Anti-T-cell reagents for human bone marrow transplantation: ricin linked to three monoclonal antibodies.

Three new reagents that react against human T cells were synthesized by covalently linking the toxin ricin to monoclonal antibodies recognizing differentiation antigens on the surface of T lymphocytes. Each of these immunotoxins selectively inhibited T-cell proliferation when the cells were incubated in the presence of lactose. Multipotent human stem cells were inhibited only at much higher concentrations. Mixtures of all three immunotoxins were more effective than any one alone. These reagents have the potential for preventing graft-versus-host disease in man.

In vitro studies of human pluripotential hematopoietic progenitors in polycythemia vera. Direct evidence of stem cell involvement.

Previous in vitro studies on committed hematopoietic progenitors have suggested that polycythemia vera (PV) is a clonal disorder arising in a pluripotential hematopoietic stem cell. In this study, recently developed technics for clonal assay of a human multipotential progenitor cell (CFU-GEMM) were used to assess the functional characteristics of CFU-GEMM in 19 PV patients. These studies showed: (a) increased numbers of detectable CFU-GEMM in blood and bone marrow samples of PV patients as compared with normals (P less than 0.002 and P less than 0.02, respectively); (b) erythropoietic differentiation of PV CFU-GEMM without exogenous erythropoietin (Ep) in culture (in marked contrast to CFU-GEMM of both normals and subjects with secondary erythrocytosis which require exogenous Ep for terminal hemoglobinization of their erythroid component), a property shown by experiments with an anti-Ep antiserum to be related to increased sensitivity of PV CFU-GEMM to Ep; (c) increased megakaryocyte formation by PV CFU-GEMM as compared with normals (P less than 0.025); and (d) a linear relationship, extrapolating to the origin, between CFU-GEMM detected and cells cultured. These studies demonstrate that at least two clinical features of PV, increased erythropoiesis and megakaryocytopoiesis, are reflected in corresponding functional characteristics of PV CFU-GEMM, and provide direct evidence of distinctive pluripotential stem cell activity in this disorder.

Studies on the liver to kidney switch of erythropoietin production.

Although the liver is the major site of erythropoietin (Ep) production in the fetus, this function is assumed by kidneys in the adult. The mechanisms underlying the liver to kidney switch of Ep formation are not understood. We studied the natural progression of this transition in sheep by measuring Ep production in response to anemia in normal and bilaterally nephrectomized fetal and newborn sheep beginning at about 80 d gestation (normal gestation: 140 d). Removal of both kidneys before induction of anemia did not affect Ep formation up to about 120 d of gestation. A significant reduction (29%, P < 0.02) in Ep synthesis was first noted at about 130 d of gestation (initiation of switch). This level of nephrectomy-induced reduction of Ep formation persisted until about 15 d after birth. Thereafter, bilateral nephrectomy caused further significant decreases (P < 0.05) in Ep production, gradually resulting in near total absence of Ep production at about day 40 postpartum (completion of switch). Chronic administration of testosterone (12 mg/wk) or estradiole benzoate (1.5 mg/d, 5 d/wk) to the fetus/newborn beginning at 85-90 d of gestation enhanced or suppressed erythropoiesis, respectively, but failed to affect the time at which the liver to kidney switch was initiated and/or completed. By contrast, a significant delay (P < 0.001) in the onset, but not completion of the switch occurred in animals that were either thyroidectomized or rendered chronically anemic beginning in the second third of the gestation period. Administration of thyroxin (1.2 mg/d, 5 d/wk) to thyroidectomized fetus/newborns not only prevented the delay in the initiation of the switch, but also accelerated the rate at which the switch was completed. These results demonstrate that in sheep (a) the liver to kidney switch of Ep production is initiated in utero during the last third of the gestation period, but is completed after birth, (b) this transition occurs gradually; the assumption of Ep producing capacity by the kidney is not preceded by an abrupt loss of hepatic Ep formation; and (c) the switch is not affected by changes in sex hormone levels during the prenatal-postnatal growth periods, but is profoundly influenced by alterations in thyroid hormone and oxygen supply-demand levels.

