Anatomic position determines oncogenic specificity in melanoma.

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.

Shariant platform: Enabling evidence sharing across Australian clinical genetic-testing laboratories to support variant interpretation.

Sharing genomic variant interpretations across laboratories promotes consistency in variant assertions. A landscape analysis of Australian clinical genetic-testing laboratories in 2017 identified that, despite the national-accreditation-body recommendations encouraging laboratories to submit genotypic data to clinical databases, fewer than 300 variants had been shared to the ClinVar public database. Consultations with Australian laboratories identified resource constraints limiting routine application of manual processes, consent issues, and differences in interpretation systems as barriers to sharing. This information was used to define key needs and solutions required to enable national sharing of variant interpretations. The Shariant platform, using both the GRCh37 and GRCh38 genome builds, was developed to enable ongoing sharing of variant interpretations and associated evidence between Australian clinical genetic-testing laboratories. Where possible, two-way automated sharing was implemented so that disruption to laboratory workflows would be minimized. Terms of use were developed through consultation and currently restrict access to Australian clinical genetic-testing laboratories. Shariant was designed to store and compare structured evidence, to promote and record resolution of inter-laboratory classification discrepancies, and to streamline the submission of variant assertions to ClinVar. As of December 2021, more than 14,000 largely prospectively curated variant records from 11 participating laboratories have been shared. Discrepant classifications have been identified for 11% (28/260) of variants submitted by more than one laboratory. We have demonstrated that co-design with clinical laboratories is vital to developing and implementing a national variant-interpretation sharing effort. This approach has improved inter-laboratory concordance and enabled opportunities to standardize interpretation practices.

Performance of somatic structural variant calling in lung cancer using Oxford Nanopore sequencing technology.

BACKGROUND: Lung cancer is a heterogeneous disease and the primary cause of cancer-related mortality worldwide. Somatic mutations, including large structural variants, are important biomarkers in lung cancer for selecting targeted therapy. Genomic studies in lung cancer have been conducted using short-read sequencing. Emerging long-read sequencing technologies are a promising alternative to study somatic structural variants, however there is no current consensus on how to process data and call somatic events. In this study, we preformed whole genome sequencing of lung cancer and matched non-tumour samples using long and short read sequencing to comprehensively benchmark three sequence aligners and seven structural variant callers comprised of generic callers (SVIM, Sniffles2, DELLY in generic mode and cuteSV) and somatic callers (Severus, SAVANA, nanomonsv and DELLY in somatic modes). RESULTS: Different combinations of aligners and variant callers influenced somatic structural variant detection. The choice of caller had a significant influence on somatic structural variant detection in terms of variant type, size, sensitivity, and accuracy. The performance of each variant caller was assessed by comparing to somatic structural variants identified by short-read sequencing. When compared to somatic structural variants detected with short-read sequencing, more events were detected with long-read sequencing. The mean recall of somatic variant events identified by long-read sequencing was higher for the somatic callers (72%) than generic callers (53%). Among the somatic callers when using the minimap2 aligner, SAVANA and Severus achieved the highest recall at 79.5% and 79.25% respectively, followed by nanomonsv with a recall of 72.5%. CONCLUSION: Long-read sequencing can identify somatic structural variants in clincal samples. The longer reads have the potential to improve our understanding of cancer development and inform personalized cancer treatment.

Whole-genome landscapes of major melanoma subtypes.

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.

Whole Genome Sequencing in Advanced Lung Cancer can be Performed Using Diff-Quik Cytology Smears Derived from Endobronchial Ultrasound, Transbronchial Needle Aspiration (EBUS TBNA).

INTRODUCTION: Maximising alternative sample types for genomics in advanced lung cancer is important because bronchoscopic samples may sometimes be insufficient for this purpose. Further, the clinical applications of comprehensive molecular analysis such as whole genome sequencing (WGS) are rapidly developing. Diff-Quik cytology smears from EBUS TBNA is an alternative source of DNA, but its feasibility for WGS has not been previously demonstrated. METHODS: Diff-Quik smears were collected along with research cell pellets. RESULTS: Tumour content of smears were compared to research cell pellets from 42 patients, which showed good correlation (Spearman correlation 0.85, P < 0.0001). A subset of eight smears underwent WGS, which presented similar mutation profiles to WGS of the matched cell pellet. DNA yield was predicted using a regression equation of the smears cytology features, which correctly predicted DNA yield > 1500 ng in 7 out of 8 smears. CONCLUSIONS: WGS of commonly collected Diff-Quik slides is feasible and their DNA yield can be predicted.

