ALBERT

Description: Introduction: FL is generally responsive to conventional-dose chemotherapy but long term disease-free survival (DFS) is uncommon. High-dose chemo-radiotherapy followed by ASCT has the potential to induce remission in this disease but the long-term benefit of this modality remains to be determined. Methods: Between 1990 and 2003, we transplanted 52 pts originally diagnosed with low-grade FL (31 grade 1, 21 grade 2). Twenty-five (48%) had biopsy-proven large cell transformation (FL grade 3 or diffuse large cell lymphoma) before ASCT. The median number of prior therapies was 2 (range: 1 to 7). Prior to ASCT, 45 pts (87%) were responsive to salvage therapy with 20 pts (38%) in CR. Five pts (10%) had chemo-resistant disease at the time of ASCT. High-dose regimens included BCNU-cyclophosphamide-etoposide (31%), melphalan/TBI (27%), and cyclophosphamide/TBI (25%). Thirty-eight pts (73%) received peripheral stem cells (PSCT) and 14 pts (27%) received autologous bone marrow (BM) with 4-hydroxyperoxycyclophosphamide (4-hc) purging in 9 cases (17%). The median age was 49 yrs (range: 29–65). Results: There was 1 treatment-related death during the first 100 days. After ASCT, 36 pts (69%) achieved a CR, 2 (4%) had a PR, and 7 (13%) had stable disease. Among those in CR, 20 (56%) had a CR pre-ASCT, 14 (41%) had a lesser response, and 1 (3%) was chemo-resistant. Median follow-up (f/u) of survivors was 5.3 yrs (range: 1.7 months to 12.4 yrs). The median overall survival (OS) has not yet been reached. The median event-free survival (EFS) is 3.4 yrs (range: 1.7 months to 12.4 yrs). Among complete responders, more than 50% are disease free at last follow-up (range 1.7 months to 12.1 yrs). Variables favorably affecting EFS and OS are age 〈 60 yrs (p = 0.007, 0.015 respectively), achievement of a CR after ASCT (p = 0.002, 0.001), absence of transformation (p = 0.038, 0.017), BM vs. PSCT (p = 0.042, 0.086), and 4-hc BM purging (p = 0.044, 0.059). Number of prior regimens, response prior to ASCT, type of preparative regimen, and addition of TBI, were not significantly associated with EFS, DFS, or OS. In multivariable analysis, achievement of CR after ASCT and age 〈 60 yrs are the only significant predictors of EFS and OS. Adjusted for age, 53% of pts with a CR after ASCT are alive and event-free at last f/u (range: 2.4 months to 12.4 yrs) (Figure 1). In contrast, the median EFS among pts without a CR is 0.5 yrs (range: 1.7 months to 5.3 yrs). Conclusion: ASCT is a reasonable therapeutic approach to FL, resulting in long term EFS for some pts, even with relapsed, refractory and/or transformed disease. In our experience, significant predictors of EFS and OS after ASCT are complete response and age

Description: Kaposi sarcoma (KS) is the most common AIDS-associated malignancy and is characterized by angiogenesis and the presence of spindle cells. Kaposi sarcoma-associated herpesvirus (KSHV) is consistently associated with all clinical forms of KS, and in vitro infection of dermal microvascular endothelial cells (DMVECs) with KSHV recapitulates many of the features of KS, including transformation, spindle cell proliferation, and angiogenesis. To study the molecular mechanisms of KSHV pathogenesis, we compared the protein expression profiles of KSHV-infected and uninfected DMVECs. This comparison revealed that heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in heme catabolism, was up-regulated in infected endothelial cells. Recent evidence suggests that the products of heme catabolism have important roles in endothelial cell biology, including apoptosis and angiogenesis. Here we show that HO-1 mRNA and protein are up-regulated in KSHV-infected cultures. Comparison of oral and cutaneous AIDS-KS tissues with normal tissues revealed that HO-1 mRNA and protein were also up-regulated in vivo. Increased HO-1 enzymatic activity in vitro enhanced proliferation of KSHV-infected DMVECs in the presence of free heme. Treatment with the HO-1 inhibitor chromium mesoporphyrin IX abolished heme-induced proliferation. These data suggest that HO-1 is a potential therapeutic target for KS that warrants further study. (Blood. 2004;103: 3465-3473)

