Roger H. Herzig

Adult Human Mesenchymal Stem Cells Added to Corticosteroid Therapy for the Treatment of Acute Graft-versus-Host Disease

The unique immunomodulatory properties of mesenchymal stem cells (MSCs) make them a rationale agent to investigate for graft-versus-host disease (GVHD). Human MSCs were used to treat de novo acute GVHD (aGVHD). Patients with grades II-IV GVHD were randomized to receive 2 treatments of human MSCs (Prochymal(R)) at a dose of either 2 or 8 million MSCs/kg in combination with corticosteroids. Patients received GVHD prophylaxis with tacrolimus, cyclosporine, (CsA) or mycophenolate mofetil (MMF). Study endpoints included safety of Prochymal administration, induction of response to Prochymal, and overall response of aGVHD by day 28, and long-term safety. Thirty-two patients were enrolled, with 31 evaluable: 21 males, 10 females; median age 52 years (range: 34-67). Twenty-one patients had grade II, 8 had grade III, and 3 had grade IV aGVHD. Ninety-four percent of patients had an initial response to Prochymal (77% complete response [CR] and 16% partial response [PR]). No infusional toxicities or ectopic tissue formations were reported. There was no difference with respect to safety or efficacy between the low and high Prochymal dose. In conclusion, Prochymal can be infused safely into patients with aGVHD and induces response in a high proportion of GVHD patients.

Chimerism and Tolerance Without GVHD or Engraftment Syndrome in HLA-Mismatched Combined Kidney and Hematopoietic Stem Cell Transplantation

Durable chimerism and donor-specific tolerance can be safely achieved without GVHD in HLA-mismatched donor/recipient pairs. Teaching Tolerance According to Greek mythology, the Chimera was a fire-breathing creature made of parts from different animals: the body of a lioness, a snake’s head at the end of the tail, and the head of the goat. Sightings of this fearsome beast portended any of a number of terrible disasters. In the context of organ transplantation, a “chimera” can indicate both desirable and disastrous outcomes. For example, hematopoietic chimerism, in which the immune cells in the graft recipient come from both the host and the donor, may promote graft tolerance, but may also cause graft-versus-host disease (GVHD), in which the donor immune cells attack the healthy tissue of the host. Leventhal et al. now report mixed chimerism and tolerance without the negative side effects of GVHD or engraftment syndrome in a phase 2 clinical trial of combined kidney and hematopoietic transplantation. Leventhal et al. used a combination of mobilized cells enriched for hematopoietic stem cells and graft-facilitating cells—which are composed largely of plasmacytoid precursor dendritic cells—with nonmyeloablative conditioning in conjunction with kidney transplant from major histocompatibility complex–mismatched, nonrelated donors and recipients. Five of eight kidney transplant recipients exhibited durable chimerism and were weaned off immunosuppressive therapies by 1 year after transplantation, with no signs of GVHD or engraftment syndrome. If confirmed in larger patient cohorts, this approach to transplantation could free some patients from the difficulties associated with lifelong immunosuppression and add transplantation as a viable option for patients for whom no matched donors exist. As with the Chimera of legend, mixed chimerism may be a harbinger of things to come—albeit hopefully a brighter future for transplant patients. The toxicity of chronic immunosuppressive agents required for organ transplant maintenance has prompted investigators to pursue approaches to induce immune tolerance. We developed an approach using a bioengineered mobilized cellular product enriched for hematopoietic stem cells (HSCs) and tolerogenic graft facilitating cells (FCs) combined with nonmyeloablative conditioning; this approach resulted in engraftment, durable chimerism, and tolerance induction in recipients with highly mismatched related and unrelated donors. Eight recipients of human leukocyte antigen (HLA)–mismatched kidney and FC/HSC transplants underwent conditioning with fludarabine, 200-centigray total body irradiation, and cyclophosphamide followed by posttransplant immunosuppression with tacrolimus and mycophenolate mofetil. Subjects ranged in age from 29 to 56 years. HLA match ranged from five of six loci with related donors to one of six loci with unrelated donors. The absolute neutrophil counts reached a nadir about 1 week after transplant, with recovery by 2 weeks. Multilineage chimerism at 1 month ranged from 6 to 100%. The conditioning was well tolerated, with outpatient management after postoperative day 2. Two subjects exhibited transient chimerism and were maintained on low-dose tacrolimus monotherapy. One subject developed viral sepsis 2 months after transplant and experienced renal artery thrombosis. Five subjects experienced durable chimerism, demonstrated immunocompetence and donor-specific tolerance by in vitro proliferative assays, and were successfully weaned off all immunosuppression 1 year after transplant. None of the recipients produced anti-donor antibody or exhibited engraftment syndrome or graft-versus-host disease. These results suggest that manipulation of a mobilized stem cell graft and nonmyeloablative conditioning represents a safe, practical, and reproducible means of inducing durable chimerism and donor-specific tolerance in solid organ transplant recipients.

