Naomi B. Haas

Effect of Granulocyte/Macrophage Colony-Stimulating Factor on Circulating CD8+ and CD4+ T-Cell Responses to a Multipeptide Melanoma Vaccine: Outcome of a Multicenter Randomized Trial

Purpose: Granulocyte/macrophage colony-stimulating factor (GM-CSF) administered locally together with vaccines can augment T-cell responses in animal models. Human experience has been limited to small and uncontrolled trials. Thus, a multicenter randomized phase II trial was done to determine whether local administration of GM-CSF augments immunogenicity of a multipeptide vaccine. It also assessed immunogenicity of administration in one versus two vaccine sites. Experimental Design: One hundred twenty-one eligible patients with resected stage IIB to IV melanoma were vaccinated with 12 MHC class Irestricted melanoma peptides to stimulate CD8+ T cells plus a HLA-DRrestricted tetanus helper peptide to stimulate CD4+ T cells, emulsified in incomplete Freunds adjuvant, with or without 110 g GM-CSF. Among 119 evaluable patients, T-cell responses were assessed by IFN- ELIspot assay and tetramer analysis. Clinical outcomes were recorded. Results: CD8+ T-cell response rates to the 12 MHC class Irestricted melanoma peptides (by day 50) with or without GM-CSF were 34 and 73, respectively (P < 0.001), by direct ELIspot assay. Tetramer analyses corroborated the functional data. CD4+ T-cell responses to tetanus helper peptide were higher without GM-CSF (95 versus 77; P = 0.005). There was no significant difference by number of vaccine sites. Three-year overall and disease-free survival estimates (95 confidence interval) were 76 (67-83) and 52 (43-61), respectively, with too few events to assess differences by study group. Conclusions: High immune response rates for this multipeptide vaccine were achieved, but CD8+ and CD4+ T-cell responses were lower when administered with GM-CSF. These data challenge the value of local GM-CSF as a vaccine adjuvant in humans. (Clin Cancer Res 2009;15(22):703644)

Randomized Multicenter Trial of the Effects of Melanoma-Associated Helper Peptides and Cyclophosphamide on the Immunogenicity of a Multipeptide Melanoma Vaccine

PURPOSE This multicenter randomized trial was designed to test whether melanoma-associated helper peptides augment CD8(+) T-cell responses to a melanoma vaccine and whether cyclophosphamide (CY) pretreatment augments CD4(+) or CD8(+) T-cell responses to that vaccine. PATIENTS AND METHODS In all, 167 eligible patients with resected stage IIB to IV melanoma were randomly assigned to four vaccination study arms. Patients were vaccinated with 12 class I major histocompatibility complex-restricted melanoma peptides (12MP) to stimulate CD8(+) T cells and were randomly assigned to receive a tetanus helper peptide or a mixture of six melanoma-associated helper peptides (6MHP) to stimulate CD4(+) T cells. Before vaccination, patients were also randomly assigned to receive CY pretreatment or not. T-cell responses were assessed by an ex vivo interferon gamma ELISpot assay. Clinical outcomes and toxicities were recorded. RESULTS Vaccination with 12MP plus tetanus induced CD8(+) T-cell responses in 78% of patients and CD4(+) T-cell responses to tetanus peptide in 93% of patients. Vaccination with 12MP plus 6MHP induced CD8(+) responses in 19% of patients and CD4(+) responses to 6MHP in 48% of patients. CY had no significant effect on T-cell responses. Overall 3-year survival was 79% (95% CI, 71% to 86%), with no significant differences (at this point) by study arm. CONCLUSION Melanoma-associated helper peptides paradoxically decreased CD8(+) T-cell responses to a melanoma vaccine (P < .001), and CY pretreatment had no immunologic or clinical effect. Prior work showed immunologic and clinical activity of 6MHP alone. Possible explanations for negative effects on CD8 responses include modulation of homing receptor expression or induction of antigen-specific regulatory T cells.

Phase 2 trial of epothilone B analog BMS-247550 (ixabepilone) in advanced carcinoma of the urothelium (E3800): a trial of the Eastern Cooperative Oncology Group.

Paclitaxel and docetaxel are active agents in advanced urothelial cancer. BMS‐247550 (ixabepilone) has activity in preclinical models in paclitaxel resistant models. A phase 2 trial of this epothilone was performed to determine the activity and toxicity of this agent in a multi‐institutional setting in patients previously treated with 1 prior chemotherapy regimen.

