A Bayesian adaptive design for biomarker trials with linked treatments.

BACKGROUND: Response to treatments is highly heterogeneous in cancer. Increased availability of biomarkers and targeted treatments has led to the need for trial designs that efficiently test new treatments in biomarker-stratified patient subgroups. METHODS: We propose a novel Bayesian adaptive randomisation (BAR) design for use in multi-arm phase II trials where biomarkers exist that are potentially predictive of a linked treatment's effect. The design is motivated in part by two phase II trials that are currently in development. The design starts by randomising patients to the control treatment or to experimental treatments that the biomarker profile suggests should be active. At interim analyses, data from treated patients are used to update the allocation probabilities. If the linked treatments are effective, the allocation remains high; if ineffective, the allocation changes over the course of the trial to unlinked treatments that are more effective. RESULTS: Our proposed design has high power to detect treatment effects if the pairings of treatment with biomarker are correct, but also performs well when alternative pairings are true. The design is consistently more powerful than parallel-groups stratified trials. CONCLUSIONS: This BAR design is a powerful approach to use when there are pairings of biomarkers with treatments available for testing simultaneously.This work was supported by the Medical Research Council (grant number G0800860) and the NIHR Cambridge Biomedical Research Centre.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/bjc.2015.27

High resolution melting for mutation scanning of TP53 exons 5-8.

BACKGROUND: p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples. METHODS: We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status. RESULTS: One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons. CONCLUSION: HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Amplicon-Dependent CCNE1 Expression Is Critical for Clonogenic Survival after Cisplatin Treatment and Is Correlated with 20q11 Gain in Ovarian Cancer

Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers

Copy Number Analysis Identifies Novel Interactions Between Genomic Loci in Ovarian Cancer

Ovarian cancer is a heterogeneous disease displaying complex genomic alterations, and consequently, it has been difficult to determine the most relevant copy number alterations with the scale of studies to date. We obtained genome-wide copy number alteration (CNA) data from four different SNP array platforms, with a final data set of 398 ovarian tumours, mostly of the serous histological subtype. Frequent CNA aberrations targeted many thousands of genes. However, high-level amplicons and homozygous deletions enabled filtering of this list to the most relevant. The large data set enabled refinement of minimal regions and identification of rare amplicons such as at 1p34 and 20q11. We performed a novel co-occurrence analysis to assess cooperation and exclusivity of CNAs and analysed their relationship to patient outcome. Positive associations were identified between gains on 19 and 20q, gain of 20q and loss of X, and between several regions of loss, particularly 17q. We found weak correlations of CNA at genomic loci such as 19q12 with clinical outcome. We also assessed genomic instability measures and found a correlation of the number of higher amplitude gains with poorer overall survival. By assembling the largest collection of ovarian copy number data to date, we have been able to identify the most frequent aberrations and their interactions

GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers

We describe methods with enhanced power and specificity to identify genes targeted by somatic copy-number alterations (SCNAs) that drive cancer growth. By separating SCNA profiles into underlying arm-level and focal alterations, we improve the estimation of background rates for each category. We additionally describe a probabilistic method for defining the boundaries of selected-for SCNA regions with user-defined confidence. Here we detail this revised computational approach, GISTIC2.0, and validate its performance in real and simulated datasets

Deregulation of MYCN, LIN28B and LET7 in a Molecular Subtype of Aggressive High-Grade Serous Ovarian Cancers

Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention

Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer

<p>Abstract</p> <p>Background</p> <p><it>MAP2K4 </it>is a putative tumor and metastasis suppressor gene frequently found to be deleted in various cancer types. We aimed to conduct a comprehensive analysis of this gene to assess its involvement in ovarian cancer.</p> <p>Methods</p> <p>We screened for mutations in <it>MAP2K4 </it>using High Resolution Melt analysis of 149 primary ovarian tumors and methylation at the promoter using Methylation-Specific Single-Stranded Conformation Polymorphism analysis of 39 tumors. We also considered the clinical impact of changes in <it>MAP2K4 </it>using publicly available expression and copy number array data. Finally, we used siRNA to measure the effect of reducing <it>MAP2K4 </it>expression in cell lines.</p> <p>Results</p> <p>In addition to 4 previously detected homozygous deletions, we identified a homozygous 16 bp truncating deletion and a heterozygous 4 bp deletion, each in one ovarian tumor. No promoter methylation was detected. The frequency of <it>MAP2K4 </it>homozygous inactivation was 5.6% overall, and 9.8% in high-grade serous cases. Hemizygous deletion of <it>MAP2K4 </it>was observed in 38% of samples. There were significant correlations of copy number and expression in three microarray data sets. There was a significant correlation between <it>MAP2K4 </it>expression and overall survival in one expression array data set, but this was not confirmed in an independent set. Treatment of JAM and HOSE6.3 cell lines with <it>MAP2K4 </it>siRNA showed some reduction in proliferation.</p> <p>Conclusions</p> <p><it>MAP2K4 </it>is targeted by genetic inactivation in ovarian cancer and restricted to high grade serous and endometrioid carcinomas in our cohort.</p

