Suppression and recovery of immunologic function after bone marrow transplantation.

Infections are common causes of death in patients undergoing bone marrow transplantation for aplastic anemia or leukemia. A better understanding of the immunologic recovery following marrow grafting may lead to clinical trials of which the goals are to improve immunologic reactivity and to decrease infection.

Hematopoietic stem-cell transplantation for treatment-related leukemia or myelodysplasia.

PURPOSE: This report describes results of related or unrelated hematopoietic stem-cell transplants in 111 patients with treatment-related leukemia or myelodysplasia performed consecutively at the Fred Hutchinson Cancer Research Center between December 1971 and June 1998, and identifies patient and treatment characteristics associated with survival and relapse. PATIENTS AND METHODS: At transplantation, 56 patients had treatment-related secondary acute myeloid leukemia (AML), 15 had refractory anemia with excess blasts in transition (RAEB-T), 23 had refractory anemia with excess blasts (RAEB), 15 had refractory anemia (RA), and two had refractory anemia with ringed sideroblasts (RARS). Conditioning regimens were total-body irradiation (TBI) and chemotherapy for 60 patients, busulfan (BU) 14 to 16 mg/kg and cyclophosphamide (CY) 120 mg/kg (BUCY) for 27 patients, BU targeted to 600 to 900 ng/mL plasma steady-state concentration with 120 mg/kg CY (BUCY-t) for 22 patients, and miscellaneous chemotherapy for two patients. The donors were HLA-identical or partially identical family members for 69 patients and unrelated donors for 42 patients. RESULTS: The 5-year disease-free survival was 8% for TBI, 19% for BUCY, and 30% for BUCY-t (P =.006). The 5-year cumulative incidence of relapse was 40% for secondary AML, 40% for RAEB-T, 26% for RAEB, and 0% for RA or RARS (P =.0009). The 5-year cumulative incidence of nonrelapse mortality after TBI was 58%; after BUCY, 52%; and after BUCY-t, 42% (P =.02). CONCLUSION: Patients at risk for treatment-related leukemia or myelodysplasia should be followed closely and be considered for stem-cell transplantation early in the course of myelodysplasia using conditioning regimens such as BUCY-t designed to reduce nonrelapse mortality.

Ovarian function following marrow transplantation for aplastic anemia or leukemia.

One hundred eighty-seven women between 13 and 49 years of age had ovarian function evaluated from 1 to 15 years (median, 4) after marrow transplant for aplastic anemia or leukemia. Among 43 women transplanted for aplastic anemia following 200 mg/kg cyclophosphamide (CY), all 27 less than 26 years of age, but only five of 16 greater than 26 years of age recovered normal ovarian function. Nine of the 43 have had 12 pregnancies, resulting in eight live births, and two elective and two spontaneous abortions. All eight children are normal. Nine of 144 women transplanted for leukemia following 120 mg/kg CY and 9.20 to 15.75 Gy total body irradiation (TBI) recovered ovarian function. Two of these nine have had three pregnancies, resulting in two spontaneous and one elective abortion. The probability of having ovarian failure was 0.35 by 7 years for patients receiving CY alone and was 1.00 at 1 year for patients receiving CY plus TBI (P less than .0001). By 7 years after transplant the probabilities of having normal ovarian function were 0.92 after CY alone and 0.24 after CY plus TBI (P less than .0001). Multivariate analysis showed that TBI was the only factor significantly influencing ovarian failure and that both TBI and greater patient age at transplant were significantly associated with a decreased probability of recovering normal ovarian function. These data demonstrate that after high-dose CY, recovery of ovarian function occurs in younger women and in a minority of older women, but after CY and TBI, recovery occurs in only a few younger women and none of the older women.

Autologous marrow transplantation for malignant lymphoma: a report of 101 cases from Seattle.

