Transplants across human leukocyte antigen barriers

Transplants Across Human Leukocyte Antigen Barriers Massimo F. Martelli, Franco Aversa, Ester Bachar-Lustig, Andrea Velardi, Shlomit Reich-Zelicher, Antonio Tabilio, Hilit Gur, and Yair Reisner Clinical experience with full haplotype-mismatched stem cell transplants has a 20-year history. Early results in leukemia patients were disappointing because of a high incidence of severe graft-versus-host disease (GvHD) in T-replete transplants or high rejection rates in T-cell– depleted transplants. The breakthrough came with introduction of a megadose T-cell– depleted progenitor cell transplant following a high-intensity conditioning regimen and the realization that donor natural killer (NK) cell alloreactivity also plays a role in facilitating engraftment and in preventing relapse. Treating end-stage patients inevitably confounded clinical outcome in early pilot studies. Today, highrisk acute leukemia patients are treated at less advanced stages of disease, receive a reasonably well-tolerated conditioning regimen, and benefit from advances in post-transplant immunological reconstitution. These factors have markedly reduced transplant-related mortality. Overall, event-free survival (EFS) and transplant-related mortality (TRM) compare favorably with reports from unrelated matched transplants. T-cell– depleted megadose stem cell transplant from a mismatched family member, who is immediately available, can now be offered as a viable option to candidates with high-risk acute leukemias. Semin Hematol 39:48-56. Copyright © 2002 by W.B. Saunders Company.

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ONE MARROW transplantation (BMT) offers a cure for many patients with leukemia or other hematological disorders. In the world registry network, which includes more than 5 million human leukocyte antigen (HLA)-typed volunteers, the odds of finding a matched unrelated donor vary according to race and range from approximately 60% to 70% for Caucasians to less than 10% for ethnic minorities.9,20 Major limitations are age restrictions and closer matching which, paradoxically, reduces the chance of finding a suitable matched donor. Another drawback for a patient who urgently needs a transplant is the time lapse from registration to identifying a donor from the potential panel and harvesting the bone marrow cells. For these reasons, BMT is not feasible for approximately half of the candidates. On the other hand, virtually all patients have an HLA-haploidentical two- or three-loci mismatched family member who is immediately available. However, unmanipulated BMT from these donors has been largely unsuccessful because of the high incidence of severe graftversus-host disease (GvHD) and graft rejection.1 In an attempt to facilitate engraftment without excessive GvHD, Henslee-Downey et al22 exploited the princiFrom the Department of Hematology, University of Perugia, Perugia, Italy; and the Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. Supported in part by a AIRC grant to M.F.M. and A.V. Address reprint requests to Massimo F. Martelli, MD, Ematologia e Immunologia Clinica, Universita` di Perugia, Policlinico Monteluce, 06100 Perugia, Italy. Copyright © 2002 by W.B. Saunders Company 0037-1963/02/3901-0006$35.00/0 doi:10.1053/shem.2002.29255

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ple of sequential immunomodulation in the recipient by combining partial ex vivo T-cell depletion of the donor bone marrow with the T10B9 monoclonal antibody and in vivo T-cell lysis with the immunotoxin H65-RTA. In pediatric patients, they reported 0.88, 0.16, 0.51, and 0.35 probabilities of engraftment, acute GvHD, chronic GvHD, and survival, respectively. Guinan et al16 attempted to tolerize alloreactive T cells in the graft by stimulating donor bone marrow cells against host cells in the presence of cytolytic T lymphocyte-A4 (CTLA-4). Primary engraftment was achieved in nine of 12 children, but three developed acute GvHD. Post-transplant immunosuppressive therapy, with its inherent risk of infection-related deaths, was necessary to prevent GvHD. Numerous clinical trials have demonstrated that extensive ex vivo T-cell depletion of bone marrow, without any post-transplant prophylaxis, prevents acute and chronic GvHD without impairing engraftment in HLA-identical sibling transplants,5,32,34 even in patients with severe combined immunodeficiency (SCID) who receive a transplant from HLA-haploidentical three-loci mismatched family members.39 Unfortunately, when tested in leukemia patients, full haplotype-mismatched T-cell– depleted BMT was associated with a high incidence of graft failure.23