Description: All genetic types of severe combined immunodeficiency (SCID) can be cured by stem cell transplantation from related donors. The survival rate approaches 80%, and most deaths result from opportunistic infections acquired before transplantation. It was hypothesized that the survival rate and kinetics of immune reconstitution would be improved for infants receiving transplants in the neonatal period (first 28 days of life), prior to the development of infections. A 19.2-year retrospective/prospective analysis compared immune function in 21 SCID infants receiving transplants in the neonatal period with that in 70 SCID infants receiving transplants later. Lymphocyte phenotypes, proliferative responses to mitogens, immunoglobulin levels, and T-cell antigen receptor excision circles (TRECs) were measured before transplantation and sequentially after transplantation. Of 21 SCID infants with transplantations in the neonatal period, 20 (95%) survive. Neonates were lymphopenic at birth (1118 ± 128 lymphocytes per cubic millimeter). Infants receiving transplants early developed higher lymphocyte responses to phytohemagglutinin and higher numbers of CD3+ and CD45RA+ T cells in the first 3 years of life than those receiving transplants late (P 

Description: MLN518 is a small molecule inhibitor of FLT3, PDGFR and c-Kit that is currently being evaluated as a therapy for AML. Previous phase I evaluation of MLN518 showed that it inhibits the phosphorylation of both wild-type and ITD-mutated FLT3 in patients’ leukemic blasts with an IC90 in the range of 100–175 ng/mL. Anti-leukemic activity was also observed, with decreases in both peripheral and bone marrow blasts. Dose-limiting toxicity, consisting of reversible general muscular weakness and/or fatigue was associated with trough plasma MLN518 concentrations 〉 1000 ng/mL. We are now conducting a phase II study of MLN518 in patients with relapsed or refractory AML and in untreated patients with AML considered unfit for standard AML therapies. Eligibility requires demonstration of the FLT3 ITD mutation in the patient’s blasts. All patients are treated with MLN518 at an initial dose of 525 mg po bid, with provision for dose reduction if MLN518-associated weakness occurs. Twenty patients have been treated with MLN518 in this study, eighteen of whom are currently evaluable (2 patients have recently started therapy). Toxicities associated with MLN518 therapy have included weakness/fatigue, QTc prolongation (relationship to MLN518 uncertain), and nausea and vomiting. MLN518 plasma concentration-time data for the first fourteen patients demonstrates that all patients achieved steady-state trough plasma concentrations 〉 150 ng/mL. Both inter- and intra-subject variability (%CV) in trough steady-state concentrations were 〈 30%. Assessment of total and phosphorylated FLT3 in leukemic blasts isolated from peripheral blood was possible in 4 patients. Western blots from blasts obtained before and after MLN518 dosing demonstrated either partial or complete inhibition of FLT3 phosphorylation with MLN518 plasma concentrations 〉 130 ng/mL. Of the eighteen evaluable patients, response could not be assessed in three because intercurrent illness and/or MLN518-associated toxicity precluded adequate treatment with MLN518 (≥ 14 days). Seven patients experienced progressive AML without evidence of any anti-leukemic effect. Two patients had stable disease for ≥ 50 days and subsequently underwent bone marrow transplantation. Although no complete or partial remissions have been observed, 6 patients have demonstrated evidence of an anti-leukemic effect with decreases in both peripheral and bone marrow blasts of 1-3 months duration. In these 6 patients the mean decrease in the absolute peripheral blast count was 92%, with a range of 85–100%. The mean decrease in the bone marrow blast percentage was 62%, with a range of 44–94%. We conclude that MLN518 has anti-leukemic activity in FLT3 ITD-mutated AML and should be further evaluated as a component of remission-induction and/or maintenance therapy in this disease.