Allogeneic bone marrow transplantation for relapsed and refractory lymphoma using genotypically HLA-identical and alternative donors.

Twenty-two patients, ages 16.6 to 43.9 years (median age, 30 years), with relapsed or refractory lymphoma were treated by allogeneic bone marrow transplantation after high-dose chemotherapy with or without total body irradiation (TBI). Seven patients had Hodgkin's disease, four had low-grade histology non-Hodgkin's lymphoma (NHL), seven had intermediate-grade NHL, and four had high-grade NHL. Of the 22 patients, 17 received T-cell (CD-3)-depleted marrow after intensive pretransplant chemoradiotherapy, and five received T-cell-replete grafts after chemotherapy-based preparative regimens. Five patients were transplanted from donors other than genotypically HLA-identical siblings: four from partially HLA-matched relatives, and one from a phenotypically HLA-identical unrelated donor. Acute graft-versus-host disease (GVHD) was less than or equal to grade II in all patients, and chronic GVHD was limited or absent in all but one patient. Of the 21 assessable patients, 17 (80.9%) achieved complete remissions. Death due to transplant-associated complications occurred in five patients, and five patients have relapsed. Thirteen patients are alive, and 12 are continuously relapse-free at a median follow-up of longer than 28 months (range, greater than 10 to greater than 58 months) from transplant. The cumulative probability of treatment failure from relapse or progression of lymphoma was 29% (95% confidence interval [CI], 12% to 51%), while the actuarial lymphoma-free (ie, event-free) survival plateau is 54.6% (95% CI, 34% to 76%). For young patients with advanced malignant lymphoma, allogeneic bone marrow transplantation appears superior to salvage chemotherapy for achievement of long-term, lymphoma-free survival and may be preferable to autologous bone marrow transplantation for selected patients.

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.

The location of the Philadelphia chromosomal breakpoint site and prognosis in chronic granulocytic leukemia.

Chronic granulocytic leukemia (CGL) is associated with a reciprocal translocation between chromosomes 9 and 22. The breakpoint sites on chromosome 22 are clustered in a limited region known as the major breakpoint cluster region (Mbcr). This region is approximately 5.8 Kb long and can be arbitrarily subdivided into five zones (1 through 5 from the 5' towards the 3' end) as defined by the particular sites of three restriction endonucleases. Using Southern blot analysis with two DNA probes, one spanning both the 5' and 3' regions of the Mbcr while the other only the 3' region, we mapped the precise location of the chromosomal breakpoints within the Mbcr in 62 patients with CGL and examined possible clinical correlations. There were 39 patients with 5' breakpoints (zones 1-3) and 23 patients with 3' breakpoints (zones 4 and 5). We found no correlation between the clinical phase of the disease at last followup and breakpoint distributions. The distributions of chronic phase duration (CPD) and survival were similar between patients with 5' breakpoints (median CPD = 4.0 years) and those with 3' breakpoints (median CPD = 5.2 years). Presenting clinical features and the rates of lymphoblastic transformation were also similar among the subgroups. Our data suggest that the precise location of the breakpoint within the Mbcr in CGL may not have clinical relevance.

Chemotherapy versus transplants for acute myelogenous leukemia in second remission.

The best therapy for persons with acute myelogenous leukemia (AML) in 2nd remission is unknown. Bone marrow transplants from an HLA-identical sibling are reported to be better than chemotherapy but this is controversial. The objective of the study was to compare 3-year leukemia-free survival (LFS) in comparable subjects receiving chemotherapy or a transplant. 485 persons with AML in 2nd remission were studied. The chemotherapy cohort included 244 persons treated on trials of the British Medical Research Council, Eastern Cooperative Oncology Group and MD Anderson Hospital. The transplant cohort included 257 persons transplanted worldwide and reported to the international Bone Marrow Transplant Registry (16 were also chemotherapy subjects.) Subjects were selected for comparable age and year of treatment. Preliminary analyses identified two factors correlated with LFS: age < or = or > 30 years and 1st remission duration < or = or > 1 year; subsequent analyses were partitioned accordingly. Three-year probabilities of treatment-related mortality with chemotherapy and transplants were 7% (95% confidence interval, 3-15%) vs 56% (49-63%). Three-year leukemia relapse probabilities were 81% (74-86%) vs 41% (33-49%). Three-year probabilities of LFS were 17% (12-23%) vs 26 (20-32%). Cohort analysis showed significantly higher LFS with transplants vs chemotherapy in persons < or = 30 years and 1st remissions > 1 year (41% (29-53%) vs 17% (7-32%); P = 0.017) and those in > 30 years with 1st remissions < or = 1 year (18% (9-29%) vs 7% (2-16%); P = 0.046). Others had comparable LFS with both treatments. These data indicate better LFS with HLA-identical sibling transplants than chemotherapy in some persons with AML in 2nd remission.

