Funded by the National Institutes of Health (NIH), the aim of the Model Organism ENCyclopedia of DNA Elements (modENCODE) project is to provide the biological research community with a comprehensive ...encyclopedia of functional genomic elements for both model organisms C. elegans (worm) and D. melanogaster (fly). With a total size of just under 10 terabytes of data collected and released to the public, one of the challenges faced by researchers is to extract biologically meaningful knowledge from this large data set. While the basic quality control, pre-processing, and analysis of the data has already been performed by members of the modENCODE consortium, many researchers will wish to reinterpret the data set using modifications and enhancements of the original protocols, or combine modENCODE data with other data sets. Unfortunately this can be a time consuming and logistically challenging proposition.
In recognition of this challenge, the modENCODE DCC has released uniform computing resources for analyzing modENCODE data on Galaxy (https://github.com/modENCODE-DCC/Galaxy), on the public Amazon Cloud (http://aws.amazon.com), and on the private Bionimbus Cloud for genomic research (http://www.bionimbus.org). In particular, we have released Galaxy workflows for interpreting ChIP-seq data which use the same quality control (QC) and peak calling standards adopted by the modENCODE and ENCODE communities. For convenience of use, we have created Amazon and Bionimbus Cloud machine images containing Galaxy along with all the modENCODE data, software and other dependencies.
Using these resources provides a framework for running consistent and reproducible analyses on modENCODE data, ultimately allowing researchers to use more of their time using modENCODE data, and less time moving it around.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We sequenced 11 germline exomes from five families with familial pancreatic cancer (FPC). One proband had a germline nonsense variant in ATM with somatic loss of the variant allele. Another proband ...had a nonsense variant in PALB2 with somatic loss of the variant allele. Both variants were absent in a relative with FPC. These findings question the causal mechanisms of ATM and PALB2 in these families and highlight challenges in identifying the causes of familial cancer syndromes using exome sequencing.
We have developed a computational framework for spatiotemporal integration of molecular and anatomical datasets in a virtual reality environment. Using two case studies involving gene expression data ...and pharmacokinetic data, respectively, we demonstrate how existing knowledge bases for molecular data can be semantically mapped onto a standardized anatomical context of human body. Our data mapping methodology uses ontological representations of heterogeneous biomedical datasets and an ontology reasoner to create complex semantic descriptions of biomedical processes. This framework provides a means to systematically combine an increasing amount of biomedical imaging and numerical data into spatiotemporally coherent graphical representations. Our work enables medical researchers with different expertise to simulate complex phenomena visually and to develop insights through the use of shared data, thus paving the way for pathological inference, developmental pattern discovery and biomedical hypothesis testing.
Abstract
We recognize that many cancers are highly complex mixtures of sub-populations of cells, which are also influenced by their microenvironment. This heterogeneity explains in part, why the ...majority of current therapeutic approaches for cancer work best when multiple agents are combined. Therefore, in an era of targeted therapeutics it becomes critical to understand the complexity of tumors both at diagnosis and over the course of therapy, including measures of heterogeneity. Here we present genomic, transcriptomic and in situ proteomic profiling of a cohort of breast cancer (BCa) lumpectomies to reveal the extent of heterogeneity in pathologically defined unifocal and multifocal cancers. Integration of molecular profiling, phylogenetic analyses and radiomics has the potential to significantly improve BCa clinical management and stratification to targeted therapies that are already available in the clinic. In this study, lumpectomies with imaging data were processed as whole mounts with serial blocks reviewed. Tissue cores were taken from at least three different regions through the lumpectomy for nucleic acid extraction and tissue microarray (TMA) construction, focusing on morphologic/histological differences in addition to the spatial orientation of the sampled region within the lumpectomy. Targeted sequencing using the Oncomine Comprehensive Assay v3 (OCAv3); transcriptional profiling using the NanoString Breast Cancer 360 Panel; in situ profiling by multiplex fluorescence immunohistochemistry (MxIF) and Digital Spatial Profiling (see abstract Spears et al) were performed. From this cohort of 60 patients, we present a subset of patients demonstrating integration of the molecular profiling to reveal the phylogenetic relationship between the multiple samplings and the impact on clinical decision making in BCa. Briefly, we identified differences in the molecular subtypes between the different sample regions from the same unifocal cancer as well as differences in the predicted responses to anti-PDL1 therapy by transcriptional profiling; while targeted sequencing of driver mutations suggested the likelihood of an ancestral tumor cell giving rise to the lesions in pathologically defined multifocal cancers. However it was evident that genes and pathways found to be aberrant in these different lesions from the same cancer could impact the response to standard BCa treatment, or the selection of targeted therapies. In situ proteomics demonstrated differences in the expression of standard BCa markers ER, PgR, HER2 and Ki67 in addition to immune markers in the tumor and tumor microenvironment. While there are clinically validated transcriptional risk test available for BCa, we have demonstrated that transcriptomics or genomics alone is insufficient for a rational stratification of patients to currently available targeted therapies; therefore supporting the need for an integrative approach.