Comparison of Preparative Regimens in Transplants for Children With Acute Lymphoblastic Leukemia

PURPOSE Preparative regimens involving total-body irradiation (TBI) produce significant late toxicities in some children who receive bone marrow transplants, including impaired growth and intellectual development. Busulfan is often used as an alternative to TBI, but there are few data regarding its relative efficacy. PATIENTS AND METHODS We compared outcomes of HLA-identical sibling transplants for acute lymphoblastic leukemia (ALL) in children (< 20 years of age) who received cyclophosphamide plus TBI (CY/TBI) (n = 451) versus those who received busulfan plus cyclophosphamide (Bu/CY) (n = 176) for pretransplant conditioning. Patients received transplants between 1988 and 1995 and their results were reported to the International Bone Marrow Transplant Registry by 144 participating institutions. The CY/TBI and Bu/CY groups did not differ in gender, immune phenotype, leukocyte count at the time of diagnosis, chromosome abnormalities, remission status, or length of initial remission. T-cell depletion was used more frequently in the CY/TBI group; the Bu/CY group included a higher proportion of children who were less than 5 years of age. The median follow-up period was 37 months. RESULTS The 3-year probabilities of survival were 55% (95% confidence interval [CI], 50% to 60%) with TBI/CY and 40% (95% CI, 32% to 48%) with Bu/CY (univariate P =.003). The 3-year probabilities of leukemia-free survival were 50% (95% CI, 45% to 55%) and 35% (95% CI, 28% to 43%), respectively (univariate P =.005). In a multivariate analysis, the risks of relapse were similar in the two groups (relative risk [RR], 1.30 for Bu/CY v CY/TBI; P =.1). Treatment-related mortality was higher in the Bu/CY group (RR, 1.68; P =.012). Death and treatment failure (relapse or death, inverse of leukemia-free survival) were more frequent in the Bu/CY group (RR, 1. 39; P =.017 for death; RR, 1.42; P =.006 for treatment failure). CONCLUSION These data indicate superior survival with CY/TBI conditioning, compared with Bu/CY conditioning, for HLA-identical sibling bone marrow transplants in children with ALL.

ALLOGENEIC BONE-MARROW TRANSPLANTATION FOR CHRONIC MYELOGENOUS LEUKAEMIA

In 117 patients with chronic myelogenous leukaemia (CML) treatment with a combination of high-dose chemoradiotherapy plus transplantation of allogeneic bone-marrow from HLA-identical, mixed-lymphocyte-culture-identical siblings resulted in an actuarial probability of 3-year survival of 63 +/- 16% (95% confidence interval) for 39 patients transplanted in chronic phases; 36 +/- 14% for 56 transplanted in accelerated phase; and 12 +/- 15% for 22 transplanted during blast crisis. Irrespective of disease status at the time of transplantation, and in contrast to chemotherapy, a plateau-effect was observed in the survival curves starting 14 to 19 months after transplantation. The actuarial probability of recurrent or persistent leukaemia at 3 years was 7 +/- 9% for patients transplanted in chronic phase, 41 +/- 19% for accelerated phase, and 41 +/- 39% for blastic phase. All relapses occurred within 18 months of transplantation. This study demonstrates that long-term disease-free survival in CML can be achieved with bone-marrow transplantation. Best results were obtained in patients transplanted during chronic phase of the disease.