Between October 1979 and January 1988, 101 patients with malignant lymphoma who failed initial induction treatment or relapsed received high-dose combination chemotherapy or chemoradiotherapy followed by infusion of autologous bone marrow. Twenty-eight of the 101 patients survive, 18 of whom are disease-free for a median of 26 (range, 12 to 66) months. The 5-year actuarial probabilities of survival, event-free survival (EFS), and relapse from transplantation were 20%, 11%, and 84%, respectively. Multivariate analysis showed that the likelihood of EFS was decreased among patients transplanted with a Karnofsky score of less than 80%. Recurrent lymphoma after transplant was the most important cause of treatment failure with 36 of 62 relapses occurring within 100 days from marrow infusion. Early, but not late relapse, was more frequent in patients transplanted for advanced lymphoma, and both early and late relapses were increased among patients with impaired pretransplant clinical performance or high-grade histology of lymphoma. Ten patients who relapsed post-transplant are alive, seven in remission. Further improvement of these results will require earlier transplantation, improved preparative regimens, or early posttransplant therapy.

New malignant diseases after allogeneic marrow transplantation for childhood acute leukemia.

PURPOSE: To determine the incidence of and risk factors for second malignancies after allogeneic bone marrow transplantation (BMT) for childhood leukemia. PATIENTS AND METHODS: We studied a cohort of 3, 182 children diagnosed with acute leukemia before the age of 17 years who received allogeneic BMT between 1964 and 1992 at 235 centers. Observed second cancers were compared with expected cancers in an age- and sex-matched general population. Risks factors were evaluated using Poisson regression. RESULTS: Twenty-five solid tumors and 20 posttransplant lymphoproliferative disorders (PTLDs) were observed compared with 1.0 case expected (P <.001). Cumulative risk of solid cancers increased sharply to 11.0% (95% confidence interval, 2.3% to 19.8%) at 15 years and was highest among children at ages younger than 5 years at transplantation. Thyroid and brain cancers (n = 14) accounted for most of the strong age trend; many of these patients received cranial irradiation before BMT. Multivariate analyses showed increased solid tumor risks associated with high-dose total-body irradiation (relative risk [RR] = 3.1) and younger age at transplantation (RR = 3.7), whereas chronic graft-versus-host disease was associated with a decreased risk (RR = 0.2). Risk factors for PTLD included chronic graft-versus-host disease (RR = 6.5), unrelated or HLA-disparate related donor (RR = 7. 5), T-cell-depleted graft (RR = 4.8), and antithymocyte globulin therapy (RR = 3.1). CONCLUSION: Long-term survivors of BMT for childhood leukemia have an increased risk of solid cancers and PTLDs, related to both transplant therapy and treatment given before BMT. Transplant recipients, especially those given radiation, should be monitored closely for second cancers.

Autologous marrow recovery and sensitization to non-HLA antigens after HLA-identical marrow transplantation for aplastic anemia.

One hundred seventy-five patients with severe aplastic anemia were treated by high-dose cyclophosphamide and HLA-A, -B, and -D-identical sibling marrow transplants. Thirty-eight patients rejected their grafts. Four of the 38 showed autologous marrow recovery as determined by blood genetic markers. The remarkable feature of one case following autologous marrow recovery was the presence of unidirectional proliferative and cytotoxic responses of circulating host lymphocytes to marrow donor lymphocytes in mixed lymphocyte culture and cell-mediated lympholysis. Presumably these responses were the result of in vivo sensitization to those non-HLA antigens for which donor and recipient differed.

Myasthenia gravis after allogeneic bone marrow transplantation: relationship to chronic graft-versus-host disease.

Three patients with chronic graft-versus-host disease (GVHD) developed myasthenia gravis (MG) 762 to 1,180 days after allogeneic bone marrow transplantation. Symptoms of MG were observed after taper or discontinuation of immunosuppressive treatment of chronic GVHD. All patients developed antibodies to acetylcholine receptor, and one had antibody formation to striated muscle. One patient died of complications of treatment of MG. The severity of disease underscores the importance of the differential diagnosis and the need for prompt therapy of this late complication after human bone marrow transplantation.

Treatment of acute graft-versus-host disease after allogeneic marrow transplantation. Randomized study comparing corticosteroids and cyclosporine.

