- Email: [email protected]
Graft failure
Graft failure Michael Potter
Incidence of graft failure Graft failure in the setting of autologous transplantation is a rare event in modern practice, with an incidence <1%.1 The position with regard to allogeneic transplantation is more complex. In the standard setting of FIC HLA-identical sibling transplantation with no T-cell depletion,
The clinical impact of graft failure The standard complications of graft failure include prolonged hospitalization, an increased risk of bacterial and fungal infection and risk of hemorrhage.5,6 This may lead to an increased risk of transplantrelated mortality. In addition, there is evidence that incomplete donor chimerism is associated with an increased relapse risk.7
Mechanisms of graft failure In the allogeneic setting, the concept of an immunologic process causing graft rejection is well established. Historically, this is best described in the setting of allogeneic transplantation for patients with severe aplastic anemia8 and may be in part attributed to alloimmunization from previous blood transfusions. An immunologic mechanism of graft failure or rejection is also associated more with recipients of HLA-mismatched and unrelated donor transplants than those with matched related donors. In the unrelated donor setting, it has been shown that HLA class II disparity between donor and recipient may not increase the rate of graft rejection but class I disparity, especially at the HLA C locus, is associated with this complication.9,10 This is believed to be predominantly a T-cell mediated process involving donor cytotoxic T-cells, although there is also evidence for a natural killer (NK) cell-mediated graft rejection process.11,12 Immunologic graft rejection is usually characterized by a transient process of donor engraftment followed by loss of donor cells and either a complete failure of hemopoiesis or a recovery of autologous reconstitution, the latter being more typical with RIC transplants. Another mechanism of graft failure relates to the composition of the donor graft. Clearly, graft failure may be due to an inadequate number of transplanted hemopoietic stem cells in both the allogeneic and autologous setting. Many studies have been published indicating the requirement for engraftment in terms of mononuclear cell (MNC) numbers, and particularly CD34+ (stem cell) numbers, infused in both the allogeneic and autologous settings.13–15 The commonly quoted threshold for prompt and reliable engraftment is the infusion of ≥2 ×
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The traditional approach to transplantation involves myeloablative conditioning with chemotherapy and/or radiotherapy resulting in a subsequent period of obligate pancytopenia followed by recovery of hemopoiesis. In the setting of myeloablative or full-intensity conditioned (FIC) transplantation, myeloid engraftment is often defined as the first day that a neutrophil count of 0.5 × 109/l is achieved and sustained. Red cell and platelet engraftment implies independence from transfusion support. A platelet count of 20 × 109/l and/or 50 × 109/l sustained and transfusion independent are useful clinical definitions of megakaryocyte engraftment. Primary graft failure implies a failure to ever achieve these target values. Secondary graft failure implies an initial achievement of these parameters but subsequent cytopenias with a neutrophil count <0.5 × 109/l and requirement for transfusions. The diagnosis of graft failure will also usually require bone marrow examination to demonstrate hypocellularity and lack of infiltration by the original malignant disease or another process. These definitions of primary and secondary graft failure are applicable to both the allogeneic and autologous settings. In recent years, however, the introduction of non-myeloablative or reduced-intensity conditioning (RIC) allogeneic transplantation requires supplementary definitions of graft failure. Patients may no longer have an obligate period of pancytopenia following conditioning, and engraftment may be a seamless transition from recipient to donor hemopoiesis. In this case, graft failure may be better defined in terms of chimerism analysis with a failure to achieve or subsequent loss of donor chimerism. The traditional definitions of graft failure may also not take into account deficiencies specific to individual lineages, e.g. red cell aplasia which is now well documented following allogeneic transplantation. There is also the concept of poor graft function, where the basic parameters of engraftment are fulfilled but blood counts may remain suboptimal for long periods of time, leading to potential complications. Both primary and secondary graft failure are usually evident within the first 6 months following transplantation, although late graft failure can occur and historically has been seen more commonly in patients with severe aplastic anemia and β-thalassemia major. In patients with malignant disease, late graft failure may herald subsequent relapse of the original clone.
the rate of graft failure is of the order of 1–2%.2 T-cell depletion increases the risk of graft failure.3 Recipients of RIC transplants are also at higher risk of this complication.4 Unrelated donor transplants and HLA-mismatched donor transplants (related/unrelated) may also be associated with a higher risk of graft failure.2 For example, in the setting of a RIC transplant or a T-cell depleted FIC transplant with an HLA-mismatched donor transplant, graft failure rates of 5–30% may occur.2–4
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106 CD34 cells per kg of recipient body weight. This may represent a gross oversimplification, however, and it is difficult to define a lower limit for successful engraftment in clinical practice. The source of stem cells also needs to be taken into account. In the field of umbilical cord transplantation, satisfactory engraftment may be obtained with CD34 counts more than 1 log below the above figure.16,17 CD34 numbers are clearly not the only determinant of engraftment in an infused stem cell product. The presence of other populations of cells, such as T-cells or even serum factors, may affect the engraftment process.16 However, clinically the pace of engraftment is more rapid with peripheral blood stem cell transplantation than bone marrow transplantation, which, in turn, exceeds that seen in the setting of umbilical cord transplantation, and this is predominantly a CD34 numbers effect. The quality of stem cell products in terms of engraftment potential may also be adversely affected by events occurring following collection of cells. Examples include prolonged storage prior to cryopreservation,18 the cryopreservation process itself and subsequent thawing procedure,19 in vitro manipulation of the graft (e.g. CD34 selection3), incubation with antibodies for T-cell depletion20 or malignant cell purging.21