The HLA Barrier in T-Cell–Depleted Transplants Resistance to engraftment is mediated primarily by host-derived cytotoxic T lymphocytes.23,38 Animal models showed that the recipient immune system that survives after lethal total body irradiation (TBI) can be suppressed by increasing the dose of TBI47 or

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Transplants Across HLA Barriers

Figure 1. Conditioning regimens during the periods March 1993 to August 1995 and October 1995 to October 2000 used for full haplotype-mismatched transplants.

by combining the standard dose with selective anti– T-cell measures with low extrahematological toxicity.12,26 Engraftment is also enhanced when myeloablative drugs (dimethyl-myleran, busulfan, or thiotepa) are combined with TBI.27,52 However, enhancing immunosuppression and myeloablation by adding antithymocyte globulin (ATG) and thiotepa to single-dose TBI and cyclophosphamide (CY), as we did in leukemia patients in 1992, still did not ensure engraftment of conventional doses of full haplotype-mismatched T-cell– depleted bone marrow cells (Aversa et al, unpublished observations). The barrier to engraftment of T-cell– depleted mismatched transplants was first overcome by the clinical application of the stem cell megadose, a principle successfully pioneered in experimental models in the late 1980s. Escalating doses of T-cell– depleted mismatched bone marrow cells were associated with full donor type engraftment in mice that had been presensitized with donor lymphocytes,6,27 or whose immune system had been partially reconstituted with graduated numbers of host T cells before the transplant,27 and in animals pretreated with sublethal doses of TBI, which spare a substantial number of recipient T lymphocytes.27,40 In 1993 the cell doseescalation concept was first tested in humans by supplementing bone marrow with granulocyte colonystimulating factor (G-CSF)–mobilized peripheral blood progenitor cells (PBPCs), both depleted of T lymphocytes by soybean agglutination and E-rosetting.3,41 The recipients were conditioned with a highly immunosuppressive and myeloablative regi-

men that included TBI in a single fraction at fast dose-rate, CY, ATG, and thiotepa. Eighty percent of the 36 adults with advanced-stage acute leukemia achieved primary sustained engraftment. Although no post-transplant immunosuppressive therapy was used as prophylaxis, the incidence of GvHD was significantly lower than in T-cell–replete mismatched transplants. Over time, modifications to our approach have led to remarkable progress. In October 1995 fludarabine was substituted for CY in the conditioning regimen (Fig 1) in order to minimize extrahematological toxicity.4 At the same time, with the aim of eliminating GvHD, we reduced the number of CD3⫹ cells in the graft to a mean of 2 ⫻ 104/kg recipient body weight (or 1 log less than in the previous study). Therefore, PBPCs were depleted of T lymphocytes by E-rosetting followed by CD34⫹ selection (Table 1). Although we employed for the first time positive selection of CD34 cells, thereby drastically eliminating potential non–T-facilitating cells, the excellent engraftment rate was not affected. Since January 1999, CD34⫹ cells have been selected in a one-step procedure using the Clinimacs device (Miltenyi Biotec, Bergisch Gladbach, Germany) (Table 1).30 We obtained the same degree of CD34⫹ cell purification and found this approach to be simpler and less timeconsuming than former methods. Furthermore, posttransplant G-CSF administration to the recipients was stopped because experimental data suggested it induces immunosuppression.33,49 Table 2 shows the effects of all these changes on

Table 1. Graft Processing and Graft Composition Years

No. of patients Methods CD34⫹ ⫻ 106/kg CD3⫹ ⫻ 104/kg

1993-1995

1995-1997

1999-2000

36 Lectin SBA E-rosette 10.8 22.4

43 E-rosette CD34⫹ selection (by Ceprate SC) 10.5 2.0

33 CD34⫹ selection (by Clinimacs) 12 1.0

Abbreviation: SBA, soybean agglutinin.

