Mature T-lineage leukemia with growth factor-induced multilineage differentiation.

We report an acute T-lymphoblastic leukemia with a predominantly mature CD3+ CD7+ WT31+ phenotype that was induced to differentiate into different cell lineages by various recombinant human growth factors. In the presence of IL-3 or GM-CSF, the leukemic cells gave rise to myeloid and monocytic cells including terminally differentiated, partially functional, segmented neutrophilic granulocytes as assessed by morphologic, cytochemical, immunophenotypic, and functional criteria. In the presence of IL-2, leukemic granulated lymphoid cells exhibiting MHC-unrestricted cytotoxicity and expressing a CD2+ CD3+ CD5+ CD7+ CD8+ CD33+ WT31+ Leu19+ phenotype arose. Leukemic cell cultures initiated with IL-3 yielded growth factor-independent cells with a mixed lineage phenotype and morphologic and cytochemical evidence of immature blasts. These were T lymphocyte and myeloid surface antigen (CD2,CD3,CD5,CD7,CD13,CD33,WT31) positive. Identical rearrangements of the constant region of the TCR-delta gene and of the joining regions of the TCR-beta, -gamma, and -delta genes were observed in the fresh and all cultured leukemic cells, indicating that they were derived from the same malignant clone. Consistent with the molecular genetic data, the cytogenetic analyses of the GM-CSF-, IL-3-cultured and the growth factor-independent leukemic cells showed the presence of multiple, closely related abnormal clones, all of which had an interstitial deletion of part of the long arm of chromosome 6 and a complex 1;10;12 translocation. In conclusion, these data demonstrate the involvement of a multipotent leukemic precursor cell in this predominantly mature CD2+ CD3+ CD5+ CD7+ WT31+ T-ALL. This multipotent leukemic precursor may be susceptible to various growth factors and respond with ordered differentiation and maturation.

Further characterization of the in vitro tumoricidal activity of staphylococcal protein A.

Treatment of plasma or serum from leukemic patients with solid phase staphylococcal Protein A induced leukemic blast cell lysis in vitro, but this effect was relatively independent of the amount of immunoglobulin G (IgG) removed. Samples with approximately equal cytotoxic activity contained markedly different IgG levels, while samples with similar IgG levels had a wide range of tumoricidal activity. Assays of plasma samples collected during a perfusion of one plasma volume through a Protein A-Sepharose column indicated that the duration of the procedure had a greater effect on cytotoxic activity than did the amount of IgG removed. Neither added leukemic nor normal IgG significantly improved blast cell viability in treated serum. Cytotoxic activity was not dialyzable and concentrated in the Mr less than 100,000 fraction of samples separated by filtration. Treated cytotoxic serum samples did not have important Clq binding activity. These results suggest that the in vitro tumoricidal activity of solid phase Protein A is probably due to a toxic substance added to serum during immunoadsorption rather than to its immunoadsorptive capacity.

Effects of plasma treatment with purified protein A and Staphylococcus aureus Cowan I on spontaneous animal neoplasms.

Eleven dogs with spontaneous neoplasms were intensively treated with an immunoadsorption system consisting of a continuous flow centrifuge, Protein A-Sepharose columns, and a semi-automatic elution system. Despite consistent and substantial lowering of immunoglobulin G levels, tumor regression was noted in only one of 11 dogs. In contrast, infusion of small volumes of plasma after incubation with heat and formalin-treated Staphylococcus aureus Cowan I resulted in a tumoricidal response in five of six animals. These results suggest that tumor necrosis is probably not induced by Protein A-mediated removal of humoral "blocking" factors.

Immunoglobulin and T-cell receptor gene rearrangement in blast crisis of chronic myelocytic leukemia.

Chronic myelocytic leukemia (CML) displays a wide repertoire in its terminal phase, with blast cells showing characteristics of myeloid, B-lymphoid, or T-lymphoid cells in some patients. Blast crisis (BC) cells from 14 patients were studied for immunoglobulin (Ig)- and T-cell-associated gene rearrangements. Five myeloid BC patients had no Ig- or T-cell-associated gene rearrangement. In contrast, all eight patients with pure lymphoid BC displayed C mu rearrangement and two also showed kappa-light chain rearrangement. One patient with mixed (lymphoid and erythroid) BC, however, showed neither Ig- nor T-cell-associated rearrangements. One patient displayed both Ig- (C mu) and T-cell-associated (T beta and T gamma) rearrangements. These cells expressed CD9, CD10, and CD24 surface antigens, but no T-cell antigens. Although most lymphoid blast crises appear to represent an early stage in B-cell differentiation, some cells have undergone apparently inappropriate gene rearrangements during differentiation. Such cells may have been immortalized while undergoing normally occurring nonproductive rearrangement or may, due to their malignant nature, display abnormal genotypic characteristics.

