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.

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.

Induction of a syngeneic graft-versus-leukemia effect following bone marrow transplantation for chronic myeloid leukemia.

Although clinical data support the concept of a graft-versus-leukemia (GVL) effect following allogeneic bone marrow transplantation (BMT), there are few data to support a similar GVL activity following syngeneic BMT in man. To identify cells with a potential antileukemic activity post-BMT, we monitored the immunological reconstitution in a patient with chronic phase chronic myeloid leukemia (CML) who received a syngeneic BMT from his identical twin brother. Peripheral blood mononuclear cells (PBMC) from the donor prior to the transplant and from the recipient posttransplant were cultured with recombinant interleukin-2 to generate lymphokine activated killer (LAK) cells. LAK cells from both sources lysed the cell line target cells K562 and LCL and also recipient and allogeneic CML target cells in a 51Cr release cytotoxicity assay. Donor-derived LAK cells did not kill normal donor marrow. LAK cells had similar effects on granulocyte-macrophage progenitor cells (CFU-GM): LAK cells from both donor pre-BMT and recipient post-BMT inhibited the proliferation of CFU-GM from the patient's CML cells, but again donor LAK cells did not inhibit the colony growth of normal donor marrow. These results suggest that a syngeneic GVL effect is inducible following BMT in man and that this activity may be truly antileukemic and spare normal marrow progenitors.

Bone marrow transplantation for chronic myeloid leukemia: the use of histocompatible unrelated volunteer donors.

We treated 17 patients with chronic myeloid leukemia (CML) by bone marrow transplantation using marrow from human leukocyte antigen (HLA)-matched unrelated donors. Patients were conditioned with a combination of in vivo monoclonal antibodies, chemotherapy with daunorubicin (n = 7) or busulfan (n = 10) and cyclophosphamide, and both total body and total lymphoid irradiation. Donor marrow was depleted of T cells by incubation with monoclonal antibodies of the Campath series. Fourteen (88%) of 16 evaluable patients had sustained engraftment. Four (27%) of the 15 evaluable patients developed acute graft-versus-host disease (GVHD) of grade II or greater, and 4 of 12 evaluable patients developed chronic GVHD. Three patients developed hematological and two developed cytogenetic evidence of relapse. Eight patients (47%) survive at a median follow-up of 32 months (range 10-51 months), giving an actuarial survival of 44%. Five patients remain alive without evidence of hematological or cytogenetic relapse, giving an actuarial disease-free survival of 27%. Pneumonitis caused or contributed to death in six of the nine patients who died. We conclude that T-cell depletion can prevent the severest forms of GVHD but also increases the risk of relapse after transplant with unrelated donors, as it does with HLA-identical siblings. Nevertheless the use of matched unrelated donors should be considered for CML patients who lack HLA-identical siblings.

Use of cyclosporin A in allogeneic bone marrow transplantation for severe aplastic anemia.

We report on 18 months of experience with cyclosporin A (Cy A) in allogeneic bone marrow transplantation for severe aplastic anemia (SAA). Twenty-three patients treated with Cy A for postgraft immunosuppression are described and compared with 14 similar patients with SAA in whom methotrexate (MTX) was used. The early results are encouraging with 73% survival in the Cy A group compared with 43% in the MTX group. The improvement is partly attributable to the low incidence of graft failure. Graft-versus-host disease (GVHD) remains a problem with an overall incidence of 70% in Cy A-treated aplastic patients, although mortality has been ony 14%. Toxicity attributable to Cy A has so far been acceptable and nephrotoxicity is usually mild and reversible. However, three aplastic patients have developed clinically significant renal impairment while receiving both Cy A and aminoglycoside antibiotics.

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.

Unrelated donor marrow transplantation between 1977 and 1987 at four centers in the United Kingdom.

Retrospectively we analyzed the histocompatibility data and clinical results of bone marrow transplantation in 51 patients who received marrow from unrelated donors (UD) from 1977 to 1987 at one of four UK BMT centers. We compared the results with those obtained in 51 transplants carried out at the same centers using HLA-identical (ID) sibling donors. Of the UD/recipient pairs 32 (63%) were serologically identical for HLA A, B, and DR antigens, and 37% showed varying degrees of mismatch. UD-BMT primary diagnoses were: severe aplastic anemia or Fanconi's anemia (n = 17), acute leukemia (n = 11), chronic myeloid leukemia (n = 21), and other conditions (n = 2). T cell depletion of the graft was associated with a significant improvement in survival in both UD and ID-BMT. Graft failure was more common in recipients of UD than of ID transplants (13 [25%] vs. 5 [10%] P = 0.05) but there was no significant difference in the frequency of acute or chronic graft-versus-host disease. Actuarial survival was superior for recipients of ID transplants (UD vs. ID: 49% vs. 78%, respectively, at 3 months; 32% vs. 63% at one year). Reduced survival for recipients of UD-BMT was confirmed in case control regression analysis (relative risk 3.0, P = 0.01). Nevertheless in patients whose only alternative is a partially mismatched family donor we think that UD-BMT is justified.