Clinical, pharmacokinetic and biological studies of topotecan

The topoisomerase I inhibtor topotecan is a potent water-soluble camptothecin derivative with activity in a wide variety of preclinical models. Topotecan exhibits schedule dependency in vivo, with the greatest activity being observed on repeated dose schedules. On the basis of the initial clinical studies that showed a short plasma halflife, we attempted to prolong drug exposure by giving topotecan as a 24-h infusion weekly. In a phase I trial, we treated 32 patients at doses ranging from 1.0 to 2.0 mg/m2. The patient population had not been heavily pretreated with chemotherapy and was of good performance status. The incidence of neutropenia, which was dose-limiting, increased sharply with relative small increments in dose. Doses greater than 1.5 mg/m2 were associated with nadirs that developed after one to three weekly treatments. A patient with metastatic colorectal cancer had a prolonged partial response. The plasma pharmacokinetics of topotecan (lactone and open forms) was characterized in 21 patients. Mean plasma steady-state drug levels were proportional to the dose and were within the range required to exert cytotoxicity in preclinical models. Plasma elimination curves were fit to a one-compartment model, in which the harmonic mean half-life of topotecan was 3.5 h. The ratio of the lactone to the total drug concentrations was constant throughout, which suggests that for this schedule the total drug concentration may be used as a measure of active lactone exposure. This conclusion is supported by the pharmacodynamic analysis, which revealed a positive correlation of both lactone and total drug steady-state concentrations with bone marrow toxicity. The further investigation of this and other infusional schedules in phase II trials will be conducted. The steady-state concentrations of total drug will be measured in several of these trials to establish its potential role in adaptive dosing using this schedule. Such a strategy is justified by the interpatient variability in toxicity and the steep dose-response curve observed in this study. Preliminary evidence of interpatient variability in the mRNA expression of topoisomerase I in the peripheral mononuclear cells and colon mucosa is presented. Trials are under way using biological endpoints for further selection of patients in whom the use of topoisomerase inhibitors may be therapeutically beneficial.

Mechanisms of apoptosis resistance and treatment strategies to overcome them in hormone‐refractory prostate cancer

New therapeutic strategies are needed to improve treatment outcomes in men with hormone‐refractory prostate cancer. A better understanding of the molecular mechanisms of cell death in response to therapeutic strategies will help avoid ineffective treatment regimens and provide a molecular basis for new therapeutic modalities targeting apoptosis‐resistant forms of prostate cancer. In this review, the authors focused on the established aberrations of apoptosis in hormone‐refractory prostate cancer, and they have described novel treatment strategies to overcome apoptosis resistance. Cancer 2008.

Immunogenicity for CD8+ and CD4+ T cells of 2 formulations of an incomplete freund's adjuvant for multipeptide melanoma vaccines.

An incomplete Freunds adjuvant (IFA) commonly used in experimental cancer vaccines has recently been reformulated. Oleic acid used in the surfactant was purified from a vegetable source (olives, IFA-VG) rather than an animal source (beef tallow, IFA-AN). To provide an insight into the adjuvant properties of the new formulation, we reviewed T-cell responses, by enzyme-linked immunospot assay, to multipeptide vaccines in 2 sequential clinical trials that spanned this transition of adjuvants. Analyses included 194 patients who received either IFA-AN or IFA-VG for all vaccines, and a subset of 93 patients best matched by study arm for vaccine antigens (12 melanoma peptides restricted by major histocompatibility complex class I, 12MP; plus a tetanus helper peptide, tet) administered with IFA but without granulocyte macrophage-colony stimulating factor. Inflammation was observed at vaccine sites clinically for almost all patients, even including ulceration in a subset with each IFA formulation. CD8+ T-cell response rates to the 12 melanoma peptides were 53% [95% confidence interval (CI), 44%, 61%)] for IFA-AN and 46% [95% CI, 32%, 59%)] for IFA-VG. In the 93 patient subset, these rates were 73% [95% CI, 61%, 83%)] and 70% [95% CI, 47%, 87%)], respectively. CD4+ T-cell responses to tetanus helper peptide were identified in 94% [95% CI, 86%, 98%)] and 96% [95% CI, 78%, 100%)], respectively. Responses to individual human leukocyte antigen (HLA)-A1, A2, and DR associated peptides were largely preserved, but reactivity trended lower for some HLA-A3 associated peptides. Despite the necessarily retrospective nature of the analysis and limitations of multiple comparisons, our summary data support the use of IFA-VG as an adjuvant with multipeptide vaccines in melanoma patients.

Phase I trial of 5-fluorouracil by 24-hour infusion weekly.

A novel schedule of 5-fluorouracil administration has been developed for biochemical modulation studies. In combination with the pyrimidine synthesis inhibitor PALA, 5-fluorouracil has been given as a 24-hour infusion, repeated weekly: a dose of 2600 mg/m2 is well tolerated. To identify a suitable dose of 5-fluorouracil as a single agent on this schedule, we treated 26 patients at doses ranging from 2800 to 3400 mg/m2 per week. Two-thirds of the patients had failed previous therapy, and most were symptomatic from their disease. Over half of the patients had metastatic colorectal cancer. The dose-limiting toxicity was diarrhea: Grade 3 or 4 toxicity occurred at every level tested. Twenty-two of the 26 patients required therapy interruption because of toxicity. The severity of this toxicity indicated that escalation of 5-fluorouracil on this schedule beyond the 2600 mg/m2 known to be tolerated in the PALA-containing regimen, would be impractical. Two patients, both with previously untreated colorectal cancer, had partial remissions lasting three and five months respectively. This dose-intense schedule of 5-fluorouracil administration will be explored further in large-scale randomized trials.

Vinblastine and estramustine phosphate in metastatic renal cell carcinoma: a phase II trial of the Fox Chase Network.