Structure and expression of clustered P element homologues in Drosophila subobscura and Drosophila guanche.

Sequence relationships and functional aspects were analysed in the P element homologues of Drosophila subobscura (Ds) and D. guanche (Dg). In both species, the P homologues are clustered at a single genomic position. They lack the characteristic terminal structures of actively transposing P elements, but they have the coding capacity for a 66-kDa 'repressor-like' protein. Two different types of cluster units (G-type and A-type) can be distinguished. The A-type unit, which is present in multiple copies, is transcribed in adult flies. In contrast, the G-type unit has a much lower copy number and is apparently not expressed. In Dg, the isolated G-type sequence carries a 420-bp insertion in the promoter region, which is probably responsible for inactivation. Sequence comparisons of different cluster units show that differentiation of the two types precedes the lineage split of these species. Substitution rates of the deduced proteins reveal two distinct subregions: high variability at the N terminus and strong sequence conservation in the rest of the protein. The variable region contains motifs characteristic of DNA-binding proteins. Adaptive diversification of the cluster units towards specific binding properties might be a plausible explanation for variability in the N-termini. Both unit types have lost the weak promoter region characteristic of P transposons. In the A-type unit, a new promoter has been formed which is apparently composed of parts of insertion sequences derived from two different mobile elements.(ABSTRACT TRUNCATED AT 250 WORDS)

Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae.

As a model system for the study of factors affecting gene expression, hepatitis B virus core antigen (HBcAg) has been expressed in the yeast Saccharomyces cerevisiae. The singularly high levels of expression achieved are approx. 40% of the soluble yeast protein. The HBcAg polypeptides are present as 28-nm particles which are morphologically indistinguishable from HBcAg particles in human plasma and are highly immunogenic in mice. The plasmid construction employed to achieve these very high levels of expression utilizes the constitutively active yeast promoter from the GAP491 gene which is fused in a way that all non-translated sequences flanking the HBcAg coding region are yeast-derived. Hybrid constructions containing 3'-nontranslated viral DNA (yeast 5') or 5'-nontranslated viral DNA (yeast 3') as well as a construction with both 5'- and 3'-nontranslated viral DNA also have been made. A comparison of these constructions for levels of HBcAg expression indicates that the strongest contributor to the high levels of protein is the presence of 5'-flanking sequences which are yeast-derived; secondarily, a significant improvement can be achieved if the 3'-flanking sequences also are yeast-derived. The high abundance of HBcAg in the highest producer is explicable in part on the basis of the very high stability in yeast cells of HBcAg polypeptides. Analysis of the HBcAg coding sequence reveals a very low index of codon bias for S. cerevisiae, largely discounting codon usage as a contributor to the high level of protein obtained.

Influence of dietary carbohydrate on skeletal muscle glucose uptake.

The effect of altering the percent of dietary carbohydrate on the rate of skeletal muscle glucose uptake was studied using the perfused rat hindlimb preparation. The rats received either a high carbohydrate (HC; 65%), mixed (M; 35%) or low carbohydrate (LC; 10%) isocaloric diet for 7 days. With 0.1 mU/ml of insulin in the perfusate, the muscle of rats on the HC diet had a 33% increase in the rate of glucose uptake and the muscle of rats fed the LC diet a 23% decrease in the rate of glucose uptake when compared to the muscle of rats fed the M diet (3.34 mumol/g/30 min). With 10.0 mU/ml of insulin in the perfusate, ie maximal insulin stimulation, the rate of glucose uptake showed a similar dietary effect as that obtained with 0.1 mU/ml insulin. Compared to the M diet (8.67 mumol/g/30 min), the rate of glucose uptake increased 26% in muscle of rats from the HC group and decreased by 20% in muscle of rats from the LC group. Diet had no effect on the rate of muscle glucose uptake in the absence of insulin. Under both maximal and submaximal insulin stimulation, glycogen accumulation was greatest in muscle from HC fed rats and least in muscle from LC fed rats. During perfusion muscle intracellular free glucose and glucose-6-phosphate accumulation for the three dietary groups was negligible. The groups did not differ significantly in their muscle hexokinase or beta-hydroxyl acyl CoA dehydrogenase activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of antibody to the PreS2 sequence of the hepatitis B virus envelope protein using an immuno-ligand assay with a silicon sensor detection system.

