pregnancy in patients transplanted prior to 1994 who survived for a minimum of 2 years is given in Table 2. These data are not comparative because the patients within the groups were not age matched. Nonetheless, it is clear that the overall incidence of pregnancy is low (< 2%) except for patients transplanted for SAA and this is in accordance with the available literature. Such data are limited in their ability to accurately predict the likely return of fertility post-HSCT, however, because many patients do not wish to become parents following the diagnosis of a potentially life-threatening illness.

Table 2. Incidence of pregnancy following stem cell transplantation
• Gonadal recovery following SCT : other studies have looked at clinical and laboratory indicators of gonadal function in post-SCT patients. In women these include amenorrhea, menopausal symptoms, raised gonadotrophin levels, and low estrogen. In men, sperm quality (motility and morphology) and quantity can be assessed, and the typical biochemical profile would be raised FSH with normal LH and normal/low testosterone levels.
• Fertility following SCT for nonmalignant disease : return of gonadal function following cyclophosphamide conditioning for SAA was noted in 56 of 103 adult female survivors in Seattle (as indicated by return of menstruation and normal gonadotrophin and estradiol levels); 28 (27%) women subsequently conceived^ref. Of 109 adult male survivors in the same study, 61% had return of sperm production and 28 (26%) subsequently fathered children. Previous data from this and from other centers indicated that gonadal recovery is usual in women younger than the age of 25 years at the time of transplantation but sharply decrease thereafter^{ref1, ref2, ref3}. In thalassemia, gonadal failure is common as a result of both transfusional hemosiderosis and conditioning with BuCy. Approximately 40% enter puberty, but pregnancies are very rare^{ref1, ref2}
• fertility following HSCT for malignant disease :
• most of the patients given TBI conditioning experience gonadal failure. Recovery of gonadal function occurs in 10% to 14% of the women, and the incidence of pregnancy is < 3%^{ref1, ref2, ref3}. In men, recovery of gonadal function has been reported in < 20% of patients, and use of increasing doses of TBI may be associated with considerably lower recovery^{ref1, ref2}. Parenting a child following administration of TBI is a rare event in men. We have identified 27 men who fathered children naturally following TBI^ref, and 5 such patients have been reported by the Seattle group^ref
• the use of busulphan and cyclophosphamide (BuCy) is also associated with a high incidence of gonadal failure in women, and there have been no pregnancies reported using BuCy for patients with leukemia^{ref1, ref2}. In men, this conditioning appears to be associated with return of gonadal function in approximately 17% of cases, which is similar to the recovery after TBI conditioning. Few patients have subsequently fathered children naturally; 20 patients were identified by the EBMT LEWP^ref and 2 patients were reported by the Seattle group^ref.
• melphalan alone or associated with VP16 or cyclophosphamide in HSCT conditioning is compatible with return of fertility in up to 50% of cases^{ref1, ref2}. Fewer studies have evaluated gonadal function following autologous HSCT, and most of these studies have involved small numbers of patients.
• The use of BEAM conditioning for autografts may be associated with a high incidence of gonadal recovery in women^ref, but in men azoospermia is almost always the rule^{ref1, ref2}. The EBMT LEWP has retrospectively identified 7 male patients who have fathered children naturally following BEAM, however^ref
• Pretransplantation counseling and treatment options : ideally, the pretransplantation counseling process should include data on the chance of gonadal failure and should assess the relevance of this situation to the patient. If the patient is of reproductive age and wishes to parent a child following SCT, it may be possible to alter the choice of conditioning protocol without compromising other factors such as survival. Clearly, this is not always an option, and discussion of management options for gonadal failure is thus essential. This should include discussion of assisted conception techniques and in women management of a premature menopause. In women with no residual ovarian function following HSCT, implantation of embryos cryopreserved prior to SCT is currently the only option for parenting a woman's own genetic child. This option, however, requires that prior to HSCT the underlying disease can tolerate a minimum 4- to 6-week delay in treatment for controlled stimulation of the ovary and egg collection. It also requires that the patient have a committed partner to provide sperm. In situations in which treatment cannot be delayed or there is no committed partner, consideration can be given to freezing ovarian tissue prior to SCT. Centers in some countries will provide this service for young women, but the patients need to be aware that currently this is a research rather than clinical tool and that to date there have been no successful pregnancies in human subjects using this methodology. Although the incidence of assisted conception increases annually, there are few published reports of pregnancy outcome following assisted conception in SC transplant recipients^{ref1, ref2, ref3}. In the pregnancy outcome study published by the EBMT LEWP, a high incidence of relapse was noted in female patients with chronic myelocytic leukemia (CML) who had conceived using assisted techniques^ref. On this basis we recommended that women with CML who wish to have assisted conception wait for 2 years post-HSCT and are demonstrated to be bcr-abl negative before implantation. Sperm cryopreserved prior to HSCT may be used post-HSCT for artificial insemination, in vitro fertilization (IVF), and embryo transfer or for in vitro injection into the cytoplasm of the oocyte (ICSI). Semen collection should ideally occur at diagnosis before chemotherapy has been given. Concurrent administration of chemotherapy is not an absolute contraindication to storage, but the patient must be informed of potential risks. Posttransplantation management should routinely include symptomatic and biochemical monitoring of gonadal function. Although the patient should be prepared for infertility, a possible need for contraception soon after SCT must also be emphasized, particularly in women who resume menstruation or patients who do not wish to become parents. Patients have conceived within 6 months of HSCT, and unexpected pregnancy may result in requests for termination. Distressing vasomotor symptoms may commence acutely post-HSCT, but this situation may be prevented by initiating SHRT. Alternatively, SHRT can be initiated at the onset of symptoms or when gonadotrophin levels indicate ovarian failure. SHRT does not suppress ovulation and will not, therefore, prevent recovery of gonadal function or pregnancy. It is, therefore, important to intermittently withdraw HRT and assess gonadotrophin levels. If there are signs of gonadal recovery, patients may benefit from contraceptive advice. Alternatively, because recovery in women is likely to be followed by a premature menopause, this period may be perceived by the patients as a window of opportunity to conceive naturally. Only regular follow-up will identify this point in time.
• quality of life (QoL) has become an essential outcome criterion for treatment strategies^ref. QoL assessment can identify rehabilitation needs and can facilitate the initiation and evaluation of rehabilitation programs. When HSCT is an alternative competing therapeutic option for a disease, eg, chronic myelogenous leukemia or acute myeloid leukemia, QoL may be integrated in the pre-HSCT counseling, in an often-complex clinical decision-making process. Finally, psychosocial and QoL indices may have a predictive value for survival in SCT^ref. Despite a clear definition of health by the WHO in 1948, as a " state of complete physical, mental and social well being, and not merely the absence of disease," QoL remains difficult to measure. It can be considered to be a multidimensional, time-dependent concept, which is both individual and subjective. Furthermore, there is poor correlation between symptoms and signs and overall QoL. Self-evaluation by the patient rather than reporting by observers is thus of central importance when assessing QoL. 2 main approaches can be used for self-evaluation of QoL: (1) psychometric tests (questionnaires) and (2) utility/preferences measures. These approaches should include items concerning physical functioning, disease/treatment-related symptoms, psychological and social functioning, sexual functioning and body image, satisfaction with health care and the doctor-patient relationship, and, more recently, spiritual concerns. Psychometric measures can be generic, disease specific, and site or therapeutic specific. Many instruments have been developed, for example the Sickness Impact Profile (SIP)^ref or SF-36^ref for generic assessment and the European Organization for Research and Treatment of Cancer Core Quality of Life (EORTC QLQ-C30)^ref or FACT^ref (Functional Assessment of Cancer Therapy Scale) for cancer patients. For HSCT patients, a specific questionnaire has been validated, the FACT-BMT^ref. These instruments are patterned around a similar concept: a core questionnaire consisting of several subunits for specific life domains, in combination with disease- or treatment-specific modules. The psychometric measures provide a descriptive profile as to how each patient is feeling, and it can be used to describe groups of patients for comparative purposes in clinical trials. However, for decision making, it may be desirable to formally compare the therapeutic benefit versus the changes in QoL for a given clinical management strategy. For example, if the more efficacious therapy is associated with poorer QoL, is it worthwhile? Various methods are available for determining patients' preferences. The utility approach, combining survival and QoL (like quality adjusted time without symptoms or toxicity, Q-TWiST) enables a comparison of different policies of management when both survival and QoL vary simultaneously by combining QoL and survival into a single summary score. These tools have been mainly used in patients with cancer, but to date there has been only one published study^ref comparing patients who underwent SCT with patients treated by chemotherapy alone. Overall, studies^{ref1, ref2} have reported good levels of function and well-being in long-term survivors of HSCT. Despite this level of functioning, there are increased reports of fatigue, lack of energy, sleep problems, and sexual dissatisfaction. Risk factors identified for impaired QoL post-SCT include older age and advanced disease at HSCT, lower level of education, and the development of cGVHD. Fatigue and sleeping disorders have been reported in up to 65% patients post-HSCT^{ref1, ref2, ref3}, and these changes may persist for several years following HSCT^{ref1, ref2, ref3, ref4}. Problems with sexuality and intimacy after HSCT are reported in approximately 25% of patients. Changes in sexual experience and lower level of sexual satisfaction have been reported in addition to disorders of sexual functioning^{ref1, ref2, ref3, ref4, ref5}. Some data suggest that women have higher prevalence rates than men^{ref1, ref2}. Neuropsychological deficits have also been investigated in patients undergoing allogeneic HSCT^{ref1, ref2, ref3}. Problems with memory can be found in nearly 20% of patients within the first year after HSCT; this problem is unrelated to the patient's emotional status or intake of psychoactive drugs. However, neuropsychological examination prior to HSCT also reveals cognitive impairments in 10% of patients^{ref1, ref2, ref3}. Patients with a history of central nervous system (CNS) disease seemed to be at higher risk of cognitive deficits, especially children treated with cranial radiation or the combination of cytotoxic drugs and irradiation^{ref1, ref2, ref3, ref4, ref5, ref6,ref7, ref8}. Lower IQ levels have been described in children 1 year following SCT, but this decrease remained stable at the 3-year follow-up^ref. Changes in IQ were minimal, however, in children transplanted at a very young age (< 3 years)^ref
• late occurrence of diabetes mellitus, systemic arterial hypertension, and cardiovascular (CV) disease in 1089 hematopoietic cell transplant (HCT) survivors who underwent HCT between 1974-1998, survived 2 years and were not currently taking immunosuppressant agents, and compared them to 383 sibling controls. All subjects completed a 255-item health questionnaire. The mean age at survey completion was 39.8 yrs for survivors and 38.6 yrs for siblings, mean follow-up was 8.3 years.
• adjusting for age, gender, race, and body mass index (BMI), survivors of allogeneic HCT were 3.65 times (95% CI: 1.82-7.32) more likely to report diabetes mellitus than siblings and 2.06 times (95% CI 1.39-3.04) more likely to report systemic arterial hypertension compared to siblings, but did not report other CV outcomes with any greater frequency. Allogeneic HCT survivors were also more likely to develop hypertension (OR= 2.31, 95% CI: 1.45-3.04) than autologous recipients. TBI exposure was associated with an increased risk of diabetes (OR=3.42, 95% CI: 1.55-7.52). Thus, HCT survivors have a higher age- and BMI-adjusted risk of diabetes and hypertension, potentially leading to a higher than expected risk of CV events with age^ref
Prevention : evidence-based rating system used in the HSCT guidelines.
• strength of the recommendation : category and definition
• A : both strong evidence for efficacy and substantial clinical benefit support recommendation for use. Should always be offered.
• B : moderate evidence for efficacy—or strong evidence for efficacy, but only limited clinical benefit—supports recommendation for use. Should generally be offered.
• C : evidence for efficacy is insufficient to support a recommendation for or against use, or evidence for efficacy might not outweigh adverse consequences, (e.g., drug toxicity, drug interactions), or cost of the chemoprophylaxis or alternative approaches. Optional.