Analysis of hereditary cancer gene variant classifications from ClinVar indicates a need for regular reassessment of clinical assertions.

The clinical classification of variants may change with new information, however, there is limited guidance on how often significant changes in variant classification occur. We used ClinVar to examine how variant classification changes over time. We developed a custom parser and accessed variant data from ClinVar between January 2015 and July 2021. The ClinVar-assigned "aggregate" classification of variants in 121 hereditary cancer genes was harmonized across releases to align to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology terms. Aggregate classification categories were grouped as: benign/likely benign (B/LB); likely pathogenic/pathogenic (LP/P); variant of uncertain significance (VUS); conflicting interpretations of pathogenicity (Conflicting); or Other. We profiled changes in aggregate variant classification between consecutive semi-annual ClinVar releases. The proportion of variants that changed aggregate classification between semi-annual ClinVar releases ranged from 0.6% to 6.4%. The most frequent changes were "VUS to conflicting," "other to LP/P," and "B/LB to Conflicting." A limited number of variants changed aggregate classification from "LP/P to B/LB," or vice versa. Our analysis indicates need for regular reassessment of clinical variant interpretations. The parser developed for this project will facilitate extraction of relevant interpretation data from ClinVar.

Considerations for using population frequency data in germline variant interpretation: Cancer syndrome genes as a model.

Aggregate population genomics data from large cohorts are vital for assessing germline variant pathogenicity. However, there are no specifications on how sequencing quality metrics should be considered, and whether exome-derived and genome-derived allele frequencies should be considered in isolation. Germline genome sequence data were simulated for nine read-depths to identify a minimum acceptable read-depth for detecting variants. gnomAD exome-derived and genome-derived datasets were assessed for read-depth, for six key cancer genes selected for variant curation by ClinGen expert panels. Non-Finnish European allele frequency (AF) or filter AF of coding variants in these genes, assigned into frequency bins using modified ACMG-AMP criteria, was compared between exome-derived and genome-derived datasets. A 30X read-depth achieved acceptable precision and recall for detection of substitutions, but poor recall for small insertions/deletions. Exome-derived and genome-derived datasets exhibited low read-depth for different gene exons. Individual variants were mostly assigned to non-divergent AF bins (>95%) or filter AF bins (>97%). Two major bin divergences were resolved by applying the minimal acceptable read-depth threshold. These findings show the importance of assessing read-depth separately for population datasets sourced from different short-read sequencing technologies before assigning a frequency-based ACMG-AMP classification code for variant interpretation.

Phenotypic and molecular dissection of metaplastic breast cancer and the prognostic implications.

Metaplastic breast carcinoma (MBC) is relatively rare but accounts for a significant proportion of global breast cancer mortality. This group is extremely heterogeneous and by definition exhibits metaplastic change to squamous and/or mesenchymal elements, including spindle, squamous, chondroid, osseous, and rhabdomyoid features. Clinically, patients are more likely to present with large primary tumours (higher stage), distant metastases, and overall, have shorter 5-year survival compared to invasive carcinomas of no special type. The current World Health Organisation (WHO) diagnostic classification for this cancer type is based purely on morphology - the biological basis and clinical relevance of its seven sub-categories are currently unclear. By establishing the Asia-Pacific MBC (AP-MBC) Consortium, we amassed a large series of MBCs (n = 347) and analysed the mutation profile of a subset, expression of 14 breast cancer biomarkers, and clinicopathological correlates, contextualising our findings within the WHO guidelines. The most significant indicators of poor prognosis were large tumour size (T3; p = 0.004), loss of cytokeratin expression (lack of staining with pan-cytokeratin AE1/3 antibody; p = 0.007), EGFR overexpression (p = 0.01), and for 'mixed' MBC, the presence of more than three distinct morphological entities (p = 0.007). Conversely, fewer morphological components and EGFR negativity were favourable indicators. Exome sequencing of 30 cases confirmed enrichment of TP53 and PTEN mutations, and intriguingly, concurrent mutations of TP53, PTEN, and PIK3CA. Mutations in neurofibromatosis-1 (NF1) were also overrepresented [16.7% MBCs compared to ∼5% of breast cancers overall; enrichment p = 0.028; mutation significance p = 0.006 (OncodriveFM)], consistent with published case reports implicating germline NF1 mutations in MBC risk. Taken together, we propose a practically minor but clinically significant modification to the guidelines: all WHO_1 mixed-type tumours should have the number of morphologies present recorded, as a mechanism for refining prognosis, and that EGFR and pan-cytokeratin expression are important prognostic markers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Telomere sequence content can be used to determine ALT activity in tumours.

