Biomedical subjects
Publications and source records attributed to R Storb.
This paper describes the development of nonmyeloablative marrow transplant programs that have little toxicity in a canine model and their translation to patients with malignant and nonmalignant hematological diseases.
We have previously described pharmacokinetic studies with a dimethylsulfoxide-based intravenous busulfan preparation in a canine model and in preliminary clinical trials. Using the same intravenous busulfan preparation, we carried out a dose escalation study to determine a marrow-ablative dose and to test the ability of autologous marrow to reconstitute hematopoiesis in dogs so treated. Busulfan was given intravenously at doses of 3.75 to 40 mg/kg. Marrow ablation was achieved at 20 mg/kg given either as a single dose or in four daily increments of 5 mg/kg each. There was a relative sparing of lymphocytes. A busulfan dose of 40 mg/kg resulted in severe central nervous system toxicity. Otherwise, nonhematopoietic toxicity was minimal and restricted to mild hepatic abnormalities. Four dogs were given busulfan at 20 mg/kg followed 30 hours later by infusion of autologous marrow, and all showed prompt and complete hematopoietic reconstitution. The area under the curve (AUC) determined by busulfan concentration in plasma over time was dose dependent, ranging from 12 to 100 microg x h/mL for busulfan doses of 3.75-20 mg/kg. There was a suggestion that the plasma half-life increased at the highest busulfan doses used. Intravenous administration of busulfan circumvented differences in bioavailability; nevertheless, considerable variations in the pharmacokinetic parameters were observed between individual animals. Thus, intravenous busulfan can be given safely and is effective in ablating hematopoiesis. However, factors other than absorption influence the AUC, and individualization of dosing may be required even with intravenous administration of the drug.
Graft-vs.-host disease (GVHD) and infection are major complications of allogeneic bone marrow transplantation. Intravenous immunoglobulin (IVIg) given at a dose of 500 mg/kg/wk has been shown to decrease the risk of acute GVHD, interstitial pneumonia, and infection in adults early after allogeneic transplantation. The current study is a controlled trial to determine whether a lower total dose of IVIg given with pretransplant loading reduces the incidence of transplant-related complications. In a randomized trial of 241 patients > or =20 years of age who were given related donor marrow allografts, 121 individuals receiving Ig prophylaxis (500 mg/kg/d loading from day -6 to -1 and then 100 mg/kg every 3 days from day 3 to 90) were compared with 120 control patients who did not receive IVIg. Randomization was stratified by human leucocyte antigen-matching, remission status of malignancy, GVHD prophylaxis, and cytomegalovirus (CMV) serology. The study was powered to detect a reduction in acute GVHD by 18% and a decrease in transplant-related mortality by 17%. Pretransplant IVIg loading and posttransplant maintenance achieved median serum IgG levels >1350 mg/dL, which were approximately twofold greater than the untreated controls (p<0.01). White blood cell and platelet recoveries were similar for the two groups, although control patients required fewer units of platelets per day (2.5 vs. 3.3, p = 0.008). No significant differences in the incidence of CMV infection, interstitial pneumonia, or bacteremia were observed. The incidence of acute GVHD did not differ between the two groups; however, acute GVHD was less frequent among IVIg recipients achieving maximum serum IgG levels >3000 mg/dL (60 vs. 79%). Neither transplant-related mortality nor disease-free survival was significantly altered by Ig prophylaxis. However, the cumulative incidence of relapse of malignancy was higher in IVIg recipients than in controls (31 vs. 18%, p = 0.03). Multivariable regression analysis demonstrated a 1.89 increased relative risk of relapse for individuals given IVIg (p = 0.021). We conclude that pretransplant loading and a shorter course and lower total dose of IVIg prophylaxis did not appear to decrease the risk of acute GVHD or mortality among adults receiving related donor marrow transplants. Note, IVIg administration may be associated with an increased risk of recurrent malignancy, a finding that warrants further investigation.
