Aplastic anaemia in association with coeliac disease: a series of three cases.

We report a series of three patients in whom the diagnoses of aplastic anaemia (AA) and coeliac disease were made concurrently. Haematological manifestations of coeliac disease are well described but this is the first report to suggest an association with aplastic anaemia. 'Silent/atypical coeliac disease', in the absence of gastrointestinal symptoms, is increasingly recognised and patients may present with generalised symptoms, such as malaise and fatigue, which are easily attributable to AA. Immunosuppressive therapy for AA could modulate the course of celiac disease. We recommend clinicians should be vigilant for signs of coeliac disease in patients with AA.

Comparison of long-term outcomes after allogeneic hematopoietic stem cell transplantation from matched sibling and unrelated donors.

Long-term survivors of hematopoietic stem cell transplants remain at risk of potentially fatal complications that detract from life quality. Long-term morbidity and mortality were compared between matched recipient cohorts surviving 2 or more years and defined by donor type, HLA matched sibling donor (MSD) or volunteer unrelated donor (URD). Patients were previously entered into the prospective multicenter International Unrelated Search and Transplant Study. Thirty-nine centers provided data on 108 URD and 355 MSD recipients surviving more than 2 years. Long-term survival, performance status, chronic GvHD (c-GvHD), secondary malignancy, endocrine dysfunction, cataracts, bone necrosis and dental pathology were compared between cohorts. Twelve year survival was 77+/-5% for the MSD and 67+/-11% for the URD cohort (P=0.1). Late death occurred in 105 of 463 recipients alive at 2 years, 73 after 355 (21%) MSD and 32 after 108 (30%) URD transplants, P=0.10. Of 105 deaths, the cause was relapse in 60 and unrelated to relapse in 45 cases. Cumulative incidence of extensive c-GvHD (P=0.002), cataracts (P=0.02) and bone necrosis (P=0.02) was higher after URD transplants. No long-term difference in endocrine dysfunction, secondary malignancy and major dental pathology was detected. This landmark study will assist physicians counseling patients pre-transplant and with their long-term care post transplant.

Bone marrow transplants from mismatched related and unrelated donors for severe aplastic anemia.

For patients with acquired severe aplastic anemia without a matched sibling donor and not responding to immunosuppressive treatment, bone marrow transplantation from a suitable alternative donor is often attempted. We examined risks of graft failure, graft-versus-host disease and overall survival after 318 alternative donor transplants between 1988 and 1998. Sixty-six patients received allografts from 1-antigen and 20 from >1-antigen mismatched related donors; 181 from matched and 51 from mismatched unrelated donors. Most patients were young, had had multiple red blood cell transfusions and poor performance score at transplantation. We did not observe differences in risks of graft failure and overall mortality by donor type. The probabilities of graft failure at 100 days after 1-antigen mismatched related donor, >1-antigen mismatched related donor, matched unrelated donor and mismatched unrelated donor transplants were 21, 25, 15 and 18%, respectively. Corresponding probabilities of overall survival at 5 years were 49, 30, 39 and 36%, respectively. Although alternative donor transplantation results in long-term survival, mortality rates are high. Poor performance score and older age adversely affect outcomes after transplantation. Therefore, early referral for transplantation should be encouraged for patients who fail immunosuppressive therapy and have a suitable alternative donor.

Incidence and specificity of HLA antibodies in multitransfused patients with acquired aplastic anemia.