The replicative immortality of human cancer cells is achieved by activation of a telomere maintenance mechanism (TMM). To achieve this, cancer cells utilise either the enzyme telomerase, or the Alternative Lengthening of Telomeres (ALT) pathway. These distinct molecular pathways are incompletely understood with respect to activation and propagation, as well as their associations with clinical outcomes. We have identified significant differences in the telomere repeat composition of tumours that use ALT compared to tumours that do not. We then employed a machine learning approach to stratify tumours according to telomere repeat content with an accuracy of 91.6%. Importantly, this classification approach is applicable across all tumour types. Analysis of pathway mutations that were under-represented in ALT tumours, across 1,075 tumour samples, revealed that the autophagy, cell cycle control of chromosomal replication, and transcriptional regulatory network in embryonic stem cells pathways are involved in the survival of ALT tumours. Overall, our approach demonstrates that telomere sequence content can be used to stratify ALT activity in cancers, and begin to define the molecular pathways involved in ALT activation.

Whole genome sequencing of melanomas in adolescent and young adults reveals distinct mutation landscapes and the potential role of germline variants in disease susceptibility.

Cutaneous melanoma accounts for at least >10% of all cancers in adolescents and young adults (AYA, 15-30 years of age) in Western countries. To date, little is known about the correlations between germline variants and somatic mutations and mutation signatures in AYA melanoma patients that might explain why they have developed a cancer predominantly affecting those over 65 years of age. We performed genomic analysis of 50 AYA melanoma patients (onset 10-30 years, median 20); 25 underwent whole genome sequencing (WGS) of both tumor and germline DNA, exome data were retrieved from 12 TCGA AYA cases, and targeted DNA sequencing was conducted on 13 cases. The AYA cases were compared with WGS data from 121 adult cutaneous melanomas. Similar to mature adult cutaneous melanomas, AYA melanomas showed a high mutation burden and mutation signatures of ultraviolet radiation (UVR) damage. The frequencies of somatic mutations in BRAF (96%) and PTEN (36%) in the AYA WGS cohort were double the rates observed in adult melanomas (Q < 6.0 × 10-6 and 0.028, respectively). Furthermore, AYA melanomas contained a higher proportion of non-UVR-related mutation signatures than mature adult melanomas as a proportion of total mutation burden (p = 2.0 × 10-4 ). Interestingly, these non-UVR mutation signatures relate to APOBEC or mismatch repair pathways, and germline variants in related genes were observed in some of these cases. We conclude that AYA melanomas harbor some of the same molecular aberrations and mutagenic insults occurring in older adults, but in different proportions. Germline variants that may have conferred disease susceptibility correlated with somatic mutation signatures in a subset of AYA melanomas.

Mixed ductal-lobular carcinomas: evidence for progression from ductal to lobular morphology.

Mixed ductal-lobular carcinomas (MDLs) show both ductal and lobular morphology, and constitute an archetypal example of intratumoural morphological heterogeneity. The mechanisms underlying the coexistence of these different morphological entities are poorly understood, although theories include that these components either represent 'collision' of independent tumours or evolve from a common ancestor. We performed comprehensive clinicopathological analysis of a cohort of 82 MDLs, and found that: (1) MDLs more frequently coexist with ductal carcinoma in situ (DCIS) than with lobular carcinoma in situ (LCIS); (2) the E-cadherin-catenin complex was normal in the ductal component in 77.6% of tumours; and (3) in the lobular component, E-cadherin was almost always aberrantly located in the cytoplasm, in contrast to invasive lobular carcinoma (ILC), where E-cadherin is typically absent. Comparative genomic hybridization and multiregion whole exome sequencing of four representative cases revealed that all morphologically distinct components within an individual case were clonally related. The mutations identified varied between cases; those associated with a common clonal ancestry included BRCA2, TBX3, and TP53, whereas those associated with clonal divergence included CDH1 and ESR1. Together, these data support a model in which separate morphological components of MDLs arise from a common ancestor, and lobular morphology can arise via a ductal pathway of tumour progression. In MDLs that present with LCIS and DCIS, the clonal divergence probably occurs early, and is frequently associated with complete loss of E-cadherin expression, as in ILC, whereas, in the majority of MDLs, which present with DCIS but not LCIS, direct clonal divergence from the ductal to the lobular phenotype occurs late in tumour evolution, and is associated with aberrant expression of E-cadherin. The mechanisms driving the phenotypic change may involve E-cadherin-catenin complex deregulation, but are yet to be fully elucidated, as there is significant intertumoural heterogeneity, and each case may have a unique molecular mechanism. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Signatures of mutational processes in human cancer.