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Table 2. Full Haplotype-Mismatched Transplantation for Acute Leukemia (October 1995-October 2000) No. of patients Median age (range), yr Disease AML ALL Status at transplant High-risk CR I CR ⱖ II Relapse Graft composition CD34⫹ (⫻ 106/kg) CD3⫹ (⫻ 104/kg) Engraftment Primary Overall Days to Neutrophils ⬎ 1.0 ⫻ 109/L Platelets ⬎ 25 ⫻ 109/L Acute GvHD ⱖ II/evaluable Chronic GvHD/evaluable

76 24 (4-58) 41 35 11 31 34 11.5 1.5 70 (92%) 75 (98%) 11 17 3/72 3/65

NOTE. Grafts were processed by the Ceprate SC system for 43 patients and by the Clinimacs device in 33.

engraftment and GvHD. In a global analysis of the 76 high-risk acute leukemia patients who have received transplants since October 1995, primary sustained engraftment was achieved in 70 (92%). Five of the six exhibiting primary rejection successfully engrafted after second transplants from different haploidentical family donors. Hematopoietic recovery was extremely rapid, with neutrophil counts reaching 1 ⫻ 109/L and platelet counts 25 ⫻ 109/L at a median of 11 days (range, 8 to 19) and 17 days (range, 12 to 84), respectively. As expected, in the last 33 patients neutrophil recovery was 4 days later than in the 43 patients who received post-transplant G-CSF. Analysis of DNA polymorphism documented full donortype chimerism in both the peripheral blood and the bone marrow of all available patients. Three patients developed acute GvHD, grade II to IV, which progressed to chronic in two. Fludarabine-based conditioning was well tolerated even in these advancedstage, heavily pretreated leukemia patients. There was no veno-occlusive disease of the liver; severe oral mucositis was minimal. Thus far, no patient has died of lung toxicity since total radiation dose on the lungs was reduced to 4 Gy. Handgretinger et al19 reported similar engraftment rates and no GvHD in children with acute leukemia after a chemotherapy-alone based conditioning regimen. In the Perugia and Tu¨bingen studies, GvHD was prevented by ex vivo T-cell depletion alone. The inocula contained a number of T lymphocytes similar to the threshold dose established to prevent GvHD in

SCID patients receiving mismatched transplants. Another factor that may have contributed to T-cell depletion in vivo, thereby reducing the incidence of GvHD, was the inclusion of ATG in the conditioning regimen, which persisted in plasma for several days. Similarly, in the Tu¨bingen trial OKT3 was administered during conditioning and after the transplant. Another inference emerging from these two reports is that whether or not TBI is part of the conditioning, the engraftment rate does not appear to be affected when a stem cell megadose is given. A feasible hypothesis to explain how CD34⫹ cell megadoses overcome the barrier of residual donorspecific host cytotoxic T lymphocyte precursors (CTL-p) after immunomyeloablative conditioning is that CD34⫹ cells exert “veto” activity; “veto” relates to the ability of cells to neutralize CTL-p directed against their antigens.11,46 Indeed, when human, purified CD34⫹ cells were added to bulky mixed lymphocyte cultures, they reduced the frequencies of CTL-p against matched stimulators but not against stimulators from a third party.36 This effect was exhibited only if the cells are added within the first 48 hours of a mixed lymphocyte culture. Therefore, the veto effect seems to be directed against CTL-p, but does not affect differentiated anti-donor CTL. Preliminary data suggest that the veto activity of human CD34⫹ cells, similarly to other veto cells described in the literature, is mediated by apoptosis.17 However, it seems that both CD8 and FasL, which are crucial for the veto activity of CD8⫹ CTLs, are probably not mediators of this activity. Very recently, Sca-1⫹Lin⫺ mouse early progenitors were found to exhibit potent veto activity, similar to human CD34⫹ cells (Gur et al, unpublished observations). Examination of such purified stem cells in cultures of T-cell receptor (TCR) transgenic CD8⫹ T cells directed against H2d suggested that the veto activity of hematopoietic stem cells is likely mediated by deletion of the effector cells specifically directed against class I molecules presented on the veto cells. Clearly, further elucidation of the possible mechanisms mediating the veto activity of early hematopoietic stem cells might be more easily explored in the mouse model, especially utilizing “knockouts” or mutants of key molecules. The tolerizing activity of the CD34⫹ cells might, in the future, be used to promote engraftment after sublethal conditioning. This has been achieved in mice by dose escalation using purified Sca-1⫹Lincells,7 but the equivalent number of stem cells is impossible to obtain in humans with present technology. However, preliminary results indicate that it might be possible to harvest 1 to 2 logs more veto cells upon short-term culture of human CD34⫹ cells (Gur et al, unpublished observations). An additional source of tolerogenic cells might be non– host-reactive CTL (generated against a third-party donor)