Seventy-seven patients (age 12 to 46 years) who underwent allogeneic marrow transplantation for hematologic malignancy or aplastic anemia and who had grade II to IV acute graft-versus-host disease despite methotrexate prophylaxis were randomly assigned to receive methylprednisolone 2 mg/kg per day intravenously (n = 39) or cyclosporine (n = 38) either 12 to 15 mg/kg per day orally or 3 to 5 mg/kg per day intravenously. In both groups, clinical and histologic evidence of graft-versus-host disease was detected at medians of 16 and 25 days, respectively. Drugs were given for a minimum of 14 days unless significant deterioration occurred. If graft-versus-host disease did not improve with this therapy, treatment with a second agent was initiated. Treatment responses were scored after reviewing clinical and laboratory data collected before, during, and after the 14-day treatment period. Possible scores were as follows: -1, worse; 0, no change; + 1, improvement in one organ system (skin, liver, gut) with no deterioration in the other two; +2, complete resolution of all involved systems. The median response score among 39 methylprednisolone-treated patients was 0. Sixteen patients (41 percent) showed response to treatment, 11 with partial and five with complete response. The median response score among 38 cyclosporine-treated patients was +1. Twenty-three patients (61 percent) showed response to treatment, 15 with partial and eight with complete response (p = 0.039). Twenty patients receiving methylprednisolone and 18 receiving cyclosporine required additional therapy. The incidence of chronic graft-versus-host disease was similar in both groups. It developed in all nonresponding patients at risk who had received secondary therapy. Among responding patients (scores +1 or +2) who were not given additional treatment, chronic graft-versus-host disease developed in eight of 11 (72 percent) receiving methylprednisolone and five of ten (50 percent) receiving cyclosporine. Survival beyond 17 months was similar in the two groups (28 percent and 24 percent, respectively). These data suggest that cyclosporine is a useful agent for the treatment of acute graft-versus-host disease, comparable in its efficacy to methylprednisolone.

Cataracts after total body irradiation and marrow transplantation: a sparing effect of dose fractionation.

We examined 277 patients, who have been followed for 1 to 12 years after marrow transplantation, for cataract development. In preparation for transplantation, 96 patients with aplastic anemia were conditioned with chemotherapy only, usually cyclophosphamide 50 mg/kg X 4 intravenously, while 181 patients (two with aplastic anemia and 179 with a hematologic malignancy) were conditioned with a regimen of total body irradiation (TBI) and chemotherapy. TBI was delivered from two opposing 60Co sources at an exposure rate of 4 to 8 cGy/min, either as a single dose of 10 Gy (105 patients) or in fractions (76 patients), usually at increments of 2 to 2.25 Gy/day for 6 to 7 days for cumulative doses of 12 to 15.75 Gy. To date, 86 patients have developed cataracts. Kaplan-Meier product limit estimates of the incidence of cataracts for patients given chemotherapy only and no TBI, single-dose TBI, and fractionated TBI are 19, 80, and 18%, respectively. On the basis of proportional hazards regression analyses, patients given single-dose TBI had a relative risk of developing cataracts that was 4.7-fold higher than in patients given fractionated TBI or chemotherapy only (p less than 0.00005), suggesting a significant sparing effect with use of TBI dose fractionation. Addition significant risk factors included the chronic use of steroids posttransplant (highly associated with the presence of chronic graft-versus-host disease), and the diagnoses of acute lymphoblastic or chronic myelogenous leukemia.

Marrow transplantation following escalating doses of fractionated total body irradiation and cyclophosphamide--a phase I trial.

Thirty-six patients with advanced hematologic malignancy were entered into a Phase I study designed to define the maximum tolerated dose of unshielded total body irradiation delivered from dual 60 Cobalt sources at an exposure rate of 8 cGy/min and given in fractions twice daily for total doses ranging from 12 Gy to 17 Gy. All patients received cyclophosphamide, 120 mg/kg administered over 2 days before total body irradiation. Allogeneic marrow was infused from HLA-identical siblings (n = 29) or one locus HLA incompatible family members (n = 3); three patients received cryopreserved autologous marrow and one patient received syngeneic marrow. The maximum tolerated dose of total body irradiation given as 2 Gy fractions twice a day was 16 Gy. One of eight patients receiving 12 Gy, none of four receiving 14 Gy, three of 20 receiving 16 Gy, and two of four receiving 17 Gy developed severe (Grade 3-4) regimen-related toxicity. The primary dose limiting toxicity was pneumonitis, followed by veno-occlusive disease of the liver, renal impairment, and mucositis. Five patients (14%) are alive, four disease-free 798-1522 days posttransplant. Twenty (56%) relapsed posttransplant. Further investigation of regimens containing 16 Gy of hyperfractionated total body irradiation is warranted to assess anti-tumor efficacy.