Host factors which may affect engraftment Patients with certain diagnoses are at higher risk of graft rejection than others. This relates to the extent to which the patients have already received myelo- or immunosuppressive therapies, the number of transfusions of cellular products received and possible stromal defects in the marrow microenvironment associated with certain diagnoses. Patients with severe aplastic anemia and β-thalassemia major may have typically received tens or hundreds of units of cellular blood products prior to transplantation. This may have the effect of alloimmunization which increases the risk of graft failure.8,22,23 Patients with myeloproliferative disorders including chronic myeloid leukemia may have received little in the way of intensive chemotherapy prior to transplantation. The increased cellularity in the bone marrow of patients with myeloproliferative disorders coupled with the lack of intensive prior chemotherapy compared to patients with acute leukemia may increase the risk of graft failure in these diseases. Stromal abnormalities occur in myelofibrosis where the normal bone marrow stroma is replaced by a dense fibrotic reaction.24 In severe aplastic anemia, it is possible that stromal defects contribute to the etiology of the disease in certain patients. If these are not corrected by transplantation, similar problems may ensue with the donorderived hemopoiesis.25 Finally, patients with moderate to massive splenomegaly at the time of transplantation may be at higher risk of graft failure.24,26 This may be as a result of preferential donor engraftment in the spleen rather than the marrow and subsequent inefficient hemopoiesis coupled with the effects of hypersplenism.26,27 The intensity of the conditioning regimen may also affect the engraftment process. In the last 10 years there has been the successful introduction of RIC transplantation with non-myeloablative chemotherapy and/or low-dose total-body irradiation regimens. In simple terms, conditioning treatment needs to have two elements: myelosuppression and immune suppression. The myelosuppressive element creates space for the incoming donor hemopoiesis and the immunosuppressive element eradicates the potential for host-versus-graft immune reactions. The former element (myelosuppression) is substantially attenuated in RIC transplantation, with an associated increase in the potential for graft failure. Therefore, this is usually compensated for by increasing the immunosuppressive element of the conditioning regimen. In practice, this has usually been achieved by the use of
fludarabine, a purine analog with very potent T-cell suppressive properties, in conjunction with non-myeloablative doses of chemotherapy – busulfan, melphalan or cyclophosphamide – or low-dose total-body irradiation.28,29 In vivo administration of T-cell antibodies such as antithymocyte globulin (ATG) or alemtuzumab (CAMPATH-1H) prior to transplantation is an alternative approach with the potential additional benefit of reduction in graft-versus-host disease (GvHD) risk due to T-cell depletion of the incoming graft.30,31 Infections which occur in the early post-transplant phase may be a cause of non- or suboptimal engraftment. Certain viruses such as parvovirus may directly infect hemopoietic cell progenitors, leading to a failure of maturation. In particular, parvovirus targets erythroid progenitors and has been associated with pure red cell aplasia.32 Many viruses, particularly those of the herpes group, have been shown to cause the potential for hemophagocytosis, which may be a cause of graft failure. Examples include the Epstein–Barr virus (EBV),33 cytomegalovirus (CMV)34 and human herpes virus 6 (HHV6).35 Other viruses which may also stimulate hemophagocytosis include adenoviral36 and influenza viral infections.37 Myelosuppressive drugs may also be a cause of graft failure or impairment. The choice of post-transplant immunosuppressive regimen may affect engraftment. Ciclosporin is generally considered to be ‘protective’ against graft failure by reducing the chance of a host-versus-graft immune-based rejection. Patients with aplastic anemia and β-thalassemia major generally receive prolonged treatment with ciclosporin A to at least 1 year post transplant to prevent late graft rejection in these high-risk disorders. Ciclosporin is often used with 3–4 injections of methotrexate. The latter increases the potency of post-transplant immune suppression and reduces the incidence of severe GvHD. However, methotrexate is also myelosuppressive and its use usually does lead to a delay in engraftment of neutrophils by around 7 days.38 Other drugs may also have myelosuppressive effects. Co-trimoxazole, used for prophylaxis against Pneumocystis infections, may lead to macrocytosis and cytopenias.39 Ganciclovir and its prodrug valganciclovir, used to prevent or treat CMV infection, are a commonly recognized cause of often severe myelosuppression.40,41 Mycophenolate mofetil, which may be used for post-transplant immune suppression or in the treatment of GvHD, may also be myelosuppressive.42 H2 antagonists have also been associated with graft failure.43 Rare causes of graft failure relate to nutritional factors such as folic acid deficiency, vitamin B12 deficiency and protein energy malnutrition. Delayed red cell engraftment, including rare cases of pure red cell aplasia, may also be seen in cases of ABO mismatched transplantation.44 Intensive T-cell depletion may delete subsets of donor T-cells which support engraftment. One such population of cells has been named ‘veto cells’.45,46 These have been described in animal models of transplantation and shown to counteract host-versus-graft immune-based rejection. This may account for the high rate of graft rejection seen in the T-cell depleted setting.
Donor factors which may affect engraftment In the unrelated donor setting, male donors are usually preferred over female donors. There are several reasons for this. The collection of cells, particularly peripheral blood progenitor cells (PBPC) by pheresis, may be technically easier because of better venous access. A higher yield of stem cells may result because of this and increased donor weight. In female donors, alloimmunization from prior pregnancies may theoretically increase the potential for graft failure.47 Donor age may also have an effect on cell yield and engraftment, and younger donors are generally preferred.
Approach to the patient with graft failure Suspicions should be raised in the patient who has not engrafted neutrophils by day 21 or certainly day 28. The latter time-point may be more appropriate in recipients receiving post-transplant methotrexate, where engraftment is typically delayed. Initially, it is recommended that the details of the infused stem cells product are reviewed, including CD34 and MNC counts. The procedures undertaken in the cell processing laboratory should also be reviewed. It is good practice in the stem cell laboratory for a pilot vial of stem cells to be cryopreserved and stored with the bags for reinfusion. In the setting of graft failure, the pilot vial may be thawed and studies of cell viability and in vitro proliferative assays performed. The patient should also undergo detailed clinical assessment. The drug chart should always be reviewed carefully. If possible, myelosuppressive drugs should be stopped. For example, in a patient requiring CMV therapy with ganciclovir or valganciclovir, foscarnet or cidofovir may be used as less myelosuppressive alternatives. Co-trimoxazole may be substituted with pentamidine for Pneumocystis prophylaxis. Relevant clinical signs include moderate or massive splenomegaly which has occasionally been described in association with graft failure, for example in myelofibrosis. Appropriate tests for virology should be arranged including PCR assessment of EBV, CMV, parvovirus, adenovirus and HHV6 in particular. A bone marrow aspirate and trephine should be performed. This may show aplasia or hypoplasia, hemophagocytosis associated with viral infection, or infiltration by underlying disease in cases of hematologic malignancy. Virus-associated hemophagocytic syndrome may respond to treatment of the underlying virus and possibly to intravenous immunoglobulin infusion. Co-trimoxazole based immune suppression may be associated with megaloblastic changes in the marrow and treated with folinic acid. Blood and/or marrow should also be sent for donor–recipient chimerism analysis. Historically, this has been assessed by ABO grouping in cases of ABO-mismatched transplants, or cytogenetic analysis including X-Y chromosome fluorescence in situ hybridization (FISH)
for sex-mismatched transplants. More recent tests include characterization of donor and recipient DNA by PCR of variable number of tandem repeats (VNTR) which allows assessment of chimerism even in sex-matched transplants. In the allogeneic setting, graft failure is usually associated with loss of donor chimerism which may be complete or partial. Complete absence of donor chimerism usually implies an immune-based graft rejection process. The presence of graft failure in association with predominant donor chimerism may suggest an extrinsic source of marrow suppression, such as by a drug or virus.
