Michael J. Vasconcelles

Phase II Study of Pemetrexed With and Without Folic Acid and Vitamin B12 as Front-Line Therapy in Malignant Pleural Mesothelioma

PURPOSE This phase II clinical study evaluated the efficacy of pemetrexed for the treatment of malignant pleural mesothelioma (MPM). PATIENTS AND METHODS Patients with a histologically proven diagnosis of MPM, chemotherapy-naive measurable lesions, and adequate organ function received pemetrexed (500 mg/m2) intravenously over 10 minutes every 3 weeks. After a protocol change, most patients also received folic acid and vitamin B12 supplementation to improve safety. RESULTS A total of 64 patients were enrolled. Nine (14.1%) of the 64 patients had a partial response. The Kaplan-Meier estimate for median overall survival was 10.7 months. Forty-three patients received vitamin supplementation for all courses of therapy, and 21 patients did not. Seven of the nine responders were vitamin supplemented. The median overall survival was 13.0 months for supplemented patients and 8.0 months for nonsupplemented patients. Vitamin-supplemented patients completed more cycles of therapy than nonsupplemented patients (median, six v two cycles, respectively). Grade 3/4 neutropenia (23.4%) and grade 3/4 leukopenia (18.8%) were the most common laboratory toxicities. Fatigue and febrile neutropenia were the most commonly reported nonlaboratory events (grade 3, 6.3%; grade 4, 0.0% each). The incidence of these toxicities was generally lower in the supplemented patients. CONCLUSION Single-agent pemetrexed for MPM resulted in a moderate response rate (14.1%) and median overall survival of 10.7 months. Patients supplemented with folic acid and vitamin B12 tolerated treatment better (less toxicity and more cycles of treatment) and had a 5-month greater median overall survival than nonsupplemented patients. These results indicate that patients with MPM could benefit from single-agent pemetrexed treatment combined with vitamin supplementation.

Clofarabine Plus Cytarabine Compared With Cytarabine Alone in Older Patients With Relapsed or Refractory Acute Myelogenous Leukemia: Results From the CLASSIC I Trial

PURPOSE To compare the receipt of clofarabine plus cytarabine (Clo+Ara-C arm) with cytarabine (Ara-C arm) in patients ≥ 55 years old with refractory or relapsed acute myelogenous leukemia (AML). PATIENTS AND METHODS Patients were randomly assigned to receive either clofarabine (Clo) 40 mg/m(2) or a placebo followed by Ara-C 1 g/m(2) for five consecutive days. The primary end point was overall survival (OS). Secondary end points included event-free survival (EFS), 4-month EFS, overall remission rate (ORR; complete remission [CR] plus CR with incomplete peripheral blood count recovery), disease-free survival (DFS), duration of remission (DOR), and safety. RESULTS Among 320 patients with confirmed AML (median age, 67 years), the median OS was 6.6 months in the Clo+Ara-C arm and 6.3 months in the Ara-C arm (hazard ratio [HR], 1.00; 95% CI, 0.78 to 1.28; P = 1.00). The ORR was 46.9% in the Clo+Ara-C arm (35.2% CR) versus 22.9% in the Ara-C arm (17.8% CR; P < .01). EFS (HR: 0.63; 95% CI, 0.49 to 0.80; P < .01) and 4-month EFS (37.7% v 16.6%; P < .01) favored the Clo+Ara-C arm compared with Ara-C arm, respectively. DFS and DOR were similar in both arms. Overall 30-day mortality was 16% and 5% for CLO+Ara-C and Ara-C arms, respectively. In the Clo+Ara-C and Ara-C arms, the most common grade 3 to 4 toxicities were febrile neutropenia (47% v 35%, respectively), hypokalemia (18% v 11%, respectively), thrombocytopenia (16% v 17%, respectively), pneumonia (14% v 10%, respectively), anemia (13% v 0%, respectively), neutropenia (11% v 9%, respectively), increased AST (11% v 2%, respectively), and increased ALT (10% v 3%, respectively). CONCLUSION Although the primary end point of OS did not differ between arms, Clo+Ara-C significantly improved response rates and EFS. Study follow-up continues, and the role of clofarabine in the treatment of adult patients with AML continues to be investigated.