BACKGROUND: This study aimed to establish the prevalence and characteristics of anti-HLA in antibody acquired aplastic anemia patients following cessation of antithymocyte globulin therapy and to characterize antibody in terms of epitope specificity. STUDY DESIGN AND METHODS: One hundred and fifty multitransfused, untransplanted patients from eight European centers were investigated by serologic methods. RESULTS: Sixty-two percent were antibody positive. Eighteen HLA-Class-I-specific antibodies (15 IgG, 3 IgM) were identified in 13 patients; 13 antibodies were specific for HLA-A epitopes and 5 for HLA-B. Epitope analysis identified significant correlation between serum reactivity and amino acid substitutions associated with HLA-Class-I epitopes. An excess of antibodies to HLA-A1-associated cross-reactive groups was identified. There was no significant difference in antibody frequency in patients taking cyclosporine compared to those who were not. CONCLUSION: Data suggested a contribution from B cell memory of alloantigens introduced during pregnancy. In some cases, antibody production continued many years after the last transfusion, and although the target varied between individual patients, the antibody to HLA was focused on a few specific Class I epitopes, the majority of which mapped to the HLA-A molecule.

In vitro expansion of cord blood does not prevent engraftment of severe combined immunodeficient repopulating cells.

This study aimed to assess the potential of human cord blood (CB) cells to engraft in the xenogenic non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model after in vitro expansion culture. We also studied the quality of human haemopoiesis arising from the transplantation of fresh or expanded cells in this model. Cord blood CD34(+) cells were cultured for 3, 7 or 10 d with stem cell factor, Flt3, thrombopoietin, interleukin 3 (IL-3), IL-6 and granulocyte colony-stimulating factor, all at 10 ng/ml in serum-replete conditions. Transplantation of mice with fresh CB containing 3 x 10(4) CD34(+) cells and 1-2 SCID repopulating cells (SRC) resulted in a median of 7.4% (0.4%-76.8%) human engraftment. When mice received the expanded product of 1-2 SRC, the ability to repopulate NOD/SCID mice was maintained even after 10 d of in vitro culture. Serial dilution of the expanded cells suggested that in vitro expansion had increased SRC numbers two- to fourfold. Expanded SRC produced long-term culture-initiating cells, clonogenic cells and CD34(+) cells in the same proportions as fresh cells after successful engraftment. Therefore, expanded SRC were able to differentiate in the same way as fresh SRC. There was a trend towards lower levels of engraftment when d 7 cultured cells were transplanted (median engraftment 0.8%, range 0.0-24.0%) compared with 1-2 fresh SRC. Our data suggest that this is owing to reduced proliferation of cultured cells in vivo. By utilizing limiting numbers of CB SRC, we confirmed that the engraftment potential of SRC in the NOD/SCID model was preserved after in vitro expansion. Furthermore, dilution experiments strongly suggest two- to fourfold expansion of SRC in vitro. These studies are relevant for developing clinical stem cell expansion strategies.

Status of umbilical cord blood transplantation in the year 2001.

Umbilical cord blood (UCB) transplantation is limited to small recipients because of the low haemopoietic cell dose. Children from ethnic minority groups may benefit most from cord blood transplantation. Cohort controlled retrospective data indicate that there is significantly less acute and chronic graft versus host disease associated with the transplantation of human major histocompatibility complex (HLA) identical sibling cord blood compared with HLA identical sibling marrow. Controlled data are not yet available to confirm this observation in unrelated donor cord blood transplantation. The difference in leukaemic relapse seen after cord blood compared with bone marrow transplantation is also unknown. Tentative recommendations for the use of umbilical cord blood for transplantation are as follows. Collection is indicated from healthy newborn siblings when urgent transplantation is required for an older child in a family. The haematologist responsible for the older child, with the approval of the family and the obstetric team, should contact the medical director of the nearest cord blood bank to discuss arrangements for the UCB to be collected and HLA typed. Antenatal blood sampling to HLA type the fetus is not recommended. Umbilical cord blood should be considered when allogeneic transplantation is the treatment of choice for a child who does not have an HLA identical sibling, or a well matched unrelated adult volunteer donor. The potential advantages and disadvantages of using an HLA haplotype matched peripheral blood stem cell family donor rather than an unrelated cord blood donation should be discussed. There are no comparative data available as yet. At present, UCB transplantation should only be considered if a suitably matched donation contains at least 2 x 10(7)/kg nucleated cells. Effectively, this means that most adults and larger children are not suitable recipients.