Management of graft failure Growth factor administration Units differ as to their policies for the routine administration of growth factors such as granulocyte-colony stimulating factor (G-CSF) to transplant recipients. Large, retrospective registry studies have provided conflicting data as to whether this practice is beneficial or detrimental to patient survival.51,52 A small advantage in terms of the pace of neutrophil engraftment may, however, be expected. In the author’s unit, the routine use of G-CSF post transplant is reserved for patients receiving grafts with low CD34 content, such as in the setting of umbilical cord transplantation. In patients with suspected primary or secondary graft failure, evidence supports a trial of G-CSF or granulocyte macrophage-colony stimulating factor (GM-CSF).53 This is more likely to be successful where the cause of graft failure is related to an extrinsic factor (such as drug or viral toxicity) than to an intrinsic, immune-based rejection. Growth factors are often very useful in the management of patients with suboptimal myeloid engraftment caused by similar toxicities. Erythropoietin generally has a very limited role in the post-transplant setting.
Manipulation of immunosuppressive drugs In the setting of allogeneic stem cell transplantation, the fact that immune suppression can be withdrawn with continued donor engraftment and absence of GvHD implies that bidirectional tolerance has been successfully achieved. Transplants have been performed without any post-transplant immune suppression, and successful donor engraftment can occur. In such cases, intensive T-cell depletion is required to prevent GvHD. In patients at high risk of immunologic graft rejection such as those with aplastic anemia or β-thalassemia major who have received intensive transfusion regimens prior to transplantation, prolonged administration of ciclosporin for at least 1 year is recommended. This implies that immunosuppressive therapy with ciclosporin is suppressing a host-versus-graft immune-based rejection. In the case of a patient who is demonstrating signs of incipient or established graft failure, increasing immune suppression would therefore seem a logical therapeutic approach. This could be achieved by increasing the dose of drugs such as ciclosporin to the high end of the therapeutic range or by the addition of other drugs such as corticosteroids or mycophenolate mofetil. While this may be successful, particularly in cases of slow/progressive loss of donor chimerism, this maneuver is unlikely to correct established graft failure demonstrated by pancytopenia/marrow aplasia or by complete loss of donor chimerism. In the setting of a selective erythroid graft failure associated with ABO mismatch (this usually occurs in a blood group O recipient receiving transplantation from a blood group A or B donor with anti-A or anti-B antibodies lysing engrafting erythroid precursor cells in the marrow), increasing immune suppression may, however, be successful.54
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The cell source is also an important consideration. The yield of CD34+ cells is usually superior in peripheral blood rather than bone marrow collections. The composition of the graft may also be different, with a higher T-cell content and increased G-CSF driven myeloid progenitors. These differences may explain the significant improvements in the rate of neutrophil and platelet engraftment in recipients of PBPC transplantation compared to bone marrow transplantation (BMT).48 In certain conditions, however, bone marrow may be a preferable source of hemopoietic progenitor cells to peripheral blood. An example of this is aplastic anemia where a superior outcome has been described with BMT. This is mainly related to a reduced incidence of chronic GvHD.49 The rate of graft failure is higher in the unrelated donor setting than the matched related donor setting.2 Disparity at major and minor histocompatibility antigen loci is clearly a risk factor for an immunebased graft rejection.2 However, as the Perugia group first demonstrated, it is possible to obtain full donor engraftment even in the haploidentical setting, provided a very immune suppressive conditioning protocol is used, together with a high CD34 content in the infused graft, together with intensive T-cell depletion to prevent the complications of acute and chronic GvHD.50 In the unrelated umbilical cord setting, satisfactory donor engraftment is usually seen despite very low doses of CD34 cells infused and usually significant HLA mismatching. This may be a result of an enhanced in vivo proliferative capacity of umbilical cord-derived stem cells, or the presence of cellular or serum factors which enhance engraftment.
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Infusion of donor lymphocytes to correct graft failure With the advent of RIC transplantation, and particularly if T-cell antibodies are used pretransplant as part of the conditioning regimen with a resulting in vivo T-cell depletion, there has been interest in monitoring lineage-specific chimerism. It has been demonstrated that progressive loss of T-cell donor chimerism can be associated with graft failure. In the RIC setting, this can be a progressive process, with or without restoration of autologous hemopoiesis. Progressive loss of donor Tcell chimerism may also be associated with the potential for increased relapse risk.7 It has been demonstrated that this progressive loss of donor T-cell chimerism can be reversed by donor lymphocyte infusions (DLI).55 DLI are often given in a graded, incremental and increasing dose schedule to minimize the risk of GvHD. In the context of complete graft failure with loss of donor myeloid cells, DLI is unlikely to restore full donor hemopoiesis.
Infusion of further donor stem cells without prior conditioning Graft failure with loss of donor hemopoiesis may also be restored by a second infusion of donor stem cells.56 In patients who have previously received bone marrow as a source of stem cells for transplantation, a second request is often made for G-CSF mobilized peripheral blood progenitor cells, as the expected dose of CD34+ cells and T-cells may be higher. A second transplant procedure with unmanipulated donor stem cells may be successful in reversing graft failure associated with a prior T-cell depleted transplant approach. Because of the high T-cell content of such infusions, however, there is a significant risk of GvHD, and consideration therefore needs to be given to appropriate post-transplant immune suppression. Alternatively, recipients may be given a T-cell antibody such as ATG or alemtuzumab (CAMPATH 1H) prior to infusion of donor stem cells. Another approach has been to CD34 select the second transplant or use other methods of in vitro Tcell depletion. In practice, the author has found that infusion of a second transplant without prior conditioning is most applicable to patients who have demonstrated graft failure with pancytopenia but still have evidence of donor hemopoiesis on chimerism analysis. In these cases, the graft failure may be a result of inadequate initial donor CD34 counts, or subsequent suppression of donor hemopoiesis by drugs or viral infections.