Citation Format: Jane Bayani, Quang M. Trinh, Mary Anne Quintayo, Cheryl Crozier, Megan Hopkins, Jingping Qiao, Alison Cheung, James Mainprize, Quaid Morris, Melanie Spears, Martin Yaffe, Lincoln Stein, John M. Bartlett. Tumour spatial heterogeneity in breast cancer and the impact on clinical management abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3131.
Abstract
Many cancers are highly complex mixtures of many sub-populations of cells, also influenced by their microenvironment. This heterogeneity explains, in part, why the majority of current ...therapeutic approaches for cancer work best when multiple agents are combined. Therefore, in an era of targeted therapeutics it becomes critical to understand the complexity of tumours both at diagnosis and over the course of therapy, including measures of heterogeneity. Here we present genomic, transcriptomic and in situ proteomic profiling of a cohort of breast cancer (BCa) lumpectomies with associated imaging data to reveal the extent of heterogeneity in pathologically defined unifocal and multifocal cancers. Integration of molecular profiling, phylogenetic analyses and radiomics has the potential to significantly improve BCa clinical management and stratification to targeted therapies that are already available in the clinic. In this study, lumpectomies were processed as whole mounts with serial blocks reviewed. Tissue cores were taken from at least three different regions throughout the lumpectomy for nucleic acid extraction and tissue microarray (TMA) construction, focusing on morphologic/histological differences in addition to the spatial orientation of the sampled region within the lumpectomy. Targeted sequencing using the Oncomine Comprehensive Assay v3 (OCAv3); transcriptional profiling using the NanoString Breast Cancer 360 Panel; and in situ profiling by multiplex fluorescence immunohistochemistry (MxIF) performed (see abstract Cheung et al). From this cohort of 60 patients, we present a subset of patients demonstrating integration of the molecular profiling to reveal the phylogenetic relationship between the multiple samplings and the potential impact on clinical decision making in BCa. We identified differences in the molecular subtypes between the different sample regions from the same unifocal cancer as well as differences in the predicted responses to anti-PDL1 therapy by transcriptional profiling. Targeted sequencing of driver mutations suggested the likelihood of an ancestral tumour cell giving rise to the lesions in pathologically defined multifocal cancers; however it was evident that genes and pathways found to be aberrant in these different lesions from the same cancer could impact the response to standard BCa treatment, or the selection of targeted therapies. In situ proteomics demonstrated differences in the expression of standard BCa markers ER, PgR, HER2 and Ki67, in addition to immune markers in the tumour and its microenvironment. While there are clinically validated transcriptional risk tests available for BCa, we have demonstrated that transcriptomics or genomics alone is insufficient for a rational stratification of patients to currently available targeted therapies, therefore, supporting the need for an integrative approach.
