ALBERT

Description: Historically, graft-versus-host disease (GVHD) beyond 100 days after hematopoietic cell transplantation (HCT) was called chronic GVHD, even if the clinical manifestations were indistinguishable from acute GVHD. In 2005, the National Institutes of Health (NIH) sponsored a consensus conference that proposed new criteria for diagnosis and classification of chronic GVHD for clinical trials. According to the consensus criteria, clinical manifestations rather than time after transplantation should be used in clinical trials to distinguish chronic GVHD from late acute GVHD, which includes persistent, recurrent, or late-onset acute GVHD. We evaluated major outcomes according to the presence or absence of NIH criteria for chronic GVHD in a retrospective study of 740 patients diagnosed with historically defined chronic GVHD after allogeneic HCT between 1994 and 2000. The presence or absence of NIH criteria for chronic GVHD showed no statistically significant association with survival, risks of nonrelapse mortality or recurrent malignancy, or duration of systemic treatment. Antecedent late acute GVHD was associated with an increased risk of nonrelapse mortality and prolonged treatment among patients with NIH chronic GVHD. Our results support the consensus recommendation that, with appropriate stratification, clinical trials can include patients with late acute GVHD as well as those with NIH chronic GVHD.

Description: Abstract 348 We previously reported results of 3 sequential trials of GVHD prophylaxis with mycophenolate mofetil (MMF) BID/TID and cyclosporine (CSP) BID with various taper schedules in patients (pts) with advanced hematologic malignancies given unrelated G-CSF-mobilized peripheral blood stem cell (PBSC) grafts after fludarabine 90 mg/m2 and 2 Gray total body irradiation. Cumulative incidences of grades II-IV acute GVHD in the 3 trials were 52, 53 and 77%, respectively. The goal of the current protocol was to evaluate, in a phase II randomized 3-arm study, which drug combination or schedule was most promising in preventing acute GVHD. Tacrolimus (Tac) was used in place of CSP and each of the 3 arms used MMF TID until day 30 and then BID, but the subsequent duration of MMF varied. In Arm1, pts received Tac until day 180 and MMF until day 96. In Arm2, Tac was given until day 150 and MMF until day 180. In Arm3, Tac was given until day 150 and MMF until day 180 with the addition of rapamycin from days -3 through 80. One hundred seventy-five pts ineligible for myeloablative conditioning were enrolled on this multi-institutional study between Jan/05 and Aug/09, and results on the first 159 pts (Arm1 n=56; Arm2 n=51; Arm3 n=52) are reported here with a median follow-up of 18.4 months for surviving pts. The median age of pts was 60 (range 13-75) yrs. Sixty-six (42%) had previous autologous (n=55) or allogeneic (n=11) HCT. All pts were matched for HLA-DRB1 and -DQB1 at the allele level: 16 had single allele mismatches at HLA-A, -B or –C and the remainder (n=143) were fully HLA-matched. Diagnoses included AML (n=72), NHL (n=36), MM (n=19), ALL (n=10), CLL (n=9), MDS (n=8), HL (n=4), and CML (n=1). Randomization was based upon transplant center (FHCRC vs other), number of prior chemotherapy treatments (0-2 vs 3+), and age (

