Autologous graft-versus-host disease: a new frontier in immunotherapy.
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Animals↗
Biomedical subjects
A M Yeager
Publications and source records attributed to A M Yeager.
Lymphoid reconstitution after transplantation of congenic hematopoietic cells in busulfan-treated mice.
The effects of pretransplant conditioning with high-dose busulfan, a myeloablative but nonimmunosuppressive alkylating agent, on reconstitution of lymphoid tissues by donor cells after bone marrow transplantation (BMT) has not been extensively examined. We used flow cytometric analyses to study the kinetics and extent of lymphocyte repopulation in C57BL/6 mice (immunophenotype Ly-5.2) given graded doses of busulfan (10 to 100 mg/kg) or total body irradiation (TBI; 900 rad) and hematopoietic cell transplantation (HCT; transplantation of bone marrow and spleen cells) from congenic Ly-5.1 donors. Mice transplanted after 10 mg/kg of busulfan had slow and incomplete lymphoid engraftment; only 6% to 11% of lymphocytes in the peripheral blood, lymph nodes, and spleen were positive for Ly-5.1 at 30 days after transplant, slightly increased to 13% to 20% at 60 days, and stabilized at 40% to 46% by 180 days after HCT. Higher doses of busulfan (20 to 100 mg/kg) provided dose-dependent congenic lymphoid reconstitution. Thirty days after HCT, the range of Ly-5.1 cells in blood, lymph nodes, and spleen of Ly-5.2 recipient mice was 43% to 54% after 20 mg/kg of busulfan, 66% to 71% after 50 to 80 mg/kg, and 77% to 85% after 100 mg/kg. Sixty days after transplant, lymphoid chimerism increased to 57% to 68% in 20 mg/kg recipients, 72% to 79% after 35 mg/kg, and 75% to 90% in animals given 50 mg/kg or greater, as seen in radiation chimeras. Despite slower early reconstitution after lower doses of busulfan, donor lymphocytes exceeded 90% to 95% by 90 to 120 days after HCT in all mice given at least 20 mg/kg. Even though busulfan lacks directly immunosuppressive properties, virtually complete sustained lymphoid reconstitution by transplanted congenic donor stem cells occurs after its administration. These observations suggest that pretreatment with busulfan may be effective in gene therapy strategies that involve infusion of autologous marrow cells into which functional genes have been inserted.
Animals↗
Hematopoietic cell transplantation after administration of high-dose busulfan in murine globoid cell leukodystrophy (the twitcher mouse).
We evaluated the effectiveness of administration of high-dose busulfan (BU), an alkylating agent with substantial myeloablative but negligible immunosuppressive properties, on the engraftment of congenic normal bone marrow and spleen cells (hematopoietic cell transplantation; HCT) in murine galactosylceramidase deficiency (the twitcher mouse), a model of human sphingolipid storage disease. Untreated mice died with extensive demyelination and failure to thrive at a median age of 40 d (range, 28-47; n = 51). The life-span of twitcher mice given HCT at age 10 d after 100 mg/kg BU was significantly prolonged (median, 94 d; range, 55-140; n = 17); these animals did not develop the hindlimb paralysis seen in untreated twitchers. Histologic examination of twitcher sciatic nerves after HCT showed remyelinated areas and decreased globoid cell inclusions. Animals given HCT after conditioning with BU had both lymphoid and hematopoietic repopulation with donor cells by 90 d after HCT, as documented by presence of donor glucose phosphate isomerase-1A activity in blood, bone marrow, spleen, and lymph nodes. After BU and HCT, galactosylceramidase activity in livers and spleens of twitcher mice reached 45 and 80% of control, respectively; only modest elevations were observed in kidneys and lymph nodes. Hydrolase activity rose to 20% in the brains and exceeded control values in the sciatic nerves of transplanted twitcher mice, indicating entry of at least some donor-derived cells and/or hydrolase across the blood-brain and blood-nerve barriers after BU and HCT.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Predictive value of colony-forming unit assays for engraftment and leukemia-free survival after transplantation of chemopurged syngeneic bone marrow in rats.