Regulation of immunoglobulin production after human marrow grafting. The role of helper and suppressor T cells in acute graft-versus-host disease.

The effect of acute graft-versus host disease (GVHD) on T4 and T8 lymphocyte regulation of in vitro immunoglobulin production was explored. The peripheral blood lymphocytes from 20 patients were studied sequentially in the first 100 days after sibling bone marrow grafting for hematologic malignancy or aplastic anemia. T and non-T lymphocytes were prepared from peripheral blood by Ficoll-Hypaque density gradient centrifugation and sheep erythrocyte rosetting. T cells were enriched for T4 or T8 cells and cocultured for six days with pokeweed mitogen and autologous non-T or T and non-T cells from unrelated normal individuals. Immunoglobulin production was assessed using a reverse hemolytic plaque assay. All three patients without acute GVHD had failure of non-T cells to secrete immunoglobulin, one had failure of helper T cell activity, and 2 developed suppressor T cells. Similarly, all six patients studied sequentially after the development of GVHD had non-T-cell failure, five developed helper T cell failure, and five had suppressor T cells. These data suggest no difference in lymphocyte function before or after the development of acute GVHD. When the T cells of these patients were split into T4 and T8 subpopulations and studied for immunoglobulin production there was helper T cell failure in 4 of 9 tests with enriched T4 populations. Five of 9 tests with T8 enriched populations showed suppressor activity. Suppressor T cell function was also seen in 4 of 9 tests with with T4-enriched populations. These data show that T cell function does not necessarily correlate with the surface phenotype during the first 100 days after grafting. A role for cytomegalovirus (CMV) infection in bringing out suppressor activity is suggested, because among patients without GVHD, 6 of 8 tests in CMV-positive patients showed suppressor cells compared with none of 4 tests in patients without CMV infection.

Antigen-specific antibody responses of lymphocytes to tetanus toxoid after human marrow transplantation.

In vitro IgG anti-tetanus toxoid (IgG anti-TT) antibody produced by peripheral blood lymphocytes (PBL) from 16 normal subjects (9 marrow donors and 7 random healthy subjects) and 17 marrow graft recipients from 45-1058 days postgrafting was measured with an enzyme-linked immunosorbent assay (ELISA). PBL from 11 of 13 seropositive (anti-TT greater than or equal to 1:1024) normal subjects produced IgG anti-TT in vitro, whereas the PBL from the 3 seronegative (IgG anti-TT less than 1:1024) normal subjects did not. In our normal subjects, there was a high correlation between seropositivity and in vitro IgG anti-TT production (P = .0048, chi 2, two-tailed). PBL from only one of 13 seropositive marrow graft recipients produced in vitro IgG anti-TT antibody. B and T cell functions of 8 marrow graft recipients were assessed by coculturing their T and B cells with those from their HLA-identical marrow donors. One short-term patient and 7 long-term patients (4 with and 3 without chronic graft-versus-host disease) were studied. Recipient B cells failed to produce antibody in the presence of donor T cells in 7 of these 8 cases. However, T cells from long-term survivors provided helper activity to immune donor B cells in 7 of 9 evaluable cases. TT-specific helper T cell activity was present in most seropositive long-term recipients, and B cells from marrow recipients failed to produce specific antibody in the presence of normal donor TT-specific helper T cells. These results, TT-specific T cell helper activity, and normal circulating serum IgG anti-TT antibody levels in marrow graft recipients without postgrafting TT boosters suggest that specific immunity had been transferred from the marrow donor to the marrow recipient.

Association of interstitial pneumonia and diminished in vitro lymphocyte blastogenesis in human marrow graft recipients.

The lymphocyte responses of 51 human marrow graft recipients were tested in vitro 18 to 45 days after marrow grafting by stimulation with lymphocytes from unrelated individuals or phytohemagglutinin (PHA). Fourteen of the 51 patients subsequently developed interstitial pneumonia (IP). The relative responses of the lymphocytes of patients who developed IP were significantly lower than those of individuals not developing IP (P less than 0.01 for allogeneic cells, P = 0.02 for PHA). These nonspecific in vitro tests of cell-mediated immunity are of value in identifying patients at risk of developing IP.