High-dose cytosine arabinoside therapy with and without anthracycline antibiotics for remission reinduction of acute nonlymphoblastic leukemia

Seventy-eight patients with acute nonlymphoblastic leukemia in relapse were treated with high-dose cytosine arabinoside (3 g/m2 intravenously (IV) every 12 hours for 12 doses) alone, or with three days of anthracycline antibiotics (doxorubicin 20 mg/m2 or daunorubicin 30 mg/m2 IV daily) after completing the course of cytosine arabinoside. Consolidation and maintenance therapy was not given. When anthracyclines were added there was no increase in frequency or severity of nonhematologic toxicity including conjunctivitis, photophobia, dermatitis, cerebellar dysfunction, and gastrointestinal disturbance. All 78 patients achieved aplasia of the bone marrow. Five patients in each group died before bone marrow recovery. The use of anthracyclines did not prolong bone marrow recovery, with both groups demonstrating adequate granulocyte and platelet counts about four weeks after beginning treatment. Forty-one (53%) of the total 78 patients achieved a complete remission. In patients not clinically resistant to conventional-dose cytosine arabinoside, both regimens were equally effective inducing a complete remission (high-dose cytosine arabinoside alone, 12/19 [63%]; with anthracycline, 11/17 [65%], P = .270); in patients clinically resistant, the regimen including anthracycline was superior (15/27 [56%] v 3/15 [20%], P = .022). The duration of unmaintained response was similar (median, five months), but the longest remissions occurred when anthracyclines were used. Thus, high-dose cytosine arabinoside is effective in producing remissions in relapsed patients with acute nonlymphoblastic leukemia, and the addition of an anthracycline enhances this effect.

Intensive melphalan chemotherapy and cryopreserved autologous bone marrow transplantation for the treatment of refractory cancer.

Thirty-three adult and pediatric patients with refractory malignancies were treated with escalating doses of melphalan (120-225 mg/m2 IV over 3 days) followed by reinfusion of previously harvested and cryopreserved autologous marrow. The hematological and nonhematological toxicities and the therapeutic effects of this regimen were evaluated. Increasing doses of melphalan did not alter the rate of decline nor the recovery of peripheral blood counts. Granulocyte (greater than 500/microL) and platelet count (greater than 20,000/microL) recovery occurred in a median of 19 (range 12-54) and 24 (range: 12-54) days after bone marrow transplantation, respectively. Five patients experienced severe infection, three of which were fatal, and one patient died due to thrombocytopenic hemorrhage. Toxicity to the gastrointestinal system was dose limiting. The maximum tolerated dose of melphalan was 180 mg/m2; only three of 24 patients experienced severe stomatitis, esophagitis, and diarrhea at this level or less, while eight of nine patients at 225 mg/m2 were affected (p less than 0.005). Administration of cyclophosphamide (300 mg/m2 IV) 1 week before melphalan therapy did not reduce the incidence of severe gastrointestinal toxicity. Plasma melphalan concentration peaked 30-60 min after infusion (4.8-11.5 micrograms/mL) but declined rapidly. Cerebrospinal fluid concentration was 10% of the corresponding plasma concentration and was undetectable at 3 hours. Antitumor responses occurred in nine of 13 patients with malignant melanoma (five complete and four partial remissions), and ranged 2-12+ months with a median of 5 months. Four of six neuroblastomas demonstrated responses (three complete and one partial remission( lasting a median of 7.5 (range: 5-10) months. Other tumors in which this regimen had activity included breast cancer and Ewings sarcoma. The overall response rate for the 33 patients was 30% complete remissions (10 patients) and 21% partial remissions (seven patients). High dose melphalan and autologous bone marrow transplantation is a promising therapy for patients with malignancies for which no effective treatment is known or for patients whose cancer is refractory to conventional therapeutic agents.