Transferrin receptor-mediated suppression of in vitro hematopoiesis by transferrin-gallium.

The expression of transferrin receptors on cells is felt to reflect iron requirements for proliferation or for hemoglobin production. We have recently shown that transferrin-gallium (Tf-Ga) complexes bind to cellular transferrin receptors and inhibit cellular iron incorporation. In this study, Tf-Ga in a dose-dependent manner inhibited the growth of erythroid (erythroid burst-forming units [BFU-E]-derived), granulocyte-macrophage (granulocyte-macrophage colony-forming units [CFU-GM]-derived) and mixed (mixed CFU [CFU-GEMM]-derived) hematopoietic colonies. Although major differences in the response of the different progenitor cells to Tf-Ga were not seen, CFU-GEMM-derived colonies appeared to be more sensitive to growth inhibition by Tf-Ga. The inhibitory effects on colony growth were reversible after 48 h of exposure of marrow cells to Tf-Ga, suggesting that the initial effects of Tf-Ga were mainly cytostatic and that continuous exposure of cells to Tf-Ga was required for maximal growth inhibition. Transferrin-iron (Tf-Fe) added to the Tf-Ga-containing cultures restored colony growth; however, this effect was best seen when Tf-Fe was added at day 0 of incubation. Tf-Fe added on days 3 or 7 failed to restore GEMM colonies and restored only a fraction of BFU-E and GM colonies. Tf-Ga appears to inhibit hematopoietic progenitor cell growth by interfering with cellular iron utilization during an early phase of progenitor cell proliferation. The use of Tf-Ga may allow further exploration of the role of iron and the Tf receptor in the regulation of hematopoietic progenitor cell growth.

Low-dose amphotericin B prophylaxis against invasive Aspergillus infections in allogeneic marrow transplantation.

PURPOSE: Invasive Aspergillus infections cause significant morbidity and mortality in marrow transplant patients. In this study, we examined whether administration of intravenous low-dose prophylactic amphotericin B could reduce the incidence and mortality associate with invasive aspergillosis in patients undergoing allogenic marrow transplantation. PATIENTS AND METHODS: The subjects of this analysis were 186 consecutive patients undergoing allogeneic marrow transplantation in an adult bone marrow transplant unit between July 1, 1985, and September 30, 1990, utilizing consistent disease-specific chemoirradiation and graft-versus-host disease protocols. The incidence, morbidity, and case fatality of invasive aspergillosis in the study group receiving amphotericin chemoprophylaxis were compared with that in two historic cohorts managed without prophylactic amphotericin B. Univariate and multivariate statistical analyses were performed to examine whether an apparent protective effect could be attributed to differences in patient and treatment variables among the cohorts and to determine potential toxicities of the chemoprophylaxis regimen. RESULTS: There was a significant reduction in both the incidence (p = 0.003) and mortality (p = 0.03) of invasive aspergillosis in patients receiving amphotericin B chemoprophylaxis as compared with those not receiving chemoprophylaxis. The prophylactic amphotericin B schedule, as employed here, was not associated with increased renal or hepatic toxicity as compared with that in historically managed patients. CONCLUSION: These data suggest that the risks of invasive aspergillosis in allogeneic marrow transplant recipients can be reduced by administration of prophylactic amphotericin B during the pretransplant and peritransplant periods.

Pulmonary function changes in long-term survivors of bone marrow transplantation.