We evaluated the efficacy of in vitro clonogenic assays for acute myeloid leukemia (AML) (CFU-Leuk) and granulocyte-macrophage progenitor cells derived from normal bone marrow (BM) (CFU-GM) to predict hematopoietic engraftment, median survival time (MST) and leukemia-free survival (LFS) in LBN rats that received injections of untreated or drug-treated AML and/or normal BM cells. Injection of untreated AML cells resulted in a log-linear relationship between AML cell dose and time of death from leukemia; LBN rats given 10(6) cells died with AML (MST, 24 days; range, 19-28) after injection. A minimum of 0.5-1.0 X 10(6) untreated normal BM cells was needed to insure satisfactory hematopoietic reconstitution in at least 50% of lethally irradiated LBN rats. After ex vivo incubation with graded concentrations of 4-hydroperoxycyclophosphamide (4HC) or bleomycin (BLEO), LBN AML or normal BM cells were cultured for CFU-Leuk or CFU-GM and injected into untreated or lethally irradiated syngeneic recipients. Over a variety of drug concentrations (4HC, 3-30 micrograms/ml; BLEO, 100-10,000 mU/ml) and cell doses (10(6)-10(7)/animal) examined, the log-kill estimates derived from in vitro CFU-Leuk assays correlated with the observed MST or LFS. Recovery of greater than 1% CFU-GM from 4HC- or BLEO-treated suspensions of normal BM was associated with satisfactory engraftment in lethally irradiated LBN rats. Clonogenic assays also predicted for engraftment and LFS in animals that received mixtures of AML and normal BM cells (1:10) treated with 4HC and/or BLEO. We conclude that CFU-Leuk and CFU-GM assays are useful screening techniques to develop and evaluate strategies for ex vivo purging with chemotherapeutic agents in this preclinical model of autologous marrow transplantation for AML.
Acute Disease↗
Analysis of factors predicting speed of hematologic recovery after transplantation with 4-hydroperoxycyclophosphamide-purged autologous bone marrow grafts.
We previously described the predictive value of graft colony-forming units granulocyte macrophage (CFU-GM) content after 4-hydroperoxycyclophosphamide (4-HC) purging for the duration of aplasia after autologous bone marrow transplantation. Despite the uniform 4-HC concentration, we observed heterogeneity in CFU-GM survival and the kinetics of engraftment. We have now analysed patient and graft characteristics for 154 patients undergoing autologous transplantation with 4-HC purged grafts to further define this heterogeneity. Patients transplanted for the treatment of malignant lymphoma reached a peripheral blood granulocyte count of greater than 0.5 x 10(9)/l (median, 20 versus 40 days; p less than 0.001) and platelet transfusion independence (median, 30 versus 70 days; p less than 0.001) significantly faster than patients transplanted for acute non-lymphoblastic leukemia. Other diagnostic groups were intermediate. These differences were independent of graft CFU-GM content. Multiple other patient and graft factors including patient age, peripheral blood counts on day of harvest, and amounts of other hematopoietic progenitors also predicted the kinetics of engraftment in univariate and multivariate analysis. Cytomegalovirus infection during the aplastic period predicted a delay in granulocyte (p = 0.024) but not platelet recovery (p = 0.174). This analysis demonstrates that multiple patient, graft, and post-transplant factors predict the engraftment capacity of autografts, and the kinetics of engraftment with 4-HC purged grafts. The multiple predictive factors explain a significant portion of the variability in engraftment kinetics observed after transplantation with 4-HC purged autografts.
Adolescent↗
Pediatric bone marrow transplantation.
