Allogeneic or autologous bone marrow transplantation for acute lymphoblastic leukemia in first complete remission

Forty-seven patients with high risk acute lymphoblastic leukemia (ALL) received an allogeneic (allo) or autologous (auto) bone marrow transplant (BMT). Patients in both groups were comparable in terms of age, initial presentation of ALL and induction chemotherapy. Allo patients were transplanted earlier (median 3 months after CR) than auto patients (median 6.5 months after CR). Auto patients received more consolidation chemotherapy before BMT. All patients received total body irradiation 2.2 Gy/day x 5 days after cyclophosphamide 60 mg/kg x 2 (18 allo and five auto) or melphalan 140 mg/m2 (seven allo and 17 auto). Prevention of graft-versus-host disease (GVHD) was by conventional immunosuppression in 17 patients and T cell depletion in eight. Seven patients (28%) developed moderate to severe acute GVHD. Auto marrow was treated in vitro in each case. Seven patients died in CR from BMT complications (five allo and two auto). The probability of relapse was 9% for patients receiving allo BMT and 52% for patients receiving auto BMT (p less than 0.01). The disease-free survival was 71% for allo BMT and 40% for auto BMT (p = NS). Early BMT is an effective form of consolidation for high risk patients with ALL in first CR. An allogeneic anti-leukemia effect was demonstrated in this study.     

Successful second allogeneic bone marrow transplantation in a relapsed acute myeloid leukemia patient with fungal liver abscess

Summary Disseminated fungal infection not infrequently complicates the course of allogeneic bone marrow transplantation (allo BMT) in severely immunocompromised patients, and the prognosis of BMT patients who develop systemic fungal infection is very poor. We describe a patient who developed disseminatedCandida albicans infection with liver abscess after the first allo BMT for acute myelogenous leukemia (FAB M2). The infection was successfully eradicated by the administration of miconazole and amphotericin B. However, 1 year after the first allo BMT, the patient suffered a relapse of acute myelogenous leukemia with fungal liver abscess. A second allo BMT, accelerating granulocyte recovery by recombinant human granulocyte colony-stimulating factor (rhG-CSF), was successfully performed and the fungal liver abscess resolved with a combination therapy of fluconazole and amphotericin B. The patient is alive and free of both leukemia and fungal disease more than 37 months after the first allo BMT and 25 months after the second allo BMT.     

Mixed chimerism in the B cell lineage is a rapid and sensitive indicator of minimal residual disease in bone marrow transplant recipients with pre-B cell acute lymphoblastic leukemia

One of the major problems after allogeneic bone marrow transplantation (BMT) is a high frequency of leukemia relapse. We have prospectively studied the presence of donor- and recipient-derived chimeric cells in bone marrow recipients with pre-B cell acute lymphoblastic leukemia (pre-B-ALL). The chimeric status of BMT recipients was compared to minimal residual disease (MRD) detection by analysis of immunoglobulin heavy chain (IgH) and T cell receptor (TcR) genes. Post-transplant blood and bone marrow samples from 12 patients with pre-B-ALL were studied. Five patients showed mixed chimerism (MC) in the CD19-positive cell fraction. Four of them have relapsed to date. The remaining patient with MC in the B cell lineage was also MRD positive in the same samples. All seven patients with donor chimerism in the B cell fraction remain in clinical remission (P = 0.01). In samples from all five patients having MC in the B cell lineage, the patient-specific IgH or TcR rearrangement was also detected. In three of four patients who relapsed, MC in the B cell lineage was seen more than 2.5 months prior to morphologically verified relapse. The results of this comparison suggest that routinely performed MC analysis of the affected cell lineage may facilitate post-BMT monitoring and rapid therapeutic decisions in transplanted patients with pre-B-ALL.     