Different behaviour of fresh and cultured CD34+ cells during immunomagnetic separation.

In-vitro expansion of human cord blood (CB) cells could enhance peripheral blood recovery and ensure long-term engraftment of larger recipients in the clinical transplant setting. Enrichment of CD34+ cells using the MiniMACS column has been evaluated for the preparation of CB CD34+ cells before and after expansion culture. Repurification of CD34+ cells after culture would assist accurate phenotypic and functional analysis. When fresh CB mononuclear cells (MNC) were separated, the MACS positive (CD34+) fraction (90.1% pure) contained a mean (+/- SD, n = 5) of 93.0 +/- 8.0% of the eluted CD34+ cells, 99.6 +/- 0.7% of the CFU-GM and all of the eluted long-term culture-initiating cells (LTC-IC). Cord blood CD34+ cells were then cultured for 14 d with IL-3, IL-6, SCF, G-CSF and GM-CSF, each at 10 ng/ml. The total cell expansion was 2490 +/- 200-fold and the CD34+ cell expansion was 49 +/- 17-fold. The percentage of CD34+ cells present after expansion culture was 1.2 +/- 0.85%. When these cells were repurified on the MiniMACS column, the MACS positive fraction only contained 40.3 +/- 13.4% of the eluted CD34+ cells which was enriched for the mature CD34+ CD38+ subset, 24.4 +/- 8.8% of the eluted CFU-GM and 79.5 +/- 11.0% of the LTC-IC. The remaining cells were eluted in the MACS negative fraction. In conclusion, repurification of cultured CD34+ cells does not yield a representative population and many progenitors are lost in the MACS negative fraction. This can give misleading phenotypic and functional data. Cell losses may be important in the clinical setting if cultured cells were repurified for purging.

Abnormal cytogenetic clones in patients with aplastic anaemia: response to immunosuppressive therapy.

We report the response to immunosuppressive therapy with antithymocyte globulin (ATG) and cyclosporin or oxymetholone in 13 cases of aplastic anaemia (AA) with an abnormal cytogenetic clone detected at or sometime after diagnosis. Blood and bone marrow examination showed no distinctive morphological features of myelodysplasia (MDS) at diagnosis. Haematological response occurred promptly in eight cases; the remainder responded after additional immunosuppression with or without oxymetholone. Three patients had a late relapse of AA, treated successfully by allogeneic bone marrow transplantation in one; the others responded to oxymetholone. Transformation to MDS or acute leukaemia was not observed after a median follow-up of 4.1 years (range 1.2-11.2). In four patients the cytogenetic clone disappeared after treatment.

Clinical application of in vitro expansion of cord blood.

Expansion of cord blood (CB) haemopoietic cells has been investigated with the aim of reducing cytopenia following transplantation. We investigated the increase in total nucleated cells, colony-forming cells (CFC), CD34+ cells and long-term culture-initiating cells (LTC-IC) by limiting dilution after a 14-day culture of CB CD34+ cells (5 x 10(3)/ml) with SCF, IL-3, IL-6, GM-CSF and G-CSF all at 10 ng/ml. On average nucleated cells increased 2500-fold, CD34+ cells 39-fold and CFU-GM 49-fold with maintenance of BFU-E. The more primitive LTC-IC expanded on average 2.5-fold. Expansion of a 20% aliquot of a CB donation could provide a 5-7-fold increase in progenitor cells, and a 1570-fold increase in post-progenitor cells compared to an untreated donation.

Is in vitro expansion of human cord blood cells clinically relevant?