Transplants Across HLA Barriers which show potent veto activity.8,37 Thus, CD34⫹ cells and/or CTL against the third party might be used to induce graft tolerance in patients with contraindications for extensive myeloablation and immunosuppression as, for example, in the elderly or with nonneoplastic hematological diseases.

Donor Natural Killer Cell Alloreactivity Donor natural killer (NK) cell alloreactivity, a biological phenomenon unique to mismatched transplants, may, besides the conditioning regimen and the megadose of stem cells, play a role in engraftment. NK cells are regulated by inhibitory receptors for major histocompatibility (MHC) class I molecules.25,31 Some of these killer cell immunoglobulin-like receptors (KIRs), are specific for epitopes shared by certain class I alleles, and each KIR is expressed by a subset of NK cells. Therefore, in the NK repertoire, some NK cells recognize, and are blocked by, specific class I alleles. These NK cells are responsible for alloreactions when the mismatched target cells do not express the specific class I alleles that block them. In mismatched transplants, host and/or donor NK cells are responsible for one of the following situations: (1) no NK alloreactivity when the HLA disparity is not recognized by host and donor NK cells; (2) a potential for NK cell-mediated graft rejection when the host NK cells do not recognize as self the donor MHC; or (3) a potential for graft-versus-host (GvH) reactions when donor NK cells do not recognize the host MHC and are consequently activated to lyse the recipient cells. For patients in the third category, reconstituting NK cells contain a high frequency of donor NK cell clones that are alloreactive towards the recipient and can lyse the recipient’s lymphohematopoietic cells, without causing GvHD.42 In our experience, these patients achieve primary sustained engraftment, confirming that donor alloreactive NK cells contribute to ablate the host immune system in vivo. The few rejections we encountered were all in patients in the first and second NK cell categories, whose donors were unable to mount NK cell alloreactions against the host.44 Biological evidence in support of these clinical observations comes from animal models.44 Even after mild host immune suppression in mice, the infusion of donor-versus-recipient alloreactive NK cells efficiently ablated the host immune and myeloid cells. Engraftment was achieved despite transplantation across major MHC barriers, and no contribution to engraftment was exerted by donor H-2d/b T cells (as they are tolerant of the H-2b host). The alloreactive NK cell infusion did not cause GvHD even under stringent conditions, as when high NK cell numbers were given to lethally irradiated animals.