Immunoglobulin production of donor origin after marrow transplantation for acute leukemia or aplastic anemia.

Four patients with acute leukemia and one with aplastic anemia were not transfused within 90 days before marrow transplantation from the HLA-identical sibling. When studied 4 to 12 1/2 months after transplantation their immunoglobulin allotypes were those of their donors.

Late effects on gonadal function of cyclophosphamide, total-body irradiation, and marrow transplantation.

One hundred thirty-seven patients had gonadal function evaluated 1-11 years after marrow transplantation. All 15 women less than age 26 and three of nine older than age 26 who were treated with 200 mg/kg cyclophosphamide recovered normal gonadotropin levels and menstruation. Five have had five pregnancies resulting in three live births, one spontaneous abortion, and one elective abortion. Three of 38 women who were prepared with 120 mg/kg cyclophosphamide and 920-1200 rad total-body irradiation had normal gonadotropin levels and menstruation. Two had pregnancies resulting in one spontaneous and one elective abortion. Of 31 men prepared with 200 mg/kg cyclophosphamide, 30 had normal luteinizing hormone levels, 20 had normal follicle-stimulating hormone levels, and 10 of 15 had spermatogenesis. Four have fathered five normal children. Thirty-six of 41 men prepared with 120 mg/kg cyclophosphamide and 920-1750 rad total-body irradiation had normal luteinizing hormone levels, ten had normal follicle-stimulating hormone levels, and 2 of 32 studied had spermatogenesis. One has fathered two normal children. It was concluded that cyclophosphamide does not prevent return of normal gonadal function in younger women and in most men. Total-body irradiation prevents return of normal gonadal function in the majority of patients.

Immunologic recovery in human marrow graft recipients given cyclosporine or methotrexate for the prevention of graft-versus-host disease.

Immunologic recovery was studied in ten patients with aplastic anemia and 23 patients with hematologic malignancy who received HLA-identical marrow grafts and cyclosporine postgrafting as prophylaxis against graft-versus-host disease. Cyclosporine , 12.5 mg/kg/day, was administered beginning on the day before marrow infusion and continued for 50 days, when it was tapered and discontinued by 6 months postgrafting . Results were compared with data from concurrent and previously described patients receiving methotrexate as prophylaxis for graft-versus-host disease. Patients treated with cyclosporine or methotrexate had lower-than-normal immunologic parameters and were not different from one another 3-5 months postgrafting . By 11 to 18 months after grafting lymphocyte counts had normalized in both groups. Serum IgA levels were low and IgG levels had normalized in methotrexate-treated patients, and IgM was normal in cyclosporine -treated patients. In vivo antibody production to T-dependent antigens and skin test responses to recall antigens continued to be impaired. The response to the neoantigen dinitrochlorobenzene was still impaired in patients treated with cyclosporine and normal in patients given methotrexate. These data suggest that immunologic recovery after marrow transplantation is similar in cyclosporine -treated and methotrexate-treated patients.

Recovery of in vivo cellular immunity after human marrow grafting. Influence of time postgrafting and acute graft-versus-host disease.

Three hundred thirty-two marrow graft recipients and 241 healthy marrow donors were studied by skin testing with recall and neoantigens. Two hundred thirty patients with leukemia and seventy-eight patients with aplastic anemia received allogeneic HLA-identical sibling marrow. Twenty-four patients with leukemia received syngeneic marrow. The conditioning regimen prior to marrow infusion consisted of 120 mg/kg cyclophosphamide and 9.2-15.75 Gy total-body irradiation (leukemia) or 200 mg/kg cyclophosphamide (aplastic anemia). The patients were skin-tested with the neoantigens dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin, and a battery of five recall antigens around 100, 150, 365, 730, 1095, 1460, and 1825 days after grafting. A binary logistic regression analysis was used to investigate the factors thought to influence immunocompetence. At 3 months postgrafting, the proportion of patients positive to DNCB was equal to that of normal marrow donors, but thereafter it was lower until 2 years. The proportion of patients positive to keyhole limpet hemocyanin was lower than normal regardless of the time after grafting. The proportion of patients positive to recall antigens was lower than that of normal marrow donors until 4 years after grafting. Patients with a history of acute graft-versus-host disease had the lowest probability of a positive reaction to recall antigens. None of the other factors was significantly associated with an increased or reduced level of response.