Peripheral blood recovery after cord blood (CB) transplantation is delayed compared with marrow. Expansion of CB haemopoietic cells has been investigated with the aim of reducing cytopenia following transplantation. Mature cells, post-progenitors, progenitors and long-term culture-initiating cells (LTC-IC) from expansion products may contribute to peripheral blood recovery. We investigated the increase in total nucleated cells, colony-forming cells (CFC), CD34+ cells and LTC-IC by limiting dilution after a 14 day culture of CB CD34+ cells (5 x 10(3)/ml) with SCF, IL-3, IL-6, GM-CSF and G-CSF all at 10 ng/ml. On average, nucleated cells increased 2500-fold, CD34+ cells 39-fold and CFU-GM 49-fold with maintenance of BFU-E. The more primitive LTC-IC expanded on average 2.5-fold but effects on long-term marrow-repopulating cells (LTRC) during culture are unknown. A practical application of in vitro expansion of CB might be to expand a 20% aliquot of a CB donation and infuse the remainder unmanipulated. This could provide a 5- to 7-fold increase in progenitor cells, an estimated 1570-fold increase in post-progenitor cells and maintenance of LTC-IC compared to an untreated donation. Combined with in vivo post-transplant growth factor therapy this could prompt early peripheral blood recovery after CB transplantation, without significant loss of LTC-IC or donor lymphocytes.

Oral squamous cell carcinoma after allogeneic bone marrow transplantation for Fanconi anaemia.

We report a case of oral squamous cell carcinoma (SCC) originating in the buccal mucosa of an 18-year-old female patient with chronic graft-versus-host disease (GVHD) 9 years after HLA-identical sibling bone marrow transplantation (BMT) for Fanconi anaemia (FA). The case highlights the problems of malignant change in FA and also the increased risk of second malignancy after BMT. The literature is reviewed with regard to previous cases and the possible aetiology of tumour formation. A high index of suspicion to any epithelial lesion in FA is appropriate so that early diagnosis may lead to improved prognosis.

Bone marrow transplantation for severe aplastic anemia: has outcome improved?

Bone marrow transplants for severe aplastic anemia were first performed in the 1970s. Transplant regimens, supportive care, and patient selection have changed substantially since then. Our objective was to determine the impact of these changes on transplant outcome. We studied 1,305 recipients of HLA-identical sibling transplants for aplastic anemia between 1976 and 1992, reported to the IBMTR by 179 centers. We compared survival of transplants performed in three intervals (1976 through 1980 [n = 186], 1981 through 1987 [n = 648], and 1988 through 1992 [n = 471]) using Cox proportional hazards regression. Five-year survival (+/-95% confidence interval) increased from 48% +/- 7% in the 1976-1980 cohort to 66% +/- 6% in the 1988-1992 cohort (P < .0001). Risks of graft-versus-host disease (GVHD) and interstitial pneumonia decreased over time, but the risk of graft failure did not. Higher long-term survival resulted primarily from decreased mortality in the first 3 months posttransplantation. Late mortality risks were low and changed little over the intervals studied. In multivariate analysis, changes in transplantation strategies accounted for most but not all of the improved outcome. Use of cyclosporine to prevent GVHD was the most important factor. Changes in patient selection did not seem to explain improved survival. Survival after HLA-identical sibling bone marrow transplantations for aplastic anemia has improved since 1976. Changes in GVHD prophylaxis account for much of this improvement. Other changes may also operate.

Donor search or autografting in patients with acute leukaemia who lack an HLA-identical sibling? A matched-pair analysis. Acute Leukaemia Working Party of the European Cooperative Group for Blood and Marrow Transplantation (EBMT) and the International Marrow Unrelated Search and Transplant (IMUST) Study.