Mechanisms of graft failure after human marrow transplantation: a review.

Graft failure (GF) following bone marrow transplantation (BMT) in man is usually attributed to allograft rejection; however, other mechanisms of GF exist. Exposure of the developing allograft to viruses, myelotoxic drugs, and damage to the host-derived marrow stroma during graft-versus-host disease (GVHD) may all cause GF. Later, in the posttransplant course, recurrence of the original disease may ultimately destroy or replace the graft. Overall allogeneic rejection remains the most important cause of GF and most in vivo and in vitro evidence points to a T cell mechanism. The incidence of rejection after clinical BMT is very variable and highly dependent on HLA and non HLA histocompatibility differences between donor and recipient, the level of recipient sensitisation pre-BMT and the immunosuppressive protocols used pre-transplant and for the prevention of GVHD.

Histocompatible unrelated donors for bone marrow transplantation.

Bone marrow transplantation using HLA one haplotype mismatched family donors has been associated with a high incidence of severe graft-versus-host disease, and an increased risk of graft failure. Recently, the use of histocompatible unrelated donors has been investigated when an HLA genotypically identical sibling is not available. Conventional histocompatibility testing of potential unrelated donors does not always predict alloreactivity in vivo. In this review, the preliminary results of histocompatible unrelated transplants are presented and methods of matching unrelated donors discussed.

The use of unrelated bone marrow donors in the treatment of patients with chronic myelogenous leukemia: experience of four marrow transplant centers.

Thirty-seven patients with chronic myelogenous leukemia who lacked an HLA-identical sibling were transplanted with bone marrow from an HLA-A,B,DR-matched, one locus-mismatched, or two locus-mismatched unrelated volunteer donor. Twenty-two were in chronic phase and 15 had advanced to either accelerated phase or blast crisis. The projected 1000-day survival is 55% for chronic phase patients and 22% for accelerated or blast phase patients. For patients transplanted during chronic phase, results appeared to be comparable whether the donor was fully HLA-matched or HLA one locus-mismatched. These results indicate that marrow grafting from either HLA-identical or HLA one locus-mismatched volunteer donors may be effective therapy for patients with chronic myelogenous leukemia who lack an acceptable related donor.

A survey of use of unrelated volunteer donor bone marrow transplantation at 46 centres worldwide, 1989-93. International Marrow Unrelated Search and Transplant (IMUST) Study.

Unrelated donor bone marrow transplantation is increasingly used to treat haemopoietic disorders where no HLA-identical sibling is available. The International Marrow Unrelated Search and Transplant Study has collected core data on consecutive unrelated donor BMT (UD-BMT) and HLA-identical sibling donor BMT (ID-BMT) performed in 46 participating centres world-wide between March 1989 and February 1993. Eighteen UD-BMT were performed in the first 6-month period in 14 participating centres, while in the last period there were 103 UD-BMT in 46 centres. The percentage of BMT recipients with the following diagnoses were: bone marrow failure UD-BMT 15% and ID-BMT 11%; AML 13% and 27%; ALL 18% and 17%; CML 48% and 31%; and other diseases 7% and 14%. Thirty-eight per cent of UD-BMT recipients had advanced disease compared with only 23% of ID-BMT recipients. Thirty six per cent of UD-BMT compared with 21% of ID-BMT recipients were under 16 years old. More extreme differences in pre-transplant clinical characteristics between UD and ID-BMT recipients were found when diagnosis and stage of disease were considered together. This survey indicates how UD and ID-BMT are currently used in the treatment of haematological disease; however, longer follow-up is required to assess the value of UD-BMT in the management of patients with bone marrow disorders.

A prospective study of factors determining the outcome of unrelated marrow donor searches: report from the International Marrow Unrelated Search and Transplant Study Working Group on behalf of collaborating centres.

