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Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours. Funding sources for this study include grants to the Pancreatic Cancer Sequencing Initiative program from the Ontario Institute for Cancer Research (OICR), through support from the Ontario Ministry of Research and Innovation, the Canada Foundation for Innovation; research award to F.N. from the OICR and the Canadian Institutes for Health Research (CIHR); Canadian Friends of the Hebrew University, the SMGS Family Foundation, NCI grant P50 CA102701 (Mayo Clinic SPORE in Pancreatic Cancer) and NCI grant R01 CA97075 (Pancreatic Cancer Genetic Epidemiology Consortium). F.N. is supported by a fellowship award from CIHR and is a recipient of a scholar’s research award from the Ontario Institute of Cancer Research (OICR), through support from the Ontario Ministry of Research and Innovation. G.Z. is a Clinician–Scientist of the Fonds de la Recherche en Sante du Quebec. P.J.C. is a Wellcome Trust Senior Clinical Fellow. T.J.H., L.D.S., J.D.M. and S.G. are recipients of Senior or Clinician–Scientist Awards from the Ontario Institute for Cancer Research

© 2014 Nature America, Inc. All rights reserved., Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA., This work was supported in part by an IDEAS grant from Princess Margaret Hospital, the Head and Neck Translational Research Program (I.W., B.A.C., P.C.B. and J.D.M.), the Ontario Institute for Cancer Research and the government of Ontario (P.C.B. and J.D.M.) and by a Terry Fox Research Institute New Investigator Award (P.C.B.). C.H. and F.-F.L. acknowledge support from the Wharton family, Joe's Team, Gordon Tozer, the Campbell Family Institute for Cancer Research and the Ministry of Health and Long-Term Planning, Canada.

Background Quantitative real-time PCR (qPCR) is the “gold-standard” technique for measuring mRNA abundances. To correctly compare samples and generate biologically valid results, qPCR data usually require comprehensive normalization to account for sample content variation between reactions. The most common normalization approaches use one or more endogenous controls (reference or house-keeping genes) to adjust the measured levels of experimental genes appropriately. Ideal reference genes are those that display minimal variation across experimental conditions, and thus can vary widely across different biological systems. In particular, toxicogenomic studies of transcriptionally-disruptive toxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. Results We examined seven candidate reference genes in 199 mice varying in genotype and time/dose of TCDD exposure. We assessed gene-stability in four ways: (1) the variance of the raw Cq values across biological replicates, (2) the fold-change from basal mRNA levels following treatment, (3) the inter- and intra-group stability evaluated using the NormFinder algorithm, (4) the comparative ΔCq method for each candidate gene. Univariate analyses showed Hprt and Eef1a1 are the two most stable individual reference genes. It has been suggested that using multiple genes would produce a more consistent normalization factor; multivariate analysis was performed using NormFinder. In general, stability increased with the number of genes used, but specific gene-combinations synergized. Conclusions We have validated seven reference genes for use in analyzing mRNA abundances in mouse models of TCDD toxicity. The use of multiple reference genes increases stability, providing more consistent normalization and more reliable results. The number of reference genes used should be maximized, based on experimental capabilities (platform, sample availability, etc.). Our results show the benefit of validating reference genes using multiple methods prior to generating large biological datasets.

Prostate tumours are highly variable in their response to therapies, but clinically available prognostic factors can explain only a fraction of this heterogeneity. Here we analysed 200 whole-genome sequences and 277 additional whole-exome sequences from localized, non-indolent prostate tumours with similar clinical risk profiles, and carried out RNA and methylation analyses in a subset. These tumours had a paucity of clinically actionable single nucleotide variants, unlike those seen in metastatic disease. Rather, a significant proportion of tumours harboured recurrent non-coding aberrations, large-scale genomic rearrangements, and alterations in which an inversion repressed transcription within its boundaries. Local hypermutation events were frequent, and correlated with specific genomic profiles. Numerous molecular aberrations were prognostic for disease recurrence, including several DNA methylation events, and a signature comprised of these aberrations outperformed well-described prognostic biomarkers. We suggest that intensified treatment of genomically aggressive localized prostate cancer may improve cure rates.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

505 Background: The International Cancer Genome Consortium and The Cancer Genome Atlas have had a global transformative impact on our understanding of cancer. These programs have mapped the genomic landscape of common and rare tumors setting the scene for a comprehensive change in the approach to cancer diagnosis and treatment. However, the task remains incomplete until these mutational events are linked to clinical outcomes in the context of current therapeutic intervention to drive future stratified medicine approaches. Methods: We performed targeted sequencing in patients from the Tamoxifen Exemestane Adjuvant Multicentre trial. DNA was extracted and a 101 gene panel analysed using a novel mutation calling pipeline. Both a priori and machine learning analyses were performed using distant recurrence free survival as the primary endpoint. Results: In 1,491 successfully analyzed samples 1,070 (71.76%) samples exhibited at least one single nucleotide mutation (range 0-94, 1.828+/-0.133, mean+/-s.e.). 98/101 genes were mutated in at least one patient. Only variants in PIK3CA, TP53, MLL3, CDH1 were detected in 5% or more of samples. Twenty genes were associated with increased risk of recurrence in multivariate analyses corrected for clinic-pathological variables, 50% of these genes were involved in transcriptional regulation or RNA/protein processing. In a multivariate analysis, two combined signalling modules were independently prognostic for residual risk following hormone therapy (HRvalidation 3.10 95%CI 1.78-5.40 and HRvalidation 2.70 95%CI 1.57-4.64). Conclusions: We successfully performed a signalling pathway-based targeted sequencing analysis within predefined signalling modules. In supervised and unsupervised analyses we identified multiple signalling cassettes linked to poor outcome in patients with ER+ve breast cancers treated with modern endocrine therapy in the context of a phase III clinical trial. These results identify novel candidates as targets to treat endocrine refractory breast cancers.