One hundred and ninety-one patients with acute leukaemia who received bone marrow from HLA-A, -B and -DR identical unrelated donors and were reported to EBMT and/or IMUST, were matched with 382 patients receiving autologous bone marrow for diagnosis, age, stage of disease and year of transplantation. Transplant-related mortality (TRM) was significantly higher in recipients of unrelated marrow compared to autograft recipients, 44 +/- 4% (+/- 95% confidence interval) and 15 +/- 3% at 2 years in the two groups, respectively (P < 10(-4)). In contrast, relapse probability was lower in recipients of unrelated marrow, being 32 +/- 5% at 2 years compared to 55 +/- 3% in recipients of autografts (P < 10(-4)). Two-year leukaemia-free survival (LFS) in patients with acute lymphoblastic leukaemia was 39 +/- 5% and 32 +/- 3% in the two groups, respectively. Among patients with acute myeloid leukaemia (AML), the corresponding figures were 36 +/- 6% and 46 +/- 5% in the two groups, respectively (P = NS). In AML in first remission (CR-1), the 2-year survival was 42 +/- 10% in recipients of unrelated bone marrow, compared to 69 +/- 8% in autograft recipients (P = 0.008). When all patients with acute leukaemia were included, the 2-year LFS was 38% in recipients of unrelated marrow, compared to 37% in autograft recipients (NS). In conclusion, this retrospective analysis supports the design of a prospective randomized study in patients with high-risk/advanced acute leukaemia who lack a suitable related bone marrow donor, to ascertain which of the two strategies, if any, should be favoured.

Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings.

PURPOSE: To compare outcomes of bone marrow transplants for leukemia from HLA-identical siblings, haploidentical HLA-mismatched relatives, and HLA-matched and mismatched unrelated donors. PATIENTS: A total of 2,055 recipients of allogeneic bone marrow transplants for chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) were entered onto the study. Transplants were performed between 1985 and 1991 and reported to the International Bone Marrow Transplant Registry (IBMTR). Donors were HLA-identical siblings (n = 1,224); haploidentical relatives mismatched for one (n = 238) or two (n = 102) HLA-A, -B, or -DR antigens; or unrelated persons who were HLA-matched (n = 383) or mismatched for one HLA-A, -B, or -DR antigen (n = 108). HLA typing was performed using serologic techniques. RESULTS: Transplant-related mortality was significantly higher after alternative donor transplants than after HLA-identical sibling transplants. Among patients with early leukemia (CML in chronic phase or acute leukemia in first remission), 3-year transplant-related mortality (+/-SE) was 21% +/- 2% after HLA-identical sibling transplants and greater than 50% after all types of alternative donor transplants studied. Among patients with early leukemia, relative risks of treatment failure (inverse of leukemia-free survival), using HLA-identical sibling transplants as the reference group, were 2.43 (P < .0001) with 1-HLA-antigen-mismatched related donors, 3.79 (P < .0001) with 2-HLA-antigen-mismatched related donors, 2.11 (P < .0001) with HLA-matched unrelated donors, and 3.33 (P < .0001) with 1-HLA-antigen-mismatched unrelated donors. For patients with more advanced leukemia, differences in treatment failure were less striking: 1-HLA-antigen-mismatched relatives, 1.22 (P = not significant [NS]); 2-HLA-antigen-mismatched relatives, 1.81 (P < .0001); HLA-matched unrelated donors, 1.39 (P = .002); and 1-HLA-antigen-mismatched unrelated donors, 1.63 (P = .002). CONCLUSION: Although transplants from alternative donors are effective in some patients with leukemia, treatment failure is higher than after HLA-identical sibling transplants. Outcome depends on leukemia state, donor-recipient relationship, and degree of HLA matching. In early leukemia, alternative donor transplants have a more than twofold increased risk of treatment failure compared with HLA-identical sibling transplants. This difference is less in advanced leukemia.

Optimal cryopreservation of human umbilical cord blood.