We have used dogs to study gene transfer into hematopoietic stem cells, because of the applicability of results in dogs to human transplantation and the availability of canine disease models that mimic human diseases. Previously we reported successful gene transfer into canine marrow repopulating cells, however, gene transfer efficiency was low, usually below 0.1% (Kiem et al, Hum Gene Ther 1996; 7: 89). In this study we have used CD34-enriched marrow cells to study different retroviral pseudotypes for their ability to transduce canine hematopoietic repopulating cells. Cells were divided into two equal fractions that were cocultivated for 72 h with irradiated packaging cells producing vector with different retroviral pseudotypes (GALV, amphotropic or 10A1). The vectors used contained small sequence differences to allow differentiation of cells genetically marked by the different vectors. Nonadherent and adherent cells from the cultures were infused into four dogs after a myeloablative dose of 920 cGy total body irradiation. Polymerase chain reaction (PCR) analysis of DNA from peripheral blood and marrow after transplant showed that the highest gene transfer rates (up to 10%) were obtained with the GALV-pseudotype vector. Gene transfer levels have remained stable now for more than 18 months. Southern blot analysis confirmed the high gene transfer rate. Interference studies on canine D17 cells revealed that 10A1 virus behaved like an amphotropic virus and was not able to use the GALV receptor. In summary, our results show improved gene transfer into canine hematopoietic repopulating cells when CD34-enriched cells are transduced by cocultivation on a GALV-pseudotype packaging cell line in combination with a GALV-pseudotype vector. Furthermore, these results demonstrate that the monoclonal antibody to canine CD34 used in this study is able to enrich for hematopoietic repopulating cells.
We evaluated the marrow toxicity of 200 and 300 cGy total-body irradiation (TBI) delivered at 10 and 60 cGy/min, respectively, in dogs not rescued by marrow transplant. Additionally, we compared toxicities after 300 cGy fractionated TBI (100 cGy fractions) to that after single-dose TBI at 10 and 60 cGy/min. Marrow toxicities were assessed on the basis of peripheral blood cell count changes and mortality from radiation-induced pancytopenia. TBI doses studied were just below the dose at which all dogs die despite optimal support. Specifically, 18 dogs were given single doses of 200 cGy TBI, delivered at either 10 (n=13) or 60 (n=5) cGy/min. Thirty-one dogs received 300 cGy TBI at 10 cGy/min, delivered as either single doses (n=21) or three fractions of 100 cGy each (n=10). Seventeen dogs were given 300 cGy TBI at 60 cGy/min, administered either as single doses (n=5) or three fractions of 100 cGy each (n=10). Within the limitations of the experimental design, three conclusions were drawn: 1) with 200 and 300 cGy single-dose TBI, an increase of dose rate from 10 to 60 cGy/min, respectively, caused significant increases in marrow toxicity; 2) at 60 cGy/min, dose fractionation resulted in a significant decrease in marrow toxicities, whereas such a protective effect was not seen at 10 cGy/min; and 3) with fractionated TBI, no significant differences in marrow toxicity were seen between dogs irradiated at 60 and 10 cGy/min. The reduced effectiveness of TBI when a dose of 300 cGy was divided into three fractions of 100 cGy or when dose rate was reduced from 60 cGy/min to 10 cGy/min was consistent with models of radiation toxicity that allow for repair of sublethal injury in DNA.
Traditional approaches to allogeneic stem cell transplantation have relied on the use of toxic high-dose conditioning therapy to achieve allogeneic engraftment and control of underlying disease. Preclinical observations have shown that, for engraftment purposes, conditioning regimens can be reduced in intensity, resulting in reduced treatment toxicities. In preclinical canine studies, the use of potent pre- and postgrafting immunosuppression allowed for reduction in conditioning regimens and development of stable mixed chimerism. If these newer approaches using attenuated conditioning regimens can be successfully applied to human transplantation, an improved safety profile will allow potentially curative treatment of patients not currently offered such therapy. Mixed chimerism per se could prove curative of disease manifestation for various nonmalignant disturbances of the hematopoietic and immune systems. For patients with malignancy, infusion of additional donor lymphocytes may be needed to effectively treat underlying disease.
A 50-year-old woman who was retrospectively diagnosed with an early asymptomatic myelodysplastic syndrome (MDS) served as a haemopoietic stem cell donor for her HLA-identical sister who had chemotherapy-refractory non-Hodgkin's lymphoma. The MDS of the donor was classified as refractory anaemia (RA) and cytogenetically characterized by deletion of the long arm of chromosome 20 [del(20q)]. Donor cell engraftment in marrow and peripheral blood was analysed over a period of 5 months after transplant using conventional cytogenetics, fluorescence in situ hybridization, and variable number of tandem repeats. Neutrophil counts >0.5 x 109/l and platelet counts >20 x 109/l were reached promptly on days 12 and 24, respectively. Throughout the period of observation the percentage of cells with the del(20q) abnormality in the recipient's marrow and peripheral blood was comparable to the proportion of these cells in the donor. These data indicate that the abnormal clone was capable of homing to the marrow, proliferating, differentiating, and therefore contributing to haemopoiesis in a relatively efficient manner. This implies that MDS progenitor cells may not have homing and growth deficiencies, a finding that has particular relevance for autologous transplantation in MDS patients where tumour cells potentially contaminate the graft.