Bone marrow transplantation is a high-visibility, high-technology discipline with a growing list of potentially curative applications in neoplastic, hematologic, immunologic, and genetic diseases of children. The clinical problems experienced by children who have received bone marrow grafts involve pediatricians in both general and subspecialty practice and require a working knowledge of the applied immunobiology of bone marrow transplantation. As the transplantation procedure has evolved from a research tool to an established therapeutic modality, collaboration between basic scientists and clinicians has led to a greater understanding of the pathophysiology of the immunobiologic events that occur after allogeneic bone marrow transplantation and, thus, to improvements in the clinical care of transplant recipients. As its applications in pediatric medicine grow, bone marrow transplantation presents additional challenges for the future: expansion of the allogeneic donor pool, use of more effective or aggressive preparative regimens and autologous bone marrow-purging strategies, more effective prevention and treatment of graft-versus-host disease, and development of gene-replacement therapy by infusion of modified autologous bone marrow cells.
Bone Marrow Transplantation↗
Development and characterization of a cyclophosphamide-resistant subline of acute myeloid leukemia in the Lewis x Brown Norway hybrid rat.
Preclinical studies of resistance to alkylating agents in the Lewis x Brown Norway hybrid (LBN) rat model of acute myeloid leukemia (AML) have hitherto been limited by the sensitivity of LBN AML cells to cyclophosphamide (CY). We developed a CY-resistant subline of LBN AML by serial intravenous (IV) passage of AML cells followed by in vivo exposure to CY (100 mg/kg) 14 days later. After 18 and subsequent passages, CY-treated AML cells remained viable despite ex vivo incubation with 70 to 100 mumol/L 4-hydroperoxycyclophosphamide (4HC) or in vivo exposure to 100 to 300 mg/kg of CY. Once established, resistance to incubation with 4HC was stable in LBN AML cells after at least six serial in vivo passages without exposure to CY. Nevertheless, both control and CY-treated AML cells demonstrated similar dose-dependent sensitivity to 100 to 500 mumol/L phosphoramide mustard (PhM), the active alkylating end-product of CY activation in vivo. Levels of aldehyde dehydrogenase (ALDH), which inactivates CY by prevention of formation of PhM, were significantly elevated in these CY-resistant AML cells: cytosolic and particulate ALDH fractions from these cells were 11 to 13 times control with NAD cofactor and propanal substrate and three to four times control with NADP cofactor and benzaldehyde substrate. Further studies with this animal model of AML, in which resistance to CY is mediated by elevated ALDH activity, may elucidate mechanisms for effective elimination of drug-resistant leukemic cells ex vivo and in vivo.
Aldehyde Dehydrogenase↗
Ex vivo chemopurging of autologous bone marrow with 4-hydroperoxycyclophosphamide to eliminate occult leukemic cells. Laboratory and clinical observations.
The application of autologous bone marrow transplantation (ABMT) in treating acute leukemias in children has been limited by the presence of residual occult viable leukemic cells in the marrow cell suspension. One approach to this problem is the ex vivo treatment ("purging") of the autograft to eradicate these tumor cells yet spare the normal lymphohematopoietic stem cells. Initial studies of acute myeloid leukemia (AML) in a rodent model demonstrated that incubation with 4-hydroperoxycyclophosphamide (4HC), a congener of cyclophosphamide and an active alkylating agent in aqueous solution, could effectively eliminate viable AML cells from marrow cell suspensions without apparent toxicity to normal stem cells. We have conducted clinical trials of ABMT with 4HC-treated marrow in children with acute leukemia in remission; marrow was collected, treated ex vivo with 4HC (100 micrograms/ml), and cryopreserved in liquid nitrogen until reinfusion. Children received pre-ABMT conditioning with either high-dose cyclophosphamide and total body irradiation (CY-TBI) for acute lymphocytic leukemia (ALL) or high-dose busulfan and cyclophosphamide (BU-CY) for AML. Of nine children who underwent ABMT with 4HC-treated marrow for ALL in second complete remission (CR2), all relapsed (eight in the marrow, one in the central nervous system) at a median of 5 months (range, 2-17) after ABMT and all have died with relapsed ALL or as a consequence of its treatment. Twenty-nine children with AML (five in CR1, 24 in CR2) received autografts with chemopurged marrow at a median remission duration of 3 months (range, 2-15). Three patients died from sepsis during aplasia; 10 children (one in CR1 and nine in CR2) relapsed with AML at a median of 7 months (range, 2-23) after ABMT, for an actuarial relapse rate of 47%. Sixteen patients with AML (four in CR1, 12 in CR2) are in unmaintained remission at a median of 16 months (range, 6-102) after ABMT, for an actuarial disease-free survival of 49%. Although ABMT with 4HC-treated marrow appears to have a limited role in the treatment of children with ALL who lack a suitable related donor, the results in AML are encouraging and compare favorably with both syngeneic and allogeneic BMT in similar groups of patients.