• D : moderate evidence for lack of efficacy or for adverse outcome supports a recommendation against use. Should generally not be offered.
• E : good evidence for lack of efficacy or for adverse outcome supports arecommendation against use. Should never be offered.
• quality of evidence supporting the recommendation : category and definition
• I : evidence from at least one properly randomized, controlled trial.
• II : evidence from at least one well-designed clinical trial without randomization, from cohort or case-controlled analytic studies (preferably from more than one center), or from multiple time-series or dramatic results from uncontrolled experiments.
• III : evidence from opinions of respected authorities based on clinical experience, descriptive
• AI recommendations :
• 1. All persons should wash their hands before entering and after leaving the rooms of HSCT recipients and candidates undergoing conditioning therapy, or before any direct contact with patients regardless of whether they were soiled from the patient, environment or objects.
• 2. All health care workers with diseases transmissible by air, droplet, and direct contact (e.g. varicella zoster virus, infectious gastroenteritis, herpes simplex lesions of lips or fingers and upper respiratory tract infections) should be restricted from patient contact and temporarily reassigned to other duties.
• 3. When a case of laboratory confirmed legionellosis is identified in a person who was in the impatient HSCT center during all or part of the 2-10 days before illness onset, or if two or more cases of laboratory-confirmed Legionnaire's disease occur among patients who had visited an outpatient HSCT center, hospital personnel in consultation with the hospital infection control team should perform a thorough epidemiologic and environmental investigation or determine the likely environmental source(s) of Legionella species (e.g. showers, tap water faucets, cooling towers and hot water tanks.
• 4. To control VRE exposure, strict adherence to standard infection control measures is necessary, as outlined in the text.
• 5. All HCWs who anticipate contact with a Clostridium difficile-infected patient or the patient's environment or possessions should put on gloves before entering the patient's room and before handling the patient's secretions and excretions.
• 6. HSCT candidates with a recently positive tuberculin skin test or a history of a positive skin test and no prior preventive therapy should be administered a chest radiograph and evaluated for active TB.
•  AII recommendations :
• 1. HCST centers should prevent birds from gaining access to hospital air-intake ducts.
• 2. Appropriate gloves should be used by all persons when handling potentially contaminated biological materials.
• 3. Work exclusion policies should be designed to encourage HCWs to report their illnesses or exposures.
• 4. Visitors who might have communicable infectious diseases (e.g. upper respiratory tract infections, flu-like illnesses, recent exposure to communicable diseases, an active shingles rash whether covered or not, a VZV-like rash within 6 weeks of receiving a live attenuated varicella vaccine, or a history of receiving an oral polio vaccine within the previous 3-6 weeks) should not be allowed in the HSCT center or have direct contact with HSCT recipients or candidates undergoing conditioning therapy.
• 5. If Legionella species are detected in the water supplying an HSCT center, the water supply should be decontaminated and eradication of Legionella should be verified.
• 6. HSCT centers should follow basic infection control practices for control of MRSA infection and colonization, including hand washing between patients and use of barrier precautions, including wearing gloves whenever entering the MRSA-infected or MRSA-colonized patient's room.
• 7. HSCT personnel should institute prudent use of all antibiotics, particularly vancomycin, to prevent the emergence of staphylococci with reduced susceptibility to vancomycin.
• 8. Use of intravenous vancomycin is associated with the emergence of VRE; vancomycin and all other antibiotics, particularly antianaerobic agents, should be used judiciously.
• 9. All patients with Clostridium difficile disease should be placed under contact precautions for the duration of the illness.
• 10. When caring for an HCST recipient or candidate undergoing conditioning therapy with upper or lower respiratory tract infection, HCWs and visitors should change gloves and wash hands in circumstances outlined in the text.
• 11. Visitors and HCWs with infectious conjunctivitis should be restricted from direct patient contact until the drainage resolves and the ophthalmology consultant concurs that the infection and inflammation have resolved to avoid possible transmission of adenovirus to HSCT recipients.
• 12. For patients with suspected or proven pulmonary or laryngeal TB, HSCT personnel should follow guidelines regarding the control of TB in health care facilities.
• EII recommendations : Bacillus of Calmette and Guerin (BCG) vaccination is contraindicated among HSCT candidates and recipients because of its potential to cause disseminated or fatal disease among immunocompromised persons.
Practices :
• room ventilation : staff in HSCT centers should prevent access of birds to hospital air intake ducts (AII), as studies have linked such exposures to the subsequent development of invasive pulmonary respiratory tract infections in HSCT recipients^{ref1, ref2}. Although well designed clinical trials have not validated this approach, it is recommended that all allogeneic recipients be placed in rooms with > 12 air exchanges per hour and point-of-use HEPA filters that are capable of removing particles 0.3 mm in diameter (Streifel AJ. Chapter 80. Design and maintenance of hospital ventilation systems and the prevention of airborne nosocomial infections. In Mayhall CG, ed. Hospital Epidemiology and Infection Control. Second edition. Philadelphia PA: Lippincott Williams & Wilkins; 1999:1211–1221) (AIII). The utility of HEPA filtration has not been established in autologous transplant recipients, but the guidelines suggest consideration of its use in high risk autologous recipients with prolonged neutropenia who are at risk of invasive aspergillosis (CIII). Although data from the early 1980s suggested the benefit of laminar air flow (LAF) rooms in allogeneic transplant recipients with aplastic anemia who received grafts from HLA-identical siblings^ref, more recent studies suggest no survival benefit from routine LAF use in all HSCT recipients^ref. Survival of patients with aplastic anemia patients undergoing HSCT has improved in recent years, and no follow-up studies have been performed to evaluate the utility of LAF use in this population. The use of LAF rooms, even if available, is therefore deemed optional (CII).
• construction, renovation, and building cleaning : this portion of the guidelines contains no AI, AII, or E level recommendations. There are five AIII level guidelines. To minimize the risk of nosocomial aspergillosis, individuals responsible for HSCT construction or renovation are advised to consult published recommendations regarding environmental controls during construction^ref. In addition, when construction or renovation is anticipated, staff in HSCT centers should intensify aspergillosis-control measures (AIII), as outlined in a series of BIII recommendations. HSCT recipients, health care workers and visitors should avoid construction and renovation areas to the extent possible (AIII). N95 respirators have been proposed for HSCT recipients during transport near construction or renovation areas (CIII), but fit-testing and training are necessary for maximal benefit; standard surgical masks afford little protection against inhalation of fungal spores and are not recommended (DIII). Newly constructed areas should be cleaned before patients are permitted to enter them (AIII)^ref as outlined in several BIII guidelines. HSCT patients should not be exposed to activities such as vacuuming of floors or carpets, which are apt to result in the aerosolization of Aspergillus spores (AIII)^ref
• isolation and barrier precautions : this section of the guidelines contains no AI, AII, or E recommendations. Members of HSCT centers are advised to follow published guidelines for hospital isolation practices, including guidelines for the prevention of nosocomial infections (AIII)^{ref1, ref2, ref3}. The utility of specific isolation and barrier precautions in the prevention of nosocomial infections in HSCT recipients has not been studied, however, and these recommendations are extrapolated from other settings.
• hand hygiene : the importance of good handwashing practices in the care of HSCT patients cannot be over-emphasized. All persons, especially HCWs, are advised to wash hands before entering and after leaving the rooms of HSCT patients and of those receiving conditioning therapy. In addition, handwashing should be performed before and after direct contact with patients irrespective of whether soiling from the patient or from objects in the environment occurs (AI)^ref. Handwashing produces a reduction in the carriage of potential pathogens on the hands and has been shown to reduce morbidity and mortality from nosocomial infections in numerous studies^ref. Handwashing may be performed with an antimicrobial soap and water, or with hygienic hand rubs (AIII). Gloves should be worn by all persons when handling potentially infected biologic material (AII). Gloves should be changed between patients or prior to touching a clean area when soiled (AIII). Items worn on the hands by HCWs such as rings, artificial nails, and adhesive bandages should be avoided, as they may serve as a nidus for pathogenic microorganisms (BII).
• equipment : this section contains no AI-II or E level recommendations. HSCT centers are advised to adhere to established guidelines for the sterilization, disinfection and maintenance of devices and equipment utilizing only EPA-registered agents (AIII)^{ref1, ref2, ref3, ref4}.
• health care workers : all HCWs with diseases transmissible by direct contact, droplet or airborne transmission should be restricted from direct patient contact and temporarily reassigned to other tasks (AI) and should follow published guidelines regarding the duration of work restriction (BIII)^ref. These include individuals with illnesses such as varicella zoster virus (VZV) infection, infectious gastroenteritis, herpes simplex infections involving the fingers or lips, and upper respiratory tract infections. Staff at HSCT centers should formulate work exclusion policies to ensure that HCWs report illnesses or potential exposures to communicable pathogens (AII). HSCT institutions should have a written policy regarding HCW immunizations that meets guidelines recommended by the CDC, the Advisory Committee on Immunization Practices, and the Healthcare Infection Control Practices Advisory Committee (BIII)^ref.
• visitors to transplant centers : visitors with potentially communicable illnesses should be barred from the HSCT center and should not have direct contact with HSCT recipients or with candidates receiving preparative therapy (AII). Such illnesses include upper respiratory tract infection, flu-like illnesses, varicella zoster infection, a VZV-like rash developing within 6 weeks of a live attenuated VZV vaccine, or a history of receiving oral polio vaccine within the preceding 6 weeks. All visitors to HSCT centers should be capable of practicing recommended handwashing guidelines irrespective of age (AIII). HSCT centers should have written policies regarding screening of visitors for potentially communicable diseases (BII).
• patient skin and oral care : this section of the guidelines contains no AI, AII or E guidelines. 6 AIII level recommendations are offered. Education regarding dental hygiene is recommended for HSCT recipients and their caregivers concerning the importance of good dental hygiene following transplantation, so as to minimize the likelihood of oral and dental infection (AIII). In addition, all HSCT candidates should be evaluated for dental disease (AIII) and likely sources of infection should be eliminated (AIII) (Schubert MM, Peterson DE, Lloid ME. Oral complications. In : Thomas ED, Blume KG, Forman SJ, eds. Hematopoietic Stem Cell Transplantation, 2nd edition. Oxford, England: Blackwell Sciences, Inc.; 1999:751–763) 10 to 14 days should elapse between completion of dental therapy and initiation of a preparative regimen (AIII). Oral hygiene should be maintained in HSCT recipients or candidates receiving conditioning therapy who have oral mucositis through the use of 4-6 oral rinses per day with normal saline, sterile water or sodium bicarbonate solutions^ref. Following urination or defecation, female HSCT recipients should wipe the perineum from front to back to prevent urethral contamination and subsequent urinary tract infection (AIII).
• prevention of intravascular catheter-related bacterial infections : this section of the guidelines contains no AI-II or E level recommendations. HSCT centers should follow published guidelines for preventing intravascular device-related infections (AIII)^ref
• control of specific nosocomial and community-acquired pathogens :
• Legionella species : legionellosis should be considered in the differential diagnosis of pneumonia in HSCT recipients (AIII). The incubation period for Legionnaires' disease (LD) is 2-10 days. Therefore, patients with laboratory-confirmed LD who are continuously hospitalized for  10 days prior to symptom onset have definite nosocomial legionellosis. Those with proven LD who have been in hospital 2–9 days prior to illness onset have possible nosocomial acquisition. If LD is confirmed in a patient hospitalized on the HSCT unit for all or part of the 2-10 days before illness onset, or if 2 or more cases of proven LD occur in patients visiting an outpatient HSCT clinic, a thorough epidemiologic and environmental investigation should be conducted in conjunction with the infection control team to determine the environmental source(s) of the outbreak (AI)^ref.20 This should include assessment of cooling towers, hot water tanks, showers and tap water faucets. Once identified, the environmental source should be removed or decontaminated (AIII). If Legionella species are found in the HSCT center water supply, it should be decontaminated (AII) and patients should not take showers (DIII) but should receive sponge baths utilizing Legionella-free water (BIII). In addition, water from faucets contaminated with Legionella species should not be used in the HSCT unit so as to prevent the creation of infectious aerosols (CIII). Only sterile water should be used for rinsing nebulation devices and other respiratory care equipment after cleaning, and for filling reservoirs of these devices (BII)^ref
• methicillin-resistant staphylococci : personnel at HSCT centers should adhere to standard infection control practices in order to optimize the control of methicillin-resistant Staphylococcus aureus (MRSA) (AII)^ref. These practices include handwashing between patient encounters, use of appropriate barrier precautions, and wearing gloves when entering the room of an MRSA-infected or colonized patient. Intravascular devices infected or colonized with MRSA should be removed (AIII).