PURPOSE: This study was undertaken to evaluate long-term pulmonary function changes in patients undergoing bone marrow transplantation (BMT), to assess their clinical significance, and to identify factors influencing these changes. METHODS AND MATERIALS: Pulmonary function tests (PFT) were evaluated before and after BMT in 111 adult patients undergoing BMT between 1985 and 1991. Forced expiratory volume at 1 s (FEV1), forced vital capacity (FVC), diffusing capacity (DLCO), and total lung capacity (TLC) were evaluated. One hundred and three patients (92.8%) received total body irradiation (TBI) to a total dose of 14 Gy in nine equal fractions. The lung dose was restricted to < 6.5 Gy in 95% of patients with partial transmission lung shielding. Seventy-eight percent of patients had acute graft-versus-host disease (aGVHD), 69% chronic graft-vs.-host disease (cGVHD), and 63% posttransplant pulmonary infection. Effects of GVHD, TBI, radiation dose to the lungs, dose rate of TBI, posttransplant pulmonary infection, Busulfan use for conditioning, age, and history of smoking were evaluated for their influence on pulmonary function. RESULTS: Posttransplant FEV1, FVC, and TLC were lower than pretransplant values (p < 0.05) at 6 months and 1 year posttransplant with subsequent recovery. DLCO was significantly lower at all posttransplant intervals. FEV1 did not fall significantly in patients without acute or chronic GVHD and recovered earlier than in patients without posttransplant pulmonary infection. Recovery of FVC, TLC, and DLCO was also delayed in patients with acute and chronic GVHD and posttransplant pulmonary infection. Multiple regression analysis revealed an association between a higher radiation dose to the lungs, and decreased FVC at 2 years (p = 0.01). Progressive obstructive pulmonary disease was not observed. CONCLUSION: An initial decline in PFTs with subsequent recovery was observed. Factors associated with delayed recovery and incomplete recovery of PFTs were GVHD, posttransplant pulmonary infection, and higher radiation dose to the lungs. The conditioning regimen used at Medical College of Wisconsin, including relatively high TBI doses with partial transmission pulmonary shielding, appears to be well tolerated by the lungs in long-term survivors. No progressive decline in PFTs or symptomatic decline in pulmonary function was observed during the time interval studied.

Technical modifications in hyperfractionated total body irradiation for T-lymphocyte deplete bone marrow transplant.

The Medical College of Wisconsin implemented a major bone marrow transplant (BMT) program in July 1985. The type of transplants to be focused on were allogeneic T-lymphocyte deplete. Total body irradiation (TBI) was initially patterned after the Memorial method. Patients received total body irradiation in a sitting position at a dose rate of 20-25 cGy/minute with 50% attenuation lung blocks used both anterior/posterior and posterior/anterior. Electron boosting was utilized for the ribs beneath the lung blocks. Occasionally, lower extremity boosting was required because of the sitting position. A dose of 14 Gy was chosen since T-lymphocyte deplete bone marrow transplant data suggest the need for higher total doses to consistently obtain engraftment. This dose was given in 3 equal daily fractions over 3 days following conditioning chemotherapy. Six of 11 patients treated in this manner developed lethal pulmonary events. In response to the pulmonary toxicity, partial lung shielding was increased to 60% attenuation. In the next 107 patients receiving this program of total body irradiation there was a reduced incidence of fatal pulmonary events (10 cases of fatal idiopathic interstitial pneumonitis and 12 cases of fatal pulmonary infections) after a median follow-up of 9 months. This was an obvious improvement over the initial group. A significant level of hepato-renal toxicity was also observed with 14 Gy total body irradiation when no liver or kidney blocking was used. Of the first 20 patients treated, three cases of fatal veno-occlusive disease resulted. Subsequently, a 10% attenuation right sided liver block was added. Five of 98 patients treated with this block have developed fatal hepatic dysfunction, (median follow-up of 7.2 months). This incidence is not statistically different from the initial group but favors the use of the liver block. Some renal toxicity was also detected with the earlier regimen, especially in pediatric patients. Partial kidney blocking has been implemented to minimize this toxicity. Our current dose rate has been reduced to 8 cGy/minute in a further attempt to reduce organ toxicity. To date, this selective blocking has not adversely affected the excellent rate (96%) of first time engraftments.