Actuarial Analysis↗
Saposins (sphingolipid activator proteins) in the twitcher mutant mouse.
The twitcher mutant mouse, the animal model of Krabbe disease (human globoid cell leukodystrophy), is characterized by apparent deficiency of galactosylceramide beta-galactosidase activity. Saposin A and C, the heat-stable small sphingolipid activator glycoproteins, stimulate the activity of galactosylceramide beta-galactosidase as well as glucosylceramide beta-glucoside. The role of these saposins in the twitcher mutation was investigated. Boiled supernatant fractions, which contained saposins, were prepared from homogenates of twitcher brain, liver, kidney, and spleen. These preparations showed an almost identical effect on the activity of purified glucosylceramide beta-glucosidase (measured by hydrolysis of 4-methylumbelliferyl-beta-glucoside) with similar preparations from control tissues. The effect on the activity of galactosylceramide beta-galactosidase as well as 4-methylumbelliferyl-beta-glucoside beta-glucosidase in the twitcher brain and liver homogenates by authentic saposin A and C was similar to that in control tissues. These results suggest that the twitcher mutation does not affect the concentrations of saposin A or C or their interaction with galactosylceramide beta-galactosidase.
Animals↗
Allogeneic bone marrow transplantation for patients with high-risk acute lymphoblastic leukemia.
Seventy-four consecutive patients with high-risk acute lymphoblastic leukemia (ALL) were given cyclophosphamide (CY; 50 mg/kg on each of 4 days) plus total body irradiation (TBI; 300 rad on each of 4 days) followed by a human leukocyte antigen (HLA)-identical allogeneic bone marrow transplant (BMT). Eighteen patients in first complete remission (CR1), 36 in CR2, 16 in CR3, and four in CR4 were transplanted. Patients in CR1 were transplanted 1 to 8 months (median, 3 months) after attaining CR. All 18 patients in CR1 had one or more poor risk factors: age more than 18 (N = 17), initial leukocyte count greater than or equal to 20,000 (N = 11), Ph 1 chromosome (N = 2), delay in attaining CR more than 6 weeks (N = 8), or extramedullary disease (N = 1). Of those transplanted in CR2, 72% had relapsed on therapy. The 5-year event-free survival (EFS) rates for patients transplanted in CR1, CR2, and CR3 are 42%, 43%, and 25%, respectively, at median follow-up times of 57, 54, and 72 months, respectively. Children aged less than 18 years transplanted in CR2 have a 5-year EFS rate of 54%. All CR4 patients died early after transplant. The actuarial probability of relapse is 20%, 26%, and 48% for those transplanted in CR1, CR2, and CR3, respectively. Although there was substantial transplant-associated mortality, it decreased over the decade of the study (P = .01). This study indicates that BMT offers an attractive alternative to postremission chemotherapy in patients in CR1 with poor prognostic factors and in patients in second remission.
Adolescent↗
Bone marrow transplantation in lysosomal storage diseases: a prelude to gene-insertion therapy.
Allogeneic bone marrow transplantation is a therapeutic option for enzyme replacement in children with lysosomal storage diseases, for which no current conventional treatments are curative. Although marrow allografting is costly, intensive, and not risk free, strides are being made in the management and prevention of procedure-related complications.
Bone Marrow Transplantation↗
Density-gradient separation of autologous bone marrow grafts before ex vivo purging with 4-hydroperoxycyclophosphamide.