Description: We previously identified the mitochondrial solute carrier, Mitoferrin1 (Slc25a37, Mfrn1) as the principal mitochondrial iron importer essential for heme and iron-sulfur (Fe-S) cluster synthesis in developing erythroblasts. Its closely related paralog, Mitoferrin2 (Slc25a28, Mfrn2) functions in an analogous role in non-erythroid cells. Zebrafish with mutations in Mfrn1 have defects in hemoglobinization and maturation of erythroid cells caused by defective acquisition of iron into the mitochondria (GC Shaw, et al., 2006 Nature 440:96–100). Mfrn1 is highly expressed in embryonic and definitive sites of hematopoiesis in zebrafish and mouse, such as the developing blood island (ICM), fetal liver and adult bone marrow. In contrast, Mfrn2 is ubiquitously expressed, including at very low levels in erythroid cells. To understand the transcriptional regulation of Mfrn1 and Mfrn2, we used bioinformatics tools to identify potential cis-regulatory motifs (CRM) within each gene from mouse. The Gateway-modified Tol-2 vector was used to rapidly clone these conserved, minimal CRM fragments from mouse upstream of the basal promoter and an EGFP-reporter. Each construct was then introduced into zebrafish embryos for transient and stable expression. Using this strategy, we identified CRM’s that recapitulate the endogenous mRNA expression pattern of Mfrn genes during zebrafish development. Germ line stable transmission of the murine Tg(Mfrn1:EGFP) reporter in zebrafish showed robust EGFP expression in erythroid progenitors and mature erythrocytes and the remarkable conservation of function for CRM’s across species. In contrast, the mouse Tg(Mfrn2:EGFP) was expressed in skeletal muscle, heart, liver, and pronephros. The Mfrn1 enhancer is located ~35 kb upstream of the transcription start site and contains two GATA consensus motifs, which bind GATA-1 by chromatin immunoprecipitation analysis. Moreover, the ~150 bp Mfrn1 enhancer fragment exhibits transcriptional activation when coupled to the minimal γ-globin promoter driving expression of a luciferase reporter in K562 cells. Site-directed mutagenesis revealed that both GATA motifs are required for robust erythroid expression. In a complementary approach, transient knockdown of GATA-1 in zebrafish embryos using anti-sense morpholinos selectively ablated Mfrn1 mRNA expression in the ICM, consistent with the epistatic relationship of GATA-1 and Mfrn1. The zebrafish transgenic lines harboring the two murine Mfrn enhancers have proven useful in studying the regulatory and developmental expression in the Mfrn genes in erythroid and non-hematopoietic organs, such as heart and liver. Our results show that the combined use of bioinformatics, Gateway-mediated cloning, and Tol-2 mediated transgenesis in zebrafish embryos is an effective approach to functionally interrogate the transcriptional activity of putative CRM’s in vivo. The conservation and faithful expression of mouse CRM’s in zebrafish demonstrate the utility of this functional approach for analyzing mammalian CRM’s.

Description: Background. We previously reported data from 103 pts receiving PBSC from HLA-matched unrelated donors after conditioning with 2 Gy total body irradiation (TBI) plus 90 mg/m2 fludarabine. Postgrafting immunosuppression included MMF (administered from day 0 until day +40 with taper through day +96), and CSP (given from day -3 to day +100, with taper through day 180) (historical group). The incidences of grade III–IV acute and chronic extensive GVHD were 14% and 48%, respectively. Several studies have suggested that CSP could prevent activation-induced cell death of T-cells, and thus potentially delay the eradication of donor-versus-host alloreactive T-cells, preventing tolerance induction. Conversely, antimetabolite inhibitors, such as MMF, could delete alloreactive T-cells by induction of activation-induced cell death and apoptosis, and thus might favor tolerance induction Pts and Methods. Here, we investigated whether postgrafting immunosuppression with prolonged MMF (given at 15 mg/kg orally thrice a day from day 0 to day +30, then at 15 mg/kg orally twice a day until day 150, and then tapered at day 150 and discontinued at day 180) and truncated CSP (abruptly discontinued on day 80), would promote tolerance induction and reduce the incidence of GVHD (current protocol, n=71) after unrelated HCT with nonmyeloablative conditioning. Results. Sustained donor engraftment was achieved in 68 pts (96%) in the current protocol, versus in 98 pts (95%) in the historical group. Grades II, III and IV acute GVHD were seen in 36 (50.7%), 11 (15.5%) and 7 (9.9%) pts, respectively, in the current protocol, versus 39 (37.9%), 11 (10.7%) and 4 (3.9%) pts, respectively, in the historical group. The incidences of grade II–IV and grade III–IV acute GVHD were 75% and 23% in the current protocol, versus 52% (P=.05) and 14% (P=.14) in the historical group. The 1-yr incidence of chronic GVHD was 46% in the current protocol, versus 48% in the historical group. Finally, the 1-yr probabilities of relapse, nonrelapse mortality and progression-free survival were 24%, 36% and 40%, respectively, in the current protocol, versus 26% (P=.9), 18% (P=.005) and 56% (P=.04) in the historical group. The increased incidence of nonrelapse mortality in the current protocol was not due only to the increased incidence of grade II–IV acute GVHD, since nonrelapse mortality remained significantly higher in the current protocol than in the historical group after adjusting for occurrence of grade II and grade III–IV acute GVHD (P=.04). Evaluation of pretransplant comorbidities is ongoing. Conclusions. Postgrafting immunosuppression with prolonged MMF and truncated CSP failed to decrease the incidence of GVHD after nonmyeloablative conditioning with URD. Ongoing efforts are directed at reducing the risk of acute GVHD after nonmyeloablative conditioning for unrelated donors by replacing tacrolimus for CSP, and by adding sirolimus to MMF and CSP.