Sensitive immunoassays are essential for establishing the efficacy of recombinant vaccines to hepatitis B virus (HBV). These experimental vaccines include the PreS2 and S domains of the HBV envelope protein. To facilitate measurement of antibody against HBV PreS2, we employed the immuno-ligand assay with silicon sensor-based detection. Labeling of immune reagents with the haptens biotin and fluorescein allows adaptation to the immunofiltration light addressable potentiometric sensor (LAPS) system. A biotinylated monoclonal anti-PreS2 antibody and anti-PreS2 in clinical serum samples competitively bind in liquid phase to a fluorescein labeled PreS2 + S antigen. Streptavidin mediates the immobilization on biotinylated nitrocellulose membranes. Fluorescein mediates binding of an anti-fluorescein urease conjugate to the immune complex. Urease serves as the signal-generating component which subsequently is measured in the LAPS reader. In comparison to a competitive RIA, the immuno-ligand assay demonstrated a four-fold improved sensitivity using a smaller sample volume. The higher sensitivity resulted in earlier detection of seroconversion during a clinical vaccine study.

bcr/abl mRNA detection following bone marrow transplantation for chronic myelogenous leukemia.

We have utilized the polymerase chain reaction (PCR) to sensitively detect persistence of the chronic myelogenous leukemia (CML) malignant clone and to study bcr/abl mRNA splicing patterns following bone marrow transplantation. Thirteen of sixteen patients displayed persistent malignant cells during post-BMT clinical remission. In two patients bcr/abl mRNA was detected 4 and 9 months prior to clinical relapse. In eleven of fourteen patients in continued clinical remission malignant cells were detected post-BMT. Ten of these eleven patients were also cytogenetically normal. Seven patients have lost all evidence of bcr/abl transcript, but only at 1-2 years posttransplant, while four have shown persistence of the bcr/abl transcript from 28 days to 3 years post-BMT and one has converted from an initially negative result at 1 year post-BMT to detectable levels of chimeric mRNA at 2 years. Thus, 8/9 patients tested at or before 6 months, 7/12 at 1 year, and 3/10 at 2 years showed persistent detectable CML cells. Intriguingly, mRNA splicing patterns changed in 5 patients following BMT, with complete loss of mRNA containing bcr exon 3 (n = 2) or new appearance of mRNA not containing bcr exon 3 (n = 2). A single patient transiently lost evidence of bcr exon 3 expression while persistently expressing the bcr exon 2/abl exon 2 splice. Our data suggest that the majority of patients harbor small numbers of malignant cells following transplantation, and that such persistence may not inevitably predict clinical relapse. Complete elimination of the malignant CML clone post-BMT may rely on immunological mechanisms (e.g., graft-vs-leukemia).

Prednisone therapy for acute graft-versus-host disease: short- versus long-term treatment. A prospective randomized trial.

We report the results of a controlled study in which BMT patients with moderate/severe acute graft-versus-host disease (GVHD) who responded to primary treatment with corticosteroids were prospectively randomized to short versus long taper of their steroid doses. Thirty patients with moderate/severe acute GVHD who responded by 14 days were eligible for random assignment of their steroid tapering schedule. Patients in the short taper group received a total PRED dose of 2275 mg/m2 over 86 days, whereas those in the long taper group received 6300 mg/m2 over 147 days. Patients in the long taper group achieved resolution of acute GVHD after a median of 30 days of therapy (range 6-30), whereas those receiving the short taper resolved after a median of 42 days (12-74) (P = 0.01). After 8 weeks of therapy, only 2 of 13 evaluable long taper and 3 of 13 short taper patients still had active GVHD. The median PRED dose required to achieve complete resolution of acute GVHD was not different between the two groups: 1300 mg/m2 for the long taper patients and 1800 mg/m2 for the short taper patients. Importantly, the incidence of chronic GVHD and survival at 6 months was similar in the 2 groups. The incidence of steroid-related complications was similar, as well. This study suggests that the rapid administration of high-dose PRED to a cumulative dose of 2000 mg/m2 might lead to complete and prompt resolution of acute GVHD in the majority of patients and that rapid PRED taper might provide a mechanism for minimizing steroid-related morbidity. Further investigation and formal studies of the dose-response relationships and kinetics of steroid administration may lead to improvement in the management of acute GVHD.