The significance of host haemopoietic cells detected by cytogenetic analysis of bone marrow from recipients of bone marrow transplants

Bone marrow from 39 patients who received a bone marrow transplant (BMT) from a matched donor of different sex were studied by chromosome analysis for evidence of mixed haemopoietic chimaerism (MC). Recipient metaphases were detected in the bone marrow of 10 patients after BMT. Patients in whom MC was detected within 6 weeks of BMT did not all have a poor outcome. Two of seven are disease-free survivors at greater than 470 and greater than 632 d. All three patients in whom MC was diagnosed more than 6 weeks after BMT subsequently relapsed. Four factors appear to be important in determining the probability of relapse when MC is detected in a patient after BMT: the timing of detection of residual recipient cells; the proportion of these cells in the bone marrow; persistence of these cells in increasing proportions; and the karyotype of the recipient metaphases detected. Cytogenetic assessment may provide the earliest indication of relapse in these patients. In addition, this study provides further evidence that cyclophosphamide and total body irradiation, as used in these patients, may be inadequate conditioning therapy for BMT.     

Treatment of autoimmune diseases in mice by a new method for allogeneic bone marrow transplantation

Remarkable advances have been made in bone marrow transplantation (BMT), which has become a powerful strategy for the treatment of leukemia, aplastic anemia, congenital immunodeficiency, and also autoimmune disease. Using various animal models, allogeneic (allo) BMT has been found to be useful in the treatment of various autoimmune diseases. In MRL/lpr mice, which are radiosensitive (<8.5 Gy) and are an animal model for autoimmune diseases, conventional BMT resulted in only transient effects; the manifestations of the autoimmune diseases recurred 3 months after BMT. Using MRL/lpr mice, we have very recently established a new strategy for allo BMT. We injected bone marrow cells (BMC) directly into the bone marrow cavity (intrabone marrow [IBM] injection) of recipients that had received fractionated irradiation. This 'IBM-BMT' was found to be effective in treating autoimmune diseases in radiation-sensitive and chimeric-resistant MRL/lpr mice. In addition, this strategy was found to be applicable for the transplantation of organs. We believe that these strategies for BMT and organ transplantation herald a new era in transplantation.     

Natural history of mixed chimerism after bone marrow transplantation with CD6-depleted allogeneic marrow: a stable equilibrium

Mixed hematopoietic chimerism (MC) is a common finding after allogeneic bone marrow transplantation (BMT), but the natural history of this phenomenon remains unclear. To understand the evolution and the implications of this finding, we performed a prospective analysis of the development of mixed chimerism in 43 patients with hematologic malignancies who received bone marrow (BM) from human leukocyte antigen (HLA)-identical sibling donors. T-cell depletion in vitro with anti-T12 (CD6) monoclonal antibody and rabbit complement was used as the only method of graft-versus-host disease (GVHD) prophylaxis. Overall, MC was identified in peripheral blood (PB) and BM in 22 of 43 (51%) patients evaluated. MC was found by restriction fragment length polymorphism (RFLP) analysis in 21 of 40 (53%) patients, by cytogenetic analysis in 6 of 29 (21%) patients, and by red blood cell phenotyping in 4 of 9 (44%) patients. RFLP studies were performed at 0.5, 1, 3, 6, 9, and 12 months post-BMT and then every 6 months, and showed a high probability of developing MC in the first 6 months after BMT followed by stabilization after 12 months. Cytogenetic analysis was less sensitive in detecting MC. Once MC was detected after BMT, the percentage of recipient cells increased very slowly over more than 3 years of follow-up, and no patient reverted to complete donor hematopoiesis (CDH). Thus, recipient and donor cells remained in a relative state of equilibrium for prolonged periods that seemed to favor recipient cells over donor cells. Patient's disease, remission status, or intensity of the transplant preparative regimen did not influence the subsequent development of mixed chimerism. Early immunologic reconstitution was the only factor that correlated with the subsequent chimeric status of the patients. The percentage and absolute number of T3 (CD3) and T4 (CD4) positive cells at day 14 after BMT were significantly higher in the patients who maintained CDH but NK cell reconstitution was similar in both groups, suggesting that early reconstitution with T cells may play a role in preventing recovery of recipient cells after BMT. GVHD was also associated with maintenance of CDH, but the probability of relapse, survival, and disease-free survival was identical in patients with MC and CDH.     