Cryopreservation techniques for umbilical cord blood (UCB) have been based on methods established for marrow (BM) and peripheral blood progenitor cells (PBPC) with varying degrees of success. The aim of this study was to optimise cryopreservation of UCB haemopoietic cells based on sound cryopreservation principles. UCB samples were cryopreserved with different combinations of DMSO and hydroxyethyl starch (HES) by a variety of freezing protocols. After cooling at 1 degree C/min in solutions containing 4% HES and various concentrations of DMSO there was a dramatic fall in CD34+ recovery from 85.4% (s.d. 28.4) to 12.2% (s.d. 10.0) as DMSO concentration was reduced from 5 to 2.5%. Varying HES concentration in solutions containing 5% DMSO did not have a significant effect on CD34+ cell recovery. Increasing cooling rate from 1 to 10 degrees C/min significantly reduced CD34+ recovery (P < 0.0001) while increasing DMSO concentration up to 10% had little effect (P = 0.8, two-way ANOVA). Good recovery of UCB CD34+ cells can be achieved with 5-10% DMSO at a controlled cooling rate of 1 degrees C/min. There was a significant difference (P < 0.0001) in the apparent recovery of CD34+ cells between paired aliquots thawed in the presence (recovery = 76.8%, s.d. 26.0) and absence (32.5%, s.d. 18.7) of DNase. In conclusion, conditions for cryopreserving UCB for clinical banking that yield optimal recovery of CD34+ cells have been established.

Spontaneous graft-versus-host disease following autologous peripheral blood progenitor cell transplantation in chronic myeloid leukaemia.

Myeloablation followed by haemopoietic reconstitution using autologous peripheral blood progenitor cells (PBPC) is applicable to some patients with CML, particularly where there is no allogeneic stem cell donor available, and interferon alpha has failed to achieve a significant cytogenetic response. Cells lacking the Philadelphia (Ph) chromosome can be collected at the early phase of myeloid recovery after intensive chemotherapy, and reconstitution after autografting can be associated with prolonged suppression of the Ph positive clone. It is possible that mechanisms other than this "in vivo purge' may contribute to disease control, for example an autologous graft-versus-leukaemia effect. We report two patients in whom significant autologous graft-versus-host disease (auto-GVHD) has occurred, which has not previously been described as a spontaneous event after PBPC autograft for CML. We postulate that mononuclear cells collected in an early phase of recovery after intense myelosuppression have the capacity to produce self-reactivity after autografting. These cells, which may include autoreactive T lymphocytes or antigen-presenting dendritic cells, might mediate a useful graft-versus-leukaemia effect.

Bone marrow transplantation for chronic myeloid leukemia with volunteer unrelated donors using ex vivo or in vivo T-cell depletion: major prognostic impact of HLA class I identity between donor and recipient.

Between August 1985 and July 1994, we performed 115 volunteer unrelated donor (VUD) bone marrow transplants (BMT) for first chronic phase (n = 86) or advanced phase (n = 29) chronic myeloid leukemia (CML). Standard serologic HLA typing of potential donors and recipients was supplemented with one-dimensional isoelectric focusing (IEF) for class I proteins, allogenotyping for DR and DQ alleles using DNA restriction fragment length polymorphism (RFLP) analysis, and the measurement of antirecipient major histocompatibility complex (MHC) cytotoxic T-lymphocyte precursor cells in the donors' blood (CTLp assay). Recipients were conditioned for transplantation with a combination of high-dose chemotherapy and total body irradiation (n = 103) or high-dose chemotherapy alone (n = 12). Twenty eight recipients received ex vivo T-cell-depleted marrow, and 84 underwent some form of in vivo T-cell depletion. The probability of severe (grades III or IV) acute graft-versus-host disease (aGVHD) was 24%, and that of extensive chronic graft-versus-host disease (cGVHD), 38%. Proportional hazards regression analysis showed an association between low frequency CTLp and a reduced incidence of severe aGVHD (relative risk [RR], 0.28; P = .0035). The probability of relapse at 3 years was 23%, with first chronic phase disease being independently associated with a lower risk of relapse (RR, 0.71; P = .01). The overall leukemia-free survival (LFS) at 3 years was 37%; the LFS for the first chronic phase and advanced phase recipients was 41% and 26%, respectively. First chronic phase disease (RR, 0.56; P = .063) and the combination of recipient cytomegalovirus (CMV) seronegativity and an IEF-matched donor (RR, 0.48; P = .011) were both associated with improved LFS. The probabilities of survival and LFS for patients under 40 years of age transplanted in first chronic phase from an IEF-matched donor were 73% and 50%, respectively. We conclude that VUD BMT is a reasonable option for patients with CML; when using ex vivo or in vivo T-cell depletion, optimal results are achieved in patients transplanted in chronic phase with marrow from donors without demonstrable class I HLA mismatch and a low CTLp frequency.