Administration of second donation of stem cells with prior conditioning Clearly, this represents another approach in cases of established graft failure. A request may be made for a second donation of stem cells from the original donor, and a second transplant performed after further conditioning therapy.57 In patients who have received an initial RIC approach, consideration may be given to increasing the intensity of immune or myelosuppression for the second transplant. For patients who have initially received a full-intensity approach, however, a second transplant procedure is usually performed with RIC, perhaps with emphasis on increased immune suppression, in order to minimize the toxicity of the second transplant procedure.57 In patients who have initially received a pan T-cell antibody in vivo prior to the first transplant or another method of T-cell depletion, second transplants for graft failure may be performed with no T-cell depletion as a means of promoting donor engraftment. Clearly, in this situation the risks of GvHD are considerably increased, and due consideration therefore needs to be given to adequate post-transplant immune suppression.
There is also the possibility of requesting a new donor for the second transplant.58 In this setting, the patient will require further conditioning therapy along the lines discussed above, and this approach is occasionally successful. A second donation of stem cells, usually from the original donor following conditioning therapy, is the author’s preferred approach in cases of established graft failure with a total absence of donor chimerism (both T-cell and myeloid). A RIC approach is generally employed in this setting. Finally, it is also possible to rescue patients with graft failure and complete loss of donor chimerism with reinfusion of prior stored autologous stem cells.58 In this case, pretransplant conditioning is not required. Patients considered at high risk of graft failure may therefore be considered for pretransplant autologous back-up harvesting of marrow or peripheral blood stem cells. This includes recipients of mismatched related or unrelated donors and umbilical cord transplants. The main issue which needs to be considered here, however, is the prospect of an autologous graft contaminated by tumor cells, and this approach is therefore only recommended in patients who are known to be in complete remission in the setting of malignant disease.
Infusion of other cell products Marrow-derived mesenchymal stem cells (MSC) have a range of biologic properties which may be of therapeutic value in the field of human transplantation. This includes the ability of MSCs to support human hemopoiesis.59,60 Another interesting observation is the inhibitory effect of MSCs in in vitro models of mixed lymphocyte culture (MLC). This suggests an immunosuppressive effect of MSCs which may be exploited to clinical advantage. Early clinical studies of infusion of MSCs derived from donors or third parties (HLA matched or mismatched) have demonstrated a potential beneficial role in the treatment of acute GvHD and in treating or reducing the risk of graft failure in certain settings.59,60 Randomized clinical trials are now under way.
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Klaus J, Herrmann D, Breitkreutz I et al. Effect of CD34 cell dose on hematopoietic reconstitution and outcome in 508 patients with multiple myeloma undergoing autologous peripheral blood stem cell transplantation. Eur J Haematol 2007;78:11–28 Ringden O, Barrett AJ, Zhang MJ et al. Decreased treatment failure in recipients of HLAidentical bone marrow or peripheral blood stem cell transplants with high CD34 cell doses. Br J Haematol 2003;121:874–885 Weaver CH, Hazelton B, Birch R et al. An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 1995;86:3961–3969 Terakura S, Azuma E, Murata M et al. Hematopoietic engraftment in recipients of unrelated donor umbilical cord blood is affected by the CD34+ and CD8+ cell doses. Biol Blood Marrow Transplant 2007;13:822–830 Wagner JE, Barker JN, DeFor TE et al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002;100:1611–1618 Antonenas V, Garvin F, Webb M et al. Fresh PBSC harvests, but not BM, show temperaturerelated loss of CD34 viability during storage and transport. Cytotherapy 2006;8:158–165 Fois E, Desmartin M, Benhamida S et al. Recovery, viability and clinical toxicity of thawed and washed haematopoietic progenitor cells: analysis of 952 autologous peripheral blood stem cell transplantations. Bone Marrow Transplant 2007;40:831–835 Hale G, Waldmann H. CAMPATH-1 monoclonal antibodies in bone marrow transplantation. J Hematother 1994;3:15–31 Alvarnas JC, Forman SJ. Graft purging in autologous bone marrow transplantation: a promise not quite fulfilled. Oncology (Williston Park) 2004;18:867–876 Lucarelli G, Galimberti M, Polchi P et al. Bone marrow transplantation in patients with thalassemia. N Engl J Med 1990;322:417–421 Novotny VM, van Doom R, Witvliet MD et al. Occurrence of allogeneic HLA and nonHLA antibodies after transfusion of prestorage filtered platelets and red blood cells: a prospective study. Blood 1995;85:1736–1741 Guardiola P, Anderson JE, Bandini G et al. Allogeneic stem cell transplantation for agnogenic myeloid metaplasia: a European Group for Blood and Marrow Transplantation, Societe Francaise de Greffe de Moelle, Gruppo Italiano per il Trapianto del Midollo Osseo, and Fred Hutchinson Cancer Research Center Collaborative Study. Blood 1999;93: 2831–2838 Weber-Mzell D, Urban C, Benesch M et al. Durable remission following a third allogeneic stem cell transplantation in a patient with repeatedly relapsing SAA. The importance of stroma cells for sustained engraftment? Pediatr Transplant 2007;11:332–335 Helenglass G, Treleaven J, Parikh P et al. Delayed engraftment associated with splenomegaly in patients undergoing bone marrow transplantation for chronic myeloid leukaemia. Bone Marrow Transplant 1990;5:247–251 Pamphilon DH, Cornish JM, Goodman S et al. Successful second unrelated donor BMT in a child with juvenile chronic myeloid leukaemia: documentation of chimaerism using the polymerase chain reaction. Bone Marrow Transplant 1993;11:81–84 Slavin S, Nagler A, Naparstek E et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 1998;91:356–363 Niederwieser D, Maris M, Shizuru JA et al. Low-dose total body irradiation (TBI) and fludarabine followed by hematopoietic cell transplantation (HCT) from HLA-matched or mismatched unrelated donors and postgrafting immunosuppression