We found in previous studies that the cytotoxicity of 4-hydroperoxycyclophosphamide (4-HC) toward normal and malignant cells in autologous bone marrow grafts was modulated by the variable erythrocyte content of the incubation mixture. This affected both the kinetics of engraftment and the probability of leukemic relapse. To decrease this variability in 4-HC cytotoxicity, we initiated a trial of density-gradient separation of the autografts to remove erythrocytes and other mature blood cells before the 4-HC treatment. A series of 64 consecutive patients was studied. Light-density (sp. gr. much less than 1.077 g/ml) cells were isolated in a 2-step procedure by Ficoll-diatrizoate separation of previously isolated buffy-coat cells. Overall cell recovery after buffy-coat and gradient separations was 28.9 +/- 11.7% ( +/- SD). These cells were then treated with 60 micrograms/ml of 4-HC. The erythrocyte content of the incubation mixtures averaged 0.8%, and did not predict percent CFU-GM survival after the 4-HC incubation. The variability in CFU-GM survival after the purge was less than for previous patients who received buffy-coat cells (with erythrocyte contents ranging from 4-22%) treated with 100 micrograms/ml of 4-HC, indicating that a more uniform purge was achieved. Although the selected dose of 4-HC (60 micrograms/ml) yielded a lower CFU-GM survival, the kinetics of engraftment did not significantly differ between the recipients of light-density or buffy-coat cells. No patient required use of a 'backup' graft because of engraftment failure.
Adolescent↗
A subline of the Brown Norway myeloid leukemia in the Lewis x Brown Norway rat: in vivo growth characteristics and development of an in vitro clonogenic assay.
A subline of Brown Norway (BN) acute myelocytic leukemia (AML) which can be propagated in suspension culture (designated IPC-81) is described. Injection into Lewis x BN F1 hybrid (LBN) rats resulted in a log-linear correlation between tumor cell dose and time till death from the onset of leukemia even after multiple (greater than 16) passages in vitro. An in vitro clonogenic assay for IPC-81 colony formation (CFU-leuk) was developed with excellent cloning efficiency (55-82%). Colonies grew without the addition of specific growth factors; syngeneic spleen-conditioned medium inhibited CFU-leuk by 40%, but co-culture with untreated normal LBN rat bone marrow cells had no effect on CFU-leuk. CFU-leuk could be detected in the bone marrow 7 to 10 days before morphologic detection of leukemia in injected animals. This cell line should prove useful in the preclinical evaluation of new strategies for treating AML and evaluating new bone marrow purging methods.
Animals↗
Autologous bone marrow transplantation.
Autologous bone marrow transplantation in the acute leukemias and lymphomas offers potentially curative treatment in patients who do not have a histocompatible, allogeneic donor. Results of marrow autografting in the lymphomas are especially encouraging, with disease-free survivals of 50-60% in patients who have failed primary and secondary treatment regimens. In the acute leukemias, one may expect 20-40% relapse-free survival after autologous marrow transplantation. We feel that ex vivo treatment ("purging") is necessary to eradicate occult tumor cells from autologous remission marrow in hematological malignancies, but this remains a controversial issue to some investigators. Preliminary studies of autologous bone marrow transplantation are promising in multiple myeloma and certain childhood tumors, and autografting is currently being explored in the treatment of other solid tumors such as adenocarcinoma of the breast.
Bone Marrow Transplantation↗
Efficacy of ex vivo purging for autologous bone marrow transplantation in the treatment of acute nonlymphoblastic leukemia.