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Between 1970 and 1996, 333 patients with severe aplastic anemia underwent HLA-matched related marrow transplant after conditioning with cyclophosphamide (CY). Thirty-five percent of patients transplanted between 1970 and 1976 (group 1), 12% of those transplanted between 1977 and 1981 (group 2), and 9% of patients transplanted between 1982 and 1997 (group 3) had graft rejection. Graft rejection occurred later among group 3 patients (median, 180 days) than among those in groups 1 and 2 (medians, 28 and 47 days, respectively; P < .001 group 3 v 2). In group 3, 92% of rejecting patients underwent a second transplant, compared with 78% and 77% in groups 1 and 2, respectively. Group 1 patients received various conditioning regimens before second transplant, whereas most patients of groups 2 and 3 received CY combined with antithymocyte globulin (ATG). Graft-versus-host disease (GVHD) prophylaxis after second transplant consisted of methotrexate (MTX) for all group 1 and 2 patients, whereas group 3 patients received MTX combined with cyclosporine (CSP). Over the three time periods studied, first graft rejection decreased from 35% to 9%, and the proportion of rejecting patients undergoing second transplants increased from 77% to 92%. The 10-year probability of survival after second transplants increased from 5% to 83%. Multivariate analysis showed MTX/CSP GVHD prophylaxis to be a significant factor accounting for the increase in patient survival after second transplant.
We report the results of a phase III open-label, randomized, multicenter trial comparing tacrolimus/methotrexate to cyclosporine/methotrexate for graft-versus-host disease (GVHD) prophylaxis after HLA-identical sibling marrow transplantation in patients with hematologic malignancy. The primary objective of this study was to compare the incidence of moderate to severe (grade II-IV) acute GVHD. Secondary objectives were to compare the relapse rate, disease-free survival, overall survival, and the incidence of chronic GVHD. Patients were stratified according to age (<40 v >/=40) and for male recipients of a marrow graft from an alloimmunized female. There was a significantly greater proportion of patients with advanced disease randomized to tacrolimus arm (P = . 02). The incidence of grade II-IV acute GVHD was significantly lower in patients who received tacrolimus than patients in the cyclosporine group (31.9% and 44.4%, respectively; P = .01). The incidence of grade III-IV acute GVHD was similar, 17.1% in cyclosporine group and 13.3% in the tacrolimus group. There was no difference in the incidence of chronic GVHD between the tacrolimus and the cyclosporine group (55.9% and 49.4%, respectively; P = .8). However, there was a significantly higher proportion of patients in the cyclosporine group who had clinical extensive chronic GVHD (P = . 03). The relapse rates of the two groups were similar. The patients in the cyclosporine arm had a significantly better 2-year disease-free survival and overall survival than patients in the tacrolimus arm, 50.4% versus 40.5% (P = .01) and 57.2% versus 46.9% (P = .02), respectively. The significant difference in the overall and disease-free survival was largely the result of the patients with advanced disease, 24.8% with tacrolimus versus 41.7% with cyclosporine (P = .006) and 20.4% with tacrolimus versus 28% with cyclosporine (P = .007), respectively. There was a higher frequency of deaths from regimen-related toxicity in patients with advanced disease who received tacrolimus. There was no difference in the disease-free and overall survival in patients with nonadvanced disease. These results show the superiority of tacrolimus/methotrexate over cyclosporine/methotrexate in the prevention of grade II-IV acute GVHD with no difference in disease-free or overall survival in patients with nonadvanced disease. The survival disadvantage in advanced disease patients receiving tacrolimus warrants further investigation.
BACKGROUND: Alloreactive donor T cells in marrow grafts mediate graft-versus-host disease (GVHD), but T-cell depletion has resulted in increased graft failure. Add-back of gene-modified alloreactive donor T cells could prevent graft rejection. After engraftment, in vivo depletion of those modified T cells with ganciclovir may control GVHD. METHODS: Canine recipient-specific donor cytotoxic T lymphocytes (CTL) were retrovirally transduced with the herpes simplex virus thymidine kinase gene. RESULTS: Gibbon ape leukemia virus-pseudotyped vector yielded primary CTL transduction efficiency of 22.9+/-9.9%. After selection and expansion, 96.7+/-0.8% of CTL expressed retrovirally transferred genes. Recipient-specific cytotoxic activity was maintained with 84.3% specific lysis. After ganciclovir treatment, herpes simplex virus thymidine kinase-transduced CTL proliferation was reduced 98.7+/-0.2% compared with controls. CONCLUSIONS: We have demonstrated efficient ex vivo transduction, expansion, maintenance of alloreactivity, and ganciclovir-mediated ablation of canine CTL, which will permit in vivo studies in the dog, a well-established model for GVHD and engraftment.