Citation Format: Jane Bayani, Quang M. Trinh, Mary Anne Quintayo, Cheryl Crozier, Megan Hopkins, Jingping Qiao, Alison Cheung, James G Mainprize, Quaid Morris, Melanie Spears, Martin J. Yaffe, Lincoln D. Stein, John M.S. Bartlett. Revealing tumour spatial heterogeneity in breast cancer and the impact on clinical management abstract. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-002.
Abstract
Due to breast screening, ductal carcinoma in situ (DCIS) accounts for approximately 25% of all newly diagnosed breast neoplasms. Believed to be a precursor to invasive carcinoma, a ...significant number of patients diagnosed with DCIS are effectively managed by surgery alone or in conjunction with radiotherapy and endocrine therapy. In general, there is an 8-11% relative risk for a subsequent invasive carcinoma over a period of 10 year, with 98% breast cancer-specific survival after 10 years of follow up. Although mastectomy, breast conserving surgery, and radiotherapy can reduce the risk of recurrence, there are ongoing lifetime consequences of treatment. Thus, there is a clinical need to identify those patients who are at risk of an invasive recurrence from those who might not recur or experience a subsequent DCIS recurrence. In this study, 60 patients with pure primary DCIS, treated with only with breast conserving therapy, across three different clinical outcome groups were examined: patients who did not experience a recurrence within 5 years (n=20); patients who experienced a recurrent DCIS within 5 years (n=20); and patients that recurred with an invasive cancer within 5 years (n=20). Pure primary DCIS lesions, as well as the matched DCIS recurrence or invasive recurrence, were macrodissected from formalin fixed paraffin embedded tissues and subjected to nucleic acid extraction. All samples were profiled using Thermo Fisher Scientific’s validated targeted sequencing panel, the Oncomine Comprehensive Assay v3.0 (OCAv3.0). This assay is comprised of common cancer driver genes shown to be prognostic and predictive to targeted therapies in use or in late-phase clinical trials, and is currently being used in the NCI-MATCH trial (NCT02465060). While the OCA panel and accompanying Oncomine Knowledgebase Reporter provides information regarding the targeted treatments linked to known actionable mutations, this study utilized all somatic mutations and copy number changes to reveal the genomic landscape of DCIS and their matched recurrences across these pan-cancer driver genes Amongst all primary DCIS samples across the three different clinical outcome groups, PIK3CA, was frequently found to be affected by SNV and Indels (32.8%) in addition to TP53 (26.2%), NF1 (21.3%), CREBBP (16.4%), ATM (14.8%), PALB2 (14.8%). DNA repair genes, including CHEK1, RAD50, RAD51B, MRE11A, BRCA1 and BRCA2, were found to be frequently subject to mutation in these primary DCIS samples ranging from 5%-15%. Similarly, copy number gains were frequently detected in HER2 (26.2%), CDK12 (18%), FGFR1 (4.9%), GNAS (4.9%), MYC, CCNE1, AR, RAD51C and RNF43 (1.6% each), and loses at H3F3A and KNSTRN (each 1.6%). Matched primary DCIS and their recurrent DCIS or invasive lesions exhibited similar changes with invasive cancers suggesting that in some cases, the primary DCIS gives rise to the invasive cancer. We will present the preliminary findings mapping the mutational and copy-number landscape of primary DCIS and matched recurrences to identify putative genomic changes defining these clinical outcome groups and to investigate the genomic progression of DCIS to invasive carcinoma.
Citation Format: Jane Bayani, Quang M Trinh, Mary Anne Quintayo, Cheryl Crozier, Ilinca Lungu, Dan Dion, Joema Felipe-Lima, Giancarlo Pruneri, Jonas Bergh, Fredrik Warnberg, Giuseppe Viale, Lincoln D Stein, John MS Bartlett. The mutational landscape of cancer driver genes in matched primary ductal carcinoma in situ and recurrent ductal carcinoma in situ or recurrent invasive cancers abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-22.
Obesity is an established risk factor for colorectal cancer (CRC), but the evidence for the association is inconsistent across molecular subtypes of the disease.