Description: αIIbβ3-mediated outside-in signaling following platelet adhesion to fibrinogen affects platelet adhesion, degranulation, and spreading, but the precise mechanisms involved remain incompletely understood. In this study, we demonstrate that the character of αIIbβ3 signaling is dependent on the surface density of the adsorbed ligand. Time-lapse total internal reflection fluorescence microscopy (TIR-FM) allowed us to visualize the interactions between fibrinogen and the αIIbβ3 integrins on the basal membrane of adhering platelets using a fluorescently-labeled antibody specific for β3. We observed significant morphological and biochemical differences in the initial interaction between αIIbβ3 and fibrinogen depending on the fibrinogen surface density. These included differences in the kinetics of filopodia and lamellipodia formation. Although filopodia started to form at the same time after initial platelet contact with both low-density (adsorbed from 3 μg/ml) fibrinogen (~ 40–50 s), new filopodia formed for a significantly shorter period of time on low- than on high-density fibrinogen [120 s (n = 79) vs. 235 s (n = 62); medians; p〈 0.001]. Lamellipodia started to form in platelets adhering to low-density fibrinogen significantly later than in platelets on high-density fibrinogen (140 s vs. 110 s; p = 0.04). Moreover, once lamellipodia started to appear, formation of new filopodia ceased in platelets adhering to low-density fibrinogen. In contrast, in platelets on high-density fibrinogen new filopodia continued to form even in the presence of lamellipodia for another 155 s. We also observed that a higher percentage of platelets adherent to high-density fibrinogen (52 ± 6 %) failed to develop a cytoplasmic Ca2+ signal compared to platelets adherent to low-density fibrinogen (13 ± 11 %; n = 3; p

Description: Malignant B-cells in Follicular Non-Hodgkin’s Lymphoma expresses a clonal idiotype immunoglobulin which can serve as the basis for a patient-specific anti-idiotype vaccine. In a previous single-arm Phase II study by Bendandi, et al (Nature Med5:1171–1177, 1999), we evaluated the ability of tumor-specific idiotype (Id) conjugated to keyhole limpet hemocyanin (KLH) administered concurrently with granulocyte-monocyte colony-stimulating factor (GM-CSF) adjuvant to induce complete remissions and molecular remissions in treated patients. The vaccine formulation induced a tumor-specific cytotoxic CD8+ and CD4+ T-cell response in patients in first complete remission after standard chemotherapy, as well as achieved molecular remissions in 8 of 11 of these patients. Data available at the time of this abstract for the 20-patient cohort, indicates a median follow-up of 9.167 years. 9 patients (45 %) remain in continuous first CR at their most recent follow-up (either in 2004 or 2005), and overall survival is 95%. The data further indicates the median disease free survival for the cohort is 96.5 months (8.04 years). To date there have been no additional reported mortalities in this cohort. As of August 2005, we report the progress of the Phase III clinical trial for this vaccine, opened in January 2000 by the NCI to evaluate the impact of this hybridoma-based Id vaccine on disease-free survival in a group of up to 375 previously untreated patients who have attained a CR or CRu from PACE [Prednisone, Doxorubicin, Cyclophosphamide, and Etoposide (ProMACE without methotrexate)] chemotherapy, and who are randomized to receive either vaccine or control. To date, 187 patients have been accrued onto the study. Of those patients, 145 (77.5%) achieved a CR or Cru and are being followed in this ongoing clinical trial.