• Staphylococci with reduced susceptibility to vancomycin : at present, staphylococci with reduced susceptibility to vancomycin are fortunately rare^ref. HSCT centers should have laboratory facilities capable of identifying and performing antimicrobial susceptibility testing on all staphylococci (AIII). In addition, routine surveillance should be performed for staphylococci with reduced susceptibility to vancomycin (AIII), defined as a vancomycin minimal inhibitory concentration (MIC)  4 µg/mL for Staphylococcus aureus and  8 µg/mL for coagulase negative staphylococci. If such isolates are identified and confirmed, the laboratory should contact hospital infection control personnel, the HSCT center, the patient's physician, the local or state health department, and the CDC's Hospital Infection Program so that a prompt epidemiologic investigation may be performed (AIII). Hospitalized patients who are infected or colonized with these organisms should be cohorted (AIII). Prudent antibiotic use by HSCT centers, especially of vancomycin, is essential to prevent the emergence of resistant staphylococci and is recommended (AII). In addition, intravascular devices colonized or infected with staphylococci with reduced susceptibility to vancomycin should be removed (AIII).
• Vancomycin resistant enterococci (VRE) : the emergence of VRE is associated with the use of vancomycin and antianaerobic agents, and these drugs should be used judiciously (AII)^ref. To limit exposure of HSCT recipients and patients receiving conditioning therapy to VRE, adherence to standard infection control measures is recommended as summarized in the following guidelines (AI). These measures include compliance with handwashing guidelines outlined above and the cohorting of VRE-colonized or infected patients. In addition, patient rooms and equipment should be disinfected with an FDA- or EPA-registered disinfectant, including surfaces of the hospital environment such as walls, floors, bed frames, doors and bathrooms. In pediatric areas, a nontoxic disinfectant should be used (BIII). Patients with VRE should be placed under contact precautions until cultures are negative and antibiotics are discontinued (BIII). Gloves should be worn in the room of VRE-infected or colonized patients and discarded prior to leaving the room
• Clostridium difficile : patients with Clostridium difficile disease should be placed under contact precautions for the duration of their illness^ref. Gloves should be worn by HCWs who anticipate contact with HSCT patients who are colonized or infected with C. difficile or with their possessions or environment; gloves should be put on before entering the patient's room (AI). Standard published guidelines for the prevention and control of C. difficile infection should be followed, including the judicious use of antibiotics (AIII)^ref. The prophylactic use of lyophilized Saccharomyces boulardii to prevent diarrhea among HSCT recipients receiving antibiotics is contraindicated, as it has no salutary effect and has been associated with the development of S. boulardii fungemia (DII)^ref
• community-acquired respiratory virus infections : community-acquired respiratory viruses (CRVs) may produce significant morbidity and mortality in HSCT recipients^ref, and may produce nosocomial infection if introduced into the HSCT center. Measures to prevent the introduction and spread of CRVs on the HSCT unit are therefore recommended. When caring for a patient at the HSCT center with upper or lower respiratory tract infection, HCWs and visitors should change gloves and wash hands after contact with a patient, after handling respiratory secretions or objects contaminated with respiratory secretions, and between contact with a contaminated body site and the respiratory tract of or respiratory device used on the same patient (AII). This practice is important because most CRVs are transmitted by contact, especially from hand to nose and eye Appropriate infection control measures to prevent nosocomial pneumonia among HSCT patients should be followed, especially during outbreaks of community or nosocomial CRVs (AIII)^ref. It is crucial to identify and place under contact precautions HSCT recipients with respiratory syncytial virus (RSV) infection so as to prevent nosocomial transmission (AIII). To minimize the risk of CRV spread on the HSCT unit, HCWs and visitors with symptoms of upper respiratory infection should be restricted from contact with HSCT patients and individuals undergoing conditioning therapy (AIII). Because of the risk of adenovirus transmission to HSCT recipients, HCWs and visitors with infectious conjunctivitis should be prohibited from contact with patients until ocular drainage resolves and an ophthalmologist agrees that inflammation and infection have subsided (AII). Finally, when suctioning patients with upper or lower respiratory tract infections, HCWs should wear surgical masks, eye protection and gowns to avoid splashes with the patient's secretions. In addition, protective clothing should be donned when entering the patient's room, removed and discarded prior to leaving, and always changed between patient rooms (AIII)
• tuberculosis : because HSCT recipients may reactivate tuberculosis in the face of immunosuppression, candidates for HSCT should be screened by history and chart review for a history of prior exposure to tuberculosis (AIII). A tuberculin skin test utilizing 5 tuberculin units of either the Tubersol or Aplisol formulations may be administered by the Mantoux method, but may be unreliable because of the patient's baseline immunosuppression. For immunocompromised individuals, a positive skin test is defined as  5 mm of induration^ref. Individuals with a recently positive skin test or a previously positive skin test and no prior preventive therapy should have a chest radiograph and an evaluation for active tuberculosis (AI). Staff at HSCT centers should follow published guidelines concerning the control of tuberculosis in health care facilities, which include the institution of airborne precautions and the use of negative pressure rooms for patients with suspected or confirmed laryngeal or pulmonary tuberculosis (AII)^ref (CDC. Guidelines for preventing the transmission of Mycobacterium tuberculosis in health care facilities, 1994. MMWR. 1994;43 (No. RR-13):1–132). With such patients, HCWs should wear N95 respirators, even in isolation rooms, when exposed to potentially infected patients (AIII). For maximal efficacy of N95 respirators, HCWs should be fit-tested and trained in their use (AIII). Immunization with the Bacillus of Calmette and Guerin (BCG) vaccine is contraindicated in HSCT candidates and recipients because of its potential to produce disseminated and fatal infection in immunocompromised hosts (EII)^ref
• infection control surveillance : this section of the guidelines contains no A or E level recommendations. HSCT centers are advised to refrain from performing routine bacterial or fungal surveillance cultures of asymptomatic HSCT recipients (DII)^ref or, in the absence of clusters of infections, of the environment or of equipment or devices used for pulmonary function testing, delivery of inhalation anesthesia, or respiratory care (DIII). In addition, in the absence of an outbreak of nosocomial fungal infection, personnel at HSCT centers should not perform routine fungal surveillance cultures of devices or dust in patient rooms (DIII). Several studies of relevance to infection control practices on HSCT units have been published in the past year. Weinstock et al reported an outbreak of nosocomial influenza A in January 1998 occurring on a 30 bed HSCT unit, and described enhanced interventions implemented to terminate the outbreak and to prevent nosocomial influenza the following season^ref. These interventions included strict isolation of patients with influenza, reverse isolation of all other patients on the HSCT unit during the outbreak, more rapid laboratory turnaround of diagnostic tests for influenza, restriction of visitors, cohorting of HCWs assigned to the HSCT unit, universal rimantadine prophylaxis of all patients and HCWs on the unit during the outbreak, and an enhanced influenza vaccine campaign for HCWs the following season.
• Mayfield et al evaluated the role of environmental disinfectants in reducing the incidence of nosocomial Clostridium difficile-associated diarrhea (CDAD)^ref. In a before and after intervention study, the incidence rate of CDAD among HSCT patients fell from 8.6 to 3.3 cases per 1000 patient days when the environmental disinfectant was switched from quaternary ammonium to 1:10 hypochlorite solutions in the rooms of patients with CDAD. When quaternary ammonium solution was re-instituted, the CDAD rate among HSCT recipients again increased to 8.1 cases per 1000 patient days.
• Laura et al performed a randomized multicenter study in HSCT patients comparing two different time interval protocols for central venous catheter (CVC) dressing changes^ref. 399 patients with tunneled (230, Group A) or non-tunneled (169, Group B) CVCs were randomized to receive dressing changes with transparent impermeable polyurethane dressings. Patients in Group A had dressings changed at 5 versus 10 days, compared with 2 versus 5 days in Group B. There was no significant increase in local infections in those individuals randomized to longer dressing change intervals, and longer intervals were associated with lower costs and less patient discomfort.
• The relative paucity of level AI, AII, BI, and E level recommendations in the infection control guidelines is striking. Among the AI and AII recommendations, most are more global and generic recommendations, derived from and applicable to other clinical settings. More evidence-based studies are necessary to define optimal infection control practices in these high-risk patients undergoing hematopoietic cell transplantation.
• prevention of viral infections : viral infections are common and potentially fatal complications after HSCT. A key aspect of prevention of viral infections is risk assessment based on viral and host factors as well as the time patterns of infections after transplant. This time pattern has changed over the past two decades due to the introduction of standard viral prevention strategies. Several important changes in transplantation techniques have occurred over the past decade, such as use of different sources of stem cells (i.e. peripheral blood or cord blood instead of marrow), graft manipulation (e.g. CD34 selection or T cell depletion), and the use of novel and less toxic conditioning regimens (i.e. non-myeloablative or regimens with reduced toxicity). All these factors may affect the posttransplant risk and thus the prevention regimens. For some of the more recent changes, little information is available on their impact of infectious risk following transplantation. Ultimately, the choice of the prevention strategy is determined by the host immune status as well as the efficacy, toxicity, feasibility, cost and ratio of treated versus untreated individuals (i.e. ‘number needed to treat’) of the intervention. All evidence ratings in the paragraphs "preventing exposure" and "preventing disease and disease recurrence" are identical with those in the published guidelines^ref. Ratings in the paragraph "update since publication of the guidelines" are assigned by the author of this chapter and have not been authorized by CDC or any of the underwriting organizations. If not otherwise noted, recommendations apply for both adult and pediatric HSCT candidates and recipients (although dosing may be different in children). For specific dosing information, the reader is referred to the original guidelines^ref. This chapter only addresses infection prophylaxis in recipients of myeloablative conditioning regimens. Infection prophylaxis in non-myeloablative conditioning regimens is subject of ongoing studies.
• HHV-5 / CMV
• preventing exposure : determination of cytomegalovirus (CMV) IgG serostatus is recommended in all transplant candidates and their prospective donors (AIII). Seronegative HSCT recipients should receive seronegative or leukocyte-depleted blood products (AI). Seronegative autograft recipients are susceptible for primary CMV infection via blood products. Thus, seronegative or leukocyte-depleted blood products may be used to prevent transfusion-transmitted CMV infection (CIII). Seronegative candidates and HSCT recipients who are sexually active and not in long-term monogamous relationships should use latex condoms during sexual contacts (AII). Handling or changing diapers or wiping oral secretions from toddlers also represents a potential risk for CMV transmission to seronegative individuals and should therefore be avoided (AII). Summary of major recommendations for prevention of cytomegalovirus (CMV).