Detection of residual leukemia after bone marrow transplant for chronic myeloid leukemia: role of polymerase chain reaction in predicting relapse

We used the polymerase chain reaction (PCR) to detect residual leukemia-specific mRNA in blood and marrow from 37 patients in complete hematologic and cytogenetic remission after allogeneic bone marrow transplant (BMT) for chronic myeloid leukemia (CML). Our two-step PCR method involved the use of "nested primers" in the second step and could detect one K562 cell diluted into 10(5) normal cells. Elaborate measures were taken to exclude false-positive and false-negative results. In nine patients whose blood and marrow were studied simultaneously the results were concordant (two positive and seven negative). Twenty-three patients transplanted in chronic phase (CP) with unmanipulated donor marrow were studied. Blood cells from nine of these patients were studied 3 to 6 months post-BMT and six were PCR positive; three were negative on subsequent studies. Blood cells from 18 patients studied between 8 months and 8 years post-BMT were all PCR negative. Nine patients transplanted in CP with T-cell-depleted marrow cells were studied. Blood from five was positive 3 to 24 months post-BMT; blood from five was negative 3 to 6 years post-BMT. Four patients no longer in first CP were studied after BMT with unmanipulated donor marrow. Blood from all four was positive 5 to 19 months post-BMT. Based on the known clinical results of transplant in these three cohorts we conclude that PCR may be positive within 6 months of BMT in patients who can expect long-lasting remission, whereas PCR positivity later after BMT may indicate that the probability of cure is reduced. Thus, the technique may prove useful for early assessment of new transplant protocols that might inadvertently increase the risk of relapse.     

Successful allogeneic bone marrow transplantation. Crucial roles of stromal cells in prevention of graft rejection.

We have previously found that a major histocompatibility complex (MHC) restriction exists between pluripotent hemopoietic stem cells (P-HSCs) and stromal cells. Based on this finding, we have recently found using chimerism-resistant mouse combinations that successful allogeneic (allo) BMT can be executed by recruiting donor bone marrow stromal cells. The strategies include donor bone grafts under the skin, injection of whole bone marrow cells (BMCs) including stromal cells via the portal vein (PV), and injection of whole BMCs directly into the bone marrow cavity (intra-bone marrow [IBM] injection). In this paper, we show how stromal cells play crucial roles in overcoming chimerism-resistant allo BMT.     

Prognostic value of hematopoietic chimerism in patients with acute leukemia after allogeneic bone marrow transplantation: a prospective study

Hematopoietic chimerism as a predictive marker for the relapse of acute leukemia after allogeneic BMT was evaluated in a prospective study. Monthly assays of hematopoietic chimerism were performed from peripheral blood samples by PCR amplification of short tandem repeats or amelogenin loci. Between December 1997 and June 1999, 33 patients enrolled and 30 were evaluable (two early deaths, one lack of informative bands for chimerism evaluation). There were 14 male and 16 female patients (15 AML and 15 ALL) with a median age of 31 years (range 16-46). Mixed chimerism (MC) was observed at least once in 14 of 30 patients (47%). There was no significant difference between 14 patients who showed MC (MC group) and 16 patients who did not show MC (complete chimerism (CC) group) in terms of age, sex, disease status at BMT, donor type, and the number of bone marrow cells infused. There was no significant difference in the neutrophil and platelet engraftment rates between the two groups. After a median follow up of 10.9 months (range 4.3-22.4), five patients in the CC group and two patients in the MC group relapsed (P = 0.27). All five patients who relapsed in the CC group maintained CC up to 1 month prior to clinical relapse. Our study demonstrated that the patients who showed MC post BMT did not have higher risk of relapse of acute leukemia when compared to patients who did not show MC. Sensitive PCR-based assays for hematopoietic chimerism applied on a monthly basis after allogeneic BMT could not predict relapse of acute leukemia.     

