ALBERT

Description: Graft rejection after marrow transplantation is generally thought to be mediated by alloreactive immune effector cells of host origin. Transfused blood products also contain immune cells capable of alloreactivity against both donor graft and host. To reduce the risk of transfusion-associated graft-versus-host disease (GVHD) and graft rejection, standard procedure is to irradiate all blood products with at least 1,500 rad before transfusion. We report a patient with chronic myelogenous leukemia who developed graft rejection and GVHD after receiving a T-cell-depleted transplant from a serologically HLA-A, B, DR/DQ matched and mixed lymphocyte culture (MLC) nonreactive unrelated donor. Cytogenetic analysis of marrow cells collected at the time of graft rejection revealed a PH1-negative female karyotype that was not consistent with donor cells. Use of specific minisatellite DNA probes (YNH 24, H-RAS, and 3′ HVR) revealed the exclusive presence of third- party (neither donor nor recipient) restriction-fragment-length polymorphisms (RFLP) in both peripheral blood and marrow. Repeat RFLP analysis 3 days later showed persistence of this unique third-party banding pattern. DNA-based HLA-typing, using polymerase chain reaction (PCR) and oligonucleotide probe hybridization, also showed these cells to be derived from an individual whose HLA-DR type was distinct from donor and recipient. Together, these findings suggested the presence of a proliferating population of transfused cells possessing alloreactivity against both donor graft and host, despite prior irradiation of all blood products with 2,000 rad. Limiting dilution analysis to assess the frequency of irradiated lymphocytes able to respond to mitogen revealed an approximate 5- to 6-log reduction at 1,500 to 2,000 rad as compared with unirradiated controls. These data indicate that a small percentage of lymphocytes can survive irradiation at these doses and suggest that existing blood-product irradiation guidelines may require reassessment, especially in T-cell-depleted transplant recipients.

Description: Allogeneic bone marrow transplantation is the treatment of choice for many childhood leukemias. The donor of choice-an HLA matched sibling-is only available about 30% of the time. Unrelated donors are an alternative choice. In this report, we describe the results of unrelated donor bone marrow transplants (BMT) in 50 children with leukemia (25 acute lymphoblastic leukemia [ALL], 3 acute myeloid leukemia [AML], 3 juvenile chronic myelogenous leukemia [JCML], 10 chronic myeloid leukemia [CML]) or myelodysplastic syndrome (MDS; 9). The median age of the 31 male and 19 female patients was 9 years (range 2 to 18). Only 13 patients were serologically matched at HLA-A, B, DR, and DQ with their donors; 6 of these were reactive in mixed lymphocyte culture. The other 37 patients were mismatched for one (36 patients) or more (1 patient) HLA antigens. Pretransplant conditioning included cytosine arabinoside, cyclophosphamide, fractionated total body irradiation (TBI) (with lung, liver, and more recently, kidney shielding), and methylprednisolone. High-risk patients also received busulfan. Graft-versus-host disease (GVHD) prophylaxis consisted of T- cell depletion with IgM monoclonal antibody T10B9 plus complement and posttransplant cyclosporine-A. Forty-nine patients (98%) engrafted. Median times to greater than 500 polymorphonuclear leukocytes (PMN)/microL and greater than 25,000 platelets/microL were 18 and 20 days, respectively. Acute GVHD 〉 or = grade II occurred in 16 patients (33%); 13 (81%) of these died. Chronic GVHD developed in 30 of 40 patients at risk, but was extensive in only 5. Event-free survival (EFS) for all patients was 44% +/- 7% (median follow-up was 49 months), and overall survival was 50 +/- 7%. Patients with low-risk disease (ALL or AML in first or second remission and CML in chronic phase) had a better EFS than children with high-risk disease (60% v 34%, P = .07). There was no significant difference in EFS between patients who were serologically matched with their donors (46%) and those who were partially mismatched (43%) (P = .97). These data compare favorably with published reports for children transplanted with HLA-matched sibling donors and should encourage earlier consideration of unrelated donor BMT in children with leukemia or myelodysplasia.