• preventing disease or disease recurrence : 1 of 2 strategies should be used to prevent CMV disease in allogeneic HSCT recipients between engraftment and day 100 after transplant (AI). The first strategy is to give ganciclovir prophylaxis from engraftment until day 100^ref. An alternative strategy is to use virologic surveillance and administer ganciclovir for early evidence of subclinical CMV infection^{ref1, ref2}. Tests used for this purpose include CMV blood cultures, the pp65 antigenemia assay, and DNA detection methods^ref. Due to lower sensitivity, blood culture methods are generally not recommended. Limited data exist on other detection methods such as detection of pp67 mRNA and DNA by the hybrid capture method^ref and no comparative study has been reported for preemptive therapy using these assays. Centers without access to highly sensitive CMV detection methods should use prophylaxis (BII). In seronegative recipients with a positive donor, the incidence of CMV infection is only 15-20% when seronegative or leukocyte-reduced blood products are given. Thus, preemptive therapy with ganciclovir rather than prophylaxis is preferred in these patients (BII). Preemptive therapy is started on the basis of any positive antigenemia assay or two positive DNA PCR test results (BII). Ganciclovir is continued for a minimum of 3 weeks with one week of induction dosing or until day 100, whichever is longer (AI). Other regimens use 2 weeks of induction dosing and maintenance until PCR negativity^ref(BIII). Ganciclovir recipients should have absolute neutrophil counts (ANC) checked at least twice a week during the first 100 days after transplant (BIII). Management of ganciclovir-related neutropenia (ANC < 1000/mm³) includes discontinuations of ganciclovir (CIII) and/or use of G-CSF (CIII). Neutropenic patients with ongoing CMV detection in blood should be treated with foscarnet (CIII). Late CMV disease is an emerging problem in HSCT recipients given ganciclovir prophylaxis or preemptive therapy^{ref1, ref2, ref3}. Risk factors for late CMV disease include chronic graft-versus-host disease (GVHD), low CD4 counts (< 50/mm³), and CMV infection before day 100. If there is continued evidence of CMV in blood by day 100, ganciclovir should be continued until CMV is no longer detectable (AI). CMV surveillance should be continued until one year after transplant in these high-risk HSCT recipients. Ganciclovir should be administered for antigenemia, viremia, or PCR test positivity (e.g. 3 weeks or until CMV is no longer detectable) (BIII).
• High-dose acyclovir given until day 30 or until engraftment has limited efficacy in preventing CMV disease, especially when antigenemia-guided preemptive therapy is given^ref; however, survival was improved in one randomized trial with extended use of high-dose acyclovir^ref. High-dose acyclovir given until day 30 does not seem to be effective in preventing CMV infection and disease in seropositive autograft recipients and is therefore not recommended (DII). Intravenous immunoglobulin is not recommended for prevention of CMV disease (DI).
• Autologous transplant recipients generally have a lower risk of CMV disease than allograft recipients. However, among recipients of CD34-selected autografts the risk may be similar to that seen after allografting^ref. Therefore, CMV prevention strategies in CD34-selected autologous transplant recipients should be similar to those applied in allogeneic transplant recipients (BII). In recipients of unmodified autologous transplants, CMV surveillance and preemptive therapy is recommended in patients with hematologic malignancies or in those who have recently received fludarabine (CIII). Quantitative rather than qualitative CMV detection methods are preferred in autograft recipients (BII). In seropositive nonmodified autograft recipients a treatment course of 3 weeks is recommended if antigenemia levels of > 5 cells per slide are detected (BII).
• New information indicates that the in vivo replication time of CMV is short and influenced by the host immune status^ref. This information can be used to optimize the duration of induction dosing. For example, viral load may continue to rise despite start of ganciclovir in transplant recipients, especially in those receiving high-dose corticosteroids or anti-thymocyte globulin (ATG)^{ref1, ref2}. In such a situation, induction dosing should be continued until a decline of viral load is documented by quantitative detection methods such as DNA PCR or the pp65 antigenemia assay. Conversely, in HSCT recipients with immediate decline of viral load, shorter induction courses may be possible. Increases of CMV loads early during preemptive therapy (first 4 weeks) are only very rarely due to antiviral resistance in ganciclovir-naïve transplant recipients^ref. However, genotypic ganciclovir resistance has been described early after transplant in T cell depleted pediatric HSCT recipients^ref. More information is now available on the optimal duration of ganciclovir preemptive therapy. In an uncontrolled series of 84 patients there was no early rebound CMV disease after discontinuation of ganciclovir following a short course of ganciclovir with 2 weeks of induction and subsequent maintenance treatment until polymerase chain reaction (PCR)-negativity^ref. Thus, discontinuation of ganciclovir based on negative PCR testing is an alternative to treatment until day 100. Continuation of surveillance and preemptive therapy as well as long-term secondary prophylaxis have been reported in uncontrolled trials^{ref1, ref2}. A randomized trial is ongoing to assess the relative benefits of these two strategies for prevention of late CMV disease. Foscarnet has been shown to be as effective as ganciclovir for preemptive therapy in a prospective randomized study. Cidofovir has been associated with moderate renal toxicity similar to that reported for foscarnet in a retrospective analysis of the clinical experience of European centers^ref; however, no recommendations about its use can be made at this time. Only limited data exist on oral ganciclovir in allogeneic HSCT recipients^ref (Spielberger R, Zaia J. Use or oral ganciclovir for the preemptive treatment of CMV following allogeneic HCT: safety and feasibility results. Blood. 2000;96:586a. (abstract)). To date, no data exist on valganciclovir in HSCT. High-dose valacyclovir (8 g per day) moderately reduced the incidence of CMV infection (and therefore the need for preemptive therapy) compared to high-dose acyclovir in a randomized placebo-controlled double-blind study and showed similar efficacy as ganciclovir when given prophylactically in another randomized trial. The incidence of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome was not increased compared to acyclovir (Ljungman P, Camara R, Milpied N, Volin L, Reilly L, Crisp A, Group VIBMTS. A randomized study of valacyclovir as prophylaxis against CMV infection and disease in BMT recipients. 25th Annual Meeting European Group for Blood and Marrow Transplantation. Hamburg, 1999). A CMV-specific monoclonal antibody (MSL-109) has not effective in preventing CMV infection^ref. The type of posttransplant immunosuppression seems to determine, and in some cases limit, the efficacy of preemptive treatment strategy. One non-randomized study showed a high CMV disease rate in cohort of allogeneic HSCT patients who received high-dose steroid prophylaxis, mycophenolate mofetil, and ATG with preemptive PCR-guided therapy based on 2 consecutive positive results two weeks apart and discontinuation based on negative PCR^ref. Thus, earlier intervention, more frequent monitoring or ganciclovir prophylaxis may be required in patients who are heavily immunosuppressed. Analysis of the impact of pretransplant CMV serostatus on survival indicates that seronegative recipients of a seropositive donor have a higher mortality than seronegative recipients of a seronegative donors (Nichols WG, Corey L, Gooley T, Davis C. The role of CMV serostatus on mortality after stem cell transplantation in the era of pre-emptive ganciclovir therapy. 8th International CMV Conference. Monterey, CA, 2001, p 64). Thus, a CMV seronegative donor should be used if possible.
• HHV-4 / EBV
• preventing exposure : serologic testing is generally not recommended; however, it may be useful in recipients of T cell depleted or pediatric HSCT. Known Epstein-Barr virus (EBV) seronegative transplant candidates should be advised of behavior that could decrease the likelihood of EBV transmission (AII), including safe hygiene practices (e.g. handwashing [BIII]), and should avoid contact with potentially infected respiratory secretions (AII).
• preventing disease and disease recurrence : data are emerging that donor-derived EBV-specific cytotoxic T lymphocytes (CTL) can be used effectively for prophylaxis in high-risk patients, i.e. T cell depleted transplant recipients. The strategy has potential but has not been rated by CDC. Posttransplant surveillance with quantitative PCR for EBV DNA and preemptive therapy with rituximab, an anti-CD20 monoclonal antibody, have been reported to be effective in reducing viral load in a small number of high-risk patients for EBV posttransplant lymphoproliferative disease^ref. However, additional studies are needed and no firm recommendations can be made at this time. Additional data have been presented on prophylactic use of donor derived EBV-specific T cells (Bollard CM, Rooney CM, Huls MH, et al. Long term follow-up of patients who received EBV specific CTLs for the prevention or treatment of EBV lymphoma. Blood2000;96 (Suppl):478a. (abstract 2057)). Although this strategy is complex it may be considered in high-risk patients.
• HHV-1 / HSV-1
• preventing exposure : determination of herpes simplex virus (HSV) IgG serostatus is recommended in all transplant candidates (AIII). Testing of donor serostatus is currently not routine in most centers since HSV transmission by marrow or stem cell products has not been documented. HSV seronegative recipients should be informed about the mode of transmission of HSV and be advised of behavior that will decrease the likelihood of exposure, e.g. avoiding potentially infectious secretions such as cervical secretions and saliva (AII). Summary of major recommendations for prevention of herpes simplex virus (HSV).
• preventing disease or and disease recurrence : acyclovir prophylaxis is recommended for all HSV seropositive allograft recipients (AI) (Table 5). Most centers administer acyclovir from the onset of conditioning until engraftment and resolution of mucositis (BIII). This may occur before day 30 after transplant, which is the time period that was used in the original randomized studies of acyclovir prophylaxis. Routine extension of HSV prophylaxis beyond day 30 is not recommended in the absence of randomized trials (DIII); however, patients with frequent recurrences might benefit from extended suppressive therapy (CIII). Although the original studies were performed with intravenous (IV) acyclovir, oral administration is often used in individuals who can take oral medication; however, adequate dosing seems important^ref. Patients receiving ganciclovir (CIII), foscarnet, or cidofovir for CMV prevention or treatment do not require concomitant acyclovir prophylaxis due to the in vitro activity of these compounds against HSV. Routine acyclovir prophylaxis in HSV seronegative recipients is not indicated (D III), even if the donor is seropositive. Valacyclovir has been used instead of oral acyclovir for prevention of HSV in HSCT (CIII). There are no data on the use of famciclovir for HSV prophylaxis in HSCT recipients; however, the drug has in vitro activity as well. Foscarnet should not be used for routine HSV prophylaxis due to substantial renal toxicity (DIII).
• HHV-3 / VZV
• preventing exposure : determination of VZV IgG serostatus is recommended in all transplant candidates (AIII). Testing of donor serostatus is currently not routinely conducted by most centers since VZV transmission by marrow or stem cell products has not been documented. VZV is highly contagious and vaccination of family members, household contacts, and prospective visitors who are known seronegative for VZV or have no history of VZV infection should be performed (AII). This is usually done at the time of first contact with the transplant center to allow for sufficient time to complete the vaccination (ideally > 4 weeks prior to start of conditioning) (BIII). Seronegative transplant candidates and recipients should be informed about VZV transmission and advised of strategies on how to avoid exposure (AII). Transplant recipients and candidates should also avoid vaccine recipients who experience a rash after vaccination (BIII). Such rash may occur between 5 and 35 day after vaccination (median 22 days)^ref.1 Summary of major recommendations for prevention of varicella-zoster virus infection.

All patients with VZV disease should be placed under airborne and contact isolation (AII). Isolation should be continued until all lesions have crusted. Airborne precautions should be instituted 10 days after an exposure of a susceptible transplant recipient to VZV and continued until 21 days after the last exposure or 28 days postexposure if the patient received varicella-zoster immunoglobulin (VZIG) (AI). Note that individuals infected with VZV can be contagious before the rash appears.