Patterns of hematopoietic chimerism following bone marrow transplantation for childhood acute lymphoblastic leukemia from volunteer unrelated donors

Hematopoietic chimerism was analyzed in serial bone marrow samples taken from 28 children following T-cell depleted unrelated donor bone marrow transplants (UD BMT) for acute lymphoblastic leukemia (ALL). Chimeric status was determined by polymerase chain reaction (PCR) of simple tandem repeat (STR) sequences (maximal sensitivity, 0.1%). At least two serial samples were examined in 23 patients. Of these, two had evidence of complete donor engraftment at all times and eight showed stable low level mixed chimerism (MC) (<1% recipient hematopoiesis). All 10 of these patients remain in remission with a minimum follow-up of 24 months. By contrast, 13 patients demonstrated a progressive return of recipient hematopoiesis. Five of these relapsed (4 to 9 months post BMT), one died of cytomegalovirus pneumonitis and seven remain in remission with a minimum follow-up of 24 months. Five children were excluded from serial analysis as two serial samples were not collected before either relapse (3) or graft rejection (2). We conclude that as with sibling transplants, ex vivo T depleted UD BMT in children with ALL is associated with a high incidence of MC. Stable donor engraftment and low level MC always correlated with continued remission. However, detection of a progressive return of recipient cells did not universally correlate with relapse, but highlighted those patients at greatest risk. Serial chimerism analysis by PCR of STRs provides a rapid and simple screening technique for the detection of relapse and the identification of patients with progressive MC who might benefit from detailed molecular analysis for minimal residual disease following matched volunteer UD BMT for childhood ALL.     

Late graft rejection and second infusion of bone marrow in children with aplastic anaemia

Late graft rejection following allogeneic bone marrow transplantation (BMT) for aplastic anaemia is a significant clinical problem and is associated with a high risk of mortality. We report two children with severe aplastic anaemia (SAA) who developed very late graft rejection 2 years and 4 months and 10 years respectively after allogeneic BMT from HLA-identical siblings. Following a second BMT from their initial donors, engraftment has been sustained in both cases. The patients are alive with full donor chimaerism, 18 and 19 years from initial transplant. These cases illustrate that graft failure can be an extremely late event after allogeneic BMT for SAA, and that long-term sustained engraftment can be achieved in these patients with second BMT from the original donors.     

The effect of bone marrow transplantation on the osteoblastic differentiation of human bone marrow stromal cells.

Osteoporosis is a serious and relatively common complication of transplantation procedures. However, little is known about the exact mechanism or severity of osteoporosis complicated by bone marrow transplantation (BMT). We conducted both ex vivo and clinical studies to identify the mechanism and extent of bone loss after BMT. In a prospective clinical study, we intended to identify the changes in bone turnover markers and bone mineral density (BMD) after BMT. During a 1-yr follow-up, BMD was measured before BMT and 1 yr after BMT in 67 patients undergoing BMT. Biochemical markers of bone formation and resorption were measured in all patients at short-term intervals during the yearlong follow-up. In ex vivo study, we cultured human bone marrow cells of normal controls and BMT recipients in osteogenic medium and compared their osteogenic potential. Using a DNA fingerprinting method, we also investigated the origin of bone marrow stromal cells that were harvested 3-4 wk after BMT. In a clinical study of 67 patients undergoing BMT, the mean serum carboxy-terminal cross-linked telopeptide of type I collagen increased progressively until 4 wk after BMT. Thereafter, it began to decrease and reached basal values after 1 yr. Serum osteocalcin decreased progressively until 3 wk after BMT and reached basal values after 3 months. One year after BMT, lumbar spine BMD had decreased by 3.3% (P < 0.05), and total proximal femoral BMD had decreased by 8.9% (P < 0.001). For the ex vivo study, bone marrow was obtained from healthy donors (n = 7) and transplant recipients (n = 7). Then, mononuclear cells including marrow stromal cells were isolated and cultured to osteoblastic lineage. Alkaline phosphatase activities of each group were measured by the time course of secondary culture, and the mineralizing potentials were compared between the two groups. Cells cultured in our system showed characteristics of osteoblast-like cells differentiated from marrow stromal cells. They were initially in a fibroblastic-like spindle shape and became cuboidal with the formation of nodules that were later confluent. The cells were stained to both alkaline phosphatase histochemistry and Von Kossa histochemistry, demonstrating that these cells were of osteoblastic lineage differentiating from marrow stromal cells. The mean time required for the near-confluence in the primary culture was 15 and 22.9 d in healthy donors and transplant recipients, respectively (P = 0.003). Alkaline phosphatase activity was significantly lower in the bone marrow recipients than in the healthy donors at d 7 and 10 of the secondary cultures. The period at which peak activity of alkaline phosphatase was reached was also delayed in the osteoblasts derived from BMT recipient bone marrow compared with those of healthy donors. Using Von Kossa histochemistry, much more mineralization was observed in osteoblasts of healthy donors than those of BMT recipients. After BMT, although the peripheral mononuclear cells in the recipients were of donor origin, the bone marrow stromal cells were of recipient origin according to the PCR analysis using YNZ 22 mini-satellite probe. In conclusion, the differentiation of bone marrow stromal cells into osteoblasts was impaired after BMT, and this might contribute to post-BMT bone loss.     