We pooled data on body mass index ...(BMI), tumor microsatellite instability status, CpG island methylator phenotype status, BRAF and KRAS mutations, and Jass classification types for 11 872 CRC cases and 11 013 controls from 11 observational studies. We used multinomial logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI) adjusted for covariables.
Higher BMI was associated with increased CRC risk (OR per 5 kg/m2 = 1.18, 95% CI = 1.15 to 1.22). The positive association was stronger for men than women but similar across tumor subtypes defined by individual molecular markers. In analyses by Jass type, higher BMI was associated with elevated CRC risk for types 1-4 cases but not for type 5 CRC cases (considered familial-like/Lynch syndrome microsatellite instability-H, CpG island methylator phenotype-low or negative, BRAF-wild type, KRAS-wild type, OR = 1.04, 95% CI = 0.90 to 1.20). This pattern of associations for BMI and Jass types was consistent by sex and design of contributing studies (cohort or case-control).
In contrast to previous reports with fewer study participants, we found limited evidence of heterogeneity for the association between BMI and CRC risk according to molecular subtype, suggesting that obesity influences nearly all major pathways involved in colorectal carcinogenesis. The null association observed for the Jass type 5 suggests that BMI is not a risk factor for the development of CRC for individuals with Lynch syndrome.
Abstract
Colorectal cancer (CRC) is a heterogenous disease that develops through somatic mutations in driver genes, leading to activation of diverse neoplastic pathways. To systematically examine if ...somatically mutated genes and pathways impact survival, we sequenced tumor and normal DNA samples for 4,512 CRC cases using a targeted panel. We performed Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CIs) for associations of disease-specific (DS)-survival with somatically mutated genes and pathways, adjusting for age and sex, and stratifying baseline hazards by study population. We assessed statistical significance using Bonferroni p-value thresholds to account for multiple testing of 214 genes (2.34x10-4) and 6 key CRC pathways (8.3x10-3). We limited analyses to non-silent mutations.
We observed that DS-survival was significantly more favorable among individuals with hypermutated (HM) tumors, primarily consisting of microsatellite unstable and POLE-mutated tumors, as compared to those with non-hypermutated (NHM) tumors (HR=0.4, 95% CI: 0.3-0.5, p=1.2x10-17). BRAF V600E mutations were associated with poorer survival (HR=2.0, 95% CI: 1.6-2.5, p=1.6x10-10). This association was more pronounced among NHM tumors (HR=2.3, 95% CI: 1.8-2.9, p=4.3x10-12). We identified suggestive associations (p-values < 5.0x10-3 in overall or stratified analyses) between DS-survival and mutations in B2M, TP53, and SMAD4. Mutations in B2M may provide more favorable prognosis for survival (HR=0.5, 95% CI: 0.3-0.8, p=4.4x10-3), with similar effect sizes in HM and NHM tumors. Poorer survival may be associated with mutations in TP53 (HR=1.2, 95% CI:1.2-1.4, p=9.3x10-4) and SMAD4 (HR=1.3, 95% CI:1.1-1.6, p=3.2x10-3) in NHM tumors.
We further observed statistically significant associations between survival and three mutated pathways: TP53/ATM (HR=1.2, 95% CI:1.1-1.4, p=9.0x10-4), receptor tyrosine kinases (RTK) and RAS (HR=1.3, 95% CI:1.1-1.5, p=4.5x10-5), and TGF-beta (HR=1.3, 95% CI:1.1-1.5, p=2.9x10-4). Findings for TP53/ATM and RTK/RAS were primarily due to mutations in one gene within each pathway (TP53 and BRAF, respectively). However, the TGF-beta pathway finding, which was more pronounced in NHM tumors, included two genes with p-values less than 0.05 (SMAD4 p=3.2x10-3 and TGFBR2 p=0.01).