Clonal hematopoiesis as defined by polymorphic X-linked loci occurs infrequently in aplastic anemia.

We evaluated the methylation status of the X-linked gene phosphoglycerate kinase (PGK1) and the DXS 255 locus detected by probe M27 beta to study clonality in acquired aplastic anemia (AA). A total of 30 females were suitable for clonal analysis of peripheral blood polymorphonuclear cells (PMN) and mononuclear cells using a polymerase chain reaction-based procedure in 24 patients and Southern blotting in 9. Overall, 10 of 30 patients exhibited an imbalanced X-inactivation pattern. However, in 4 patients, analysis of constitutional DNA suggested a skewed methylation pattern and 2 further cases had to be excluded because of the lack of an appropriate control. A truly clonal pattern was thus established in 4 of 30 (13%) patients. In 7 patients who later developed clonal disorders of hematopoiesis, X-inactivation analysis did not predict this event in any case. In patients with a paroxysmal nocturnal hemoglobinuria phenotype, there was no correlation between the proportion of phosphatidylinositol glycan anchored protein (PIG-AP)-deficient blood cells and the corresponding X-inactivation pattern. X-inactivation analysis detected clonal hematopoiesis in only 3 of 10 patients with a deficiency in PIG-AP in the cell population under study, but sorting of nucleated cells on the basis of PIG-AP expression showed the clonal nature of PIG-AP-deficient cells. We conclude that the majority of patients with AA show polyclonal hematopoiesis using X-linked clonal analysis, but that minor clonal populations, such as PIG-AP-deficient cells, may not be detected unless sorted cell populations are separately analyzed.

Cytotoxic T lymphocyte precursor frequency analyses in bone marrow transplantation with volunteer unrelated donors. Value in donor selection.

Between May 1989 and February 1994, we performed 48 volunteer unrelated donor BMTs for first chronic phase chronic myeloid leukemia using in vivo T cell depletion for acute graft-versus-host disease (aGvHD) prophylaxis. In 40 cases, adequate material was available to measure the frequency of antirecipient MHC cytotoxic T lymphocyte precursor (CTLp) cells in the blood of potential donors. This supplemented standard serological typing, one-dimensional isoelectric focusing for class I proteins, and allogenotyping for DR and DQ alleles using DNA RFLP analysis in the donor selection process. All recipients were conditioned with cyclophosphamide 120 mg/kg, TBI 1320 cGy, and intravenous Campath 1G. GvHD prophylaxis consisted of CsA, short-course methotrexate, and intravenous Campath 1G. Minimum follow-up in all surviving recipients was 100 days. The development of aGvHD and the probability of leukemia-free survival were compared between the high frequency group (CTLp > 1 in 100,000) (n = 15) and the low frequency group (CTLp < 1 in 100,000) (n = 25). There was a trend for increasing grade of aGvHD, which was statistically significant in the high frequency group when compared with the low frequency group (P = 0.003). Both a high frequency of CTLp (relative risk [RR] = 9.0, P = 0.016) and HLA mismatch (RR = 6.7, P = 0.023) were predictors of severe aGvHD (grade III or IV). Multivariate analysis showed that CTLp group (RR = 3.4, P = 0.015) and CMV status (RR = 3.9, P = 0.008) were predictors of leukemia-free survival. Further investigation showed an interaction between the two, such that CMV seropositive recipients in the high frequency group had a relative risk of 9.4 (P = 0.0001) of treatment failure (death or relapse) when compared with other combinations. We conclude that with our present GvHD prophylaxis regimen, CTLp frequency analysis predicts post-BMT outcome and is a valuable aid in donor selection.