Minimal residual disease after allogeneic bone marrow transplantation for chronic myeloid leukaemia: a metaphase-FISH study

Metaphase-FISH was adopted for the detection of proliferating Philadelphia-positive (Ph⁺) residual leukaemic cells in 25 patients with chronic myeloid leukaemia treated with allogeneic bone marrow transplantation (BMT). Patients were followed up during their clinical remission for 4–50 months (median 17 months) after BMT. 80 bone marrow samples were studied. For most of the cases no fewer than 1000 metaphases were analysed. Six patients (24%) showed residual Ph⁺ cells during the first 6 months and two others by the end of the first year after BMT. Three patients relapsed during the study and in two of them residual Ph⁺ cells were detected during the first 6 months after BMT. In 17 patients no Ph⁺ cells were detected at any stage of follow-up and 16 (94.1%) of them continue in complete clinical and haematological remission. Our results indicate that metaphase-FISH is a reliable tool in the quantitation of proliferating residual leukaemic cells. We suggest that consecutive findings of equal amounts of residual leukaemic cells do not necessarily predict a relapse. However, their presence calls for follow-up at shorter intervals where an increasing number of these cells predicts an ensuing relapse.     

Cytogenetic events after bone marrow transplantation for chronic myeloid leukemia in chronic phase

Forty-eight patients treated by allogeneic bone marrow transplantation (BMT) for Philadelphia (Ph) chromosome-positive chronic myeloid leukemia in chronic phase had serial cytogenetic studies of marrow performed at intervals after transplant. Twenty patients received marrow cells from donors of opposite sex. Ph+ marrow metaphases were identified in 24 of 48 (50%) of patients after BMT; they were first seen early (within 1 year) in 16 cases and late (greater than 1 year after BMT) in eight cases. Ph-positivity after BMT occurred more commonly in recipients of T-depleted than nondepleted marrow (19 of 28 v 5 of 20). In 4 cases the Ph+ metaphases were found only transiently after BMT; in 11 cases the Ph+ metaphases have persisted but hematologic relapse has not ensued; in 9 cases the finding of Ph+ metaphases coincided with or preceded hematologic relapse. Chromosomes in cells of donor origin had morphological abnormalities in two cases. No relapses were identified in cells of donor origin. Our data suggest that the relationship between cells of recipient and donor origin is complex: cure of leukemia may depend on factors that operate for some months or years after BMT.     

Evolution of mixed chimerism after allogeneic bone marrow transplantation as determined on granulocytes and mononuclear cells by the polymerase chain reaction.