Phase I/II study of vaccination with electrofused allogeneic dendritic cells/autologous tumor-derived cells in patients with stage IV renal cell carcinoma.

In the present study, we assessed the feasibility, toxicity, immunologic response, and clinical efficacy of vaccination with allogeneic dendritic cell (DC)/tumor fusions in patients with metastatic renal cell carcinoma (RCC). Patients with stage IV RCC with accessible tumor lesions or independent therapeutic indications for nephrectomy were eligible for enrollment. Tumors were processed into single cell suspensions and cryopreserved. DCs were generated from adherent peripheral blood mononuclear cells isolated from normal volunteers and cultured with granulocyte macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-α. DCs were fused to patient derived RCC with serial electrical pulses. Patients received up to 3 vaccinations at a fixed dose of 4×107 to 1×108 cells administered at 6-week intervals. Twenty-four patients underwent vaccination. Twenty-one and 20 patients were evaluable for immunologic and clinical response, respectively. DCs demonstrated a characteristic phenotype with prominent expression of HLA class II and costimulatory molecules. A mean fusion efficiency of 20% was observed, determined by the percent of cells coexpressing DC and tumor antigens. No evidence of significant treatment related toxicity or auto-immunity was observed. Vaccination resulted in antitumor immune responses in 10/21 evaluable patients as manifested by an increase in CD4 and/or CD8+ T-cell expression of interferon-γ after ex vivo exposure to tumor lysate. Two patients demonstrated a partial clinical response by Response Evaluation Criteria in Solid Tumors criteria and 8 patients had stabilization of their disease. Vaccination of patients with RCC with allogeneic DC/tumor fusions was feasible, well tolerated, and resulted in immunologic and clinical responses in a subset of patients.

Identification and Characterization of a Low Oxygen Response Element Involved in the Hypoxic Induction of a Family ofSaccharomyces cerevisiaeGenes: IMPLICATIONS FOR THE CONSERVATION OF OXYGEN SENSING IN EUKARYOTES

An organisms ability to respond to changes in oxygen tension depends in large part on alterations in gene expression. The oxygen sensing and signaling mechanisms in eukaryotic cells are not fully understood. To further define these processes, we have studied the Δ9 fatty acid desaturase gene OLE1 inSaccharomyces cerevisiae. We have confirmed previous data showing that the expression of OLE1 mRNA is increased in hypoxia and in the presence of certain transition metals.OLE1 expression was also increased in the presence of the iron chelator 1,10-phenanthroline. A 142-base pair (bp) region 3′ to the previously identified fatty acid response element was identified as critical for the induction of OLE1 in response to these stimuli using OLE1 promoter-lacZ reporter constructs. Electromobility shift assays confirmed the presence of an inducible band shift in response to hypoxia and cobalt. Mutational analysis defined the nonameric sequence ACTCAACAA as necessary for transactivation. A 20-base pair oligonucleotide containing this nonamer confers up-regulation by hypoxia and inhibition by unsaturated fatty acids when placed upstream of a heterologous promoter in alacZ reporter construct. Additional yeast genes were identified which respond to hypoxia and cobalt in a manner similar toOLE1. A number of mammalian genes are also up-regulated by hypoxia, cobalt, nickel, and iron chelators. Hence, the identification of a family of yeast genes regulated in a similar manner has implications for understanding oxygen sensing and signaling in eukaryotes.