• preventing disease or and disease recurrence : VZIG should be administered to a seronegative recipient at risk for complications of VZV (i.e. < 24 months after transplant, or  24 months if on immunosuppressive therapy and/or having chronic GVHD) as soon as possible after exposure (< 96 hours) (AII) (Table 6). Seronegative recipients or candidates undergoing conditioning therapy should also receive VZIG if they are exposed to a VZV vaccinee having a varicella-like rash (BIII); these recommendations may be extended to seropositive recipients (CIII). If a second exposure occurs less than 21 days after a dose of VZIG in a susceptible individual (see above), a second dose of VZIG should be administered. Any HSCT recipient or candidate undergoing conditioning therapy who experiences a VZV-like rash should be started immediately on preemptive intravenous acyclovir until  2 days after all lesions are crusted (BIII). Virologic confirmation may be useful in situations where the appearance of the rash is atypical. A rash following exposure to a VZV vaccinee should be treated similarly (BII). Long-term acyclovir prophylaxis for 6 months to prevent VZV infection is generally not recommended (DIII); however, such prophylaxis could be considered in HSCT recipients with severe, long-term immunodeficiency (CIII). VZV vaccine use is contraindicated for individuals < 24 months after HSCT (EIII). Use of VZV vaccine among HSCT recipients is restricted to research protocols for individuals > 24 months after HSCT who are presumed immunocompetent (i.e., off immunosuppressive therapy and free of chronic GVHD). Long-term suppressive prophylaxis with acyclovir with doses higher (800 mg twice daily) and of longer duration (one year) than previously reported appears to be successful in preventing VZV reactivation (Bowden RA, Rogers KS, Meyers JD. Oral acyclovir for the long-term suppression of varicella zoster virus infection after marrow transplantation. 29th Interscience Conference on Antimicrobial Agents and Chemotherapy. Anaheim, CA, 1989, p abstract 62). Valacylovir (500 mg twice daily for prophylaxis, 1000 mg 3 times daily for recipients with stable non-disseminated disease) is being increasingly used due to its higher bioavailability. Initial concerns of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome which had been observed in HIV-infected individuals, have not been observed in two large randomized trials at much higher doses than those used for VZV (Ljungman P, Camara R, Milpied N, Volin L, Reilly L, Crisp A, Group VIBMTS. A randomized study of valacyclovir as prophylaxis against CMV infection and disease in BMT recipients. 25th Annual Meeting European Group for Blood and Marrow Transplantation. Hamburg, 1999; Winston DJ, Yeager AM, Chandrasekar PH, et al. Randomized comparison of oral valaciclovir and intravenous ganciclovir for prophylaxis of cytomegalovirus disease and complications after allogeneic bone marrow transplantation. 2nd International Conference on Transplant Infectious Diseases. Stockhom, Sweden, 2000) Whether seropositive transplant recipients can be reinfected with a second strain of virus is poorly defined. However, this may occur in the context of severe immunosuppression such T cell depletion, high-dose steroid therapy, or use of ATG. Thus, exposure should also be avoided in highly immmunosuppressed seropositive recipients, and postexposure prophylaxis with VZIG or acyclovir^ref or valacyclovir (1000 mg three times daily) given from day 2-22 of exposure may be considered. As VZV vaccination is more commonly used in the general population, an increased frequency of previously vaccinated transplant candidates can be expected. The risk of reactivation of vaccine strains after transplantation is currently unknown. Also, whether transplant recipients who have been vaccinated prior to transplant are more susceptible to infections with wild-type VZV than those who have been naturally infected with VZV is unknown. Because the vaccine is not 100% protective even in immunocompetent individuals^ref, an exposure to wild-type VZV in a HSCT recipient is likely to carry a high risk and should be treated similar to exposures in seronegative HSCT recipients or candidates, especially when intense immunosuppression is administered (e.g. T cell depletion, high-dose corticosteroids, or ATG).
• respiratory virus infections
• preventing exposure : respiratory viruses (respiratory syncytial virus [RSV], parainfluenza viruses, influenza viruses, adenovirus) are highly contagious. Strict infection control practices are advised (AIII). A targeted surveillance system that evaluates HSCT recipients with upper respiratory symptoms for the presence of respiratory viruses should be in place (AIII). Isolation should be instituted in all symptomatic patients, even before virologic confirmation (AIII) (Table 7). Summary of major recommendations for prevention of community respiratory virus infection. Influenza vaccination is strongly advised for all staff members, family members and close household contacts during each influenza season before HSCT and at least 24 months after HSCT (AI). During a nosocomial influenza A outbreak situation of strains that are not covered by the vaccine health care workers, family members, and close household contact should be offered chemoprophylaxis with amantidine or rimantidine (for influenza A) (BIII) or with neuraminidase inhibitors (zanamavir, oseltemavir) if amantidine/rimantidine is not tolerated, the outbreak strain is resistant to these agents, or the outbreak is due to influenza B (BI). Neuraminidase inhibitors have not been evaluated in children.
• preventing disease or diseases recurrence : respiratory secretions of HSCT candidates and recipients with upper respiratory symptoms should be evaluated for the presence of respiratory viruses (BIII). Several strategies have been proposed to prevent progression from upper to lower tract RSV infection, including aerosolized ribavirin alone and combination with RSV-specific antibodies or palivizumab, an RSV-specific monoclonal antibody. Controlled clinical trials are currently underway to establish the efficacy of these strategies, thus, no firm recommendations can be made. Intravenous ribavirin is not recommended for preemptive therapy (DIII). Amantidine or rimantidine chemoprophylaxis is recommended in influenza A outbreak situations for all HSCT recipients < 6 months after transplant (BIII). After 6 months, vaccination should be resumed (BIII). Chemoprophylaxis for all HSCT recipients < 24 months or > 24 months if treated for chronic GVHD has also been recommended during outbreak situations due to the poor immunologic response to the vaccine. However, no formal recommendation regarding such prophylaxis can be made because of lack of data. Influenza vaccinations during the first 6 months after transplant are unlikely to be effective (DII). Neuraminidase inhibitors are now available for treatment and prevention of influenza infection in immunocompetent subjects. However, no formal evaluation has been performed in immunocompromised patients. Thus, no recommendations can be made. Palivizumab, a RSV-specific monoclonal antibody, has been licensed for prevention in high-risk immunocompetent children; however, no prevention studies have been performed in HSCT recipients. A study of palivizumab for treatment of RSV infection showed no significant toxicity (Boeckh M, Berrey MM, Bowden RA, Crawford SW, Balsley J, Corey L. Phase I evaluation of the RSV-specific humanized monoclonal antibody palivizumab (MEDI-493) in hematopoietic stem cell transplant recipients. J Infect Dis 2001). Some centers use palivizumab for prophylaxis in pediatric (age < 2 years) HSCT recipients and candidates throughout the respiratory virus season. Whether rhinovirus is a significant pathogen in HSCT recipients remains controversial^ref (Boeckh M, Hayden F, Corey L, Kaiser L. Detection of rhinovirus RNA in bronchoalveolar lavage in hematopoietic stem cell transplants recipients with pneumonia. 40th Interscience Conference on Antimicrobial Agents and Chemotherapy. Toronto, 2000, p 262 (abstract 190)).
• prevention of fungal infection : a primary goal in the practice of transplant medicine is the prevention of infection. This is particularly true when one considers fungal infections in HSCT recipients. Fungal infection in this patient population is difficult to diagnose and treat, and, not surprisingly, is associated with a prohibitively high mortality. Factors contributing to this mortality include the following^ref:
• diagnosis is difficult, as conventional microbiologic approaches are both insensitive and often nonspecific. Invasive sampling of tissues is often necessary for diagnosis, or, alternatively, an empiric therapeutic approach is employed.
• because of the impaired inflammatory response present during the neutropenic phase preengraftment and during the treatment of GVHD postengraftment, clinical signs and symptoms of invasive fungal infection can be greatly blunted until disease is relatively far advanced. As a result, the microbial burden is usually far greater at the time of diagnosis than in nonimmunocompromised patients, a potentially important prognostic factor in determining the outcome of therapy.
• outcome is directly related to how early the therapy is instituted, with the previously listed two factors making early diagnosis difficult.
• currently licensed antifungal therapies are not ideal, having limited spectra of activity (e.g., fluconazole), a propensity for selecting resistance (e.g., flucytosine), or a particular ability to induce systemic (e.g., infusion toxicity resulting in a cytokine release syndrome with amphotericin) or organ toxicity (e.g., renal toxicity from amphotericin and hepatic toxicity from certain azoles).
• the treatment of invasive fungal infection needs to be more prolonged than that needed in the management of invasive bacterial infection.
Fungal infections of major concern can be divided into three general categories: invasive infection due to Candida and Aspergillus species, which account for > 90% of the fungal infections occurring in this patient population; infection due to the geographically restricted systemic mycoses (e.g., Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma capsulatum); and invasive infection due to the so-called "newly emerging fungi" (e.g., Fusarium, Paecilomyces, the zygomycetes, and such dematiaceous fungi as Scedosporium, Scopulariopsis, and Dactylaria)^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8}
• risk factors in the pathogenesis of fungal infection : the occurrence of invasive fungal infection in the HSCT patient is largely determined by the interaction of three factors: the presence of technical/anatomic abnormalities that compromise or attenuate the functioning of the primary mucocutaneous barrier to infection; excessive environmental exposures to potential fungal pathogens; and the net state of immunosuppression. The major technical/anatomic abnormalities that occur in the HSCT patient are those that arise from the placement of a central venous catheter for blood sampling and fluid administration and post-chemotherapy mucositis- both of which providing a portal of entry for Candida isolates. In addition, serious injury to the skin (e.g., water immersion injury, dermal or subcutaneous injury due to extravasation of noxious chemicals into soft tissues because of malfunction of vascular access devices) will commonly become secondarily infected by Candida, Aspergillus, and even zygomycetes species. Invasion of the skin and blood vessels can then occur, followed by secondary dissemination from the cutaneous portal of entry. The creation of devitalized tissue, the occurrence of undrained fluid collections, and the development of significant tissue injury (especially of the lungs) increases the risk of secondary fungal invasion^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). In the hospital setting, two epidemiologic patterns of exposure are observed: domiciliary and nondomiciliary. The term domiciliary refers to exposures occurring where the patient resides in the hospital. There is often clustering of cases in time and space. Although such exposures are usually due to a contaminated air supply, with molds such as Aspergillus being the major concern, person-to-person spread (usually on the hands of medical personnel) of Candida species, often azole resistant strains, can also occur^ref. Nondomiciliary exposures can occur anywhere within the hospital as the patient travels for an essential procedure (the operating room, the radiology or endoscopy suite, or the halls of the hospital where construction is taking place). Nondomiciliary exposures are probably more common than domiciliary, but are often more difficult to define because of the lack of clustering of cases. Indeed, the leading clue to the presence of such an environmental problem is the occurrence of a case of invasive fungal infection at a point in time when the net state of immunosuppression should not be great enough for such an infection to occur without a particularly intense exposure^{ref1, ref2} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press; Rubin RH. Infection in solid organ transplant recipients. In Rubin RH YL, ed. Clinical Approach to Infection in the Compromised Host. New York: Plenum Press, 2001:in press) Particularly notable contributors to the net state of immunosuppression for the HSCT recipient include neutropenia, GVHD and its treatment, and immunomodulating viral infection. In addition, such metabolic factors as protein-calorie malnutrition, uremia, and, perhaps, hyperglycemia contribute to the net state of immunosuppression. The patient receiving an allograft is far more immunosuppressed than the autologous HSCT recipient since the duration of neutropenia is greater and GVHD has a sustained effect in the allogeneic recipient. Reflecting this difference, the incidence of invasive fungal infection is far higher among the allogeneic population. Not unexpectedly, preventative strategies that are shown to be effective in the allogeneic HSCT recipient can be assumed to be effective in the autologous recipient; unfortunately, the converse is not necessarily true (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• prevention of fungal infection in the HSCT recipient : as delineated in 2 recent publications^{ref1, ref2}, there is a dearth of well designed, randomized, concurrently controlled clinical trials, not because of a lack of will on the part of investigators, but rather because of the inherent challenges in approaching fungal infection in this patient population: confounding factors (e.g., duration of neutropenia and other markers of the severity of the underlying disease) can obscure the efficacy of one or another antifungal regimen. In addition, the underlying disease for which the HSCT has been performed can be the cause of mortality, rather than failure of the antifungal program (other endpoints such as fever can also be problematic). The diagnosis of invasive fungal infection remains a problematic endpoint as well. Thus, the evidence relies less on randomized clinical trials and more on comparative studies that employ historical controls and careful clinical observation. Studies carried out by Benson^ref and Sacks^ref and their colleagues are reassuring in demonstrating that in other therapeutic areas, studies that employed historical controls yielded comparable results to those obtained in concurrently controlled clinical trials in at least 75% of instances.