Despite our large sample size, relatively few somatically mutated genes were significantly associated with DS-survival. It may be that pathway-level testing affords more power for analyses and that larger sample sizes are needed. It is of interest that non-silent mutations in B2M, whose product is a component of the class I major histocompatibility complex, was associated with improved DS-survival, though our finding was not statistically significant after multiple testing correction so further exploration and replication is needed.
Citation Format: Tabitha A. Harrison, Syed H. Zaidi, Conghui Qu, Amanda I. Phipps, Robert S. Steinfelder, Quang M. Trinh, Sonja I. Berndt, Daniel D. Buchanan, Peter T. Campbell, Andrew T. Chan, Kim F. Doheny, David A. Drew, Jane C. Figueiredo, Steven J. Gallinger, Marios Giannakis, Andrea Gsur, Marc J. Gunter, Michael Hoffmeister, Wen-Yi Huang, Paul J. Limburg, Victor Moreno, Polly A. Newcomb, Shuji Ogino, Ross L. Prentice, Tameka Shelford, Wei Sun, Stephen N. Thibodeau, Li Hsu, Ulrike Peters. Associations of somatically mutated genes and pathways with colorectal cancer specific survival in 4,500 colorectal cancer patients abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB090.
Abstract
Introduction: Male breast cancer (BCa) is a rare disease accounting for less than 1% of all breast cancers (BC) and 1% of all cancers in males. The clinical management is largely ...extrapolated from female BCa. Few studies have examined the genomic landscape of male BCas, with six male BCas included in The Cancer Genome Atlas (TCGA). Familial studies of male BCas have shown genomic changes similar to female BCa, while a larger targeted sequencing study of 59 male breast cancers identified recurrent mutations affecting PIK3CA and GATA3. To date, there is still limited information regarding the genomic landscape of male BCas; particularly in the context of identifying targeted treatments. To reveal genomic changes that characterize male BCas in the context of known cancer driver genes linked to prognosis and targeted agents, we performed a targeted sequencing study on 248 male BCas from the International Male Breast Cancer Program. Methods: 248 primary M0, ER+ve, HER2-ve male BCas enrolled in the Part 1 (retrospective joint analysis) International Male Breast Cancer Program of 1483 patients diagnosed between 1990-2010 (Cardoso et al. Annals of Oncology, 2018) were processed for nucleic acid extraction from formalin-fixed paraffin embedded (FFPE) tissues. Using the Thermo Fisher Scientific Oncomine Comprehensive Assay v3 (OCAv3), a validated targeted sequencing panel currently used in the NCI-MATCH trial (NCT02465060), we evaluated mutational and copy number variations (CNVs) of genes that are prognostic or predictive to targeted therapies currently in use in the clinic or late-stage clinical trials. The OCAv3 DNA pan-cancer panel assays 115 genes for determining mutational status (48 full coding and 67 hotspot) as well as copy-number assessment in 43 genes. The OCAv3 uses Ampliseq-based technology linked to the Oncomine NGS workflow to identify actionable mutations and CNVs Results: Of the 248 samples assayed, 216 passed strict quality control parameters (87.1%). Using the Oncomine NGS workflow, actionable mutations at ≥5% variant allele frequency (VAF) were most frequently identified in PIK3CA (29.2%), BRCA2 (11.1%), NF1 (11.6%), indels in TP53 (10.6%), ATR (5.6%), ATRX (5.1%), indel BRCA2 (5.1%), TP53 point mutations (4.6%), MET (4.6%), ATM (4.6%), NOTCH2 (4.6%), CHEK1 (4.2%), FANCI (4.2%), PTEN (3.2%) with a number of additional genes identified at lower frequencies. Gene amplifications were most frequently detected in MYC (24.5%), FGFR1 (14.8%), CCND1 (12%), FGF3 (9.7%), FGF19 (9.7%), MDM2 (6.5%), CDK4 (1.4%), FGFR3, MDM4, ERBB2 (0.9% each), and FLT3, AR, MYCL, CDK6, IGF1R, FGFR4, KRAS, AKT3 and ESR1 (0.5% each). Although the results here describe the mutations and copy-number changes deemed to be actionable, further analysis of all non-actionable somatic mutations and CNVs will be presented and compared to female BCas previously assayed using the same panel. Conclusion: In this targeted sequencing study of the largest series of male BCas to our knowledge, we have revealed that PIK3CA continues to be a frequently altered gene in both male and female BCas. However, there is an enrichment of mutations in genes related to DNA repair in male BCs. Interestingly, while MYC is commonly amplified in female BCa, a higher frequency of amplified cases were seen in male BCas, in contrast to female BCas. Together with our previously generated transcriptional profiling data in this data set, we believe that both common and unique biological processes comprising male and female BCas will ultimately improve their clinical management and move towards the goal of precision medicine.