Description: Clinical staging of lymphomas has a critical role in determining appropriate therapy and in therapeutic response assessment. While anatomic imaging by CT or MRI, together with physical examination and bone marrow biopsy (conventional staging modalities or CSM), have historically provided the basis for staging and response assessment, positron emission tomography using 18-fluoro-2-deoxyglucose (FDG-PET) has increasingly been employed in the management of patients (pts) with lymphomas. Studies comparing FDG-PET with anatomic imaging have generally found that FDG-PET identifies more sites of disease; however, the impact of FDG-PET imaging on assignment of clinical stage and treatment decisions is unclear. We retrospectively examined the impact of FDG-PET imaging on assignment of disease stage and treatment decisions compared with CSM alone in 91 pts with large B-cell lymphoma (LBCL; n=30), follicular lymphoma (FL; n=29) and Hodgkin’s lymphoma (HL; n=32). 25/30 LBCL (83%), 26/32 HD (81%) and 17/29 FL pts (59%) were evaluated at initial diagnosis by FDG-PET imaging and CSM, while others were evaluated during subsequent relapse. FDG-PET and CSM defined the same stage in 69/91 pts (76%). Stage assignment was most concordant in LBCL with only 5/30 (17%) of cases having different stages, while stage assignments were discrepant in 7/32 (22%) of cases of HL and in 10/29 (34%) of cases of FL. Of the 22 pts with discrepant results, FDG-PET resulted in upstaging of disease in 11 pts and downstaging in 11 pts. Independent confirmatory tests resolving the discrepancy were obtained in 3 of 11 pts upstaged (27%) and 7 of 11 pts downstaged by FDG-PET (64%). Abnormal FDG uptake in lymph nodes accounted for all cases upstaged by FDG-PET in which no confirmatory test was available (8/8 pts). Among 10 cases resolved by biopsy, discrepant sites included bone marrow (n = 4), bone (n = 1), pelvic or abdominal structures (n = 3), and lymph nodes (n = 2). FDG-PET results were true positive in 2/10, true negative in 1/10, false positive in 1/10, and false negative in 6/10 cases (including 4 cases with BM involvement). Incorporation of FDG-PET led to treatment changes in a minority of patients (2%). We conclude that FDG-PET is an efficient single imaging modality in pts with LBCL, FL and HL, providing information complementary to CSM. However, changes in treatment based on FDG-PET results are rare. Discrepancies between FDG-PET and CSM should be resolved by biopsy when feasible, particularly in cases in which treatment would be altered.

Description: The childhood leukemia, juvenile myelomonocytic leukemia (JMML), is a lethal disease of young children characterized by spontaneous growth of peripheral blood hematopoietic progenitors and hypersensitivity of hematopoietic progenitors to the cytokine GM-CSF. Notably, hematopoietic progenitors from JMML patients do not typically demonstrate hypersensitivity to the cytokine IL-3. Mutations in the RAS and NF1 genes have long been recognized as pathogenic in this disease, and more recently, mutations in the PTPN11 gene, which encodes Shp-2, a non-receptor protein tyrosine phosphatase, have also been found commonly in leukemic cells from children with JMML. We hypothesized that the mutant Shp-2 molecules observed in JMML patients would induce hypersensitivity of hematopoietic progenitors to GM-CSF. To examine this hypothesis, we subcloned the WT Shp-2 cDNA and three mutant Shp-2 cDNAs found in leukemic cells from children with JMML (E76K, D61V, and D61Y), into the murine stem cell retroviral plasmid pMIEG3 in tandem with EGFP. Each vector was sequenced to verify the desired point mutation and to rule-out unwanted mutations. Murine bone marrow low density mononuclear cells were subjected to fibronectin-assisted retroviral transduction and sorted for EGFP positive cells using fluorescence activated cell sorting. EGFP positive cells were plated into progenitor assays with increasing concentrations of GM-CSF (0, 0.01 0.1, 1, and 10 ng/mL). As predicted, transduction with each of the Shp-2 mutations induced hypersensitivity to GM-CSF compared to vector alone or to WT Shp-2, as evidenced by significantly higher % maximal colony formation at each GM-CSF dose tested (Figure 1). We next conducted progenitor assays using EGFP positive cells with increasing concentrations of IL-3 (0. 0.01, 0.1, 1, and 10 U/mL). Although hematopoietic progenitors from JMML patients have not been described to be hypersensitive to IL-3, surprisingly, we have observed in preliminary studies that introduction of each of the Shp-2 mutations induced hypersensitivity to IL-3 in a similar fashion to that observed with GM-CSF (Figure 2). These data demonstrate that somatic PTPN11 mutations observed in children with JMML induce hematopoietic cell growth aberrancies in a manner overlapping with the classical description of JMML, yet also induce unique hematopoietic cell growth characteristics based on the observed hypersensitivity to IL-3. These findings suggest that signaling pathways in addition to the Ras-MAPK cascade may be dysregulated in PTPN11 mutation-bearing hematopoietic cells and provide a novel model for the investigation of new therapeutics in JMML. Current studies are ongoing to examine the effect of these mutations on in vivo hematopoiesis and leukemogenesis.