• prevention of exposure to potentially invasive fungal species : aerosols containing excessive numbers of the geographically restricted systemic mycoses, as well as such molds as Aspergillus species, constitute a significant risk. It is generally recommended that HSCT recipients be educated to avoid circumstances in which aerosols of fungal spores might be encountered (BIII): areas of high dust exposure, urban renewal and other construction projects, chicken coops, bat caves, avocational (e.g., gardening) and vocational activities that require digging up soil, marijuana smoking, and the preparation and handling of foods that contain molds (e.g., blue cheese). Similarly, HSCT recipients should avoid exposures to naturopathic medicines, which can be contaminated with molds (CIII)^{ref1, ref2, ref3, ref4, ref5, ref6}. Although invasive candidal infection in the HSCT patient is almost always of endogenous origin, the endogenous candidal flora is modified within the hospital. A particular issue is the person-to-person transfer of Candida species (which are frequently azole-resistant) from the hands of medical personnel. Appropriate handwashing procedures by medical is strongly recommended to prevent the introduction of more difficult to treat Candida species into the "endogenous" flora (AIII)^{ref1, ref2} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). Of even greater importance are exposures to air contaminated with fungal spores, which result in invasive infection with such molds as Aspergillus species and such newly emerging species as Fusarium and the Mucorales. The portals of entry of these infections are the lungs and nasal sinuses. Special effort is required to protect HSCT recipients from contact with air containing excessive fungal spore counts (BIII)^{ref1, ref2, ref3} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• high efficiency (> 90%) particulate air (HEPA) filtration in patient rooms.
• controlled air pressures, such that the air pressure in patients' rooms, are positive in relation to the corridor, and that air flows from the rooms into the corridors.
• correctly sealed rooms, including correctly sealed windows and electrical outlets.
• high rates of room air exchange (i.e., > 12 air changes/hour).
• the construction of effective barriers between patient care and renovation or construction areas (e.g., sealed plastic) that prevent dust from entering patient care areas and that are impermeable to Aspergillus species.
• careful attention to travel routes within the hospital to avoid areas of renovation and construction. Ideally, patients leaving their rooms should be wearing highly effective masks as a further barrier to the inhalation of fungal spores.
Although HEPA filters and air pressure systems are essential and effective cleaning procedures to minimize fungal spores are important, routine surveillance cultures of the air or of the patient are not recommended (DIII)^ref
• minimization of deficits in host defenses against IFI : the first principle of practice is the eradication of all treatable infection prior to transplant. In patients with hematologic malignancies, it is not uncommon that previous attempts to induce remission with cytotoxic chemotherapy were complicated by invasive fungal infections due to Candida species or molds, especially Aspergillus. Such patients are still candidates for HSCT provided certain requirements are fulfilled (BIII): the patient has had a complete response clinically, microbiologically, and radiologically to effective antifungal chemotherapy; the patient can be continued on such therapy at least until engraftment occurs post-HSCT; and any residual anatomic abnormality (e.g., an infarcted area of lung due to invasive Aspergillus infection) or devitalized tissue has been considered for surgical resection (CIII)^{ref1, ref2, ref3, ref4} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). The first host defense deficit usually encountered by HSCT recipients is neutropenia secondary to the preparative regimen. Growth factors (e.g., GM-CSF and G-CSF) reduce the duration of neutropenia, although there is currently no evidence that this shortened effect has translated into a lower incidence of invasive fungal infection. Therefore, these agents cannot be recommended for the routine prevention of invasive fungal infection (DIII)^{ref1, ref2} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). CMV infection is an important cause of morbidity and mortality with both direct and indirect effects. The direct infectious disease effects of CMV in this population include life-threatening pneumonia and a systemic multisystem disease. CMV infection also contributes to the net state of immunosuppression, with an increased susceptibility to both yeast and mold infection being noted during active CMV disease. Accordingly, control of CMV replication is recommended, not only for its direct benefits but also to decrease the incidence of invasive fungal infection (BIII)^{ref1, ref2} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). Effective control of GVHD can reduce occurrence of invasive fungal infection^{ref1, ref2, ref3} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). A major lesson from the solid organ transplant experience is the need for so-called steroid sparing therapy; that is, the use of additional drugs such as cyclosporine +/- mycophenolate permit a decrease in prednisone dose and lower the risk of opportunistic infection, including that caused by fungi.10 In addition, the correction of metabolic abnormalities such as protein-calorie malnutrition, uremia, and hyperglycemia should decrease the net state of immunosuppression and the subsequent risk of invasive fungal disease (Rubin RH. Infection in solid organ transplant recipients. In Rubin RH YL, ed. Clinical Approach to Infection in the Compromised Host. New York: Plenum Press, 2001:in press)
• antifungal therapy in the prevention of invasive fungal infection : there are 3 different modes in which antifungal drugs can be administered to prevent invasive fungal infection: a prophylactic mode, an empiric mode, and a preemptive mode.28 In HSCT recipients the administration of topical (non-absorbable) antifungal agents (e.g., clotrimazole, nystatin, amphotericin B) orally or to the skin can be shown to decrease the colonizing fungal burden, particularly with yeasts, when applied to specific skin sites or the gastrointestinal tract. In addition, mucosal manifestations of superficial yeast infection (e.g., oral thrush, pharyngeal, and esophageal candidiasis) are probably decreased by topical therapy (CIII). However, such topical therapy does not reduce the incidence of either invasive candidiasis or invasive mold infection in HSCT patients and cannot be recommended for these purposes (DII)^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• prophylaxis of invasive yeast infection : the efficacy of oral fluconazole, a well absorbed triazole antifungal agent with particular efficacy against C. albicans and C. tropicalis, appears to be dose dependent. At a dose of 100-200 mg/day orally, fluconazole has variable effects in the prevention of invasive candidal infection. Indeed, this regimen has been associated with the emergence of infection due to such invasive species of Candida as C. krusei and C. glabrata, which are fluconazole resistant. Therefore, this regimen is not recommended for the prevention of invasive yeast infection (nor, since molds are universally resistant to fluconazole, for invasive mold infection) in HSCT recipients (DII). In contrast, at a dose of 400 mg/day there are convincing data that fluconazole administered orally or intravenously, beginning at the time of HSCT and continuing until at least the time of engraftment, provides clinically important protection against invasive yeast infection (AII). When breakthrough candidal infection does occur despite such a regimen, it should be assumed that the isolate is fluconazole resistant. The increasing incidence of C. krusei, C. glabrata, and other non-albicans strains is in large part related to the widespread use of fluconazole prophylaxis. Overall, however, the incidence of invasive candidiasis appears to be significantly decreased by fluconazole prophylaxis (at a dose of 400 mg/day) in allogeneic HSCT recipients.1,11,29,30 In general, the risk of invasive yeast infection in recipients of autologous HSCT is low enough not to require fluconazole prophylaxis (DIII). However, certain autograft patients are at higher risk and do merit prophylaxis employed for allogeneic recipients (BIII): autologous patients with underlying hematologic malignancies who will have prolonged neutropenia and mucosal damage from intense conditioning regimens; and those who have been receiving fludarabine or 2-CDA before the transplant^ref
• antifungal prophylaxis of invasive mold infection : no regimen has been shown to be clearly effective in the prevention of invasive mold infection, whether the species of mold be Aspergillus or one of the newly emerging molds such as Fusarium; hence, the importance of the previously described environmental protection strategies. Regimens studied thus far include moderate dose (0.5 mg/kg/day) amphotericin B, low dose amphotericin B (0.1–0.25 mg/kg/day), intranasal amphotericin B spray, lipid formulations of amphotericin B, aerosolized amphotericin B, and itraconazole. Similarly, there is no convincing evidence that these strategies prevent the newly emerging molds (DIII)^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press) As mentioned above, increasing success is being achieved in patients with previous invasive Aspergillus infection, which has been brought under control with an amphotericin preparation. HSCT, both allogeneic and autologous, has been successfully carried out in these individuals under the cover of antifungal prophylaxis, usually with an amphotericin preparation^{ref1, ref2, ref3, ref4} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• antifungal prophylaxis of the geographically restricted systemic mycoses : although case reports or small series of HSCT patients infected with one of the endemic mycoses (particularly coccidioidomycosis and histoplasmosis) have been reported, this has not been a significant clinical problem. Indeed, prophylactic antifungal therapy against the endemic mycoses is not recommended (DIII)^{ref1, ref2, ref3, ref4}.
• empiric therapy of fungal infection : the importance of neutropenia, particularly profound (absolute neutrophil count < 100/mm³) and prolonged (> 7 days) in the pathogenesis of invasive fungal infection has long been recognized. Because of the previously discussed difficulty in establishing the diagnosis of invasive fungal infection and the need for early initiation of therapy, empiric therapy of the febrile, neutropenic HSCT has become the standard of care. Such empiric therapy is defined as the initiation of systemic antifungal therapy to neutropenic patients who remain febrile after four days of empiric broad spectrum antibacterial therapy, and whose clinical and laboratory evaluation (including multiple blood cultures) remain negative. Amphotericin B was established over the past two decades as the therapy of choice for this purpose, with a significantly lower incidence of invasive fungal infection observed among patients receiving amphotericin B^{ref1, ref2, ref3, ref4, ref5}. More recently, liposomal amphotericin B has been shown to have equal efficacy to conventional amphotericin B, with significantly less infusion related toxicity (fever and chills) and dose related nephrotoxicity, but with significantly greater cost (BI)^{ref1, ref2}. Perhaps of greatest interest is the report of a multicenter, randomized trial in which standard liposomal amphotericin therapy at a dose of 3 mg/kg/day was compared to voriconazole at a dose of 3 mg/kg twice daily intravenously (or 200 mg orally) following two loading doses of 6 mg/kg 12 hours apart in a standard febrile neutropenia protocol. Voriconazole, a new broad-spectrum triazole agent available in oral and intravenous formulations, was equal or superior (fewer breakthrough cases of invasive fungal infection) to liposomal amphotericin. The major side effect from the voriconazole was transient visual disturbances, whereas febrile reactions and nephrotoxicity were significantly more common among the recipients of liposomal amphotericin (Walsh TJ, Finberg, R., Arndt, C., et al. Voriconazole versus liposomal amphotericin B for empirical antifungal therapy in persistantly febriele neutropenic patients: a prosepective, randomized, multicenter, international trial, 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, ON, Canada, 2000). Thus, empiric therapy of the febrile, neutropenic HSCT patient has been accepted as standard care. Amphotericin B, liposomal amphotericin B, and voriconazole have comparable efficacy. However, voriconazole appears to have less toxicity and the added convenience of an oral formulation.