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Treatment options for patients diagnosed with chronic myelogenous leukemia (CML) in chronic phase (CP) who lack a suitable related donor for marrow transplantation include hydroxyurea, interferon-alpha (IFN-alpha), or transplantation from an unrelated donor (URD). Most studies support the view that treatment with IFN-alpha results in prolonged survival compared with hydroxyurea therapy. Some patients are offered URD transplantation as a second-line treatment; however, the impact of pretransplant IFN-alpha on the outcome of URD transplantation is uncertain. To address this question, we evaluated the effect of pretransplant IFN-alpha therapy in 184 patients undergoing URD transplantation for CML in CP at a single center. Of the 184 patients, 114 did not receive IFN-alpha, whereas 22, 23, and 25 patients received IFN-alpha for, respectively, 1 to 5, 6 to 12, and more than 12 months before transplant. Pretransplant IFN-alpha therapy administered for > or = 6 months was associated with an increased risk of severe (grades III-IV) acute graft-versus-host disease (GVHD; relative risk [RR], 3.0; 95% confidence interval [CI], 1.4 to 6.2; P = .004) and mortality (RR, 2. 1; 95% CI, 1.3 to 3.5; P = .003) relative to less than 6 months or no IFN-alpha therapy. Increased mortality occurred between 100 and 365 days after transplant (P = .005), was limited to patients with severe acute GVHD, and was due to chronic GVHD refractory to immunosuppressive therapy. Other variables associated with mortality included HLA-DRB1 or DQB1 (but not HLA-A or B) mismatched donors, age greater than 50 years, weight > or = 110% of ideal body weight, and the absence of cytomegalovirus (CMV) or fungal prophylaxis. For patients treated with IFN-alpha for less than 6 months before transplant, who were < or = 50 years of age, received a HLA-A, B, DRB1, and DQB1 matched URD transplant, and received CMV and fungal prophylaxis after transplant (n = 48), survival was 87% +/- 5% at 5 years. These data provide a rationale for immediate transplantation in preference to extended treatment with IFN-alpha when the patient is < or = 50 years of age and has an HLA-compatible unrelated volunteer donor.
Preparative regimens for marrow allografts in thalassemia have two objectives. One is eradication of diseased marrow and the other suppression of host-versus-graft (HVG) reactions so that the allograft survives. A common regimen to accomplish these goals has combined high-dose busulfan with cyclophosphamide. Postgrafting immunosuppression with cyclosporine/methotrexate has been used for GVHD prevention. Some patients may die from regimen-related toxicity. Overall event-free survival is 75%. Occasional patients have become mixed donor/host hematopoietic chimeras and, yet, disease symptoms have abated. This has raised the possibility of developing safer and less toxic transplant programs that result in stable mixed hematopoietic chimerism. We have devised such a program in dogs consisting of a nonlethal dose of total body irradiation (200 cGy) before and a novel combination of mycophenolate mofetil and cyclosporine after transplant. Mixed donor/host chimerism (> or = 50% donor cells in all lineages) has persisted for > 80 weeks, even though immunosuppression was discontinued after five weeks.
Allogeneic marrow transplantation is curative therapy for thalassemia, but fewer than 30% of patients have an HLA-identical sibling marrow donor. Selection of alternative donors of hematopoietic stem cells (unrelated individuals or HLA-nonidentical family members) has been aided by establishment of world-wide donor registries now exceeding 3.6 million volunteers and by DNA-based HLA typing to more closely match potential donors. Coupled with improved methods to control graft-versus-host disease and prevent fungal and cytomegalovirus infection, remarkable progress has been made in alternative donor transplantation. For patients 50 years of age or younger, with recently diagnosed chronic myelogenous leukemia (CML) in chronic phase, 1- and 5-year survivals after HLA-A, B, DRB1 identical unrelated marrow transplantation in Seattle are 82% and 74%, respectively. These results are essentially identical to outcome in similar patients given HLA-matched sibling allografts. However, the world-wide number of alternative donor transplants for thalassemia remains limited to date: 4 unrelated and 60 HLA-nonidentical related transplants have been reported to the IBMTR since 1969 with actuarial overall survival of 75%. Using the paradigm of CML, it is likely that access to curative therapy of thalassemia will improve with optimal HLA typing and donor selection early in the course of disease.
