PTEN , PIK3CA and PKCβII . Primer sequences are available in supplemental Table 1. PCR ampliconswere subjected to direct sequencing using same primers for PCR amplification and ABI BigDye Terminator kit v1.1 according PS-341 to the manufacturer’s instructions. Sequence variations were determined by using Sequencher software 4.7 compared with GenBank genomic sequences for each gene. All of the sequence variations were confirmed by multiple, independent PCR amplifications and repeated sequencing reactions. Copy number assessments for PTEN and AKT2 Copy number analysis of AKT2, PTEN was performed using quantitative PCR . CustomTaqMan assays were obtained fromABI : All probes were labeled with FAM as the reporter dye and TAMRA as the quencher.
Each sample was run in triplicate on an ABI Prism7900HT analyzer using Caveolin 1 as a Itraconazole clinical trial reference gene. A standard curve was generated using normal human genomic DNA and data was analyzed using the standard curve method. Enzyme linked immunosorbent assay for VEGF VEGF A concentrations were quantified using the Quantikine Human VEGF ELISA kit, catalog DVE00 and the VEGF165 standard in pre cycle 1 and precycle 2 plasma specimens according to the kit instructions. Statistical design The study was designed to detect cytostatic activity by examining delays in tumor progression through the frequency of patients PFS at 6 months and cytotoxic activity through the frequency of tumor response. Activity by either measure was considered sufficient to declare the drug worthy of further investigation in a phase III trial.
A bivariate 2 stage design was used to limit needless exposure when the drug was inactive . With 27 eligible and evaluable patients, the study required more Itraconazole structure than 4 patients PFS at 6 months or more than 3 patients with responses before the study could proceed to the second stage accrual. The cumulative targeted sample size for the second stage was 52 patients. The study would require more than 12 patients PFS at 6 months or more than 8 patients with responses before the agent would be declared interesting. The sample sizes were targeted so that the probability of declaring the regimen active when in fact it is inactive was limited to 10% with approximately 90% power where pr and ps are the probabilities of response and PFS at 6 months respectively.
The null probabilities were obtained from an analysis of historical controls where the agents under investigation were deemed to have minimal activity . The frequency and severity of adverse events were evaluated with National Cancer Institute’s Common Terminology Criteria for Adverse Events and tabulated according to the organ system affected. Itraconazole solubility Cox proportional hazards models were used where feasible to assess any possible association between a biomarker of interest and PFS or OS . Associations within biomarkers and clinical characteristics were examined with Kendall’s Tau b, Spearman’s correlation coefficient, and Fisher’s Exact Test as feasible . Clinical characteristics examined with biomarker data included response, PFS ≥6 months, performance status, age group, number of prior regimens, and grade infectious diseases of tumor tissue. Since all except one of the patients had disease progression, exact Wilcoxon or Kruskal Wallis tests were conducted .
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