Influence of renal shielding on the incidence of late renal dysfunction associated with T-lymphocyte deplete bone marrow transplantation in adult patients.

Late renal dysfunction following allogeneic bone marrow transplantation has been described by a number of centers including our own. Total body irradiation appears to play a major causative role in the development of this syndrome. In an effort to decrease the incidence of this renal toxicity we have added customized partial transmission renal blocking to our total body irradiation regimen. This partial renal shielding decreases the total dose to the kidneys from 14 Gy to 12 Gy. This report compares 71 adult patients who have received total body irradiation associated with bone marrow transplantation using renal shielding with 72 adult patients who were treated without the shielding; all of the patients have survived a minimum of 100 days post-BMT. Eighteen months following BMT, 26% of patients who did not have renal shielding have developed late renal dysfunction compared to only 6% of patients with renal shielding (p less than .05). Median follow-up in the nonblocked patients is 536 days post-transplant versus 341 days for the blocked patients. This added renal blocking has not adversely affected engraftment rates or relapse rates to date. Customized renal shielding as part of 14 gray total body irradiation in preparation for bone marrow transplantation appears to have decreased the incidence of late renal dysfunction in this group of adult patients and should be considered for all patients undergoing total body irradiation in conjunction with bone marrow transplantation.

Preservation of lymphokine-activated killer activity following T cell depletion of human bone marrow.

T cell depletion has decreased the incidence and severity of graft-versus-host disease following transplantation of allogeneic bone marrow. In the treatment of leukemia, decreased GVHD has often been associated with diminished antileukemia or graft-versus-leukemia (GVL) reactivity resulting in higher relapse rates. However, we have not seen a loss of the GVL effect following transplantation of marrow grafts depleted of CD3+ T cells. This suggests that non-T-cell effectors may play a role in preventing leukemic relapse. To study whether natural killer and lymphokine-activated killer (LAK) activity in BM was compromised by T cell depletion, the effect of T-cell-specific monoclonal antibodies against CD3 and CD6 determinants alone, or in combination, on the generation and expansion of NK/LAK cells was examined in vitro and compared to the effect of T depletion on mitogen-driven T cell proliferation. Limiting dilution analysis revealed that T depletion with CD3 and/or CD6 specific antibodies significantly reduced the number of proliferating T lymphocytes but did not significantly affect the frequency of cells able to expand and mediate LAK activity. Bone marrow, depleted of CD3+ or CD6+ T cells, generated levels of LAK activity equivalent to non-T-cell-depleted bone marrow following long-term culture in recombinant interleukin 2. CD3- NKH-1+ cells were the predominant population in rIL-2 expanded marrow cultures prior to transplant and in the peripheral blood of patients who had received a CD3-depleted marrow graft 21-65 days earlier. These studies show that it is possible to selectively reduce GVH-reactive T cells in allogeneic bone marrow while retaining non-T-effector cells with potential to mediate an antileukemia effect in vivo.

Phase I study of safety and pharmacokinetics of a human anticytomegalovirus monoclonal antibody in allogeneic bone marrow transplant recipients.

This study examined the safety and pharmacokinetic profile of a potentially therapeutic and fully human anti-CMV monoclonal antibody (SDZ MSL-109) in a phase I dose escalation trial in patients receiving allogeneic bone marrow transplants. Fifteen adult marrow transplant patients, twelve with chronic myelogenous leukemia and three with acute nonlymphocytic leukemia, in cohorts of five patients each, were administered monoclonal antibody intravenously at doses of 50, 250, and 500 micrograms/kg at approximately three-week intervals for six months. Administration of the monoclonal antibody was associated with minimal side effects and no dose-related toxicity. Antibody elimination curves in all dose groups were consistent with a two-compartment model with an alpha half-life at the low, middle, and high dose groups of 1.03, 0.82, and 0.79 days, and a beta half life of 13.9, 14.0, and 16.5 days, respectively. The volume of distribution decreased with repetitive dosing to approximate the plasma volume in each patient and the pharmacokinetic profile was comparable to that of human IgG. There was no host antiidiotypic or antiallotypic antibody formation, indicating that MSL-109 was not immunogenic. Further studies are warranted to assess the potential efficacy of human monoclonal anti-CMV disease in marrow transplant recipients and other patients with immunodeficiency disorders.