We reviewed the records and reevaluated 212 patients with aplastic anemia transplanted at the Fred Hutchinson Cancer Research Center (FHCRC) between 1970 and 1993 who survived >/=2 years and who have been followed for up to 26 years. Parameters analyzed included hematopoietic function, chronic graft-versus-host disease (GVHD), skin disease, cataracts, lung disease, skeletal problems, posttransplant malignancy, depression, pregnancy/fatherhood, and the return to work or school, as well as patient self-assessment of physical and psychosocial health, social interactions, memory and concentration, and overall severity of symptoms. Survival probabilities at 20 years were 89% for patients without (n = 125) and 69% for patients with chronic GVHD (n = 86) (the status was uncertain in 1 surviving patient). All patients had normal hematopoietic parameters. Skin problems occurred in 14%, cataracts in 12%, lung disease in 24%, and bone and joint problems in 18% of patients. Eleven patients (12%) developed a solid tumor malignancy and 19% of patients experienced depression. Chronic GVHD was the dominant risk factor for late complications. Seventeen patients died at 2.5 to 20.4 years posttransplant; 13 of these had chronic GVHD and related complications. At 2 years, 83% of patients had returned to school or work; the proportion increased to 90% by 20 years. At least half of the patients preserved or regained the ability to become pregnant or father children. Patients rated their quality of life as excellent and symptoms as minimal or mild. In conclusion, marrow transplantation in patients with aplastic anemia established long-term normal hematopoiesis. No new hematologic disorders occurred. The major cause of morbidity and mortality was chronic GVHD. However, the majority of patients who survived beyond 2 years returned to a fully functional life.
Primary graft rejection after marrow transplantation occurs more frequently in patients receiving HLA-haploidentical compared with HLA-identical sibling transplants. Both human and experimental animal data suggest that the cells responsible for this phenomenon are either host natural killer (NK) cells, T cells, or both. To investigate the mechanisms of graft rejection, we have developed a canine model of marrow transplantation, which uses DLA-nonidentical unrelated donors in the absence of postgrafting immunosuppression. In this model most animals rejected their marrow grafts after a preparative regimen of 9.2 Gy total body irradiation (TBI). However, engraftment of DLA-nonidentical marrow can be facilitated when the recipients are pretreated with monoclonal antibody (MoAb) S5, which recognizes CD44. In this report, we extended these observations by first cloning the canine CD44 and, next, mapping the epitope recognized by S5, which was located in a region conserved among human and canine CD44 and was distinct from the hyaluronan binding domain. However, in vitro binding of S5 caused a conformational change in CD44, which allowed increased hyaluronan binding. Then, we reexamined the in vivo model of marrow transplantation and compared results with MoAb S5 to those with two other anti-CD44 MoAbs, IM7 and S3. Only MoAb S5 significantly increased the engraftment rate of DLA-nonidentical unrelated marrow, whereas the two other anti-CD44 MoAbs were ineffective. The enhanced in vivo effect was not related to differences in the MoAbs' avidities, since both S5 and IM7 had equivalent binding to CD44, but most likely related to the specific epitope that S5 recognizes. Thus, this study shows that the effect of the anti-CD44 MoAb S5 in facilitating engraftment is epitope specific and if one is to use an anti-CD44 to facilitate engraftment of marrow in humans, one cannot assume that any anti-CD44 would work.
Mycophenolate mofetil (MMF) was evaluated either alone or combined with cyclosporine (CSP) for preventing graft-versus-host disease (GVHD) in dogs given 9.2 Gy total body irradiation and DLA-nonidentical unrelated marrow grafts. Marrow autograft studies showed gut toxicity as limiting MMF side effects. Four groups were studied for GVHD prevention: six dogs in group 1 received MMF 10 mg/kg twice daily subcutaneously (SC) on days 0 to 27. They died between 8 to 28 days from infection or GVHD; survival was better than that of 72 controls given no immunosuppression (P = .04), but not different from 19 dogs given CSP. Four dogs in group 2 received MMF as described, along with CSP at 10 to 15 mg/kg twice daily on days 0 to 27. They died at 6 to 98 days from CSP-associated toxicity, weight loss, or infection. Nine dogs in group 3 received MMF SC twice daily 6 mg/kg/d for 3 days, followed by 10 mg/kg twice daily until day 27, along with CSP as described; four died between 7 to 106 days with intussusception, infection, or GVHD, and five became long-term survivors. Six dogs in group 4 received shortened MMF (21 days) and reduced doses of CSP given through day 100. Three died with GVHD or infection between days 38 to 119, and three became long-term survivors. Results support the notion of synergism between MMF and CSP, as evidenced by stable graft-host tolerance in greater than 50% of dogs.