It is well known that metastatic renal cell carcinoma (RCC) exhibits constitutive resistance to chemotherapeutic agents. Antimicrotubule agents such as vinblastine are associated with low but reproducible response rates (approximately 12%) in patients with RCC. Estramustine has been shown to potentiate the antimicrotubule effects of vinblastine. The authors sought to increase the activity of vinblastine in RCC through the addition of estramustine.

A Phase II trial of weekly infusional 5-fluorouracil in combination with lowdose leucovorin in patients with advanced colorectal cancer

Exogenous leucovorin is a source of reduced folate which enhances the inhibition of thymidylate synthase that results from 5-fluorouracil (5-FU) administration. Extracellular reduced folate concentrations of 1 μM have been reported to yield maximal enzyme inhibition in several cell lines treated with 5-FUin vitro. Clinical studies indicate that low doses of leucovorin have equivalent efficacy to higher doses in successfully modulating 5-FU in the treatment of colorectal cancer. Based on pharmacokinetics at higher doses, steadystate total plasma reduced folate concentrations of 1 μM would be expected from the administration of leucovorin 50 mg/m2 by 24 h infusion. This dose was admixed with 5-FU 2300 mg/m2 and administered by 24 h infusion weekly to 38 patients with advanced colorectal cancer, of whom 32 are evaluable for response. Disease sites included liver (33 patients), lung (12 patients), and bone (4 patients). Toxicity was mild to moderate, except for grade 3 diarrhea in 5 patients, and chest pain in 2 patients. Among the 32 evaluable patients, there were 14 partial remissions for a total response rate of 44% (95% confidence interval 27–61%). The median duration of response was seven (range 1 to 20+) months, and median duration of survival 16 months. These results support the use of low doses of leucovorin to modulate weekly infusional 5-FU in colorectal cancer, and provide a basis for the integration of this regimen with other modulators of 5-FU.

Paired specimens: an opportunity to answer some important questions.

One debate about renal cell carcinoma (RCC) with sarcomatoid change rests on whether these aggressive tumors exhibit a phenotype reflective of the surrounding environment or are in fact clonal events with separate features from the original tumor that require a different therapeutic approach. Well characterized microscopically, RCCs with these features contain spindle cells with high-grade pleomorphic nuclei, are highly cellular, and can resemble primary sarcomas by appearance. Immunohistochemical analysis reveals both epithelial andmesenchymal differentiation. Notably, spindle cells can be present in a variety of tumors (angiomyolipomas, primary sarcomas, high-grade clear cell, or unclassified) other than those with frank sarcomatoid change. However, RCCs with sarcomatoid change can usually be distinguished from primary or metastatic sarcomas by their expression of homatropine methylbromide, Melan A, cytokeratin (AE1/AE3), epithelial membrane antigen, and vimentin, and their lack of expression of desmin, S-100, muscle-specific actin, and smooth muscle actin in most cases. Architecturally, they can appear with a fibrosarcoma-like pattern, a storiform pattern, rhabdoid pattern, or undifferentiated. In addition, the sarcomatoid portion can be clearly demarcated or admixed with the epithelial elements. Limited analyses of RCCs with sarcomatoid features have reported expression of vascular endothelial growth factor (VEGF), kit, S6 kinase, fascin, hypoxia-inducible factor-1-alpha, carbonic anhydrase IX, glucose-transport protein 1, and p53 mutations. Most of these series have not attempted to analyze the regions of sarcomatoid change separately from the epithelial subtype. In addition, RCCs with sarcomatoid change show extensive chromosomal rearrangements, including those of the underlying subtype. The American Joint Committee on Cancer and Heidelberg both dropped sarcomatoid RCC as a distinct subtype because it was frequently accompanied by other subtypes: conventional, papillary, chromophobe, and collecting duct. One review of 101 tumors with sarcomatoid differentiation by de Peralta-Venturina et al reported a majority arising in the presence of clear cell morphology (n1⁄4 80), with the rest being papillary (n1⁄4 8), chromophobe (n1⁄4 7), collecting duct (n 1⁄4 2), and unclassified (n1⁄4 4). In a comparator population of 952 RCCs at 2 institutions, sarcomatoid features appeared in 8% of clear cell, 3% of papillary, 9% of chromophobe, 29% of collecting duct, and 11% of unclassified RCCs. Although not significant, this suggests a pairing of this poor prognostic feature with less common subtypes. Unquestionably, RCCs that contain these features are more aggressive and are associated with poorer prognosis. A higher percentage of sarcomatoid features has been associated with poorer survival in some but not all series and is not independent of stage. The morphologic features do not appear to be predictive of outcome. Shuch and colleagues (in this issue of Cancer) addressed the question of clonality versus phenotype using a unique approach. Whole kidney tumors and available resected tumor metastases from 32 patients at a single institution with a known diagnosis of RCC with sarcomatoid differentiation were analyzed for presence and percentage of sarcomatoid features in both the primary tumors and the metastases. A total of 52 metastatic sites were evaluated, the majority of them synchronous. The majority of the metastases were regional lymph nodes, but lung, liver, bone, and brain metastases were