Aerosolized pentamidine as pneumocystis prophylaxis after bone marrow transplantation is inferior to other regimens and is associated with decreased survival and an increased risk of other infections

Pneumocystis carinii pneumonia (PCP) is a life-threatening but preventable infection that may occur after bone marrow transplantation (BMT). Although various prophylactic regimens have been used in this setting to prevent active infection, their efficacy, toxicity profile, and impact on outcomes are poorly described in this patient group. We undertook a retrospective cohort study in which we reviewed the records of 451 adult patients who underwent BMT for hematologic malignancies, aplastic anemia, or myelodysplasia over a 7-year period at the Brigham and Womens Hospital. Post-BMT PCP prophylaxis consisted of aerosolized pentamidine (AP) 150 mg every 2 weeks or 300 mg per month, trimethoprim/sulfamethoxazole (TMP/SMX) 160/800 mg orally b.i.d. 3 times per week, or dapsone 100 mg orally each day. Prophylaxis was continued for 1 year post-BMT in all patients when clinically feasible. One hundred twenty-one patients were unevaluable because of death or relapse <60 days after BMT (n = 89), loss to follow-up upon hospital discharge (n = 20), or other reasons (n = 12). Three eligible patients did not receive any prophylaxis and were not further evaluated. Of the 327 patients analyzed, 133 underwent autologous BMT, 4 syngeneic BMT, 159 related allogeneic BMT, and 31 unrelated allogeneic BMT. Graft-versus-host disease prophylaxis in the 190 patients receiving allogeneic BMT consisted of T-cell depletion with anti-CD5 and complement in 58 patients and cyclosporine/methotrexate or FK506 with or without steroids in 132 patients. Eight of 327 (2.4%) documented PCP cases were identified, 0 of 105 in patients receiving only TMP/SMX. Four cases occurred in patients receiving only AP (4/44, 9.1%; odds ratio [OR] relative to TMP/SMX 23.4, 95% confidence interval [CI] 1.2, 445.2); 1 in patients receiving only dapsone (1/31, 3.2%; OR not significant); 2 in patients receiving more than 1 prophylactic regimen (2/147 1.4%; OR not significant); and 1 >1 year post-BMT in a patient who was off PCP prophylaxis. Although the patients receiving only AP had a significantly lower probability of treatment-related toxicity than those receiving TMP/SMX (OR 0.19 [95% CI 0.04, 0.851), the probability of their acquiring other serious non-PCP infections was increased (OR 2.2 [95% CI 1.0, 4.6]), and the probability of their dying by 1 year post-BMT was significantly higher (OR 5.2 [95% CI 2.4, 26.6]), even when adjusted for variables such as type of BMT (autologous versus allogeneic; high versus low risk) and sex. Although AP is associated with fewer toxicities, the data show that it is inferior to TMP/SMX in preventing PCP in the post-BMT setting and is associated with an increased risk of other infections and a higher mortality at 1 year after BMT.

MGA2 Is Involved in the Low-Oxygen Response Element-Dependent Hypoxic Induction of Genes in Saccharomyces cerevisiae

ABSTRACT Eukaryotes have the ability to respond to changes in oxygen tension by alterations in gene expression. For example,OLE1 expression in Saccharomyces cerevisiae is upregulated under hypoxic conditions. Previous studies have suggested that the pathway regulating OLE1expression by unsaturated fatty acids may involve Mga2p and Spt23p, two structurally and functionally related proteins. To define the possible roles of each of these genes on hypoxia-inducedOLE1 expression, we examined OLE1expression under normoxia, hypoxia, and cobalt treatment conditions in Δmga2 or Δspt23 deletion strains. The results of OLE1promoter-lacZ reporter gene and Northern blot analyses showed that hypoxia- and cobalt-induced OLE1 expression was dramatically decreased in a Δmga2 strain but not in a Δspt23 strain. Further analyses using low-oxygen response element (LORE)-CYC1-lacZ fusion reporter assays and electrophoretic mobility shift assays (EMSAs) demonstrated that MGA2 significantly affects the LORE-dependent hypoxic induction pathway of gene expression. When MGA2 was supplied by a plasmid, the LORE-dependent hypoxia-inducible reporter expression was recovered, as was the hypoxia-inducible complex in EMSAs in the S. cerevisiae Δmga2 strain. Supershift analysis of EMSAs using crude extracts containing mycMga2p indicated that Mga2p is a component of the LORE-binding complex. Another LORE-dependent, hypoxia-inducible gene, ATF1, was similarly affected in the Δmga2 strain. These results indicate thatMGA2 is required for the LORE-dependent hypoxic gene induction in S. cerevisiae.