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

Prostate cancer (CaP) remains the most common male malignancy worldwide, leading to over 300,000 deaths per year. In Western countries, most prostate tumours are diagnosed while they are confined to the prostate and have relatively indolent histology, as assessed by the Gleason Score (GS). CaP is a C-class tumour, characterized by large number of driver copy-number aberrations and genomic rearrangements. Therefore, while previous sequencing studies have focused largely on the coding regions of late-stage disease, herein we comprehensively characterized the copy-number profiles of 250 localized prostate cancers and analyzed the whole genomes of 124 matched tumour/normal pairs derived from patients with GS6 and GS7 prostate cancer. Using this – the largest whole-genome sequencing dataset of prostate cancer to date – we confirm the C-class character of the disease and identify strong genomic subtypes that stretch across multiple types of somatic alteration, including SNVs, CNAs and genomic rearrangements. We provide the first assessments of localized hyper-mutation phenomena (chromothripsis and kataegis) in prostate cancer, and identify specific genes driving higher levels of these hyper-mutations. We identify unexpected biases in the location and role of both non-coding SNVs and genomic rearrangements, including clear association with epigenetic processes, and with genome-wide profiling of methylation in 92 samples. Finally, we demonstrate a stark paucity of clinically-actionable mutations in localized GS6 and GS7 disease, even lacking those common in high-risk localized disease, indicating that novel therapeutic development against the recurrent targets identified here will be key to allowing less-aggressive, targeted treatment of early-stage disease. Citation Format: Michael E. Fraser, Veronica Y. Sabelnykova, Takafumi N. Yamaguchi, Alice Meng, Lawrence E. Heisler, Junyan Zhang, Julie Livingstone, Vincent Huang, Andre P. Masella, Fouad Yousif, Michael Xie, Nicholas J. Harding, Xihui Lin, Haiying Kong, Stephenie D. Prokopec, Alejandro Berlin, Dominique Trudel, Xuemei Luo, Timothy E. Beck, Richard de Borja, Alister D'Costa, Robert E. Denroche, Natalie S. Fox, Emilie Lalonde, Ada Wong, Taryne Chong, Michelle Sam, Jeremy Johns, Lee Timms, Nicholas Buchner, Michele Orain, Valerie Picard, Helene Hovington, Kenneth C. Chu, Christine P'ng, Bryan Lo, Francis Nguyen, Kathleen E. Houlahan, Christopher Cooper, Shaylan K. Govind, Clement Fung, Louis Lacombe, Colin C. Collins, Yves Fradet, Bernard Tetu, Theodorus van der Kwast, John McPherson, Thomas J. Hudson, Rob G. Bristow, Paul Boutros. The mutational landscape of localized gleason 6 and 7 prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2966. doi:10.1158/1538-7445.AM2015-2966

Men with localized prostate cancer vary widely in clinical outcome, with a 30-50% failure rate after primary treatment. There is thus significant interest in developing genomically refined prognostic groups. We sought to evaluate the extent of genetic heterogeneity, both between patients (inter-prostate) and within individual prostate glands (intra-prostate) to assess the impact of genetic heterogeneity on risk stratification within a tight clinical cohort. Copy number aberrations (CNAs) from 75 Gleason 7 patients were determined by OncoScan SNP microarrays. We measure the percentage of genome involved in a CNA, termed percent genome aberration (PGA), a proxy for genomic instability. Additionally, whole genome sequencing was applied to 10 intermediate-risk prostate tumours and matched blood, including multiple manually macro-dissected regions from 5 of the prostates (range 2 to 9). Somatic single nucleotide variants (SNVs) and genomic rearrangements (GR) were extracted from each patient. We find a high degree of inter-prostatic heterogeneity between the 75 Gleason 7 patients, with the number of CNAs per patient ranging from 0 to 929, corresponding to PGA 0 to 16.7%. Known prognostic markers can differentiate between patients at higher risk for biochemical recurrence, but only account for a fraction of the cohort. Notably, when these prognostic genes are examined within multiple regions of five independent tumours, they differ in copy number between cancerous regions of the same prostate. For example, TP53 is deleted in 1/2, 1/3, 4/9, 0/4, and 4/5 prostate regions. Indeed, phylogenetic analysis of geographically distinct regions revealed multi-clonal disease in two of the five patients; separate analyses based on SNVs, CNAs, and GRs all concluded that these patient have two genetically distinct cancers within their prostate. We demonstrate dramatic levels of inter- and intra- prostate genetic heterogeneity within pathologically identical or similar cancers. The observed intra-prostatic genomic heterogeneity, both in terms of multi-focal and multi-clonal disease, has critical implications for clinical management. Prognostic information obtained by biopsy may be inconsistent depending on the site of biopsy, and applying personalized medicine to prostate cancer will be challenging. This study highlights the need for further evaluation of how intra-prostatic heterogeneity is related to patient prognosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B129. Citation Format: Emilie Lalonde, Paul C. Boutros, Michael Fraser, Richard de Borja, Nicholas J. Harding, Dominique Trudel, Alice Meng, Pablo H. Hennings-Yeomans, Andrew McPherson, Amin Zia, Jianxin Wang, Timothy Beck, Natalie S. Fox, Taryne Chong, Michelle Sam, Jeremy Johns, Lee Timms, Nicholas Buchner, Sohrab Shah, Cenk Sahinalp, Thomas J. Hudson, John D. McPherson, Theodorus van der Kwast, Robert G. Bristow. Clinical implications of inter- and intra- prostatic heterogeneity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B129.