Varicella vaccine (VARIVAX) in healthy children and adolescents: results from clinical trials, 1987 to 1989.

A total of 3303 healthy children and adolescents, aged 12 months to 17 years, were vaccinated with one of five production lots of a live attenuated varicella vaccine (VARIVAX) containing 1000 to 1625 plaque-forming units per dose. The vaccine was generally well tolerated. Ninety-six percent (2381/2475) of vaccinees responded to vaccination by producing antibody as measured by a glycoprotein-based enzyme-linked immunosorbent assay; 99% (569/576) of those tested maintained antibody at 1 year following vaccination. The incidence of varicella following household exposure in vaccinees was approximately 12%; household contact historically results in 87% infection. Nearly all of the vaccinees who had varicella after vaccination had a clinically modified disease.

Mediation of the neuroprotective action of R-phenylisopropyl-adenosine through a centrally located adenosine A1 receptor.

1. Systemic injections of kainic acid, 10 mg kg-1, into adult rats resulted in lesions in the hippocampus, as assessed by peripheral benzodiazepine ligand binding. Co-administration of clonazepam at 1 mg kg-1 or 0.2 mg kg-1 prevented major seizures associated with kainate injections, but did not alter significantly the production of hippocampal damage. 2. The co-administration of the adenosine A1 agonist R-phenylisopropyladenosine (R-PIA, 25 micrograms kg-1, i.p.) abolished the lesions induced by kainic acid. 3. The presence of the selective A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (250 or 50 micrograms kg-1, i.p.) abolished the R-PIA neuroprotective action. 4. The A1/A2 antagonist, 8-(p-sulphophenyl)theophylline (20 mg kg-1, i.p.) which cannot cross the blood brain barrier, did not alter significantly the neuroprotective action of R-PIA, indicating that the neuroprotective action of the purine may be predominantly central. 5. The time course of the neuroprotection was also examined. R-PIA was effective when administered 2 h before or after kainate administration. 6. The results emphasise the potential utility of systemically active adenosine A1 receptor ligands in reducing CNS gliosis induced by the activation of excitatory amino acid receptors.

Modified cases of chickenpox after varicella vaccination: correlation of protection with antibody response.

Four thousand forty-two healthy children and adolescents, ages 12 months to 17 years, were vaccinated with a single dose of live attenuated varicella vaccine (VARIVAX; Merck Sharp and Dohme Research Laboratories) containing approximately 1000 to 1625 plaque-forming units/dose during clinical trials conducted from 1987 to 1989. Clinical follow-up of vaccinees revealed that 2.1 and 2.4% of vaccinees developed modified cases of varicella in the first and second years, respectively, after vaccination. Most of those who developed varicella postvaccination had an attenuated illness, characterized by fewer lesions and a lower incidence of fever (greater than or equal to 100 degrees F, oral) than after natural infection. The likelihood of developing varicella postvaccination decreased (P less than 0.0001) as the 6-week postvaccination glycoprotein-based enzyme-linked immunosorbent assay titer increased. In addition the number of lesions in these cases tended to decrease (P = 0.07 for Year 1 and P = 0.02 for Year 2) as the 6-week glycoprotein-based enzyme-linked immunosorbent assay titer increased. Thus the 6-week postvaccination glycoprotein-based enzyme-linked immunosorbent assay titer can be used as a surrogate marker for protection from natural disease.

Autologous transplantation for CML using marrow treated ex vivo with recombinant human interferon gamma.