Outcomes of searches for unrelated bone marrow donors were analysed to identify factors influencing the probability of identifying an HLA-matched donor and proceeding to an unrelated donor bone marrow transplant (UD-BMT). Between March 1989 and January 1991, 649 unrelated marrow donor searches were entered into the study. Searches were referred from transplant centres in Europe and North America to the Anthony Nolan Research Centre panel and/or the British Bone Marrow and Platelet Donor Panel, two predominantly Caucasian donor registries. Patient immunogenetic and clinical characteristics were documented on study search request forms and search outcomes were monitored at the donor registry. Data were analysed using proportional hazards regression. Major factors predicting favourable search outcomes were patient common Caucasian HLA phenotype and Caucasian ethnic group. Probability of search failure was increased by advanced disease, rare Caucasian HLA phenotype and referral to the smaller registry. Search failure frequently occurred at the mixed lymphocyte reaction stage. This study illustrates the relative roles of clinical, immunogenetic and registry factors in determining the outcome of searches. The information may be used to devise strategies to locate HLA-matched donors for a higher proportion of patients for whom UD-BMT is the preferred 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.

Recurrent graft failure following syngeneic bone marrow transplantation for aplastic anaemia.

We present the case of a 60-year-old woman with drug-induced aplastic anaemia with a healthy monozygotic twin. Proof of monozygosity was confirmed by studies using the hypervariable minisatellite probe to obtain identical DNA fingerprints in donor and recipient. In vitro co-culture studies performed showed no evidence of a recipient-derived cellular or humoral inhibitor of donor haemopoiesis. Despite this, there was no engraftment following simple marrow infusion without preconditioning. A second syngeneic transplant following high dose cyclophosphamide therapy produced trilineage engraftment but severe thrombocytopenia developed at 3 months, followed later by pancytopenia with generalized marrow failure. Following a third syngeneic transplant with cyclophosphamide and total lymphoid irradiation there was good initial engraftment but graft failure occurred at 14 weeks. A fourth transplant using Campath 1G as preconditioning resulted in no engraftment and the patient died of septicaemia 8 weeks following her fourth transplant. We suggest that the cause of the recurrent aplastic anaemia in this case was a defect of marrow stroma as neither an inhibitor of donor haemopoiesis nor an intrinsic defect of donor stem cell growth could be demonstrated in vitro.

Cytotoxic T lymphocyte precursor (CTL-p) frequency analysis in unrelated donor bone marrow transplantation: two case studies.

HLA 'matched' unrelated donor bone marrow transplantation (BMT) is associated with an increased incidence and severity of acute graft-versus-host disease (GVHD) in comparison with HLA-identical sibling transplants. Using a limiting dilution analysis system for quantitating frequencies of alloreactive cytotoxic T lymphocyte precursors (CTL-p), we previously demonstrated a correlation between CTL-p frequency and HLA disparity between responder and stimulator, and between CTL-p frequency and the incidence of acute GVHD following HLA A, B, DR matched unrelated donor BMT. In this study we assayed CTL-p frequencies in two HLA 'matched' unrelated donor/patient pairs, with single HLA antigenic mismatches detected by allogenotyping or isoelectric focusing but not by HLA serology, and demonstrated that the CTL-ps were specifically directed at the mismatched antigen. Both class I and class II antigens were detected. These data, and our previous work, suggest that high CTL-p frequencies in HLA 'matched' unrelated pairs are indicative of HLA antigenic variants undetected by serology but recognized by molecular typing, and that these are responsible for the value of the assay in predicting acute GVHD after BMT. We propose that this assay system be used in aiding final donor selection before unrelated or mismatched related donor BMT.

Bone marrow transplantation for Fanconi's anaemia: the Hammersmith experience 1977-89.

Twenty-one patients with Fanconi's anaemia (FA) were treated by allogeneic bone marrow transplantation (BMT). Two, transplanted before 1980, received high dose cyclophosphamide conditioning and both died. Subsequently 19 patients received conditioning with low dose cyclophosphamide 5 mg/kg x 4 and total body irradiation 200 cGy x 3. Ten of 19 received HLA identical sibling marrow (ID-BMT) and nine marrow from alternative donors (MM-BMT). Marrow was T cell depleted in 9/19 cases. Sustained engraftment was observed in 13 cases (eight ID-BMT, five MM-BMT). Nine patients developed greater than or equal to grade II acute graft-versus-host disease (GVHD) (six ID-BMT, nine MM-BMT). Chronic GVHD occurred in 5/11 evaluable patients. Overall survival of the low dose cyclophosphamide group was 9/19 (47%) at a median follow-up of 1257 days post-BMT (110-1825). Six of 10 (60%) survived after ID-BMT compared with two of nine (22%) after MM-BMT. We conclude that allogeneic BMT using a low dose cyclophosphamide protocol is a satisfactory treatment for FA patients who have a normal HLA identical sibling. The results of MM-BMT have been poor, and must improve before these transplants can be generally recommended for treatment of FA.

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.