All cancers are caused by somatic mutations; however, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single cancer class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, 'kataegis', is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer, with potential implications for understanding of cancer aetiology, prevention and therapy.

Diff-Quik Cytology Smears from Endobronchial Ultrasound Transbronchial Needle Aspiration Lymph Node Specimens as a Source of DNA for Next-Generation Sequencing Instead of Cell Blocks.

BACKGROUND: Next-generation sequencing (NGS) in lung cancer specimens from endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) is usually performed on formalin-fixed paraffin-embedded cell block material. OBJECTIVES: Since DNA can be damaged by this process, we investigated the potential of using DNA extracted from Diff-Quik cytology smears made for rapid on-site evaluation during EBUS-TBNA. METHODS: In a prospective study, 67 patients undergoing diagnostic EBUS-TBNA were ana-lysed. We compared cell blocks and smears for DNA yields and sequencing (TruSeq Amplicon Cancer Panel) outcomes. Smears were also evaluated for tumour cell fraction and overall cellularity (cell count). RESULTS: Primary lung cancer was diagnosed in 64 patients and metastatic malignancy in 3 patients. The DNA yield from smears was significantly higher than that obtained from matched cell blocks (mean 1,740 vs. 434 ng; p = 0.001). For 33 cases with matched smears and cell blocks the mutation profiles were similar. Smears with abundant malignant cells (using a cut-off of > 25% tumour cell fraction and > 1,000 cells) accurately predicted high (> 50 ng) DNA yield and therefore success in triaging samples to sequencing. In terms of tissue workflow, using only smears as source DNA for sequencing was an improvement in the use of only cell blocks (54/67 [80.6%] vs. 41/67 [61.2%]); however, the use of cell blocks when smears were not available or did not yield sufficient DNA further improved the success rate to 62/67 (92.5%) cases. CONCLUSION: We recommend smears in laboratory workflows as the primary source of DNA for NGS following an EBUS procedure.

Unexpected UVR and non-UVR mutation burden in some acral and cutaneous melanomas.

Ultraviolet radiation (UVR) mutagenesis causes nearly all cutaneous melanomas, however, since UVR signatures are largely absent in acral melanoma, as well as melanoma in sun-protected sites, the cause of these melanomas is unknown. Whole-genome sequencing data generated as part of the Australian Melanoma Genome Project was supplemented with a detailed histopathological assessment with the melanomas then classified as UVR or non-UVR related, based on their mutation signatures. The clinicopathological characteristics of melanomas with mutation signatures for their subtype were compared. Three (of 35=8.6%) acral melanomas, all clinically and pathologically verified as arising from acral or subungual locations, had predominant UVR mutation burden, whereas four (of 140=2.9%) cutaneous melanomas showed predominant non-UVR mutations. Among the acral melanomas, the few that were UVR dominant occurred in younger patients, had a higher mutation load and a proportion of mutation burden due to UVR, which was similar to that in melanomas from intermittently UVR-exposed skin. Acral melanomas with a UVR signature occurred most frequently in subungual sites and included tumors harboring BRAF or NF1 mutations. Cutaneous melanomas dominated by non-UVR signatures had lower mutation burdens counts and their primary tumors were thicker and had more mitoses than in other cutaneous melanomas. No histopathological features predicted UVR dominance in acral melanomas or non-UVR dominance in cutaneous melanomas. Our finding of acral/subungual melanomas with predominant UVR mutagenesis suggests that the nail plate and acral skin do not provide complete protection from UVR. Our data also confirm that cutaneous melanomas not caused by UVR are infrequent. Identifying where mutation burden is discordant with primary tumor anatomical site is likely to be clinically significant when determining treatment options for metastatic acral and cutaneous melanoma patients.

Whole exome sequencing of an asbestos-induced wild-type murine model of malignant mesothelioma.