GSVD Comparison of Patient-Matched Normal and Tumor aCGH Profiles Reveals Global Copy-Number Alterations Predicting Glioblastoma Multiforme Survival

Despite recent large-scale profiling efforts, the best prognostic predictor of glioblastoma multiforme (GBM) remains the patient's age at diagnosis. We describe a global pattern of tumor-exclusive co-occurring copy-number alterations (CNAs) that is correlated, possibly coordinated with GBM patients' survival and response to chemotherapy. The pattern is revealed by GSVD comparison of patient-matched but probe-independent GBM and normal aCGH datasets from The Cancer Genome Atlas (TCGA). We find that, first, the GSVD, formulated as a framework for comparatively modeling two composite datasets, removes from the pattern copy-number variations (CNVs) that occur in the normal human genome (e.g., female-specific X chromosome amplification) and experimental variations (e.g., in tissue batch, genomic center, hybridization date and scanner), without a-priori knowledge of these variations. Second, the pattern includes most known GBM-associated changes in chromosome numbers and focal CNAs, as well as several previously unreported CNAs in 3% of the patients. These include the biochemically putative drug target, cell cycle-regulated serine/threonine kinase-encoding TLK2, the cyclin E1-encoding CCNE1, and the Rb-binding histone demethylase-encoding KDM5A. Third, the pattern provides a better prognostic predictor than the chromosome numbers or any one focal CNA that it identifies, suggesting that the GBM survival phenotype is an outcome of its global genotype. The pattern is independent of age, and combined with age, makes a better predictor than age alone. GSVD comparison of matched profiles of a larger set of TCGA patients, inclusive of the initial set, confirms the global pattern. GSVD classification of the GBM profiles of an independent set of patients validates the prognostic contribution of the pattern

Copy number signatures and mutational processes in ovarian carcinoma.

The genomic complexity of profound copy number aberrations has prevented effective molecular stratification of ovarian cancers. Here, to decode this complexity, we derived copy number signatures from shallow whole-genome sequencing of 117 high-grade serous ovarian cancer (HGSOC) cases, which were validated on 527 independent cases. We show that HGSOC comprises a continuum of genomes shaped by multiple mutational processes that result in known patterns of genomic aberration. Copy number signature exposures at diagnosis predict both overall survival and the probability of platinum-resistant relapse. Measurement of signature exposures provides a rational framework to choose combination treatments that target multiple mutational processes.NIHR, Ovarian Cancer Action, Cancer Research UK Cambridge Centre, Cambridge Experimental Cancer Medicine Centr

Array-Comparative Genomic Hybridization Reveals Loss of SOCS6 Is Associated with Poor Prognosis in Primary Lung Squamous Cell Carcinoma

BACKGROUND: Primary tumor recurrence commonly occurs after surgical resection of lung squamous cell carcinoma (SCC). Little is known about the genes driving SCC recurrence. METHODS: We used array comparative genomic hybridization (aCGH) to identify genes affected by copy number alterations that may be involved in SCC recurrence. Training and test sets of resected primary lung SCC were assembled. aCGH was used to determine genomic copy number in a training set of 62 primary lung SCCs (28 with recurrence and 34 with no evidence of recurrence) and the altered copy number of candidate genes was confirmed by quantitative PCR (qPCR). An independent test set of 72 primary lung SCCs (20 with recurrence and 52 with no evidence of recurrence) was used for biological validation. mRNA expression of candidate genes was studied using qRT-PCR. Candidate gene promoter methylation was evaluated using methylation microarrays and Sequenom EpiTYPER analysis. RESULTS: 18q22.3 loss was identified by aCGH as being significantly associated with recurrence (p = 0.038). Seven genes within 18q22.3 had aCGH copy number loss associated with recurrence but only SOCS6 copy number was both technically replicated by qPCR and biologically validated in the test set. SOCS6 copy number loss correlated with reduced mRNA expression in the study samples and in the samples with copy number loss, there was a trend for increased methylation, albeit non-significant. Overall survival was significantly poorer in patients with SOCS6 loss compared to patients without SOCS6 loss in both the training (30 vs. 43 months, p = 0.023) and test set (27 vs. 43 months, p = 0.010). CONCLUSION: Reduced copy number and mRNA expression of SOCS6 are associated with disease recurrence in primary lung SCC and may be useful prognostic biomarkers