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Post-transplant Leukemia Relapse Cure of leukemia by allogeneic hematopoietic transplantation is achieved through the concerted action of two mechanisms: a lethal radiotherapy- and/or chemotherapy-based conditioning regimen, and the ability of the immune cells in the graft to recognize and eliminate the leukemia cells that survive the conditioning regimen.29 As a rule, this graft-versusleukemia (GvL) effect is mediated by T-cell alloreactions directed against host MHC antigens, which have a broad tissue distribution. Extensive T-cell depletion is essential to the success of mismatched transplants because the high frequency of alloreactive donor T cells in unmanipulated transplants is associated with a high incidence of severe GvHD—raising the question of the likelihood that T-cell– depleted mismatched transplants can exert a GvL effect. Recent observations showed that donor-versus-recipient NK cell alloreactivity contributes to disease eradication.42 As already mentioned, when donor NK cells do not recognize the host MHC, engrafted stem cells give rise to a transient (1 to 3 months) wave of reconstituting NK cells, whose repertoire is identical to the original displayed by the donor, including high-frequency donor-versus-recipient alloreactive NK clones. In vitro leukemia killing assays have demonstrated that most common acute lymphoblastic leukemias (ALL) are not susceptible to lysis by alloreactive NK clones, and this phenomenon is associated with no expression of LFA-1, an adhesion molecule needed for NK binding to target. Remarkably, 100% of acute myeloid leukemias (AML) and chronic myeloid leukemias that do express LFA-1 are killed by alloreactive NK clones. This finding suggests that KIR epitope mismatching in the GvH direction could predict antimyeloid leukemic effects in vivo.43 In our series of 112 acute leukemia patients transplanted since 1993, most relapses occurred in subjects with ALL, particularly in those in relapse at time of transplant. The 7-year probability of relapse was 0.70 for these very high-risk patients. Strikingly, the incidence of leukemia relapse in patients with AML was very low (0.23), despite approximately 50% being in relapse at the time of transplant (Fig 2). Retrospective evidence of the impact of NK alloreaction in relapse was obtained when donor-recipient pairs were divided into two groups: with or without KIR epitope incompatibility in the GvH direction. In transplants with KIR epitope mismatches in the GvH direction, relapse of AML was almost completely controlled. On the other hand, as predicted by the in vitro resistance to alloreactive NK cells, relapses in ALL were equally distributed between the two groups (Fig 3).43 It is interesting to compare these results with the relapse rates in T-cell– depleted transplants from

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Figure 2. Probability of leukemia relapse in 112 high-risk acute leukemia patients transplanted since March 1993. Sixteen AML patients (. . . . . .) were in remission (5 in first and 11 in second complete remission [CR]) and 37 had advanced disease (third CR, primary induction failure or relapse). Six ALL patients (——) were in first CR, 9 in second CR, and 44 had advanced-stage disease (4 in third CR, 40 in relapse).

HLA-matched family members. In our series of 30 patients with AML who received a lectin-separated BMT after a conditioning regimen similar to our mismatched protocol, the 10-year probability of relapse was 0.12 in 19 patients in first or second remission at transplant and 0.33 for 11 patients in relapse at transplant.3 Similar results have been reported elsewhere.34 These relapse rates can be explained by the intense myeloablative conditioning regimen which compensated for the lack of T-cell–mediated GvL effect. Most of our mismatched transplant recipients (47/53) were at high risk of leukemia relapse (in relapse or chemoresistant relapse, after second complete remission). When the graft did not have any potential for NK cell alloreactions, the recurrence rate overlapped with the rate in the matched patients in relapse at transplant. Strikingly, when NK cell alloreactivity was present, only one in 23 patients relapsed, a rate similar to the matched recipients transplanted in remission.

rates and similar patterns of immune reconstitution are common to other T-cell–depleted transplants, such as T-cell– depleted matched unrelated transplants.50 Several mechanisms are responsible for the posttransplant immune deficiency. Tissue damage by conditioning regimens prevents T-cell homing to peripheral lymphoid tissues, where generation and maintenance of T-cell memory take place.45 In adults, because thymic function is declining, early immune recovery stems from expansion of the mature T cells in the graft, and months later, from de novo production of naive T cells.13,21 In unmanipulated transplants, peripheral T-cell expansion is antagonized by the immunosuppressive GvHD prophylaxis. In Tcell– depleted transplants, the number of T cells in the graft has to be extremely low in order to prevent GvHD, and their homeostatic expansion could be antagonized by ATG used in the conditioning regimen.4,30 Thus, immune recovery is inevitably slow. Another aspect of post-transplant immune deficiency that has emerged from recent studies is the impact of G-CSF in transplant recipients. Generally given to hasten neutrophil recovery, G-CSF blocks interleukin-12 (IL-12) production in antigen-presenting cells (APCs) and decreases pathogen-specific T-cell responses in donor cells in vitro and in vivo. We no longer administer G-CSF to recipients.53 The engraftment rate remained unchanged, and IL-12 production by APCs was restored to normal much sooner, with CD4⫹ cell numbers and function markedly improving. Most of the post-transplant CD4⫹ T-cell clones exhibited protective Th1/Th0 cytokine production features: all clones expressed functional IL-12 receptors and few produced IL-4 and IL-10. These behavior patterns were very different from those observed in recipients who had received GCSF, whose CD4⫹ clones had clearly exhibited nonprotective type 2 functional features.