with cyclosporine and mycophenolate mofetil (MMF) can induce durable complete chimerism and sustained remissions in patients with hematological diseases. Blood 2003;101:4620–4629 Kottaridis PD, Milligan DW, Chopra R et al. In vivo CAMPATH-1H prevents graft-versushost disease following nonmyeloablative stem cell transplantation. Blood 2000;96: 2419–2425 Hale G, Jacobs P, Wood L et al. CD52 antibodies for prevention of graft-versus-host disease and graft rejection following transplantation of allogeneic peripheral blood stem cells. Bone Marrow Transplant 2000;26:69–76 Plentz A, Hahn J, Holler E et al. Long-term parvovirus B19 viraemia associated with pure red cell aplasia after allogeneic bone marrow transplantation. J Clin Virol 2004; 31:16–19 Kawabata Y, Hirokawa M, Saitoh Y et al. Late-onset fatal Epstein-Barr virus-associated hemophagocytic syndrome following cord blood cell transplantation for adult acute lymphoblastic leukemia. Int J Hematol 2006;84:545–548 Steffens HP, Podlech J, Kurz S et al. Cytomegalovirus inhibits the engraftment of donor bone marrow cells by downregulation of hemopoietin gene expression in recipient stroma. J Virol 1998;72:5006–5015 Johnston RE, Geretti AM, Prentice HG et al. HHV-6-related secondary graft failure following allogeneic bone marrow transplantation. Br J Haematol 1999;105:4041– 4043 Levy J, Wodell RA, August CS, Bayever E. Adenovirus-related hemophagocytic syndrome after bone marrow transplantation. Bone Marrow Transplant 1990;6:349–352
Incidence of graft failure Graft failure in the setting of autologous transplantation is a rare event in modern practice, with an incidence <1%.1 The position with regard to allogeneic transplantation is more complex. In the standard setting of FIC HLA-identical sibling transplantation with no T-cell depletion,
The clinical impact of graft failure The standard complications of graft failure include prolonged hospitalization, an increased risk of bacterial and fungal infection and risk of hemorrhage.5,6 This may lead to an increased risk of transplantrelated mortality. In addition, there is evidence that incomplete donor chimerism is associated with an increased relapse risk.7
Mechanisms of graft failure In the allogeneic setting, the concept of an immunologic process causing graft rejection is well established. Historically, this is best described in the setting of allogeneic transplantation for patients with severe aplastic anemia8 and may be in part attributed to alloimmunization from previous blood transfusions. An immunologic mechanism of graft failure or rejection is also associated more with recipients of HLA-mismatched and unrelated donor transplants than those with matched related donors. In the unrelated donor setting, it has been shown that HLA class II disparity between donor and recipient may not increase the rate of graft rejection but class I disparity, especially at the HLA C locus, is associated with this complication.9,10 This is believed to be predominantly a T-cell mediated process involving donor cytotoxic T-cells, although there is also evidence for a natural killer (NK) cell-mediated graft rejection process.11,12 Immunologic graft rejection is usually characterized by a transient process of donor engraftment followed by loss of donor cells and either a complete failure of hemopoiesis or a recovery of autologous reconstitution, the latter being more typical with RIC transplants. Another mechanism of graft failure relates to the composition of the donor graft. Clearly, graft failure may be due to an inadequate number of transplanted hemopoietic stem cells in both the allogeneic and autologous setting. Many studies have been published indicating the requirement for engraftment in terms of mononuclear cell (MNC) numbers, and particularly CD34+ (stem cell) numbers, infused in both the allogeneic and autologous settings.13–15 The commonly quoted threshold for prompt and reliable engraftment is the infusion of ≥2 ×
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MANAGEMENT OF POST-TRANSPLANT COMPLICATIONS
The traditional approach to transplantation involves myeloablative conditioning with chemotherapy and/or radiotherapy resulting in a subsequent period of obligate pancytopenia followed by recovery of hemopoiesis. In the setting of myeloablative or full-intensity conditioned (FIC) transplantation, myeloid engraftment is often defined as the first day that a neutrophil count of 0.5 × 109/l is achieved and sustained. Red cell and platelet engraftment implies independence from transfusion support. A platelet count of 20 × 109/l and/or 50 × 109/l sustained and transfusion independent are useful clinical definitions of megakaryocyte engraftment. Primary graft failure implies a failure to ever achieve these target values. Secondary graft failure implies an initial achievement of these parameters but subsequent cytopenias with a neutrophil count <0.5 × 109/l and requirement for transfusions. The diagnosis of graft failure will also usually require bone marrow examination to demonstrate hypocellularity and lack of infiltration by the original malignant disease or another process. These definitions of primary and secondary graft failure are applicable to both the allogeneic and autologous settings. In recent years, however, the introduction of non-myeloablative or reduced-intensity conditioning (RIC) allogeneic transplantation requires supplementary definitions of graft failure. Patients may no longer have an obligate period of pancytopenia following conditioning, and engraftment may be a seamless transition from recipient to donor hemopoiesis. In this case, graft failure may be better defined in terms of chimerism analysis with a failure to achieve or subsequent loss of donor chimerism. The traditional definitions of graft failure may also not take into account deficiencies specific to individual lineages, e.g. red cell aplasia which is now well documented following allogeneic transplantation. There is also the concept of poor graft function, where the basic parameters of engraftment are fulfilled but blood counts may remain suboptimal for long periods of time, leading to potential complications. Both primary and secondary graft failure are usually evident within the first 6 months following transplantation, although late graft failure can occur and historically has been seen more commonly in patients with severe aplastic anemia and β-thalassemia major. In patients with malignant disease, late graft failure may herald subsequent relapse of the original clone.