Description: We retrospectively evaluated 18fluoro-2-deoxyglucose positron emission tomography (FDG-PET) scans in 172 patients with lymphoma and correlated results with pathologic diagnosis using the World Health Organization (WHO) classification system. In total, FDG-PET detected disease in at least one site in 161 patients (94%) and failed to detect disease in 11 patients (6%). The most frequent lymphoma diagnoses were diffuse large B-cell lymphoma (LBCL; n = 51), Hodgkin lymphoma (HL; n = 47), follicular lymphoma (FL; n = 42), marginal zone lymphoma (MZL; n = 12), mantle cell lymphoma (MCL; n = 7), and peripheral T-cell lymphoma (PTCL; n = 5). FDG-PET detected disease in 100% of patients with LBCL and MCL and in 98% of patients with HL and FL. In contrast, FDG-PET detected disease in only 67% of MZL and 40% of PTCL. Comparison with bone marrow biopsies showed that FDG-PET was not reliable for detection of bone marrow involvement in any lymphoma subtype.

Description: Acquired and congenital defects in iron metabolism from either deficiency or excess are one of the most common human diseases. Here we present the characterization of the zebrafish frascati mutation, which results in a profound hypochromic anemia and a developmental arrest at the pro-erythroblast stage. Using a positional cloning strategy, we have identified the gene disrupted frascati in mutants as a novel member of the mitochondrial solute transporter family. Members of this family of solute carriers have related tripartite sequence and structure. They function in transporting various metabolites and substrates across the inner mitochondrial membrane. We have verified the identity of the gene in zebrafish by the following criteria: (a) tissue-restricted expression in erythroid progenitors, (b) identification of missense mutations from the frascati five alleles, (c) rescue of anemia by over-expression frascati of cRNA in mutant embryos, and (d) mimicry of anemia using inactivating antisense morpholinos in wildtype embryos. We have also identified the functional ortholog in the mouse which has an analogous tissue and developmental expression pattern. The frascati ortholog in the mouse is highly expressed in fetal liver and adult bone marrow and spleen. The murine frascati transcript and protein are induced during terminal erythroid differentiation in MEL cells treated with either DMSO or HMBA. The over-expression of the mouse frascati cRNA in zebrafish frascati mutant embryos rescued their anemia with equal efficacy as the zebrafish clone. Given the identity of the gene and the requirement for iron in heme biosynthesis in the mitochondria of the developing erythron, we injected exogenous iron-dextran into frascati embryos. The embryos injected with iron-dextran were allowed to develop to 3 days post-fertilization, then stained for hemoglobinized cells with o-dianisidine and genotyped. Using this assay, the anemia caused by frascati the mutation could be partially rescued with exogenous iron supplementation. We therefore propose that the frascati gene functions as the essential transporter for iron importation into the mitochondria for heme biosynthesis and subsequent hemoglobin production in developing erythroid progenitors. Insight into the function of frascati the gene will be directly relevant to our understanding of human disorders of iron deficiency anemia and iron-overload sideroblastic anemia.