Description: Between January 1988 and March 1993, 48 patients received T-cell- depleted marrow grafts from unrelated donors as treatment for chronic myelogenous leukemia (CML). The median age of the population was 31.7 years (range 5.4 to 53) with 17 of 48 patients greater than 40 years of age. Twenty-seven patients were transplanted in chronic phase, 17 in accelerated phase, and 4 in blast crisis. All patients received a standardized preparative regimen of cyclophosphamide, high-dose cytosine arabinoside, methylprednisolone, and total body irradiation. Marrow grafts were depleted of mature T cells with the alpha beta T- cell receptor antibody T10B9 as graft-versus-host disease (GVHD) prophylaxis. All patients also received posttransplant cyclosporine therapy. Twenty-eight of 48 patients were mismatched with their donors for one or more HLA-A, B, DR, or DQ loci by either serology or high- resolution oligonucleotide genotyping. Nine of 28 were mismatched at multiple HLA loci. Durable engraftment was achieved in 94% (45/48) of patients. The actuarial probability of developing grades II to IV and grades III to IV acute GVHD were 39.6% (95% confidence interval (CI) 26.9 to 53.0) and 8.3% (95% CI 6.1 to 10.9) for the entire cohort. There was no difference in the incidence of grades II to IV acute GVHD between patients receiving matched (36.8%) or mismatched (41.4%) marrow grafts (P = .77). The actuarial probability of relapse at 2 years was 8.8% (95% CI 2.1 to 21.6) for the entire cohort and 18% (95% CI 4 to 41) for patients transplanted in either the accelerated or blast crisis phase (advanced disease). One cytogenetic relapse has occurred among patients transplanted in the chronic phase. The probability of disease- free survival at 2 years was 52% (95% CI 24 to 70) for patients transplanted in chronic phase and 46% (95% CI 25 to 73) for patients transplanted with advanced disease. No difference in disease-free survival was observed between patients receiving matched (49%) or mismatched (51%) marrow grafts (P = .90). This study shows that patients receiving unrelated T-cell-depleted marrow grafts for CML can achieve durable engraftment with a low incidence of severe GVHD and apparent preservation of graft-versus-leukemia reactivity. These data also suggest that T-cell depletion may allow patients who might otherwise experience unacceptable toxicity from GVHD-related complications caused by older age or increased HLA disparity to benefit from unrelated marrow grafts.

Description: Disease relapse after allogeneic bone marrow transplantation (BMT) is a major cause of treatment failure and is thought to evolve from clinically occult residual disease in the recipient. However, the demonstration of minimal residual disease (MRD) in individual patients is of uncertain prognostic significance because the detection of residual disease has not consistently correlated with subsequent relapse. Moreover, the optimal therapeutic approach in patients with MRD after allogeneic BMT is unknown. The study of these issues has been hindered by the lack of clinically relevant animal models. In this report, we characterize a novel murine model for the study of MRD after allogeneic BMT. This model was designed to simulate high-risk BMT in humans in which patients receive transplants in relapse and disease recurrence in the major cause of treatment failure. The H-2-compatible, mixed lymphocyte culture nonreactive murine strains, AKR (H-2k) and CBA (H-2k), were chosen to parallel marrow transplants from HLA-matched siblings, which represent the majority of allo-transplants in humans. Male AKR leukemia cells were used in female donor/host chimeras permitting the Y chromosome to serve as a leukemia-specific marker for MRD. Detection of residual male leukemia cells in the peripheral blood of the primary host was facilitated by use of the polymerase chain reaction (PCR) and sequence-specific oligonucleotide probe hybridization (SSOPH). Use of PCR/SSOPH was highly predictive of clinical outcome (relapse or cure) in animals receiving transplants (P 〈 .00002) and detected disease recurrence earlier than comparative flow cytometric analysis studies. This murine model will be useful in evaluating the efficacy of therapeutic strategies aimed at reducing disease relapse posttransplant and can be adapted to other transplant murine tumor systems for the study of MRD.

Description: Graft rejection after marrow transplantation is generally thought to be mediated by alloreactive immune effector cells of host origin. Transfused blood products also contain immune cells capable of alloreactivity against both donor graft and host. To reduce the risk of transfusion-associated graft-versus-host disease (GVHD) and graft rejection, standard procedure is to irradiate all blood products with at least 1,500 rad before transfusion. We report a patient with chronic myelogenous leukemia who developed graft rejection and GVHD after receiving a T-cell-depleted transplant from a serologically HLA-A, B, DR/DQ matched and mixed lymphocyte culture (MLC) nonreactive unrelated donor. Cytogenetic analysis of marrow cells collected at the time of graft rejection revealed a PH1-negative female karyotype that was not consistent with donor cells. Use of specific minisatellite DNA probes (YNH 24, H-RAS, and 3′ HVR) revealed the exclusive presence of third- party (neither donor nor recipient) restriction-fragment-length polymorphisms (RFLP) in both peripheral blood and marrow. Repeat RFLP analysis 3 days later showed persistence of this unique third-party banding pattern. DNA-based HLA-typing, using polymerase chain reaction (PCR) and oligonucleotide probe hybridization, also showed these cells to be derived from an individual whose HLA-DR type was distinct from donor and recipient. Together, these findings suggested the presence of a proliferating population of transfused cells possessing alloreactivity against both donor graft and host, despite prior irradiation of all blood products with 2,000 rad. Limiting dilution analysis to assess the frequency of irradiated lymphocytes able to respond to mitogen revealed an approximate 5- to 6-log reduction at 1,500 to 2,000 rad as compared with unirradiated controls. These data indicate that a small percentage of lymphocytes can survive irradiation at these doses and suggest that existing blood-product irradiation guidelines may require reassessment, especially in T-cell-depleted transplant recipients.