Graft-versus-leukemia effects in T lineage and B lineage acute lymphoblastic leukemia

T and B lineage ALL cells express different levels of HLA-class II antigens, which may serve as targets for graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL). The object of this study was to determine whether GVL effects after HLA-identical sibling bone marrow transplantation differed in T and B lineage ALL. We studied 1132 patients with ALL of T lineage (n = 416) or of B lineage (cALLa+) (n = 716) transplanted in first (n = 605) or second (n = 527) remission with bone marrow from an HLA-identical sibling donor, between 1982 and 1992, and reported to the IBMTR by 165 teams. Cox proportional hazards regression models were used to determine the relative risk (RR) of relapse in patients with acute (grades II–IV) or chronic GVHD vs patients without GVHD. Acute and chronic GVHD were considered as time-dependent covariates. Patients transplanted in first and second remission were analyzed separately. GVHD decreased relapse risks to a similar extent in T and B lineage ALL. For first remission transplants, relative risks of relapse for patients with vs those without GVHD was 0.34 for T lineage ALL and 0.44 for B lineage ALL. Corresponding relative risks in second remission transplants were 0.54 and 0.61. This study confirms earlier findings of an antileukemia effect of GVHD in ALL. This effect was similar in T lineage and B lineage ALL, despite probable differences in HLA-class II antigen expression.

Autotransplants for Hodgkin's disease in first relapse or second remission: A report from the autologous blood and marrow transplant registry (ABMTR)

Although patients with relapsed Hodgkins disease have a poor prognosis with conventional therapies, high-dose chemotherapy and autologous hematopoietic stem cell transplantation (autotransplantation) may provide long-term progression-free survival. We reviewed data from the Autologous Blood and Marrow Transplant Registry (ABMTR) to determine relapse, disease-free survival, overall survival, and prognostic factors in this group of patients. Detailed records from the ABMTR on 414 patients with Hodgkins disease in first relapse (n = 295) or second complete remission (CR) (n = 119) receiving an autotransplant from 1989 to 1995 were reviewed. Median age was 29 (range, 7–64) years. Median time from diagnosis to relapse was 18 (range, 6–219) months; median time from relapse to transplant was 5 (range, <1–215) months. Most patients received high-dose chemotherapy without total body irradiation for conditioning (n = 370). The most frequently used high-dose regimen was cyclophosphamide, BCNU, VP-16 (CBV) (n = 240). The graft consisted of bone marrow (n = 246), blood stem cells (n = 112), or both (n = 56). Median follow-up was 46 (range, 5–96) months. One hundred-day mortality (95% confidence interval) was 7 (5–9)%. One hundred and sixty-five of 295 patients (56%) transplanted in relapse achieved CR after autotransplantation. Of these, 61 (37%) recurred. Twenty-four of 119 patients (20%) transplanted in CR recurred. The probability of disease-free survival at 3 years was 46 (40–52)% for transplants in first relapse and 64 (53–72)% for those in second remission (P < 0.001). Overall survival at 3 years was 58 (52–64)% after transplantation in first relapse and 75 (66–83)% after transplantation in second CR (P < 0.001). In multivariate analysis, Karnofsky performance score <90% at transplant, abnormal serum LDH at transplant, and chemotherapy resistance were adverse prognostic factors for outcome. Progression of Hodgkins disease accounted for 69% of all deaths. Autotransplantation should be considered for patients with Hodgkins disease in first relapse or second remission. Future investigations should focus on strategies designed to decrease relapse after autotransplantation, particularly in patients at high risk for relapse. Bone Marrow Transplantation (2001) 27, 387–396.

High-dose weekly intravenous immunoglobulin to prevent infections in patients undergoing autologous bone marrow transplantation or severe myelosuppressive therapy: A study of the American bone marrow transplant group