Description: Elucidating the mechanisms that regulate T cell activation and tolerance in vivo will provide insights into the maintenance of physiologic homeostasis and will facilitate development novel strategies for induction of transplantation tolerance. Transient activation of the small GTPase Rap1 is one of the physiologic consequences of TCR ligation and is mandatory for β1 and β2 integrin-mediated adhesion. In contrast, sustained increase of active Rap1 inhibits T cell activation and IL-2 transcription in vitro. In order to understand the role of Rap1 in the immune responses of the intact host we generated transgenic (Tg) mice, which express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg mice had no defects in thymocyte development or maturation. Rap1E63-Tg thymocytes were capable of activating Ras and Erk1/2 and, compared to wild type (WT) thymocytes, displayed enhanced LFA-1:ICAM-1-mediated adhesion and increased proliferation in response to anti-CD3. Surprisingly, although lymph node and splenic CD4+ cells from the Rap1E63-Tg mice also displayed increased LFA-1:ICAM-1-mediated adhesion, they had significantly impaired activation of Erk1/2 and dramatically reduced proliferation and IL-2 production in response to anti-CD3 and WT antigen presenting cells (APC). The defective responses of CD4+ T cells suggest that Rap1E63-Tg mice may have impaired helper function in vivo. To address this issue we immunized Rap1E63-Tg and WT mice with TNP-OVA, a T-cell dependent antigen. Total IgG, IgG1 and IgG2a were dramatically reduced, indicating that Rap1E63-Tg mice had a defect in immunoglobulin class switching, consistent with defective helper T cell-dependent B cell activation. Because these results suggest that Rap1E63-Tg CD4+ cells may have an anergic phenotype, we tested rechallenge responses. We immunized Rap1E63-Tg and WT mice with TNP-OVA in vivo and subsequently we rechallenged T cells in vitro with WT APC pulsed with OVA. Compared with WT, Rap1E63-Tg T cells had dramatically reduced proliferation, IFN- γ and IL-2 production on rechallenge, findings consistent with T cell anergy. Using suppression subtraction hybridization we determined that Rap1E63 induced mRNA expression of CD103, a marker that defines a potent subset of regulatory T cells (Treg). Strikingly, Rap1E63-Tg mice had a 5-fold increase of CD103+CD25+CD4+ Treg compared to WT mice. Rap1E63-Tg CD103+CD25+CD4+ Treg expressed the highest level of Foxp3 among all T cell subsets and had the most potent inhibitory effect on proliferation and IL-2 production when added into cultures of WT CD4+CD25− cells. Importantly, removal of the CD103+ cells significantly restored Erk1/2 activation, proliferation and IL-2 production of Rap1E63-Tg CD4+ T cells. Generation of CD103+ Treg occurs after thymic development and requires encounter of peripheral autoantigen. Consistent with this, differences in CD103+ Treg were detected only between lymph node and splenic cells and not between thymocytes from Rap1E63-Tg and WT mice. Since generation of CD103+ Treg depends on the strength of TCR signal, these results suggest that by enhancing adhesion, active Rap1 regulates the generation of Treg. Moreover, these results provide evidence that active Rap1 is a potent negative regulator of immune responses in vivo and have significant implications for the development of immune-based therapies geared towards tolerance induction.

Description: High-dose melphalan followed by ASCT is a common component of the early treatment for patients with multiple myeloma. Daily subcutaneous injections of filgrastim (Neupogen) at 5 ug/kg/day until ANC 〉 500/ul are routinely administered at our center from day +4 following ASCT, in order to accelerate hematopoietic recovery and lessen neutropenic complications. Pegfilgrastim (Neulasta) as a single 6 mg fixed dose subcutaneous injection has been shown to have similar efficacy and ease of use when compared to filgrastim in the non-transplant setting, but little data is available in the transplant setting. We began using pegfilgrastim day +1 following ASCT for patients with multiple myeloma and performed a retrospective cohort study comparing those who received filgrastim (n=6) with those who received pegfilgrastim (n=11). Transplants occurred between July 2002 and January 2004 and included all patients transplanted for myeloma in that time period for whom sufficient data was available. All patients had at least 2 x 106 CD34+ cells/kg peripheral stem cells harvested after cytoxan and filgrastim mobilization. Main outcome measures were: days from stem cell infusion to WBC nadir, days to ANC〉500/ul, and days to ANC〉1000/ul. Subjects were excluded if CBCs were drawn less frequently than every four days. There were no significant differences between the filgrastim and pegfilgrastim groups with respect to the following demographic variables: age, gender, hemoglobin, creatinine, calcium, albumin and beta-2 microglobulin at diagnosis. The groups were also balanced with respect to SPEP, UPEP, presence of lytic lesions and number of prior lines of therapy. The median number of CD34+ cells infused was similar: 5.7 x 106 in the filgrastim group vs 4.8 x 106 in the pegfilgrastim group (p=0.28). After transplant, median number of days to WBC nadir in the filgrastim group (FG) was 7 (range 5–9) vs 6 (range 5–8) in the pegfilgrastim group (PG) (p=0.31). However, median number of days to ANC〉500/ul in the FG was 11.5 (range 11–17) vs 10 (range 9–12) for PG (p=0.02). Similarly, median number of days to ANC〉1000/ul was 12 (range 11–17) for FG vs 11 (range 10–13) for PG (p=0.03). Five of six patients in the FG had neutropenic fever after transplant, compared to five of eleven patients in the PG (p=0.30). Currently, no significant differences in infection or relapse rates between groups have been noted and there were no deaths in either group. In this retrospective cohort study, pegfilgrastim was safe and at least equivalent to filgrastim for accelerating hematopoiesis after ASCT for multiple myeloma. Furthermore, there was no significant difference in the incidence of neutropenic fever, infection and survival, suggesting a similar clinical utility.