• preemptive therapy of fungal infection : preemptive therapy is defined as the administration of an antimicrobial regimen to a patient population felt to be at high risk of life threatening infection before the onset of clinically recognizable disease^ref. In the case of invasive fungal infection in the HSCT patient, 3 opportunities for preemptive therapy have been defined:
• Colonization of the respiratory tract with Aspergillus species as demonstrated by positive cultures of respiratory secretions (sputum or bronchoalveolar lavage specimen) carries a > 50% risk of subsequent or concurrent invasive disease and is an indication for the initiation of preemptive therapy to eradicate such colonization^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). In contrast, colonization with Candida species does not correlate with the risk of invasive disease and is not an indication for antifungal therapy. There are currently two problems with this approach: the absence of such early colonization in a significant percentage of patients with invasive pulmonary aspergillosis and the lack of clear-cut information as to which is the best regimen for accomplishing preemptive therapy—amphotericin B, liposomal amphotericin, or voriconazole. Itraconazole appears to be less desirable for this task given less reliable pharmacokinetics and penetration into respiratory secretions^ref
• Protocol high resolution CT scans of the lungs at regular intervals in HSCT patients during periods of high risk for invasive aspergillosis can result in earlier diagnosis of disease, and the opportunity for preemptive therapy. A particular morphology of the lung lesion, the so-called "halo sign," is particularly suggestive of invasive aspergillosis (although Aspergillus infection is the most common cause of a halo sign, such other opportunistic infections as nocardiosis, mucormycosis, and others can also cause a halo sign). Antifungal therapy with an amphotericin preparation or one of the newer drugs (see below) is indicated in this circumstance^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• Detection of fungal antigens (e.g., Aspergillus galactomannan) or fungal metabolites (e.g., arabinitol) could give early evidence of invasive infection at a time when therapy would be most effective. Of particular interest are PCR based assays that will detect both yeast and mold infection. Linkage of such results to optimized antifungal strategies appears to be the wave of the future once these assays achieve adequate sensitivity and specificity^{ref1, ref2, ref3, ref4} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• new antifungal drugs in the prevention of IFI : the licensure of the lipid associated amphotericin preparations has provided drugs with efficacy comparable to conventional amphotericin, but with considerably less toxicity, although cost issues remain of concern^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). Also of great interest are two drugs that are in the late stages of clinical development. One of these, caspofungin acetate, the first of the echinocandins to be released for human use, has been approved for the salvage therapy of invasive aspergillosis. However, the preclinical data demonstrating broad-spectrum activity against yeasts and molds, and its potential utility in combination with other antifungal agents, suggest further benefits of this new agent^{ref1, ref2}. Particularly appealing is its side effect profile, both in terms of infusion related and organ specific toxicity. A potential disadvantage is its availability only in a parenteral formulation. Voriconazole, a triazole with fungicidal activity against Aspergillus species and potent fungistatic activity against a wide variety of yeasts and molds, including several of the newly emerging fungi resistant to presently available agents, appears especially well suited to the management and prevention of fungal infection in the HSCT patient^{ref1, ref2, ref3}. Particularly noteworthy is a large randomized study of invasive aspergillosis in immunocompromised patients, a large percentage of whom were HSCT recipients. In this study, voriconazole was significantly more efficacious than amphotericin, with significantly less toxicity (Herbrecht R, Denning D.W. , Patterson, T.F., et al. Open, randomized comparison of voriconazole and amphotericin B followed by other licensed antifungal therapy for primary therapy of invasive aspergillosis. 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2001). Table 8 provides a summary of major recommendations for prevention of fungal infection after hematopoietic cell transplantation. Summary of major recommendations for prevention of invasive fungal infection.

The prevention of invasive fungal infection in the HSCT recipient remains an important goal of transplant medicine. Accomplishing this task has been made difficult by the lack of sensitivity of diagnostic techniques and the lack of an ideal antifungal agent. Three different strategies have emerged to accomplish the task of antifungal prevention: prophylactic, empiric, and preemptive. Prophylactic fluconazole at a dose of 400 mg/day has been shown to decrease both the rate of invasive yeast infection and mortality. Thus far, no prophylactic strategy has been shown to decrease the incidence of invasive mold infection, and the emphasis is on environmental protection. Empiric therapy in the febrile neutropenic patient has been established as a standard of care, with amphotericin B, lipid associated amphotericin, and voriconazole appearing to offer comparable efficacy. Preemptive therapy based on microbiologic markers, particularly non-culture based microbiologic assays, appears to be an approach for the future.
• prevention of bacterial infections : bacterial infections may complicate HSCT at any time but pose a particular risk during the neutropenia produced by cytoreduction. The main sources of infection include central venous catheters, mouth flora, and gut flora via bacterial translocation. As with all patient care, attention to proper handwashing is strongly recommended to minimize risk of a bacterial infection (AIII) (Table 9)^ref

Summary of major recommendations for prevention of bacterial infections. In general, many decisions regarding use of prophylactic agents for bacterial infections must weigh the potential immediate benefit (prevention of infection) against the longer-term consequence (development of antibiotic resistance). Because of the potential severity of antibiotic-resistant infections, particularly among HSCT recipients, every effort should be made to use preventative antibacterials with restraint. The widespread development of VRE in most US hospitals is a recent reminder of how devastating drug-resistant bacteria may be.
• neutropenia : the neutropenia from cytoreduction results in risk for predictable organisms from bowel and mouth flora. Gut flora decontamination is not recommended (DIII) prior to HSCT given the lack of clear efficacy and its potential for fostering development of resistant organisms^{ref1, ref2, ref3, ref4}. Because of limited data, no recommendation can be made regarding routine use of antibiotics for bacterial prophylaxis in afebrile and asymptomatic neutropenic patients. In particular, vancomycin should not be used in this setting (DIII), since overuse of this drug may encourage development of VRE^ref. Routine use of growth factors may shorten neutropenia but does not appear to change the rate of serious bacterial infection and so is not recommended to prevent bacterial infections^ref
• central venous catheters : guidelines for care of central venous catheters recently have been promulgated to reduce infectious and other complications^{ref1, ref2}. Handwashing is recommended prior to changing dressings or manipulation at the site. Use of silver or antibiotic-impregnated catheters may result in lower rates of infection although no studies have been performed on indwelling or implantable devices in HSCT^{ref1, ref2}. Similarly, chlorhexidine-impregnated gauze dressings may reduce infection rates, although no studies of this approach have been completed in HSCT^ref. Antibiotic ointments at the exit site are not recommended^ref. Catheters should be removed as soon as is practical; for patients with transfusion requirements, however, the central venous catheter may be required for months.
• chronic graft-versus-host disease : patients with chronic GVHD are at risk for invasive infection from encapsulated organisms, particularly S. pneumoniae, although infection with Haemophilus influenzae and Neisseria meningitis may also occur. Appropriate prophylactic antibiotics directed at these pathogens should be selected based on local resistance patterns. These should be used until resolution of chronic GVHD. Intravenous immunoglobulin (IVIG) is not recommended (DII), unless the patient is among the 20-25% of HSCT recipients with unrelated marrow grafts who experience severe hypogammaglobulinemia in the first 100 days post-HSCT (CIII)^{ref1, ref2}
• specific organisms
• Streptococcus pneumoniae infection is a particular risk for HSCT recipients with chronic GVHD or those with profound hypogammaglobulinemia^{ref1, ref2}. 3 distinct strategies can help prevent invasive S. pneumoniae infection in specific groups: In patients with chronic GVHD, antibiotic prophylaxis is recommended for as long as GVHD therapy is administered (BIII)^{ref1, ref2}. Penicillin is the drug of choice in communities where S. pneumoniae remains likely to be penicillin-susceptible^ref. Trimethoprim-sulfamethoxazole, if given daily for pneumocystis prophylaxis, is adequate to prevent S. pneumoniae as well. However, no data were found to support its use for preventing S. pneumoniae disease. Vaccination with the currently available 23-valent vaccine may be of benefit to any HSCT patient and should be administered 12 and 24 months post transplant (BIII)^ref. The role of the new conjugate 7- valent vaccine has not been determined for HSCT recipients. Among those profound hypogammaglobulinemia, use of IVIG may be effective (CIII)^{ref1, ref2}.
• Streptococcus viridans : mucositis may facilitate translocation of S. viridans from the mouth into the blood. A full dental evaluation should therefore be conducted for all patients prior to initiating HSCT conditioning (AIII). Routine use of prophylaxis directed at S. viridans is not recommended (DIII) as such a strategy has not been shown to be effective and may promote the emergence of antibiotic-resistant organisms. At certain institutions, frequent cases of overwhelming shock due to S. viridans have been described^ref. In such a circumstance, prophylaxis might be considered (CIII). Selection of the best agent for prophylaxis should be made based on the hospital antibiotic susceptibility profile.
• Haemophilus influenzae : pediatric household contacts of HSCT recipients should be current with H. influenzae type B (Hib) vaccine (AII). Exposure to infected household contacts and persons in the house who themselves had been exposed to an active case of invasive H. influenzae should be minimized. Hib conjugate vaccine should be administered to all HSCT recipients at 12, 14, and 24 months post-HSCT (BII) because HSCT recipients have low levels of H. influenza capsular antibody post-transplant.16 This approach is particularly important among persons with chronic GVHD, whose risk for invasive H. influenzae is increased.
• prevention of protozoal infections
• Pneumocystis carinii pneumonia (PCP) : the potential risk of nosocomial PCP remains controversial. Although some hospitals place patients with proven PCP into contact precaution isolation, the value of this strategy has not been established. When possible, HSCT recipients should avoid contact with persons with PCP (CIII). Prophylaxis against PCP should be given to all allogeneic recipients from the time of engraftment until 6 months after HSCT (AII). Prophylaxis should be extended for those with chronic GVHD or those receiving immunosuppressive therapy for other indications until resolution of the underlying problem (BII)^{ref1, ref2}. In some centers, prophylaxis is given for 1-2 weeks prior to transplant. However, because no studies have been performed on this approach, no recommendations can be made. Summary of major recommendations for prevention of protozoal infections. Trimethoprim-sulfamethoxazole is the preferred prophylactic agent (AII). For those unable to receive trimethoprim-sulfamethoxazole, alternative agents include dapsone 100 mg daily^ref; atovaquone 1500 mg daily^ref, or aerosol pentamidine^ref (BIII). Although these second-line agents have not been formally compared, daily dapsone appears the preferred agent. Dapsone given three times weekly should not be used^ref. Leucovorin (folinic acid) is often administered with trimethoprim-sulfamethoxazole but no studies support the use of this costly medication^ref. PCP among autologous transplant recipients is rare but does occur^ref. Patients for whom prophylaxis might be considered are those who have received fludarabine or 2-CDA, those with underlying lymphoma or leukemia, or those receiving intense conditioning regimens or graft manipulation (BIII). No recommendation can be given for other patients. In determining the best approach for a given transplant center, the risk of an adverse event related to trimethoprim-sulfamethoxazole, such as fever and rash or myelosuppression, should be weighed against the potential benefit of preventing this rare but sometimes fatal pneumonia.
• Toxoplasma gondii : all HSCT recipients should be advised regarding the risk of acquiring toxoplasmosis via ingestion of undercooked meat or through exposure to cat feces (BIII). Toxoplasmosis is a substantial risk (8.5% incidence) for T-cell depleted graft recipients if the host is seropositive for T. gondii pre-transplant^ref.24 Importantly, however, cases of toxoplasmosis post-HSCT have been reported when both host and donor are seronegative for T. gondii, reflecting the limited sensitivity of the serologic test^ref.24 Disseminated toxoplasmosis does occur, although less commonly, in other transplant setting^ref. Prophylaxis might be considered for those with a history of invasive toxoplasmosis, particularly chorioretinitis, and for T. gondii seropositive patients with acute GVHD, but data demonstrating efficacy are limited (CIII). Trimethoprim-sulfamethoxazole is recommended for prophylaxis (BII). For those in whom trimethoprim-sulfamethoxazole cannot be given, clindamycin-pyrimethamine or pyrimethamine/sulfadoxine (Fansidar)^ref should be effective (CIII). Duration of prophylaxis should parallel that of PCP prophylaxis (CIII). Secondary prophylaxis should be given for the duration of immunosuppression (BIII). Autologous transplant recipients do not have discernible risk and should not receive prophylaxis (DIII) nor should they be screened for serologic evidence of previous infection (DIII).
• Strongyloides stercoralis : HSCT recipients should avoid situations, such as exposure to potentially infected soil that might result in acquisition of S. stercoralis (AIII). A thorough travel and residence history should be obtained for all HSCT candidates (BIII)^ref. Areas with risk for S. stercoralis include the Southeastern US, the tropics, the sub-tropics, and Europe. Any potential HSCT recipient with unexplained peripheral eosinophilia or those with travel or residence to endemic areas should be screened by serologic test for asymptomatic S. stercoralis (BIII). If the serologic screen is unavailable, examination of three stool specimens can be performed, although this approach is less sensitive (BIII). Patients with a positive serology, unexplained eosinophilia despite a negative serology, or a compelling history, should receive prophylaxis. Ivermectin is the preferred drug; if it is unavailable, thiabendazole may be given (BIII). No recommendation can be made for secondary prophylaxis. All infected patients should have  3 negative stools after completion of treatment, prior to beginning HSCT (AIII).