This work has been funded by the Breast Cancer Research Foundation (BCRF).
Citation Format: Jane Bayani, Coralie Poncet, Cheryl Crozier, Quang M Trinh, Megan Hopkins, Aime Lambert Uwimana, Tammy Piper, Carrie Cunningham, Monika Sobol, Stefan Aebi, Kim Benstead, Oliver Bogler, Lissandra Dal Lago, Florentine Hilbers, Ingrid Hedenfalk, Larissa Korde, Barbro Linderholm, John Martens, Lavinia Middleton, Melissa Murray, Catherine Kelly, Cecilia Nilsson, Monika Nowaczyk, Stephanie Peeters, Aleksandra Peric, Peggy Porter, Carolien Schröder, Isabel T Rubio, Kathryn J Ruddy, Christi van Asperen, Danielle Van Den Weyngaert, Carolien HM van Deurzen, Elise van Leeuwen-Stok, Joanna Vermeij, Eric Winer, Lincoln D Stein, Sharon H Giordano, Fatima Cardoso, John MS Bartlett. The genomic landscape of male breast cancers using the oncomine comprehensive assay for actionable mutations abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-03.
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Background: Hormone receptor positive breast cancer is a therapeutic challenge. Despite optimal anti-endocrine therapies, most breast cancer deaths follow a diagnosis of early ...luminal cancer. To understand the impact of multiple aberrations in the context of current therapy, we assessed the prognostic ability of genomic signatures as a putative stratification tool to targeted therapies. Methods: This a priori study is based on molecular pathways which might predict response to targeted therapies. DNA from 420 patients from the phase III TEAM pathology cohort were used. Patients with a distant recurrence within 5 years were matched by clinical variables to those disease-free at follow up. Copy number analysis was performed using the Affymetrix Oncoscan Assay. Targeted sequencing was performed in a subset of samples for genes based on signaling cassettes mined from the ICGC. Pathways were identified as aberrant if there were copy number variations (CNVs) and/or mutations in any of the pre-determined pathway genes: 1) CCND1/CCND2/CCND3/CDK4/CDK6; 2) FGFR1/FGFR2/FGFR2/FGFR4; and 3) AKT1/AKT2/PIK3CA/PTEN. Kaplan-Meier and log-rank analyses were used for DFS between groups. Hazard ratios were calculated using the Cox proportional hazard models adjusted for age, tumour size, grade, lymph node and HER2 status. Results: 390/420 samples passed informatics QC filters. For the CCND/CDK pathway, patients with no CNV changes experienced a better DFS (HR = 1.7, 95% CI 1.3-2.3, p < 0.001). For the FGFR/FGF pathway, a similar outcome is seen among patients without CNVs (HR = 1.5, 95% CI 1.1-2.0; p = 0.005). For AKT/PIK3CA, a decrease in DFS was seen in those with aberrations (HR = 1.4, 95% CI 1.0-1.8, p = 0.03). Conclusions: We demonstrated that CNVs of genes within CDK4/CCND, PIK3CA/AKT and FGFR pathways are independently linked to high risk of relapse following endocrine treatment, with implications for identifying those patients who are at high-risk for recurrence despite optimal anti-endocrine therapy and linking molecular features driving these cancers to targeted therapies.
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