Disordered salivary immunoglobulin secretion and sodium transport in human chronic graft-versus-host disease.

Whole saliva samples and lip biopsies were collected from 12 allogeneic bone marrow transplant recipients who developed extensive chronic graft-versus-host disease (GVHD) and from 10 healthy allogeneic and syngeneic recipients without GVHD. Six of ten biopsies from patients with chronic GVHD had lichenoid stomatitis or sialadenitis, or both, with sialodochitis. Seven of nine biopsies from patients free of chronic GVHD were entirely normal, and two had either mild glandular or mucosal changes. Salivary gland involvement in chronic GVHD was associated with decreased or absent levels of salivary IgA and inorganic phosphate, decreased salivary flow rates, and increased concentrations of salivary sodium, albumin, and IgG. The most striking abnormalities were found in patients with histologic evidence of sialadenitis. In contrast, marrow transplant recipients without chronic GVHD had normal salivary immunoglobulin and electrolyte levels. Secretory IgA deficiency may contribute to the frequent sinobronchial infections observed in patients with chronic GVHD.

T cell reconstitution after bone marrow transplantation into adult patients does not resemble T cell development in early life.

Immune reconstitution after marrow transplantation has some characteristics of immune development in early life. Here we provide phenotypic data suggesting this may not be true for T cells (particularly CD4+ T cells) in the case of marrow transplantation into adults. T cells from 35 adult patients at 1 year after transplant, 14 normal neonates and 22 normal adults were studied by 3-color flow cytometry. Marked disparity between the phenotype of neonatal vs post-transplant T cells was found. Of the total CD4+ T cells, the neonates had supranormal percentages of CD45RAhigh, L-selectin+, CD29low/-, CD11alow/- and CD28+ cells whereas most patients at 1 year after transplant had subnormal percentages of these CD4+ T cell subpopulations. (sub/supra-normal denotes below/above normal adult values). Absolute blood counts of naive (CD45RAhigh, L-selectin+, CD29low/- and CD11alow/-) CD4+ T cells correlated inversely with patient age. Neonates had also supranormal percentages of CD45RAhigh, L-selectin+, CD29low/-, CD11alow/- and CD28+ CD8+ T cells whereas the patients (particularly those with chronic GVHD) tended to have subnormal percentages of these CD8+ T cell subpopulations. Contrary to the CD4+ T cells, there was no correlation between the absolute counts of CD45RAhigh, L-selectin+, CD29low/- or CD11alow/- CD8+ T cells and patient age. Whereas the vast majority of neonatal T cells were CD38high, most patient and normal adult T cells were CD38- or CD38intermediate (both CD4+ and CD8+ T cells). We conclude that, in contrast to early life, the production of naive CD4+ T cells is deficient in adult (and particularly elderly) transplant recipients.

Low IgG production by mononuclear cells from marrow transplant survivors and from normal neonates is due to a defect of B cells.

Low IgG production is a characteristic feature of both the immunodeficiency of recipients of bone marrow transplant (BMT) and the physiologic immunodeficiency of newborns. We tried to determine whether this is due to a B cell defect and whether this can be corrected in vitro by IgG production-promoting cytokines CD40 ligand (CD40L) and interleukin 10 (IL-10). Highly purified circulating B cells from patients at 1 year after transplant, normal neonates and normal adults were cultured with and without anti-mu plus CD40L plus IL-10. Proliferation was measured on day 4 and IgM and IgG production were measured on day 9. Proliferation and IgM production of B cells from patients and from neonates were somewhat low (43-67% of normal). In contrast, IgG production by B cells from patients and from neonates was markedly low (< or = 13% of normal). Decreased IgG production correlated with decreased percentage of B cells negative for membrane IgD (mIgD). We conclude that the low IgG production of normal neonates and patients at 1 year after transplant is, at least in part, caused by a defect of circulating B cells and that this defect cannot be corrected by CD40L and IL-10. Quantitative deficiency of switched mIgD-) B cells probably accounts for this defect.