Control of oral mucositis and candidiasis in marrow transplantation: a prospective, double-blind trial of chlorhexidine digluconate oral rinse.

Conditioning chemoradiotherapy damages the mucosal barrier of the mouth and throat and often produces severe oral inflammation and infection. In a prospective, double-blind, randomized study, we examined the use of a chlorhexidine digluconate mouthrinse for prophylaxis against oral mucosal complications in 51 bone marrow transplant patients. Use of chlorhexidine mouthrinse produced significant reductions in the incidence and severity of oral mucositis. Mucositis also resolved more quickly in patients receiving chlorhexidine. Concomitant reductions in total oral streptococci (p less than 0.02-p less than 0.001) and oral candida (p less than 0.004) were seen in patients using chlorhexidine. Persistent clinical oral candidiasis (thrush) was observed in 15 to 27 control group patients (56%), but only transiently in two (8%) of 24 patients who used chlorhexidine rinse (p less than 0.001). Five of 27 control group patients (19%) had candidemia, while no candidemia was observed in the chlorhexidine group (p less than 0.03). Three deaths from disseminated candidiasis occurred in the placebo group; none occurred in patients who received chlorhexidine. Prophylactic use of chlorhexidine mouthrinse produces reductions in oral soft tissue disease and oral microbial burden in patients undergoing bone marrow transplantation. The reductions in mucositis and in oral candida infections observed with prophylactic chlorhexidine mouthrinse represent a significant advantage for patients undergoing marrow transplantation.

Allogeneic marrow transplantation using T cell depletion for patients with juvenile chronic myelogenous leukemia without HLA-identical siblings.

Six children with clinical and hematologic features of juvenile chronic myelogenous leukemia (JCML) underwent bone marrow transplantation (BMT) using T cell-depleted marrow from non-HLA-matched related or closely HLA-matched unrelated donors. Patient ages ranged from 1.2 to 5 years. Four patients received cytoreductive chemotherapy prior to BMT conditioning, and four had undergone pretransplant splenectomies. The donor-recipient matching included: four transplants disparate at one HLA locus (three from unrelated donors and one from a related donor), one transplant disparate at two HLA loci, and one transplant from a one haplotype-mismatched donor. All patients were MLC reactive with their donors. Graft-versus-host disease (GVHD) prophylaxis consisted of in vitro T cell depletion with a monoclonal antibody directed against CD3, and complement in conjunction with cyclosporin A begun on day -1. Conditioning included busulfan, cytosine arabinoside, cyclophosphamide, methyl-prednisolone, and hyperfractionated total body irradiation. All patients engrafted, with median time to neutrophils greater than 500 x 10(6)/l and platelets greater than 25 x 10(9)/l of 20 and 21 days, respectively. Acute GVHD was less than or equal to grade II in all patients. Two patients died of infection (Candida, CMV) at days 74, 157. One patient relapsed at day 177, and subsequently died on day 939. Three patients are alive and disease free at 180 +, 1610 + and 2400 + days from BMT. Although intensive chemotherapy may play a role in providing transient disease control in patients with JCML, allogeneic BMT is the only curative therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence in situ hybridization with X and Y chromosome probes for cytogenetic studies on bone marrow cells after opposite sex transplantation.

We investigated the efficacy of fluorescence-labelled chromosome probes (CEP-X/Y) for the X and Y chromosomes to study patients who have had opposite sex BMT. These probes hybridize to the centromere region of the X chromosome and nearly the entire long arm of the Y chromosome. These probes are direct-labelled and produce X and Y signals that can be simultaneously viewed and readily distinguished from each other by color and size after only five brief washes. We investigated BM specimens from 20 normal donors and 16 patients who had undergone an opposite sex BMT. We found no significant interinvestigator differences with respect to scoring XX or XY interphase cells. The 'normal range' for XX cells in males was up to 0.628% and for XY cells in females it was up to 0.299%. Each of the specimens from the patients who underwent BMT had a significant number of donor cells compared with normal range. We suggest that an economical, rapid and accurate cytogenetic test can be achieved by using these probes as an adjunct to conventional cytogenetics.