Mga2p Processing by Hypoxia and Unsaturated Fatty Acids in Saccharomyces cerevisiae: Impact on LORE-Dependent Gene Expression

ABSTRACT In Saccharomyces cerevisiae, OLE1 encodes a Δ9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane. Previous studies have demonstrated that (i) OLE1 expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the OLE1 promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of OLE1. We now report that LORE-CYC1 basal promoter-lacZ fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE. Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE. Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of OLE1 hypoxic induction is mediated through Mga2p. Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs. Hypoxic induction of OLE1, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids. Data from this study suggest that the Mga2p-LORE interaction plays an important role in OLE1 expression under both normoxic and hypoxic conditions.

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function.

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.

Interim safety and efficacy results from a phase I/II study of vaccination with electrofused allogeneic dendritic cells/autologous tumor-derived cells in patients with stage IV renal cell carcinoma

2526 Background: Vaccination with dendritic cell (DC)/tumor cell fusions can induce specific anti-tumor immunity in preclinical studies and has shown clinical activity. We report results of a fully enrolled, ongoing phase I/II study of electrofusion vaccine (EFV), comprised of allogeneic DCs (alloDC) and autologous tumor-derived cells administered subcutaneously to patients with Stage IV renal cell carcinoma (RCC). METHODS Tumors were processed and cryopreserved following acquisition from the primary or a metastatic site. Volunteer donor peripheral blood mononuclear cells were used to generate alloDC. At 6 week intervals, patients received up to 3 freshly prepared, irradiated EFV, within a constant dose range of 4 x 107 to 1 x 108 total cells. Study endpoints were safety, and tumor and immunologic response assessments. RESULTS Of the 30 enrolled patients, data are available on 21; 19 of the 21 were eligible for vaccination. The median age is 58 years (12 M, 9 F); mean number of metastatic sites is 2.4. Fifteen patients received prior treatments, including high dose IL-2. The mean fusion efficiency for 50 vaccine preparations is 20.7%, as determined by cell coexpression of specific DC and tumor markers. The only serious adverse event (excessive intraoperative bleeding) considered related to the study occurred during a tumor acquisition procedure. Adverse events assessed by the investigators as related to EFV and occurring in more than 1 patient were injection site reactions (n=5), fatigue (n=3) and coughing (n=3), all Common Toxicity Criteria grade I or II. Tumor responses to date (9 patients) are 1 partial response, 3 stable disease. Complete analyses of safety, immunologic reactivity (autologous tumor-induced delayed-type hypersensitivity and intracellular interferon γ production) and tumor response assessments will be presented. CONCLUSIONS These interim data suggest a vaccination strategy employing alloDC electrofused with tumor-derived cells in patients with Stage IV RCC is feasible, well-tolerated and may show evidence of tumor response. [Table: see text].

Inhibition of human polymorphonuclear leukocyte respiratory burst activity and aggregation by 6-ketocholestanol

6-ketocholestanol, a naturally occurring oxygenated sterol, when incubated with human neutrophils (PMN), can inhibit superoxide and hydrogen peroxide generation in a dose-dependent fashion. This is accompanied by inhibition of stimulated PMN aggregation without alteration in cellular viability. This inhibitory effect is not affected by washing of the cells, and cannot be blocked by the addition of free cholesterol to the medium. These data are consistent with prior observations which showed an inhibitory effect on PMN chemotaxis by certain oxygenated sterol compounds, and support the hypothesis that certain oxygenated sterols can affect a variety of human PMN functions by a mechanism that may involve perturbation of the plasma membrane.