Description: Background: 4-HPR is a cytotoxic retinoid with broad anti-cancer activity in preclinical studies, but oral capsule 4-HPR had limited bioavailability and activity in clinical trials. An intravenous intralipid emulsion formulation of 4-HPR (ILE 4-HPR) was developed to increase 4-HPR systemic exposure. Methods: ILE 4-HPR was administered as a continuous intravenous infusion for 5 of every 21 days and systemic toxicities, clinical response, and pharmacokinetics (PK) assessed. Simon design dose escalation proceeded with a 100% increase per dose level until moderate toxicity in 2 patients or 1 dose-limiting toxicity (DLT). Ten dose levels were planned starting at 80 mg/m2/day, increasing until 1810 mg/m2. Plasma 4-HPR levels were measured by high performance liquid chromatography. Results: To date, 17 patients have been enrolled. At dose level 10 (1810 mg/m2/day), 2 patients experienced a DLT of grade 4 hypertriglyceridemia, 1 patient with transient grade 2 pancreatitis. A de-escalation to dose level 9 (1280 mg/m2/day) enrolled 5 patients: 1 had asymptomatic Grade 4 hypertriglyceridemia, 1 patient experienced pleural effusions that resolved after pleurocentesis. A de-escalation to dose level 8 (905 mg/m2/day) enrolled 5 pts: two patients experienced asymptomatic Grade 4 hypertriglyceridemias that resolved after stopping the infusion; enrollment is ongoing. PK showed a linear relationship of dose to plasma level, with steady-state 4-HPR levels of 25 μM (640 mg/m2, level 7); 54 μM (1280 mg/m2, level 9) and 62 μM (1810 mg/m2, level 10). Responses to date include a transient response in a non-Hodgkins lymphoma at 320 mg/m2, in two angioimmunoblastic T-cell lymphomas, an 8-month partial response at 1810 mg/m2/day and a 4+ month unconfirmed complete response at 905 mg/m2/day, and in a histone deacetylase inhibitor-refractory cutaneous T cell lymphoma, a 10+ month molecular complete response at 1280 mg/m2. All patients were heavily pretreated. The DLT of hypertriglyceridemia is likely related to the intralipid formulation vehicle accounting for 5/6 DLTs observed, all reversible. Conclusions: ILE 4-HPR can be safely administered and obtained 4-HPR plasma levels 6 to 7 times higher than previously obtained by oral capsule 4-HPR. Durable clinical activity was observed in T cell lymphomas in the dose range 905–1810 mg/m2. Supported in part by NCI U01CA62505 and the NCI RAID program.

Description: Dendritic cells (DCs) play a key role in the pathogenesis of HIV infection. HIV interacts with these cells through 2 pathways in 2 temporal phases, initially via endocytosis and then via de novo replication. Here the transcriptional response of human DCs to HIV-1 was studied in these phases and at different stages of the virus replication cycle using purified HIV-1 envelope proteins, and inactivated and viable HIV-1. No differential gene expression was detected in response to envelope. However, more than 100 genes were differentially expressed in response to entry of viable and inactivated HIV-1 in the first phase. A completely different set of genes was differentially expressed in the second phase, predominantly in response to viable HIV-1, including up-regulation of immune regulation genes, whereas genes encoding lysosomal enzymes were down-regulated. Cathepsins B, C, S, and Z RNA and protein decreased, whereas cathepsin L was increased, probably reflecting a concomitant decrease in cystatin C. The net effect was markedly diminished cathepsin activity likely to result in enhanced HIV-1 survival and transfer to contacting T lymphocytes but decreased HIV-1 antigen processing and presentation to these T cells.