BACKGROUND: Malignant mesothelioma (MM) is an aggressive cancer of the pleural and peritoneal cavities caused by exposure to asbestos. Asbestos-induced mesotheliomas in wild-type mice have been used extensively as a preclinical model because they are phenotypically identical to their human counterpart. However, it is not known if the genetic lesions in these mice tumours are similar to in the human disease, a prerequisite for any new preclinical studies that target genetic abnormalities. METHODS: We performed whole exome sequencing of fifteen asbestos-induced murine MM tumour cell lines from BALB/c, CBA and C57BL/6 mouse strains and compared the somatic mutations and copy number variations with those recurrently reported in human MM. We then catalogued and characterised the mutational landscape of the wild-type murine MM tumours. Quantitative RT-PCR was used to interrogate the expression of key MM genes of interest in the mRNA. RESULTS: Consistent with human MM tumours, we identified homozygous loss of the tumour suppressor Cdkn2a in 14/15 tumours. One tumour retained the first exon of both of the p16INK4a and p19ARF isoforms though this tumour also contained genetic amplification of Myc resulting in increased expression of the c-Myc proto-oncogene in the mRNA. There were no chromosomal losses in either the Bap1 or Nf2 regions. One tumour harbored homozygous loss of Trp53 in the DNA. Mutation rates were similar in tumours generated in the CBA and C57BL/6 strains when compared to human MM. Interestingly, all BALB/c tumour lines displayed high mutational loads, consistent with the known mutator phenotype of the host strain. The Wnt, MAPK and Jak-STAT signaling pathways were found to be the most commonly affected biological pathways. Mutations and copy number deletions also occurred in the Hedgehog and Hippo pathways. CONCLUSIONS: These data suggest that in the wild-type murine model asbestos causes mesotheliomas in a similar way to in human MM. This further supports the notion that the murine model of MM represents a genuine homologue of the human disease, something uncommon in cancer, and is thus a valuable tool to provide insight into MM tumour development and to aide the search for novel therapeutic strategies.

Computational approaches to identify functional genetic variants in cancer genomes.

The International Cancer Genome Consortium (ICGC) aims to catalog genomic abnormalities in tumors from 50 different cancer types. Genome sequencing reveals hundreds to thousands of somatic mutations in each tumor but only a minority of these drive tumor progression. We present the result of discussions within the ICGC on how to address the challenge of identifying mutations that contribute to oncogenesis, tumor maintenance or response to therapy, and recommend computational techniques to annotate somatic variants and predict their impact on cancer phenotype.

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance.

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.

PGTools: A Software Suite for Proteogenomic Data Analysis and Visualization.

We describe PGTools, an open source software suite for analysis and visualization of proteogenomic data. PGTools comprises applications, libraries, customized databases, and visualization tools for analysis of mass-spectrometry data using combined proteomic and genomic backgrounds. A single command is sufficient to search databases, calculate false discovery rates, group and annotate proteins, generate peptide databases from RNA-Seq transcripts, identify altered proteins associated with cancer, and visualize genome scale peptide data sets using sophisticated visualization tools. We experimentally confirm a subset of proteogenomic peptides in human PANC-1 cells and demonstrate the utility of PGTools using a colorectal cancer data set that led to the identification of 203 novel protein coding regions missed by conventional proteomic approaches. PGTools should be equally useful for individual proteogenomic investigations as well as international initiatives such as chromosome-centric Human Proteome Project (C-HPP). PGTools is available at http://qcmg.org/bioinformatics/PGTools.

Genome-wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT-ROBO, ITGA2 and MET signaling.

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-ß, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

Copy number and targeted mutational analysis reveals novel somatic events in metastatic prostate tumors.

Advanced prostate cancer can progress to systemic metastatic tumors, which are generally androgen insensitive and ultimately lethal. Here, we report a comprehensive genomic survey for somatic events in systemic metastatic prostate tumors using both high-resolution copy number analysis and targeted mutational survey of 3508 exons from 577 cancer-related genes using next generation sequencing. Focal homozygous deletions were detected at 8p22, 10q23.31, 13q13.1, 13q14.11, and 13q14.12. Key genes mapping within these deleted regions include PTEN, BRCA2, C13ORF15, and SIAH3. Focal high-level amplifications were detected at 5p13.2-p12, 14q21.1, 7q22.1, and Xq12. Key amplified genes mapping within these regions include SKP2, FOXA1, and AR. Furthermore, targeted mutational analysis of normal-tumor pairs has identified somatic mutations in genes known to be associated with prostate cancer including AR and TP53, but has also revealed novel somatic point mutations in genes including MTOR, BRCA2, ARHGEF12, and CHD5. Finally, in one patient where multiple independent metastatic tumors were available, we show common and divergent somatic alterations that occur at both the copy number and point mutation level, supporting a model for a common clonal progenitor with metastatic tumor-specific divergence. Our study represents a deep genomic analysis of advanced metastatic prostate tumors and has revealed candidate somatic alterations, possibly contributing to lethal prostate cancer.