We used an in vitro measure of drug activity to predict the efficacy of ex vivo purging of leukemic cells from autologous bone marrow grafts. We previously found that the myeloid progenitor cell (CFU-GM) content of the marrow grafts after ex vivo purging with 4-hydroperoxycyclophosphamide (4-HC) correlates with time to hematologic recovery after autologous bone marrow transplantation in patients with acute nonlymphoblastic leukemia. We observed that variable red blood cell concentration of the bone marrow incubation mixture results in differential cytotoxic activity of 4-HC. The CFU-GM content of the graft after the ex vivo treatment is a measure of this 4-HC activity. We analyzed the disease-free survival of 45 patients with acute nonlymphoblastic leukemia undergoing autologous bone marrow transplantation with 4-HC purged grafts. Patients who relapsed after transplantation had 4.2 +/- 1.1% of graft CFU-GM surviving the ex vivo purge, compared with 1.1 +/- 0.4% for patients who achieved a sustained remission (P = .06). Twenty-three patients with a CFU-GM content after 4-HC purging of greater than 1% of the pretreatment value had an actuarial disease-free survival of 12%, compared to 36% for 22 patients with a less than or equal to 1% CFU-GM content after purging (P = .006). Therefore, percent CFU-GM survival as a measure of 4-HC cytotoxicity identified a group of patients with insufficient purging. Although no randomized clinical trials have documented the need for ex vivo purging, our results suggest that effective bone marrow purging is important for the optimal application of autologous transplantation in the treatment of acute nonlymphoblastic leukemia.
Age Factors↗
Interstitial pneumonitis following autologous bone marrow transplantation.
Interstitial pneumonitis (IP) occurred in 20 of 143 (14%) patients who received cytoreductive therapy followed by autologous bone marrow transplantation (BMT) as treatment for malignancy. IP occurred at a median onset time of 41 days (5 to 624 days). All but three of the episodes were fatal. Of the thirteen cases in which tissue was examined, half were idiopathic; the remainder were due to various infectious agents. The actuarial incidences of idiopathic (7%) and CMV IP (2%) in these marrow autograft recipients were lower than the incidences of idiopathic (19%) and CMV IP (17%) in comparably treated recipients of allogeneic BMT (P less than or equal to 0.001 for both comparisons).
Adolescent↗
Successful marrow transplantation for acute myelocytic leukemia following therapy for Hodgkin's disease.
Five patients with acute myelocytic leukemia (AML) after combined modality therapy for Hodgkin's disease (HD) were treated with cyclophosphamide and busulfan followed by bone marrow transplantation (BMT). Four patients received allogeneic transplants from histocompatibility locus antigen (HLA)-compatible siblings and the fifth patient received an autologous marrow treated with 4-hydroperoxycyclophosphamide. Two patients died of complications of acute graft-v-host disease (GVHD) despite prophylaxis with either low-dose cyclophosphamide or cyclosporine. The remaining three patients were alive and disease-free 382, 617, and 620 days after transplant. These initial results are encouraging and more patients with treatment-related AML need to be evaluated with both allogeneic and autologous BMT to fully elucidate the potentially curative role of this intensive therapy in an otherwise fatal hematologic malignancy.
Adolescent↗
Gastrointestinal inflammation after bone marrow transplantation: graft-versus-host disease or opportunistic infection?
Gastrointestinal inflammation after allogeneic bone marrow transplantation may be due to acute graft-versus-host disease (GVHD) and/or superinfection with opportunistic organisms. Twenty-eight patients with barium studies suggesting gastrointestinal inflammation after bone marrow transplantation and either acute GVHD, viral infection, or both were studied to characterize the radiographic appearances of each disease and to determine whether acute GVHD could be distinguished from viral superinfection on the basis of radiographic findings. Thirteen patients had minimal or no acute GVHD, with viral infection proved in eight and strongly suspected in four others; the remaining patient was thought to have nonspecific inflammatory bowel disease. Five patients had pure acute GVHD, and 10 patients had viral enteritis superimposed on acute GVHD. Radiographic abnormalities were found in the gastrointestinal tract in both acute GVHD and viral infection and were more extensive than previously reported. Findings were similar in both entities, although gastric abnormalities were not seen in pure acute GVHD but only in viral infection, either alone or together with acute GVHD. Prolonged small bowel barium coating occurred in both viral infection and acute GVHD. Fold thickening evolved into fold effacement with a shaggy contour in two patients with viral infection. Colonic findings in all groups mimicked ulcerative colitis. Our data indicate that differentiation between acute GVHD and viral enteritis is not possible on the basis of radiographic findings alone. Both entities should be considered when gastrointestinal inflammation occurs after bone marrow transplantation.
Adolescent↗