To evaluate the clinical relevance of mixed chimerism (MC) after allogeneic bone marrow transplantation (BMT), we developed a method based on amplification of DNA minisatellites by the polymerase chain reaction (PCR). This sensitive method by which MC lower than 1% can be detected is applicable to any patient-donor pair. Furthermore, because the analysis requires only small amounts of DNA, it allowed us to analyze samples early after BMT and during graft rejection. Results were obtained within 48 hours after blood sampling. Determination of MC in granulocytes (GR) and in mononuclear cells (Mnc) was performed in 20 patients treated for various hematologic malignancies. In patients who received untreated BM, recipient cells disappeared rapidly after BMT. In patients transplanted with T-cell-depleted BM, MC occurred in 15 of 16 cases. The percentage of host Mnc was always significantly higher than the percentage of host GR. The evolution of MC in patients who received T-cell-depleted marrow showed distinct patterns depending on whether patients remained in continuous complete remission, relapsed, or rejected their grafts. During complete remission, a relatively stable and significant number of host cells could be detected during the first 2 years after transplantation. Thereafter, their number decreased, but even after 4 years, low numbers of host cells could persist. When the patients relapsed, an increase in host Mnc was monitored without significant changes in the number of donor Mnc. In contrast, after the relapse, donor GR were no longer detected. Two cases of graft rejection were studied. Directly after the onset of the rejection, donor GR and Mnc disappeared rapidly. During that period, no significant changes in the number of host Mnc were detected.     

Successful Bone Marrow Transplantation for Severe Aplastic Anemia in a Patient with Persistent Human Parvovirus B19 Infection

Persistent infection with human parvovirus B19 (B19) is primarily associated with chronic bone marrow failure in immunocompromised patients, but occasionally this organism may also affect immunocompetent hosts. B19 is also suggested as a causative agent of organ failure during bone marrow transplantation (BMT).We herein report the case of a 9-year-old girl with no previous history of immunodeficiency who developed severe aplastic anemia concurrent with B19 persistent infection. Both immunoglobulin (Ig)M antibody to B19 and B19 DNA identified by real-time polymerase chain reaction were found in the patient's serum at time of diagnosis of aplastic anemia. No giant proerythroblasts were found in her bone marrow at diagnosis. Although intravenous administration of Ig (IVIg) reduced serum B19 DNA, the aplastic status of her bone marrow did not improve. Both aplastic anemia and persistent B19 viremia were successfully treated by BMT from an HLA-identical sibling donor. Serum B19 DNA increased temporarily after BMT; however, neither organ nor marrow failure was observed. B19 DNA disappeared from the serum 2 months after BMT, suggesting that a normal immune response was restored by BMT and terminated the B19 viremia. During BMT, use of high-titer IVIg for B19 might prevent B19-associated organ failure.     

A systematic approach to molecular quantitative determination of mixed chimaerism following allogeneic bone marrow transplantation: an analysis of its applicability in a group of patients with severe aplastic anaemia

Mixed chimaerism (MC) following allogeneic bone marrow transplantation (allo-BMT) is defined as the persistent cohabitation of haematopoietic cells from recipients and donors. Its kinetics, clinical implications and more efficient laboratory approaches for MC detection are the object of ongoing research in view of the possibility of developing useful markers. Here we describe a sequential analysis of chimaerism using variable number of tandem repeat (VNTR) polymerase chain reaction (PCR) followed by quantitative, fluorescent labelled, short tandem repeat (STR) PCR. A set of four, highly discriminative VNTR and four STR markers was used to assess chimaerism. Sensitivity and regression analysis indicated that this approach was reliable for routine application in a single BMT centre. We studied 12 patients with severe aplastic anaemia (SAA) who had received allo-BMT, and had been conditioned with cyclosphosphamide (Cy) with or without anti-thymocyte globulin (ATG). We found a 50% prevalence of MC in the whole group, with levels between 4% and 37% of recipient cells. A sustained stable MC pattern after BMT was characteristic of the Cy-only conditioned patients but was also recorded in one patient treated with the Cy + ATG regime who showed a sustained MC pattern over a period of 24 months post-BMT. In none of our patients, MC was associated with an increased risk of graft rejection in a median follow-up of 39.5 months.     