Treatment with intense myelosuppressive therapy (including bone marrow transplantation) has improved survival in patients with various malignant neoplasms [1, 2]. Unfortunately, this treatment increases the incidence of infectious complications, primarily during the period of myelosuppression [3]. Various methods have been used to limit infection during myelosuppression [4-7]. Despite these precautions, bacteremia and fungemia continue to occur in at least one third of patients with sustained neutropenia. Intravenous immunoglobulin (IVIG) therapy prevents infections in patients with inborn B-cell deficiencies and hypogammaglobulinemia secondary to hematologic disorders such as chronic lymphocytic leukemia [8-10]. Intravenous immunoglobulin has also been used successfully to treat immune thrombocytopenic purpura, alloimmunity to platelets, and other immune-mediated disorders by a mechanism of immune system modulation [11]. After allogeneic bone marrow transplantation, IVIG is commonly used to prevent graft-versus-host disease [12]. During these bone marrow transplant trials, a reduction in bacterial infection was also observed in patients who were not necessarily hypogammaglobulinemic. This finding was initially reported in small anecdotal series but was later confirmed by large prospective studies [12-17]. This effect of IVIG was observed during the pre-engraftment (neutropenic) and myelosuppression recovery phases. Most patients in these studies were undergoing allogeneic bone marrow transplantation, for which graft-versus-host disease and its treatment contribute to the rate of infection [18]. Intravenous immunoglobulin is not routinely used during autologous bone marrow transplantation or severely myelosuppressive therapy because prevention of graft-versus-host disease is unnecessary. Because IVIG prevents infection after allogeneic bone marrow transplantation, it might also do so in other patients undergoing intense myelosuppression and thus may serve as a general prophylactic agent for infections. Intravenous immunoglobulin is expensive and thus should not be used indiscriminately. We designed a prospective study that randomized patients who were expected to develop severe and sustained myelosuppression to receive IVIG or no treatment. We specifically wished to determine whether IVIG could reduce the incidence of severe infections in patients with neutropenia but without allogeneic cofactors such as graft-versus-host disease. We therefore sought to determine whether the benefits of IVIG after allogeneic bone marrow transplantation occur as a direct effect of the drug or as an indirect result of a reduced incidence of graft-versus-host disease. Methods Study Design We conducted a stratified, randomized comparison of patients who either underwent autologous bone marrow transplantation or received substantial myelosuppressive therapy for acute leukemia or other malignant conditions. The protocol and consent forms were approved by the Institutional Review Boards of the three participating institutions: Baylor University Medical Center, Dallas, Texas; The University of Louisville, Louisville, Kentucky; and Vanderbilt University, Nashville, Tennessee. Patients were stratified for treatment (autologous bone marrow transplantation or myelosuppressive therapy) and were randomized at each study center by a computer-generated scheme to receive IVIG or no treatment. Neither tumor-specific cytoreductive therapy nor state of disease were used as strata. Patients with an ongoing infection, those younger than 17 years, and those with a previous intolerance to IVIG were ineligible for the study. The main end points were the development of proven clinical infection, positive blood cultures for bacteria or fungi, and survival until hospital discharge. Other analyses included the number of platelet transfusions and the development of clinical alloimmunity to platelet transfusion. Patients Between February 1990 and December 1991, 170 patients entered the study. All patients were evaluable for efficacy and were included in the analysis. The distribution of study patients is shown in (Table 1). The duration of neutropenia, the most important determinant for infection, was similar between the two groups (P > 0.2). Patients in the treatment arm and those in the control arm had statistically similar distributions of overall cytotoxic regimens and disease diagnoses (data not shown). Table 1. Patient Characteristics Treatment Protocol The IVIG used (Sandoglobulin, Sandoz Pharmaceuticals, East Hanover, New Jersey) was commercially purchased, reconstituted as a 5% solution, and administered intravenously at an initial rate of 0.02 mL/kg per minute for 30 minutes and, if tolerated, was increased every 30 minutes to a maximum rate of 0.08 mL/kg per minute. Administration of IVIG was not blinded, and controls received no placebo. Immunoglobulin was given at a weekly dose of 500 mg/kg beginning at the start of cytotoxic treatment. It was discontinued when severe side-effects occurred or when neutropenia resolved (as defined by a neutrophil count of more than 500 109/L [500/L] for 1 day). Supportive Care The patients were hospitalized in HEPA-filtered single rooms, observed strict hand-washing rules, and received low-bacterial diets. Prophylactic oral antibacterial agents were allowed, but prophylactic parenteral antibacterial drugs were not. Patients who were seropositive for Herpes simplex virus received prophylactic