the rate of graft failure is of the order of 1–2%.2 T-cell depletion increases the risk of graft failure.3 Recipients of RIC transplants are also at higher risk of this complication.4 Unrelated donor transplants and HLA-mismatched donor transplants (related/unrelated) may also be associated with a higher risk of graft failure.2 For example, in the setting of a RIC transplant or a T-cell depleted FIC transplant with an HLA-mismatched donor transplant, graft failure rates of 5–30% may occur.2–4
PART
Introduction
CHAPTER 37
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5
106 CD34 cells per kg of recipient body weight. This may represent a gross oversimplification, however, and it is difficult to define a lower limit for successful engraftment in clinical practice. The source of stem cells also needs to be taken into account. In the field of umbilical cord transplantation, satisfactory engraftment may be obtained with CD34 counts more than 1 log below the above figure.16,17 CD34 numbers are clearly not the only determinant of engraftment in an infused stem cell product. The presence of other populations of cells, such as T-cells or even serum factors, may affect the engraftment process.16 However, clinically the pace of engraftment is more rapid with peripheral blood stem cell transplantation than bone marrow transplantation, which, in turn, exceeds that seen in the setting of umbilical cord transplantation, and this is predominantly a CD34 numbers effect. The quality of stem cell products in terms of engraftment potential may also be adversely affected by events occurring following collection of cells. Examples include prolonged storage prior to cryopreservation,18 the cryopreservation process itself and subsequent thawing procedure,19 in vitro manipulation of the graft (e.g. CD34 selection3), incubation with antibodies for T-cell depletion20 or malignant cell purging.21
Host factors which may affect engraftment Patients with certain diagnoses are at higher risk of graft rejection than others. This relates to the extent to which the patients have already received myelo- or immunosuppressive therapies, the number of transfusions of cellular products received and possible stromal defects in the marrow microenvironment associated with certain diagnoses. Patients with severe aplastic anemia and β-thalassemia major may have typically received tens or hundreds of units of cellular blood products prior to transplantation. This may have the effect of alloimmunization which increases the risk of graft failure.8,22,23 Patients with myeloproliferative disorders including chronic myeloid leukemia may have received little in the way of intensive chemotherapy prior to transplantation. The increased cellularity in the bone marrow of patients with myeloproliferative disorders coupled with the lack of intensive prior chemotherapy compared to patients with acute leukemia may increase the risk of graft failure in these diseases. Stromal abnormalities occur in myelofibrosis where the normal bone marrow stroma is replaced by a dense fibrotic reaction.24 In severe aplastic anemia, it is possible that stromal defects contribute to the etiology of the disease in certain patients. If these are not corrected by transplantation, similar problems may ensue with the donorderived hemopoiesis.25 Finally, patients with moderate to massive splenomegaly at the time of transplantation may be at higher risk of graft failure.24,26 This may be as a result of preferential donor engraftment in the spleen rather than the marrow and subsequent inefficient hemopoiesis coupled with the effects of hypersplenism.26,27 The intensity of the conditioning regimen may also affect the engraftment process. In the last 10 years there has been the successful introduction of RIC transplantation with non-myeloablative chemotherapy and/or low-dose total-body irradiation regimens. In simple terms, conditioning treatment needs to have two elements: myelosuppression and immune suppression. The myelosuppressive element creates space for the incoming donor hemopoiesis and the immunosuppressive element eradicates the potential for host-versus-graft immune reactions. The former element (myelosuppression) is substantially attenuated in RIC transplantation, with an associated increase in the potential for graft failure. Therefore, this is usually compensated for by increasing the immunosuppressive element of the conditioning regimen. In practice, this has usually been achieved by the use of
fludarabine, a purine analog with very potent T-cell suppressive properties, in conjunction with non-myeloablative doses of chemotherapy – busulfan, melphalan or cyclophosphamide – or low-dose total-body irradiation.28,29 In vivo administration of T-cell antibodies such as antithymocyte globulin (ATG) or alemtuzumab (CAMPATH-1H) prior to transplantation is an alternative approach with the potential additional benefit of reduction in graft-versus-host disease (GvHD) risk due to T-cell depletion of the incoming graft.30,31 Infections which occur in the early post-transplant phase may be a cause of non- or suboptimal engraftment. Certain viruses such as parvovirus may directly infect hemopoietic cell progenitors, leading to a failure of maturation. In particular, parvovirus targets erythroid progenitors and has been associated with pure red cell aplasia.32 Many viruses, particularly those of the herpes group, have been shown to cause the potential for hemophagocytosis, which may be a cause of graft failure. Examples include the Epstein–Barr virus (EBV),33 cytomegalovirus (CMV)34 and human herpes virus 6 (HHV6).35 Other viruses which may also stimulate hemophagocytosis include adenoviral36 and influenza viral infections.37 Myelosuppressive drugs may also be a cause of graft failure or impairment. The choice of post-transplant immunosuppressive regimen may affect engraftment. Ciclosporin is generally considered to be ‘protective’ against graft failure by reducing the chance of a host-versus-graft immune-based rejection. Patients with aplastic anemia and β-thalassemia major generally receive prolonged treatment with ciclosporin A to at least 1 year post transplant to prevent late graft rejection in these high-risk disorders. Ciclosporin is often used with 3–4 injections of methotrexate. The latter increases the potency of post-transplant immune suppression and reduces the incidence of severe GvHD. However, methotrexate is also myelosuppressive and its use usually does lead to a delay in engraftment of neutrophils by around 7 days.38 Other drugs may also have myelosuppressive effects. Co-trimoxazole, used for prophylaxis against Pneumocystis infections, may lead to macrocytosis and cytopenias.39 Ganciclovir and its prodrug valganciclovir, used to prevent or treat CMV infection, are a commonly recognized cause of often severe myelosuppression.40,41 Mycophenolate mofetil, which may be used for post-transplant immune suppression or in the treatment of GvHD, may also be myelosuppressive.42 H2 antagonists have also been associated with graft failure.43 Rare causes of graft failure relate to nutritional factors such as folic acid deficiency, vitamin B12 deficiency and protein energy malnutrition. Delayed red cell engraftment, including rare cases of pure red cell aplasia, may also be seen in cases of ABO mismatched transplantation.44 Intensive T-cell depletion may delete subsets of donor T-cells which support engraftment. One such population of cells has been named ‘veto cells’.45,46 These have been described in animal models of transplantation and shown to counteract host-versus-graft immune-based rejection. This may account for the high rate of graft rejection seen in the T-cell depleted setting.
Donor factors which may affect engraftment In the unrelated donor setting, male donors are usually preferred over female donors. There are several reasons for this. The collection of cells, particularly peripheral blood progenitor cells (PBPC) by pheresis, may be technically easier because of better venous access. A higher yield of stem cells may result because of this and increased donor weight. In female donors, alloimmunization from prior pregnancies may theoretically increase the potential for graft failure.47 Donor age may also have an effect on cell yield and engraftment, and younger donors are generally preferred.