Description: Disease relapse after allogeneic bone marrow transplantation (BMT) is a major cause of treatment failure and is thought to evolve from clinically occult residual disease in the recipient. However, the demonstration of minimal residual disease (MRD) in individual patients is of uncertain prognostic significance because the detection of residual disease has not consistently correlated with subsequent relapse. Moreover, the optimal therapeutic approach in patients with MRD after allogeneic BMT is unknown. The study of these issues has been hindered by the lack of clinically relevant animal models. In this report, we characterize a novel murine model for the study of MRD after allogeneic BMT. This model was designed to simulate high-risk BMT in humans in which patients receive transplants in relapse and disease recurrence in the major cause of treatment failure. The H-2-compatible, mixed lymphocyte culture nonreactive murine strains, AKR (H-2k) and CBA (H-2k), were chosen to parallel marrow transplants from HLA-matched siblings, which represent the majority of allo-transplants in humans. Male AKR leukemia cells were used in female donor/host chimeras permitting the Y chromosome to serve as a leukemia-specific marker for MRD. Detection of residual male leukemia cells in the peripheral blood of the primary host was facilitated by use of the polymerase chain reaction (PCR) and sequence-specific oligonucleotide probe hybridization (SSOPH). Use of PCR/SSOPH was highly predictive of clinical outcome (relapse or cure) in animals receiving transplants (P 〈 .00002) and detected disease recurrence earlier than comparative flow cytometric analysis studies. This murine model will be useful in evaluating the efficacy of therapeutic strategies aimed at reducing disease relapse posttransplant and can be adapted to other transplant murine tumor systems for the study of MRD.

Description: Between January 1988 and March 1993, 48 patients received T-cell- depleted marrow grafts from unrelated donors as treatment for chronic myelogenous leukemia (CML). The median age of the population was 31.7 years (range 5.4 to 53) with 17 of 48 patients greater than 40 years of age. Twenty-seven patients were transplanted in chronic phase, 17 in accelerated phase, and 4 in blast crisis. All patients received a standardized preparative regimen of cyclophosphamide, high-dose cytosine arabinoside, methylprednisolone, and total body irradiation. Marrow grafts were depleted of mature T cells with the alpha beta T- cell receptor antibody T10B9 as graft-versus-host disease (GVHD) prophylaxis. All patients also received posttransplant cyclosporine therapy. Twenty-eight of 48 patients were mismatched with their donors for one or more HLA-A, B, DR, or DQ loci by either serology or high- resolution oligonucleotide genotyping. Nine of 28 were mismatched at multiple HLA loci. Durable engraftment was achieved in 94% (45/48) of patients. The actuarial probability of developing grades II to IV and grades III to IV acute GVHD were 39.6% (95% confidence interval (CI) 26.9 to 53.0) and 8.3% (95% CI 6.1 to 10.9) for the entire cohort. There was no difference in the incidence of grades II to IV acute GVHD between patients receiving matched (36.8%) or mismatched (41.4%) marrow grafts (P = .77). The actuarial probability of relapse at 2 years was 8.8% (95% CI 2.1 to 21.6) for the entire cohort and 18% (95% CI 4 to 41) for patients transplanted in either the accelerated or blast crisis phase (advanced disease). One cytogenetic relapse has occurred among patients transplanted in the chronic phase. The probability of disease- free survival at 2 years was 52% (95% CI 24 to 70) for patients transplanted in chronic phase and 46% (95% CI 25 to 73) for patients transplanted with advanced disease. No difference in disease-free survival was observed between patients receiving matched (49%) or mismatched (51%) marrow grafts (P = .90). This study shows that patients receiving unrelated T-cell-depleted marrow grafts for CML can achieve durable engraftment with a low incidence of severe GVHD and apparent preservation of graft-versus-leukemia reactivity. These data also suggest that T-cell depletion may allow patients who might otherwise experience unacceptable toxicity from GVHD-related complications caused by older age or increased HLA disparity to benefit from unrelated marrow grafts.