Bone marrow transplantation from related donors other than HLA-identical siblings: effect of T cell depletion.

Results of 470 bone marrow transplants from related donors other than genotypically HLA-identical siblings (alternative related donors) were analysed to identify factors associated with transplant outcome and to determine whether T cell depletion improved results. As compared to 3648 transplant from HLA-identical siblings, alternative related donor transplants were associated with increased graft failure, increased acute graft-versus-host disease (GVHD), and lower disease-free survival. The likelihood of adverse outcome correlated with increasing donor-recipient HLA-disparity. In multivariate analysis of alternative related donor transplants, donor age greater than or equal to 30 years, (relative risk [RR] 1.7, p less than 0.006), intermediate and advanced leukemia (RR 1.5 and 1.6, p less than 0.01 and p less than 0.003), infection pretransplant (RR 1.7, p less than 0.005) and 2- and 3-locus donor-recipient HLA-disparity (RR 1.3, p less than 0.04) were associated with increased risks of treatment failure. The 2-year probability of leukemia-free survival after alternative related donor transplants (n = 43) with none of these adverse prognostic features was 44% (95% confidence interval 28-59%) compared to 56% (95% confidence interval 52-59%) for similar patients receiving HLA-identical sibling transplants (n = 868, univariate p less than 0.03). T cell depletion increased graft failure and decreased acute GVHD after alternative related donor transplants but did not improve leukemia-free survival.

Impact of cytogenetic abnormalities on outcome of bone marrow transplants in acute myelogenous leukemia in first remission.

This study analyzed the impact of cytogenetic abnormalities on outcome of 1516 HLA-identical sibling bone marrow transplants for acute myelogenous leukemia (AML) in first remission reported to the International Bone Marrow Transplant Registry by 188 centers. 708 patients (47%) had cytogenetic studies performed. Transplant outcome in these subjects was similar to the 808 in whom cytogenetic studies were not performed. One or more cytogenetic abnormalities were detected in 284 (40%) of subjects studied. Relapse rates were higher and leukemia-free survival lower in patients with poor prognosis abnormalities vs those with no abnormality or with good or intermediate prognosis abnormalities (relative risk of relapse 2.40, P < 0.01; relative risk of treatment failure 1.68, P < 0.03). We conclude that cytogenetic abnormalities correlated with increased relapse in patients treated with chemotherapy. HLA-identical sibling transplants are similar.

Therapeutic plasma exchange does not appear to be effective in the management of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome following bone marrow transplantation.

Recognition of thrombotic thrombocytopenic purpura (TTP)/hemolytic uremic syndrome (HUS) following BMT has increased in recent years. The pathogenesis and etiology may be related to endothelial cell damage secondary to irradiation and/or CsA. Optimal management of this condition remains unclear. Due to similarity between this syndrome and classical TTP, patients with TTP/HUS following BMT are commonly treated with therapeutic plasma exchange (TPE). We describe our experience with 9 such patients who were treated with TPE (8 cases) and immunoadsorption with a Staphylococcal Protein A column (1 case). The exchanges were done with fresh frozen plasma and/or cryoprecipitate-depleted frozen plasma. Out of 8 patients treated with TPE, 6 died within 2 months of TPE due to secondary infections, metabolic disturbances and progression of TTP/HUS. Of these 6 patients, 5 had no hematological response, while 1 had hematological improvement. Two patients are alive 4 and 3 years later, however, they had shown only minimal hematological response at the end of 28 and 20 TPE, respectively. Their renal function remains stable but severely reduced. The ninth patient who received Staphylococcal Protein A column treatment died within 5 days of treatment without hematological improvement. Thus, in contrast to its effectiveness in classical TTP, TPE does not appear to be as effective in the management of well established TTP/HUS following BMT.