Approach to the patient with graft failure Suspicions should be raised in the patient who has not engrafted neutrophils by day 21 or certainly day 28. The latter time-point may be more appropriate in recipients receiving post-transplant methotrexate, where engraftment is typically delayed. Initially, it is recommended that the details of the infused stem cells product are reviewed, including CD34 and MNC counts. The procedures undertaken in the cell processing laboratory should also be reviewed. It is good practice in the stem cell laboratory for a pilot vial of stem cells to be cryopreserved and stored with the bags for reinfusion. In the setting of graft failure, the pilot vial may be thawed and studies of cell viability and in vitro proliferative assays performed. The patient should also undergo detailed clinical assessment. The drug chart should always be reviewed carefully. If possible, myelosuppressive drugs should be stopped. For example, in a patient requiring CMV therapy with ganciclovir or valganciclovir, foscarnet or cidofovir may be used as less myelosuppressive alternatives. Co-trimoxazole may be substituted with pentamidine for Pneumocystis prophylaxis. Relevant clinical signs include moderate or massive splenomegaly which has occasionally been described in association with graft failure, for example in myelofibrosis. Appropriate tests for virology should be arranged including PCR assessment of EBV, CMV, parvovirus, adenovirus and HHV6 in particular. A bone marrow aspirate and trephine should be performed. This may show aplasia or hypoplasia, hemophagocytosis associated with viral infection, or infiltration by underlying disease in cases of hematologic malignancy. Virus-associated hemophagocytic syndrome may respond to treatment of the underlying virus and possibly to intravenous immunoglobulin infusion. Co-trimoxazole based immune suppression may be associated with megaloblastic changes in the marrow and treated with folinic acid. Blood and/or marrow should also be sent for donor–recipient chimerism analysis. Historically, this has been assessed by ABO grouping in cases of ABO-mismatched transplants, or cytogenetic analysis including X-Y chromosome fluorescence in situ hybridization (FISH)
for sex-mismatched transplants. More recent tests include characterization of donor and recipient DNA by PCR of variable number of tandem repeats (VNTR) which allows assessment of chimerism even in sex-matched transplants. In the allogeneic setting, graft failure is usually associated with loss of donor chimerism which may be complete or partial. Complete absence of donor chimerism usually implies an immune-based graft rejection process. The presence of graft failure in association with predominant donor chimerism may suggest an extrinsic source of marrow suppression, such as by a drug or virus.
Management of graft failure Growth factor administration Units differ as to their policies for the routine administration of growth factors such as granulocyte-colony stimulating factor (G-CSF) to transplant recipients. Large, retrospective registry studies have provided conflicting data as to whether this practice is beneficial or detrimental to patient survival.51,52 A small advantage in terms of the pace of neutrophil engraftment may, however, be expected. In the author’s unit, the routine use of G-CSF post transplant is reserved for patients receiving grafts with low CD34 content, such as in the setting of umbilical cord transplantation. In patients with suspected primary or secondary graft failure, evidence supports a trial of G-CSF or granulocyte macrophage-colony stimulating factor (GM-CSF).53 This is more likely to be successful where the cause of graft failure is related to an extrinsic factor (such as drug or viral toxicity) than to an intrinsic, immune-based rejection. Growth factors are often very useful in the management of patients with suboptimal myeloid engraftment caused by similar toxicities. Erythropoietin generally has a very limited role in the post-transplant setting.
Manipulation of immunosuppressive drugs In the setting of allogeneic stem cell transplantation, the fact that immune suppression can be withdrawn with continued donor engraftment and absence of GvHD implies that bidirectional tolerance has been successfully achieved. Transplants have been performed without any post-transplant immune suppression, and successful donor engraftment can occur. In such cases, intensive T-cell depletion is required to prevent GvHD. In patients at high risk of immunologic graft rejection such as those with aplastic anemia or β-thalassemia major who have received intensive transfusion regimens prior to transplantation, prolonged administration of ciclosporin for at least 1 year is recommended. This implies that immunosuppressive therapy with ciclosporin is suppressing a host-versus-graft immune-based rejection. In the case of a patient who is demonstrating signs of incipient or established graft failure, increasing immune suppression would therefore seem a logical therapeutic approach. This could be achieved by increasing the dose of drugs such as ciclosporin to the high end of the therapeutic range or by the addition of other drugs such as corticosteroids or mycophenolate mofetil. While this may be successful, particularly in cases of slow/progressive loss of donor chimerism, this maneuver is unlikely to correct established graft failure demonstrated by pancytopenia/marrow aplasia or by complete loss of donor chimerism. In the setting of a selective erythroid graft failure associated with ABO mismatch (this usually occurs in a blood group O recipient receiving transplantation from a blood group A or B donor with anti-A or anti-B antibodies lysing engrafting erythroid precursor cells in the marrow), increasing immune suppression may, however, be successful.54
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The cell source is also an important consideration. The yield of CD34+ cells is usually superior in peripheral blood rather than bone marrow collections. The composition of the graft may also be different, with a higher T-cell content and increased G-CSF driven myeloid progenitors. These differences may explain the significant improvements in the rate of neutrophil and platelet engraftment in recipients of PBPC transplantation compared to bone marrow transplantation (BMT).48 In certain conditions, however, bone marrow may be a preferable source of hemopoietic progenitor cells to peripheral blood. An example of this is aplastic anemia where a superior outcome has been described with BMT. This is mainly related to a reduced incidence of chronic GvHD.49 The rate of graft failure is higher in the unrelated donor setting than the matched related donor setting.2 Disparity at major and minor histocompatibility antigen loci is clearly a risk factor for an immunebased graft rejection.2 However, as the Perugia group first demonstrated, it is possible to obtain full donor engraftment even in the haploidentical setting, provided a very immune suppressive conditioning protocol is used, together with a high CD34 content in the infused graft, together with intensive T-cell depletion to prevent the complications of acute and chronic GvHD.50 In the unrelated umbilical cord setting, satisfactory donor engraftment is usually seen despite very low doses of CD34 cells infused and usually significant HLA mismatching. This may be a result of an enhanced in vivo proliferative capacity of umbilical cord-derived stem cells, or the presence of cellular or serum factors which enhance engraftment.
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Infusion of donor lymphocytes to correct graft failure With the advent of RIC transplantation, and particularly if T-cell antibodies are used pretransplant as part of the conditioning regimen with a resulting in vivo T-cell depletion, there has been interest in monitoring lineage-specific chimerism. It has been demonstrated that progressive loss of T-cell donor chimerism can be associated with graft failure. In the RIC setting, this can be a progressive process, with or without restoration of autologous hemopoiesis. Progressive loss of donor Tcell chimerism may also be associated with the potential for increased relapse risk.7 It has been demonstrated that this progressive loss of donor T-cell chimerism can be reversed by donor lymphocyte infusions (DLI).55 DLI are often given in a graded, incremental and increasing dose schedule to minimize the risk of GvHD. In the context of complete graft failure with loss of donor myeloid cells, DLI is unlikely to restore full donor hemopoiesis.