Description: Autologous hematopoietic cell transplantation (HCT) followed by nonmyeloablative allogeneic HCT (auto/alloHCT) provides cytoreduction and graft-versus-myeloma effects. We report on long-term outcomes of 102 patients with multiple myeloma who received auto/alloHCT with a median follow-up of 6.3 years. Treatment consisted of high-dose melphalan and autograft followed by 2-Gy total body irradiation, with or without fludarabine, and alloHCT from human leukocyte antigen-identical siblings. Postgrafting immunosuppressive agent was cyclosporine or tacrolimus and mycophenolate mofetil. Forty-two percent of patients developed grade 2 to 4 acute graft-versus-host disease (GVHD) and 74% extensive chronic GVHD. Five-year nonrelapse mortality after allografting was 18%, 95% related to GVHD or infections. Among 95 patients with detectable disease, 59 achieved complete remissions. Median time to progression was 5 years. Median overall survival (OS) was not reached. Median progression-free survival (PFS) was 3 years. Five-year OS and PFS were 64% and 36%, respectively. Seventy-three patients receiving autoHCT within 10 months from treatment initiation had 5-year OS of 69% and PFS of 37%. In multivariate analysis, β-2-microglobulin of more than 3.5 μg/mL at diagnosis and auto/alloHCT more than 10 months after treatment initiation correlated with shorter OS (P = .03 and P = .02) and PFS (P = .04 and P = .03), whereas Karnofsky scores less than 90% at allotransplantation correlated with shorter PFS only (P = .005). Long-term disease control and GVHD remain key issues.

Description: We previously described a zebrafish mutant, frascati (frs), which exhibits profound hypochromic anemia and erythroid maturation arrest due to defects in mitochondrial iron uptake. Through positional cloning, we showed that the frs gene encodes a novel member of the vertebrate mitochondrial solute carrier family (SLC25), mitoferrin (mfrn, slc25a37). Mfrn, which is highly expressed in fetal and adult hematopoietic tissues of zebrafish and mouse, functions as the major mitochondrial iron importer essential for heme biosynthesis in vertebrate erythroblasts (Shaw GC, et al. 2006 Nature 440:96–100). To study the function of Mfrn in mammalian organisms, we identified an embryonic stem (ES) cell clone that harbors a gene trap b-geo cassette in intron 1 that inactivates the Mfrn locus. Homozygous disruption of the Mfrn locus results in embryonic lethality at E11.5 from profound anemia due to a failure of primitive erythropoiesis, confirming the requirement of Mfrn in mammalian development . Circumventing the embryonic lethality, we generated Mfrn−/− ES cells to study the role of Mfrn in definitive erythropoiesis by in vitro differentiation of embryoid bodies and mixed chimera assays. Mfrn−/− ES cells were defective in promoting the growth, differentiation, and hemoglobinization of both primitive and definitive erythroblasts by in vitro differentiation of embryoid bodies. In mixed chimera studies, Mfrn−/− ES cells failed to contribute to the erythroid compartment of adult mosaic mice, whereas measurable contribution of Mfrn−/− donor cells could be assayed in the non-erythroid, leukocyte compartment. Transcriptome microarray analysis, using the mouse Affymetrix GeneChip and the custom IronChip, revealed unexpected down-regulation of transcripts for heme-biosynthetic enzymes in Mfrn−/− erythroblasts. The block in protoprophyrin synthesis, as well as mitochondrial heme synthesis, could be partially rescued by the addition of aminolevulinic acid (ALA) to Mfrn−/− erythroblasts in vitro. Our data demonstrate that mitochondrial iron homeostasis, working through the Mfrn iron importer, coordinately regulates the synthetic pathways for porphyrin and heme in developing mammalian erythroblasts.