Slow evolution of chronic myeloid leukaemia relapsing after BMT with T-cell depleted donor marrow

Thirty-three patients with Philadelphia positive (Ph+) chronic myeloid leukaemia (CML) treated in chronic phase by bone marrow transplantation (BMT) with T-cell depleted HLA-identical sibling marrow were evaluable for relapse at a median follow up of 41 months (range 16-59 months). Twenty-six (78%) had Ph+ marrow metaphases demonstrated at some time post BMT. The subsequent pattern of disease was variable. In 15 of these cases haematological relapse occurred within 24 months of BMT. Four patients proceeded to haematological relapse more slowly. Seven patients had only cytogenetic evidence of relapse. Of the 19 patients with haematological relapse, five received second transplants and two survive; 13 of the other 14 survive in chronic phase at median times from allografting and from recognition of haematological relapse of 41 months (range 25-59 months) and 18 months (range 5-36 months) respectively. For these 13 patients disease progression after relapse seems to be relatively indolent. In the four patients we could study, blood lymphocytes were almost all of donor origin. We suggest that even in patients with cytogenetic or haematological evidence of relapse after T-cell depleted BMT, leukaemic cell proliferation may still be restrained to some extent by a graft-versus-leukaemia effect mediated by donor-derived lymphoid cells.     

Myeloid mixed chimerism is associated with relapse in bcr-abl positive patients after unmanipulated allogeneic bone marrow transplantation for chronic myelogenous leukemia

BACKGROUND AND OBJECTIVE: Although bcr-abl polymerase chain reaction (PCR) positivity after bone marrow transplantation (BMT) for chronic myelogenous leukemia (CML) is significantly related to relapse, the predictive value of the assay is not very high and therefore most investigators consider that qualitative RT-PCR data alone are too imprecise to enable clinical decisions to be taken in individual cases. To define the clinical outcome of bcr-abl positive patients after unmanipulated BMT better, we sought the origin of hematopoiesis and traced its evolution over time. DESIGN AND METHODS: Forty-nine patients received allogeneic BMT for CML (39 in chronic phase and 10 in accelerated phase/blast crisis). Median follow-up was 61 months (range 4-92). mRNA and DNA were used to assess bcr-abl and chimerism status respectively. Quantitative VNTR-PCR on total cells and lymphoid or myeloid population allowed us to assign and measure the origin of hematopoiesis. RESULTS: Both bcr-abl positivity and the presence of mixed chimerism (MC) were significantly associated with relapse (p = 0.0009 and p < 0.0001 respectively). Relapse was observed in one of 39 patients with complete donor chimerism and in 6 of 9 patients with MC. These six cases showed increasing levels of host hemopoiesis and bcr-abl positivity in the CD15-positive population prior to relapse. The other three cases had decreasing or stable low-level MC which was restricted to the T-cells as well as bcr-abl negativity. INTERPRETATION AND CONCLUSIONS: Whereas the simple detection of bcr-abl fails to identify patients who will relapse with certainty, the assessment of MC by VNTR-PCR does identify patients headed to relapse. Confirmation of myeloid involvement and increasing levels over time further elucidates the clinical outcome of bcr-abl positive patients after BMT.     

New strategies for BMT,organ transplantation,and regeneration therapy

Bone marrow transplantation (BMT) is becoming a powerful strategy for the treatment of hematologic disorders, congenital immunodeficiencies, metabolic disorders and also autoimmune diseases. We have previously found using various animal models for spontaneous autoimmune diseases, that allogeneic bone marrow transplantation (allo BMT) can be used to prevent and treat various autoimmune diseases. In addition, we have found that autoimmune diseases are stem cell disorders. However, in MRL/lpr mice, which are radiosensitive (<8.5 Gy), we found that conventional BMT had only a transient effect on autoimmune diseases, which were found to recur. Therefore, we concentrated on discovering new strategies to prevent and treat autoimmune diseases in the radiosensitive and chimeric-resistant MRL/lpr mouse. Using MRL/lpr mice, we established a new method for allo BMT. In this method, whole bone marrow cells (BMCs), containing a small number of T cells and mesenchymal stem cells (MSCs), were directly injected into the bone marrow cavity (intra-bone marrow [IBM]-BMT). MRL/lpr mice treated with IBM-BMT survived more than 2 years without showing the symptoms of autoimmune diseases. To apply this BMT method to humans, we have also established a new method for BMC harvesting using cynomolgus monkeys. In this method, BMCs are harvested from the long bones using a "Perfusion Method" (PM) and the whole BMCs (including MSCs) are then injected directly into the IBM. We believe that this new method will become a powerful strategy for the treatment of various intractable diseases, including age-associated diseases such as osteoporosis.