Infusion of further donor stem cells without prior conditioning Graft failure with loss of donor hemopoiesis may also be restored by a second infusion of donor stem cells.56 In patients who have previously received bone marrow as a source of stem cells for transplantation, a second request is often made for G-CSF mobilized peripheral blood progenitor cells, as the expected dose of CD34+ cells and T-cells may be higher. A second transplant procedure with unmanipulated donor stem cells may be successful in reversing graft failure associated with a prior T-cell depleted transplant approach. Because of the high T-cell content of such infusions, however, there is a significant risk of GvHD, and consideration therefore needs to be given to appropriate post-transplant immune suppression. Alternatively, recipients may be given a T-cell antibody such as ATG or alemtuzumab (CAMPATH 1H) prior to infusion of donor stem cells. Another approach has been to CD34 select the second transplant or use other methods of in vitro Tcell depletion. In practice, the author has found that infusion of a second transplant without prior conditioning is most applicable to patients who have demonstrated graft failure with pancytopenia but still have evidence of donor hemopoiesis on chimerism analysis. In these cases, the graft failure may be a result of inadequate initial donor CD34 counts, or subsequent suppression of donor hemopoiesis by drugs or viral infections.
Administration of second donation of stem cells with prior conditioning Clearly, this represents another approach in cases of established graft failure. A request may be made for a second donation of stem cells from the original donor, and a second transplant performed after further conditioning therapy.57 In patients who have received an initial RIC approach, consideration may be given to increasing the intensity of immune or myelosuppression for the second transplant. For patients who have initially received a full-intensity approach, however, a second transplant procedure is usually performed with RIC, perhaps with emphasis on increased immune suppression, in order to minimize the toxicity of the second transplant procedure.57 In patients who have initially received a pan T-cell antibody in vivo prior to the first transplant or another method of T-cell depletion, second transplants for graft failure may be performed with no T-cell depletion as a means of promoting donor engraftment. Clearly, in this situation the risks of GvHD are considerably increased, and due consideration therefore needs to be given to adequate post-transplant immune suppression.
There is also the possibility of requesting a new donor for the second transplant.58 In this setting, the patient will require further conditioning therapy along the lines discussed above, and this approach is occasionally successful. A second donation of stem cells, usually from the original donor following conditioning therapy, is the author’s preferred approach in cases of established graft failure with a total absence of donor chimerism (both T-cell and myeloid). A RIC approach is generally employed in this setting. Finally, it is also possible to rescue patients with graft failure and complete loss of donor chimerism with reinfusion of prior stored autologous stem cells.58 In this case, pretransplant conditioning is not required. Patients considered at high risk of graft failure may therefore be considered for pretransplant autologous back-up harvesting of marrow or peripheral blood stem cells. This includes recipients of mismatched related or unrelated donors and umbilical cord transplants. The main issue which needs to be considered here, however, is the prospect of an autologous graft contaminated by tumor cells, and this approach is therefore only recommended in patients who are known to be in complete remission in the setting of malignant disease.
Infusion of other cell products Marrow-derived mesenchymal stem cells (MSC) have a range of biologic properties which may be of therapeutic value in the field of human transplantation. This includes the ability of MSCs to support human hemopoiesis.59,60 Another interesting observation is the inhibitory effect of MSCs in in vitro models of mixed lymphocyte culture (MLC). This suggests an immunosuppressive effect of MSCs which may be exploited to clinical advantage. Early clinical studies of infusion of MSCs derived from donors or third parties (HLA matched or mismatched) have demonstrated a potential beneficial role in the treatment of acute GvHD and in treating or reducing the risk of graft failure in certain settings.59,60 Randomized clinical trials are now under way.
References 1. Wannesson L, Panzarella T, Mikhael J, Keating A. Feasibility and safety of autotransplants with noncryopreserved marrow or peripheral blood stem cells: a systematic review. Ann Oncol 2007;18:623–632 2. Ottinger HD, Ferencik S, Beelen DW et al. Hematopoietic stem cell transplantation: contrasting the outcome of transplantations from HLA-identical siblings, partially HLAmismatched related donors, and HLA-matched unrelated donors. Blood 2003;102: 1131–1137 3. Urbano-Ispizua A, Rozman C, Pimentel C et al. The number of donor CD3+ cells is the most important factor for graft failure after allogeneic transplantation of CD34+ selected cells from peripheral blood from HLA-identical siblings. Blood 2001;97: 383–387 4. Baron F, Baker JE, Storb R et al. Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 2004;104:2254–2262 5. Offner F, Schoch G, Fisher LD et al. Mortality hazard functions as related to neutropenia at different times after marrow transplantation. Blood 1996;88:4058–4062 6. Morrison VA, Haake RJ, Weisdorf DJ. Non-candida fungal infections after bone marrow transplantation: risk factors and outcome. Am J Med 1994;96:497–503 7. Mattsson J, Uzunel M, Tammik L et al. Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation. Leukemia 2001;15: 1976–1985 8. Storb R, Thomas ED, Weiden PL et al. Aplastic anemia treated by allogeneic bone marrow transplantation: a report on 49 new cases from Seattle. Blood 1976;48:817–841 9. Petersdorf EW, Longton GM, Anasetti C et al. Association of HLA-C disparity with graft failure after marrow transplantation from unrelated donors. Blood 1997 1;89:1818– 1823 10. Petersdorf EW, Kollman C, Hurley CK et al. Effect of HLA class II gene disparity on clinical outcome in unrelated donor hematopoietic cell transplantation for chronic myeloid leukemia: the US National Marrow Donor Program Experience. Blood 2001;98:2922–2929 11. Murphy WJ, Kumar V, Bennett M. Rejection of bone marrow allografts by mice with severe combined immune deficiency (SCID). Evidence that natural killer cells can mediate the specificity of marrow graft rejection. J Exp Med 1987;165:1212–1217 12. Jukes JP, Wood KJ, Jones ND. Natural killer T cells: a bridge to tolerance or a pathway to rejection? Transplantation 2007;84:679–681
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