acyclovir. All administered blood products were leukofiltered, and patients undergoing autologous bone marrow transplantation also received irradiated blood products. During periods of neutropenia, patients with fever greater than 38 C had two blood cultures taken and received empiric broad-spectrum antibacterial therapy as determined by the study center. Patients whose fever persisted were recultured. If fever persisted for 3 days and no bacterial cause was found, amphotericin B was administered at a dose of 0.5 mg/kg per day. Definitions and Evaluation of Infection The duration of neutropenia was defined as the interval from the first day the absolute neutrophil count decreased below 500 109/L (500/L) until the first day the count exceeded 500 109/L (500/L). In patients with neutropenia, the interval was measured from the first day of cytotoxic therapy until recovery from neutropenia. Each platelet transfusion, whether with single-donor platelets or random-donor pooled platelets, was denoted as one episode. Clinical alloimmunity was diagnosed when platelet counts measured 1 hour after transfusion increased by less than 5000 109/L (< 5000/L) per unit of random- or single-donor platelets transfused on two consecutive occasions. The diagnosis of bacteremia and fungemia required one or more positive blood cultures in patients with suspected infection. The diagnosis of clinical infection required evidence of a localized tissue infection with supporting features such as fever, chills, pain, or erythema with or without isolation of a pathogen. Fever without localized evidence of infection or without positive blood cultures was not considered to represent clinical infection. Statistical Analysis Assuming an infection rate of 40%, the study was designed to detect an anticipated decrease to 20% with a power of 0.80 and an -error of 0.05. Results were analyzed according to the intention to treat. For comparisons of patient groups, the Pearson chi-square test or the Mann-Whitney rank-sum test were used. The Pearson chi-square test with confirmation by the confidence interval method of Simon was used to evaluate study end points [19]. Confidence intervals of 95% were used. Binary logistic regression was used to evaluate the influence of various clinical and laboratory parameters on the end points of bacteremia or fungemia. These parameters included age, diagnosis, study center, duration of neutropenia, baseline IgG value, use of prophylactic oral antibacterials, and use of IVIG. To evaluate hypogammaglobulinemia, 5 g/L (500 mg/dL) was chosen as the lower limit of normal. To evaluate the duration of neutropenia, a threshold of 7 days was chosen. The trial ended with the resolution of neutropenia because this patient population rarely experiences serious infections after leukocyte recovery and because survival after hematopoietic recovery is largely determined by the underlying disease. Survival was reported using actual proportions. Results Infections Proven clinical infections were frequent, as shown in Table 2. Of all study patients, 43.5% had documented clinical infections. Bacteremia and fungemia occurred in 35% and 7.6% of patients, respectively. The incidences of proven clinical infection, bacteremia, and fungemia were 43%, 35%, and 6% in the IVIG group and 44%, 34%, and 9% in the control group, respectively. These differences were not statistically significant (P > 0.2). Analysis of bacteremia by organism (gram positive, gram negative, and mixed) showed no statistical difference. The most common infection in the study was bacteremia due to coagulase-negative Staphylococci. This organism was isolated in 58% of all cases of bacteremia and was the sole organism in 38% of all cases of bacteremia. Twenty-eight percent of the documented bloodstream infections were polymicrobial. Table 2. Treatment Results Only 8% of patients in this study had hypogammaglobulinemia. The distribution of these patients was similar in the IVIG (9%) and control (8%) groups. In multiple regression analysis, the pretreatment value of IgG (< 5 g/L [500 mg/dL] compared with > 5 g/L) did not predict the development of bacteremia or fungemia. Bloodstream infections were frequent, but most were controlled by broad-spectrum antibiotics. Death from infection occurred in 3.5% of study patients (4.9% in the IVIG group compared with 2.3% in the control group), yielding a difference of 2.6% (95% CI, 3.0% to 8.2%; P > 0.2). Platelet Transfusion Patients in the IVIG and

Allogeneic marrow transplantation for refractory Hodgkin's disease.

Eight patients with refractory Hodgkins disease received intensive combination chemotherapy conditioning with cyclophosphamide, carmustine (BCNU), and etoposide (VP 16-213), and allogeneic marrow transplants. All patients achieved complete responses. Three patients relapsed; two died of Hodgkins disease and one of chronic graft-v-host disease (GVHD) and infection. In all, four patients died due to transplant-related toxicity. One patient developed a fatal B-cell lymphoproliferative disorder soon after transplantation, and died without evidence of Hodgkins disease. One patient is alive and free of progression 29 months after transplantation. These data indicate that allogeneic marrow transplantation may be considered as therapy for selected patients with advanced Hodgkins disease and, despite substantial toxicity, will occasionally result in long-term responses. Better patient selection would likely improve results.