Six chronic and four advanced patients with Ph1 positive chronic myelogenous leukemia (CML) received autologous bone marrow transplantation (ABMT) using bone marrow treated ex vivo with recombinant human interferon gamma (rIFN gamma). In three cases 100% Ph1 negative bone marrow hematopoiesis was demonstrated 4 to 8 weeks after ABMT. In three additional cases, a maximum of 40-45% bone marrow metaphases were Ph1 negative 2 to 12 weeks following ABMT. In four cases, no or minimal evidence of Ph1 negative hematopoiesis could be demonstrated posttransplant. Seven patients survive in chronic phase at a median of 375 days (range 100-495 days). Five patients have not required therapy, while two patients receive low doses of rIFN alpha to control peripheral blood counts. Ex vivo treatment of CML bone marrow with rIFN gamma followed by ABMT in patients dependent upon or refractory to therapy prior to transplantation is associated with transient complete or partial Ph1 negative bone marrow hematopoiesis in the majority of patients post-transplant and with return of a chronic phase characterized by no need for therapy or by response to low doses of rIFN alpha. Modification of the ABMT procedure and efforts to maintain cytogenetic and hematologic remission following ABMT may lead to prolonged survival or cure.

Relapse of acute leukemia presenting as acute cholecystitis following bone marrow transplantation.

Though included in the differential diagnosis of jaundice and abdominal pain, acute acalculous cholecystitis is an uncommon hepatobiliary complication of bone marrow transplantation. Leukemic infiltration of the gallbladder presenting as acute cholecystitis is rare. We describe two cases of acute cholecystitis following marrow transplantation that represented an unexpected relapse with leukemic infiltration of the gallbladder wall.

Prevention of cytomegalovirus infection following bone marrow transplantation: a randomized trial of blood product screening.

From 1983 to 1987, cytomegalovirus seronegative allogeneic bone marrow recipients were randomized to receive screened cytomegalovirus (CMV) seronegative or unscreened blood products and 125 patients were available for analysis. CMV infection occurred in 18% of patients in the screened versus 38% in the unscreened blood product group. However, only two of 64 patients in the screened group and seven of 61 in the unscreened group developed culture or biopsy-proven CMV infections. Bone marrow donor CMV seropositivity was associated with an increased risk of developing CMV infection (21% with seronegative and 46% with seropositive donor), and CMV infection was not prevented by blood product screening if the bone marrow donor was sero = positive (62% for screened, 42% for unscreened group, p = 0.80). One year survival censored for relapse was 52% in the screened group versus 68% in the unscreened group (p = 0.08). Gram negative bacteremia complicated bone marrow transplantation (BMT) in 35% of patients receiving screened and 15% of those receiving unscreened blood products (p = 0.02). Relapse did not differ in the screened and unscreened groups. By multivariate analysis, high risk disease (p = 0.0002), CMV infection (p = 0.004), screened blood products group (p = 0.011), recipient age greater than 17 (p = 0.027), chronic graft-versus-host disease (p = 0.014) and gram negative bacteremia (p = 0.004) independently had a negative influence on survival. We conclude that blood product screening was effective in preventing CMV infections following BMT if both the recipient and bone marrow donor were CMV seronegative.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute graft-versus-host disease following unrelated donor marrow transplantation: failure of conventional therapy.

The response to therapy of acute graft-versus-host disease (GVHD) is uncertain in recipients of unrelated donor (URD) bone marrow transplant (BMT). We analysed the outcome of treatment in 42 patients with moderate/severe acute GVHD. Initial therapy consisted of prednisone 60 mg/m2 orally daily for 7 days (n = 42), followed by anti-thymocyte globulin (ATG) 15 mg/kg i.v. twice daily for 8-10 doses after prednisone failure (n = 22). A clinical Stage Score for acute GVHD was determined initially and after 7, 14, 21, and 28 days of prednisone or ATG. Treatment failure represented worsening score after 7 days, involvement of a new organ or failure to improve after 14-28 days. Prednisone treatment led to 10 of 41 (24%) patients improving, while secondary therapy with ATG led to four of 21 (19%) improving. Of 42 patients treated, only nine (21%) achieved a complete and continuing response of acute GVHD by day +100. Neither age, diagnosis, recipient/donor gender status, histocompatibility nor GVHD prophylaxis regimen was associated with more frequent responses. Response to GVHD therapy was significantly correlated with survival at 100 days and 1 year post-BMT. We conclude that prednisone and ATG used for treatment of acute GVHD following URD BMT are associated with a high failure rate and that more aggressive therapy is warranted in these patients.