• Trypanosoma cruzii, which causes Chagas' disease, can be transmitted by blood transfusion and possibly via HSCT. It may also be transmitted congenitally. Because the infection is difficult to treat, potential donors should be screened for IgG against T. cruzii if they were born in, received a blood transfusion in, or ever lived for  6 months in Chagas' disease endemic areas, such as parts of Mexico, Central and South America (BIII)^ref (Altclas J, Sinagra A, Jaimovich G, et al. Reactivation of chronic Chagas' disease following allogeneic bone marrow transplantation and successful pre-emptive therapy with benznidazole. Transplant Infectious Dis. 1999;1:135–137). Persons with active diseases should not serve as donors (DIII).
• guidelines for active and passive immunizations for HSCT : for the vast majority of individuals, immunizations are safe, effective and inexpensive means to prevent infectious disease^{ref1, ref2} (Centers for Disease Control and Prevention. General recommendations on immunization. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1994;43(RR-1):1–38). While it is difficult to overestimate the benefit of vaccines on world health, misconceptions and missed opportunities may abrogate this protective advantage^{ref1, ref2} (Plotkin SL, Plotkin SA. A short history of vaccination. In Plotkin SA, Mortimer EA Jr, eds. Vaccines. W B Saunders: Philadelphia; 1988). This appears especially true among individuals undergoing myeloablative HSCT, where care by multiple providers, faulty assumptions regarding immunocompetence or persistence of protective immunity and lack of immunization schedules contribute to underutilization or variability in best practices^{ref1, ref2, ref3}
• immunization of the immunocompromised host : myeloablative conditioning before HSCT impairs immune function, fosters opportunistic infection and dampens immunogenic responsiveness to vaccination^{ref1, ref2}. With passage of time, most patients recover adequate immune function, although the tempo of recovery can be delayed by older age, T-cell depletion, GVHD, anti-thymocyte globulin or other immunosuppressive agents^{ref1, ref2, ref3} (Lum LG. The kinetics of immune reconstitution after human marrow transplantation. Blood). Persisting immunodeficiency contributes to serious late infections, especially with encapsulated organisms^{ref1, ref2, ref3}. Since vaccine efficacy is lessened during the immunocompromised period but the tempo of immune recovery varies widely, it is understandable that standardized data on antibody response to vaccination following HSCT are limited. Moreover, since the number of HSCT patients enrolled in controlled vaccination trials has been small, published efficacy measuring the frequency of late infections has been scanty. Thus, guidelines of level II and III recommendations are based on either surrogate markers of protective antibody titers or clinical experience extrapolated from other patient groups^{ref1, ref2} (Centers for Disease Control and Prevention. Use of vaccines and immune globulins in person with altered immunocompetence. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1993;42(RR-4):10–18). Antibody titers persisting from childhood immunizations decrease after HSCT to barely protective or unprotective levels. This decline has been shown in allogeneic and autologous recipients of blood and marrow grafts^{ref1, ref2, ref3, ref4, ref5}. In general, HSCT recipients respond better to vaccinations if several are given at and after 1 year posttransplant^{ref1, ref2, ref3, ref4, ref5}. Unfortunately, responses are poorer for unconjugated pneumococcal polysaccharide vaccine (PPV) and unconjugatged Haemophilus influenzae type b (Hib) vaccine^{ref1, ref2, ref3, ref4}. Booster immunizations result in anamnestic antibody responses, but this benefit may be lost in those with chronic GVHD^{ref1, ref2, ref3}
• active immunization for HSCT recipients : as shown in Table 11, guidelines are provided for allogeneic and autologous recipients given myeloablative conditioning (Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the Infectious Diseases Society of America, and the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2000;6:705–706). Major recommendations for vaccinations for hematopoietic stem cell transplant (HSCT) recipients, including both allogeneic and autologous recipients.For these guidelines, HSCT recipients are presumed immunocompetent at  24 months after HSCT if they are not on immunosuppressive therapy and do not have graft-versus-host-disease (GHVD).
• Diphtheria, tetanus and pertussis : vaccinations at 12, 14 and 24 months posttransplant are recommended for diphtheria and tetanus toxoid (BII) and pertussis in children < age 7 years (BIII)^{ref1, ref2, ref3, ref4}. Patients should be reimmunized for tetanus-diphtheria toxoids every 10 years (Centers for Disease Control and Prevention. Use of vaccines and immune globulins in person with altered immunocompetence. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1993;42(RR-4):10–18)
• Haemophilus influenzae type b. Hib conjugate vaccine is given 3 times beginning one year after HSCT (BII)^{ref1, ref2, ref3}. This is recommended for patients of any age (Centers for Disease Control and Prevention. Use of vaccines and immune globulins in person with altered immunocompetence. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1993;42(RR-4):10–18)
• Hepatitis B : immunization with the recombinant vaccine is recommended for those who are otherwise susceptible: children < age 18 or adults with risk factors for hepatitis B (BIII)^ref (Nagler A, Klan Y, Adler R, et al. Successful immunization of autologous bone marrow transplantation recipients against hepatitis B virus by active vaccination. Bone Marrow Transplant. 1995;15: 4750478). Persons who fail to exhibit a response should receive a second 3-dose series.
• Pneumococcal : the 23-valent PPV had been recommended at 12 and 24 months after HSCT along with chemoprophylaxis in individuals with chronic GVHD (BIII)^ref. Recently, a 7-valent conjugate vaccine has been licensed. No data are currently available in HSCT recipients, but one strategy could be to give one or more does of the 7-valent conjugate vaccine with subsequent broader coverage with the 23-valent product^ref.
• Influenza : beginning before HSCT and resuming > 6 months after transplant, individuals should receive life-long seasonal immunization (BII)^ref (Centers for Disease Control and Prevention. General recommendations on immunization. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1994;43(RR-1):1–38; Centers for Disease Control and Prevention. Use of vaccines and immune globulins in person with altered immunocompetence. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1993;42(RR-4):10–18). Children < age 9 receiving vaccine for the first time require two doses. Those < 12 years old should receive only split-virus vaccines^ref. For optimal protection, immunization and chemoprophylaxis can be combined.
• polio : inactivated polio vaccine is recommended following allo and auto transplant (BII)^{ref1, ref2}. More information is needed regarding optimal schedules of vaccination.
• measles, mumps, rubella : MMR is a live-attenuated viral product and is indicated only in immunocompetent recipients at least 24 months posttransplant who are not receiving immunosuppressive drugs and are free of GVHD (BIII)^{ref1, ref2, ref3} (Centers for Disease Control and Prevention. Use of vaccines and immune globulins in person with altered immunocompetence. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1993;42(RR-4):10–18). No data are available for use before 24 months posttransplant.
• varicella : insufficient data are available to recommend this vaccine (EIII)^ref
• others : vaccinations for HSCT patients travelling to endemic areas are either contraindicated (live-attenuated vaccines) or not rated due to limited experience (Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the Infectious Diseases Society of America, and the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2000;6:705–706)
• active immunization for family members and healthcare workers : as shown in Table 12, a number of recommendations apply to close contacts of the patient in order to prevent spread of infectious disease (Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the Infectious Diseases Society of America, and the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2000;6:705–706) The published evidence supporting these recommendations is provided by the Centers for Disease Control and Prevention^ref. Major recommendations for vaccinations for family, close contacts, and health-care workers (HCWs) of hematopoietic stem cell (HSCT) recipients.
• passive immunization for HSCT recipients (Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the Infectious Diseases Society of America, and the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2000;6:705–706) : CMV immunoglobulin is not indicated (DI) but VZIG is recommended (AII)^ref. Intramuscular immunoglobulin may be of benefit for those who travel to areas endemic for hepatitis A or were exposed to measles but not vaccinated after HSCT (BIII)^ref (Centers for Disease Control and Prevention. Prevention of Hepatitis A through active or passive immunization. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1999;48 (RR-12): 1–37). Intravenous immunoglobulin can be administered to patients with persisting hypogammaglobulinemia (Ig < 400 mg/dl) and recurrent bacterial infection (CIII)^{ref1, ref2, ref3, ref4, ref5}. Major recommendations for use of passive immunization for hematopoietic stem cell transplant (HSCT) recipients exposed to vaccine-preventable diseases.
There are no significant immunological or alterations in clinical benefit of post-transplant G-CSF administration in T-repleted allotransplant recipient^ref.
Whereas active HBV infection was non significantly (P=0.13) higher (11%) in the BMT patients in comparison to 3% in untransplanted patients, 53% of the HHV-5 / CMV-seropositive recipients developed CMV reactivation compared to 11% of the CMV-seronegative recipients who developed CMV seroconversion. In addition, 38% of the HBV-seropositive recipients developed HBV reactivation in comparison to 0% of the HBV-seronegative recipients. Moreover, 39% of the HCV-seropositive recipients developed HCV reactivation in comparison to 6% of the HCV-seronegative recipients who developed HCV seroconversion. Previous exposure to CMV, HBV, and HCV infections in the recipients of BMT patients influences the risk of developing those viral infections^ref.
Costs of allogeneic HSCT : the total median cost of 5 posttransplant years was € 139,414 (52,095-345,640) or 167,296 US$ (the rate of 1 € is approximately 1.2 US$). The costs were highest during the first year-median inpatient and outpatient costs € 100,650 and € 13,066, respectively. The total costs during the first year were higher in patients with aGvHD grades III-IV (relative hazards [RH] 1.35, P = 0.003), bacteremia (RH 1.33, P = 0.005), veno-occlusive disease of the liver (RH 1.32, P = 0.005), prophylaxis with G-CSF (RH 1.31, P = 0.01), acute leukemia (RH 1.32, P = 0.008), and treatment in hospital instead of at home (RH 1.20, P < 0.07). During the early transplant period, a second transplantation (RH 1.28, P = 0.014) and hemorrhagic cystitis (HC; RH 1.24, P = 0.03) were also associated with higher costs. The total 5-year cost declined with longer survival rates (r = 0.4028, P < 0.001) and RIC (RH 0.79, P=0.024). Higher costs of ASCT were associated with retransplantation, acute leukemia, G-CSF prophylaxis, hospital care, myeloablative conditioning, and major transplant-related complications^ref

• Bone Marrow Donors Worldwide (BMDW) (updated every 4 months)
• European Marrow Donor Information System (EMDIS)
• World Marrow Donor Association (WMDA)
• International Bone Marrow Transplant Registry (IBMTR)/Autologous Blood and Marrow Transplant Registry (ABMTR)
• European Group for Blood and Marrow Transplantation (EBMT)
• Total Body Irridation (TBI) Registry
• European MArrow DOnor Registries (MADO)
• Anthony Nolan Bone Marrow Trust (ANBMT)
• National Marrow Donor Program (NMDP)
• ABO Mismatch Reporting Center
• British Society of Blood and Marrow Transplantation (BSBMT)
• American Society for Blood and Marrow Transplantation (ASBMT)
• Canadian Blood and Marrow Transplant Group (CBMTG)
• European Council for Accreditation in Hematology (ECAH)
• HAPLO.ORG - Haploidentical Stem Cell Transplantation
• International NetCord Organisation (NETCORD)
• International Society for Hemotherapy and Graft Engineering (ISHAGE)
• BMT Net: "a central resource of all blood and marrow transplantation -related topics"
• BMT Information
• journals :
• Biology of Blood and Marrow Transplantation
• Bone Marrow Transplantation
• Marrow Transplantation Reviews: Issues in Hematology, Oncology and Immunology

• E. Donnall Thomas (Editor), Karl G. Blume (Editor), Stephen J. Forman (Editor), Frederick R. Appelbaum (Editor), Stephen J. Jorman : Thomas' Hematopoietic cell transplantation ; 3rd edition (March 2004)
• E, Donnall Thomas (Editor), Karl G. Blume (Editor), Stephen J. Forman (Editor) : Hematopoietic Cell Transplantation; 2nd edition (January 2004)

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