Intermediate risk prostate cancer (CaP) with Gleason score (GS) of 7 show up to 100x variability in genetic instability. As CaP is multifocal and likely multiclonal, there is a need to characterize heterogeneity for patient stratification, which would increase the ability to act on genomic information by adding adjuvant therapies to offset systemic occult metastases that currently limit cure in ∼30% of patients. Individual genetic portraits could be used to improve cure on combined clinical-molecular staging criteria. We undertook a pilot study to assess the genetic heterogeneity of potentially curable GS=7 CaP. We selected 10 men with GS=7 CaP; 5 treated with external beam radiotherapy (frozen pre-treatment biopsies) and 5 treated with radical prostatectomy (RadP, frozen tumour). Additionally, DNA from 18 distinct formalin-fixed, paraffin-embedded (FFPE) foci from the 5 RadP were analysed. Each of these 28 foci were subjected to whole-genome sequencing (WGS) and OncoScan SNP arrays to yield comprehensive genetic profiles. mRNA expression was evaluated on frozen RadP by microarray. Germline DNA from whole-blood was also analysed. Following independent pathology reviews and manual macro-dissection of tumour areas of ≥70% cellularity, WGS (≥50x tumour, ≥30x germline) was performed on as little as 50 ng genomic DNA, and OncoScan arrays were performed using as little as 30ng DNA using either amplified or innate genomic DNA. Regions of CaP in FFPE RadP were recorded using a tissue map to identify independent malignant foci, and ERG immunostaining was performed to assist in the identification. In cases where ERG-positive and -negative foci were adjacent, ERG staining was repeated on an un-stained slide to confirm separate foci based on 3D multi-section analyses. ERG fusion status was also assessed in frozen samples by aCGH or IHC. Validation of SNVs via SNP array and deep-resequencing showed ∼99% accuracy. Tumour cellularity was estimated using Qpure and was >60% for all samples. Phylogenetic techniques were used to demonstrate clear multi-clonality in two tumours. Across all tumours, ∼50% of SNVs were specific to an individual tumour-region. Phylogenies were confirmed with both SNVs and CNAs, but CNAs generally exhibited greater concordance amongst different regions of the same tumour. Some previously observed recurrent mutations were previously identified as recurrent in CaP (e.g. SPOP), and the overall mutation rate for intermediate-risk CaP was only somewhat below that reported for castrate-resistant disease (11,230 somatic SNVs per tumour). Our studies support the concept that a complete characterization of inter- and intra-CaP heterogeneity is possible in fresh and archival tissues; the latter is important for correlations to clinical outcome. These approaches can then be streamlined for high-throughput analyses within personalized medicine laboratories leading to “point of care” molecular tests and individualization of therapy. Citation Format: Michael E. Fraser, Richard de Borja, Dominique Trudel, Nicholas J. Harding, Pablo H. Hennings-Yeomans, Alice Meng, Emilie R. Lalonde, Andrew Brown, Natalie S. Fox, Taryne Chong, Amin Zia, Michelle Sam, Jianxin Wang, Michelle A. Chan-Seng-Yue, Jeremy Johns, Lee Timms, Nicholas Buchner, Ada Wong, Fouad Yousif, Rob Denroche, Gaetano Zafarana, Maud HW Starmans, Hanbert Chen, Shaylan Govind, Francis Nguyen, Melania Pintilie, Neil Fleshner, Stanislav Volik, Lakshmi Muthuswamy, Colin C. Collins, Thomas J. Hudson, Lincoln D. Stein, Timothy Beck, John D. McPherson, Theodorus van der Kwast, Paul C. Boutros, Rob G. Bristow. A molecular portrait of potentially curable prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2003. doi:10.1158/1538-7445.AM2013-2003