Autologous stem cell transplantation for refractory and relapsed Hodgkin's disease: factors predictive of prolonged survival.

We analyzed patient, disease, and treatment related factors associated with long-term disease-free survival (DFS) in 62 patients with refractory or recurrent Hodgkin's disease treated with high-dose cyclophosphamide (6000 mg/m2), carmustine (BCNU; 300 mg/m2), and etoposide (900 mg/m2) (CBV) followed by autologous stem cell transplantation. There were no deaths resulting from toxicity of the preparative regimen, and all patients survived the peritransplant period. At 28 days post-transplant, the complete response (CR) rate was 76%. Patients who achieved a CR had a 50% estimated 3-year DFS (95% CI, 35-64%). Twenty-three (37%) patients remain in continuous clinical remission 1.3 to 7.7 years (median 3.8 years) after transplantation. In a univariate analysis, factors significantly associated with improved DFS included absence of B symptoms (fever, night sweats and unexplained weight loss) at transplant, response to pre-transplant salvage chemotherapy, less tumor bulk at time of transplant, and fewer prior treatment regimens. Stepwise multivariate analysis showed that the absence of B symptoms at time of transplant was independently and significantly associated with improved DFS after transplantation. CBV with autologous stem cell support can produce durable remissions with acceptable toxicity in a substantial proportion of patients who are asymptomatic at time of transplant. Earlier application of transplantation or development of additional effective antineoplastic modalities will be required to improve the results of transplantation for patients with advanced Hodgkin's disease.

Autologous bone marrow transplantation for non-transformed low-grade non-Hodgkin's lymphoma.

The purpose of this study was to evaluate autologous bone marrow transplantation (ABMT) for low-grade non-Hodgkin's lymphoma (NHL). Between 1983 and 1994, 34 patients with non-transformed low-grade NHL received high-dose chemoradiotherapy plus stem cell rescue with marrow or peripheral blood. At transplantation (5-182 months (median 32) after diagnosis), 2 patients were in complete remission (CR), 21 in partial remission (PR) or minimal disease; 11 had bulky/resistant disease. Four patients died from transplant-related causes and 10 from lymphoma progression. After 40 months median follow-up, 20 survive between 3+ and 88+ months after ABMT. One-year survival is 81% (95% confidence interval, 65-96%) and 5-year survival 37% (14-60%). The probability of relapse at 1 and 2 years is 53% (34-72%) and 75% (54-96%), respectively. Twenty-nine patients were in CR following ABMT and 11 survive in continuous CR 3 to 54 months later. Disease-free survival at 1 and 2 years is 35% (19-52%) and 18% (2-31%). Resistant disease was the only factor significantly associated with poor overall (relative risk = 3.0, P = 0.04) and disease-free survival (RR = 5.4, P = 0.003), while resistant disease and high LDH were associated with increased relapse. ABMT yields a high CR rate for patients with progressive low-grade NHL, but relapse is frequent. Even longer follow-up is required to determine its effectiveness in extending survival of patients with pre-transplant responsive disease.

Predictive factors for chronic graft-versus-host disease after histocompatible sibling donor bone marrow transplantation.

We analyzed factors associated with the development of chronic graft-versus-host disease (GVHD) in 469 consecutive patients receiving matched sibling allogeneic bone marrow transplantation (BMT). Overall, 41 +/- 6% (95% confidence interval) developed chronic GVHD between 2 and 50 months after BMT. Multivariate analysis showed that previous acute GVHD was the dominant independent risk factor predisposing to chronic GVHD (Relative Risk 4.82, p < 0.0001). In addition, recipient age of > or = 18 years and male recipient with female donor were also independent predictive factors for development of chronic GVHD. When acute GVHD and recipient age were considered, groups with distinctive risks of chronic GVHD were identified. Patients under 18 without previous grade II-IV acute GVHD had only a 10 +/- 5% risk of chronic GVHD. Patients over 18 with no prior grade II-IV acute GVHD had a 31 +/- 12% risk while patients with advanced acute GVHD, regardless of age, had the highest risk and 70 +/- 8% developed chronic GVHD. It is important to consider these factors when designing and assessing clinical trials of chronic GVHD prophylaxis and treatment.