Post-transplant Immunodeficiency A major clinical problem is the slow recovery of the antimicrobial and antiviral responses. In fact, 24 of the 34 nonleukemic deaths in our last two studies, which included 76 patients, were due mainly to bacterial or fungal infections. The incidence of infectionrelated deaths is linked to the delay in immune reconstitution and because most patients had a long history of disease, were heavily pretreated, and/or had relapsed by the time of transplant. Indeed, multivariate analyses showed that history of infections and colonization at transplant were the most significant factors for infection-related deaths (Aversa et al, unpublished observations). Relatively high infection-related mortality

Figure 3. Clinical impact of donor-v-recipient NK cell alloreactivity on relapse in AML and ALL.

Transplants Across HLA Barriers

In order to speed immunoreconstitution, several approaches can be envisaged. One is the use of nonalloreactive T cells generated by purging IL-2 receptor (CD25) mixed lymphocyte response (MLR)reactive T cells.10 Another involves the infusion of lymphocytes cocultured with irradiated cells from the recipients in the presence of CTLA-4 immunoglobulin,14,15 an agent that inhibits B7/CD28-mediated costimulation. We have recently developed a strategy to transfer donor pathogen-specific immune responses safely across the HLA barrier.35 Large numbers of donor T-cell clones raised against Aspergillus fumigatus and cytomegalovirus (CMV) antigens were screened for cross-reactivity to host alloantigens by MLR. Non– host-reactive clones, presumably devoid of GvHD potential, were pooled and infused into recipients at a dose of 1 to 5 ⫻ 105/kg body weight on day 15 after transplant. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was also given, in an attempt to improve APC function. Untreated patients developed Aspergillus- and CMV-specific Tcell responses in vitro more than 9 months posttransplant. All patients who had received the infusions exhibited significant Aspergillus- and CMVspecific responses within 3 weeks, and no patient developed GvHD. Preliminary data already show a beneficial effect on CMV reactivation.

Conclusions The use of megadose stem cell transplants from haploidentical donors has ensured a high rate of engraftment without GvHD in acute leukemia patients. Experimental and clinical results on the CD34⫹ cell veto activity, and on donor NK cell alloreactivity suggest the following working hypothesis. After transplantation of purified CD34⫹ cells, the likelihood of activation of antidonor CTL-p is proportional to the level of residual host T cells and inversely correlated to the number of veto cells. Veto activity can be contributed initially by the CD34⫹ cells infused and later by the CD33⫹ progeny of these cells, which grow exponentially within the first few days post-transplant. In addition, when using donors of HLA genotypes which allow the generation of alloreactive NK cells during the first few days after transplant, such cells can eradicate mature antidonor CTL that were able to escape the veto effect of the CD34⫹ cells. The establishment of the haploidentical graft is therefore greatly dependent not only on the ability of the inoculum of the CD34⫹ cells to veto antidonor CTL-p, but also on their ability to seed the bone marrow and to generate as rapidly as possible the derivative cells required to complete the eradication of host antidonor T cells. Clearly, all of these mechanisms are interdependent with the myeloabla-

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tion and immunosuppression induced by the conditioning regimen. However accurate this working hypothesis may be, clinical experience clearly shows that mismatched transplant has become a feasible option for patients with high-risk acute leukemia who urgently need a transplant and who do not have a matched donor. Indeed, our results in terms of transplant-related mortality (TRM) and event-free survival (EFS) compare favorably with those reported for patients at the same stage of disease who receive transplants from matched unrelated donors. In the Seattle experience with 161 AML patients who received T-replete transplants from unrelated matched donors, survival correlated with the stage of the disease at transplant48: only 7% of the 81 patients in relapse, 19% of the 16 who never achieved a remission, 28% for the 40 transplanted in second complete remission, and 50% of the 16 in first complete remission survive leukemia-free at 5 years. Age also influenced outcome, with only 14% of adults surviving leukemia-free, as compared to 32% of those under 18 years of age. Similar data are reported by the National Marrow Donor Program (NMDP) in 756 AML patients transplanted from matched unrelated donors.28,51 EFS correlates with the age of patients (25% and 35% in adults and children, respectively) and stage of disease, with the difference being even more marked with advanced-stage disease at transplant (7% EFS for adults and 23% for children). The 53 patients with AML we transplanted after March 1993 have a 0.35 probability of EFS at 7 years (Fig 4). Only six were under 18 years of age, 47 had advanced-stage disease, and 30 were in relapse. Despite these poor-prognostic factors, TRM was 44%, which overlaps with the cumulative 43% nonleukemic mortality reported in the 26 children and 135 adults in the Seattle series.48 Almost all of our 59 patients with ALL were at very

Figure 4. Probability of EFS in 112 high-risk acute leukemia patients transplanted since March 1993. The 53 high-risk AML patients (37 in advanced stage at transplant) have a 0.35 probability of surviving event-free at 6 years (. . . . . .); the 59 ALL (44 in advanced stage) have a 0.13 probability (——) (P ⬍ .01).

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high risk for leukemia relapse and nonleukemic death because they had a long history of disease, and many were in chemoresistant relapse at transplant. Their probability of EFS is 0.13 at 7 years. A similar probability of leukemia-free survival has been reported in 321 patients with ALL not in remission at transplant.2 In full haplotype-mismatched transplants, one major factor has undoubtedly confounded interpretation of the outcome: most recipients had advancedstage disease at the time of transplant. Among 37 children transplanted in Tu¨bingen, the 21 in remission at transplant have a 0.39 probability of EFS as compared to 0.13 for those not in remission at transplant.18,24 Similarly, from among 112 acute leukemia patients (53 AML, 59 ALL) we have transplanted since 1993, the 27 (18 AML, 9 ALL) in first hematological and cytogenetic remission (n ⫽ 7) or second stable complete hematological remission at transplant (n ⫽ 20) have a 0.45 probability of EFS at 7 years (Fig 5). Eleven of these 27 patients, who did not receive post-transplant G-CSF, have an even better probability of EFS with a low TRM (0.10). Although the number of patients is low, improvements in clinical outcome are being achieved. Mismatched transplantation should be offered not as a last resort but as a reasonable option in the early stages of disease to high-risk acute leukemia patients without a matched donor. Furthermore, progress in our knowledge of the biology underlying the tolerizing activity of other facilitating cells that could be added to the transplants (expanded CD34⫹ cells, nonalloreactive CTL, NK alloreactive cells) might be exploited to make conditioning regimens less toxic,

Figure 5. Effect of disease status on the probability of EFS. The 27 patients (18 AML, 9 ALL) who were at either first hematological and cytogenetic remission or stable second remission (early-stage disease) at transplant have a 0.45 probability of EFS (. . . . . .). The 85 with advanced-stage disease have a 0.14 probability of EFS (——) (P ⬍ .007).

and thus extend full haplotype-mismatched transplants to the elderly and to patients with nonmalignant hematological disorders.

Acknowledgment We are indebted to Dr Geraldine Boyd for assistance in the preparation of the manuscript.

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