Citations

Background BRCA1 interacting protein C-terminal helicase 1 (BRIP1) is one of the Fanconi Anaemia Complementation (FANC) group family of DNA repair proteins. Biallelic mutations in BRIP1 are responsible for FANC group J, and previous studies have also suggested that rare protein truncating variants in BRIP1 are associated with an increased risk of breast cancer. These studies have led to inclusion of BRIP1 on targeted sequencing panels for breast cancer risk prediction. Methods We evaluated a truncating variant, p.Arg798Ter (rs137852986), and 10 missense variants of BRIP1, in 48 144 cases and 43 607 controls of European origin, drawn from 41 studies participating in the Breast Cancer Association Consortium (BCAC). Additionally, we sequenced the coding regions of BRIP1 in 13 213 cases and 5242 controls from the UK, 1313 cases and 1123 controls from three population-based studies as part of the Breast Cancer Family Registry, and 1853 familial cases and 2001 controls from Australia. Results The rare truncating allele of rs137852986 was observed in 23 cases and 18 controls in Europeans in BCAC (OR 1.09, 95% CI 0.58 to 2.03, p=0.79). Truncating variants were found in the sequencing studies in 34 cases (0.21%) and 19 controls (0.23%) (combined OR 0.90, 95% CI 0.48 to 1.70, p=0.75). Conclusions These results suggest that truncating variants in BRIP1, and in particular p.Arg798Ter, are not associated with a substantial increase in breast cancer risk. Such observations have important implications for the reporting of results from breast cancer screening panels.

Abstract Many somatic mutations have been detected in pancreatic ductal adenocarcinoma (PDAC), leading to the identification of some key drivers of disease progression, but the involvement of large genomic rearrangements has often been overlooked. In this study, we performed mate pair sequencing (MPseq) on genomic DNA from 24 PDAC tumors, including 15 laser-captured microdissected PDAC and 9 patient-derived xenografts, to identify genome-wide rearrangements. Large genomic rearrangements with intragenic breakpoints altering key regulatory genes involved in PDAC progression were detected in all tumors. SMAD4, ZNF521, and FHIT were among the most frequently hit genes. Conversely, commonly reported genes with copy number gains, including MYC and GATA6, were frequently observed in the absence of direct intragenic breakpoints, suggesting a requirement for sustaining oncogenic function during PDAC progression. Integration of data from MPseq, exome sequencing, and transcriptome analysis of primary PDAC cases identified limited overlap in genes affected by both rearrangements and point mutations. However, significant overlap was observed in major PDAC-associated signaling pathways, with all PDAC exhibiting reduced SMAD4 expression, reduced SMAD-dependent TGFβ signaling, and increased WNT and Hedgehog signaling. The frequent loss of SMAD4 and FHIT due to genomic rearrangements strongly implicates these genes as key drivers of PDAC, thus highlighting the strengths of an integrated genomic and transcriptomic approach for identifying mechanisms underlying disease initiation and progression. Cancer Res; 76(3); 749–61. ©2015 AACR.

Abstract The prevalence of germline pathogenic mutations in a comprehensive panel of cancer predisposition genes is not well-defined for patients with pancreatic ductal adenocarcinoma (PDAC). To estimate the frequency of mutations in a panel of 22 cancer predisposition genes, 96 patients unselected for a family history of cancer who were recruited to the Mayo Clinic Pancreatic Cancer patient registry over a 12-month period were screened by next-generation sequencing. Fourteen pathogenic mutations in 13 patients (13.5%) were identified in eight genes: four in ATM, two in BRCA2, CHEK2, and MSH6, and one in BARD1, BRCA1, FANCM, and NBN. These included nine mutations (9.4%) in established pancreatic cancer genes. Three mutations were found in patients with a first-degree relative with PDAC, and 10 mutations were found in patients with first- or second-degree relatives with breast, pancreas, colorectal, ovarian, or endometrial cancers. These results suggest that a substantial proportion of patients with PDAC carry germline mutations in predisposition genes associated with other cancers and that a better understanding of pancreatic cancer risk will depend on evaluation of families with broad constellations of tumors. These findings highlight the need for recommendations governing germline gene-panel testing of patients with pancreatic cancer. Cancer Epidemiol Biomarkers Prev; 25(1); 207–11. ©2015 AACR.

We used a target-centric strategy to identify transporter proteins upregulated in pancreatic ductal adenocarcinoma (PDAC) as potential targets for a functional imaging probe to complement existing anatomical imaging approaches. We performed transcriptomic profiling (microarray and RNASeq) on histologically confirmed primary PDAC tumors and normal pancreas tissue from 33 patients, including five patients whose tumors were not visible on computed tomography. Target expression was confirmed with immunohistochemistry on tissue microarrays from 94 PDAC patients. The best imaging target identified was SLC6A14 (a neutral and basic amino acid transporter). SLC6A14 was overexpressed at the transcriptional level in all patients and expressed at the protein level in 95% of PDAC tumors. Very little is known about the role of SLC6A14 in PDAC and our results demonstrate that this target merits further investigation as a candidate transporter for functional imaging of PDAC.

Abstract Introduction: Triple negative breast cancers (TNBC) are characterized as lacking estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression and TNBC patients have higher rates of recurrence and death compared with other breast cancer subtypes. For TNBC patients who fail standard chemotherapy, there are a lack of novel drug therapies, given the absence of well-defined molecular targets. Recently, a microarray meta-analysis identified 7 triple negative subtypes, including the validation of the luminal androgen receptor (LAR) positive subtype [Lehmann, 2011]. However, microarray technology is dependent on probe-target specificity and the 7 subtypes have yet to be validated using RNA sequencing data, and the presences of recurrent genomic alterations in the 7 subtypes are unknown. METHODS: We obtained 1106 breast cancer RNA-Seq bam files from The Cancer Genome Atlas (TCGA) and aligned with Tophat v1.3. The PAM50 intrinsic gene signature was used to extract a cohort of 128 TNBC samples. Consensus clustering of genes, greater than 75th percentile of variance, was performed using Kmeans clustering in Spearman's correlation space. A nearest centroid prediction model was developed from genes differentially expressed among the clusters. Eighty independent TNBC RNA sequencing samples were obtained (British Colombia; BC) [Shah, 2012] which were calibrated to our TNBC conditional quantile normalized cohort and sub-typed by our model. RESULTS: Using RNA-Seq gene expression count data, we identified 5 clusters, all of which were stable, including the LAR cluster. Signaling pathway impact analysis (SPIA) implicated cytokine-cytokine receptor interaction, leukocyte transendothelial migration, and regulation of actin cytoskeleton pathways commonly altered in the non-LAR TNBC subtypes. In contrast, cell cycle, ECM-receptor interaction, endocrine regulated calcium reabsorption, and insulin signaling pathways were altered in the LAR versus non-LAR subtypes. Neuroactive ligand-receptor interactions were observed to be altered commonly between all sub-types. We then applied our model to the Shah, et al cohort. In this cohort, the LAR subtype was consistent with Shah's classification of ‘other’ TNBC and contained no basal samples by PAM50 intrinsic modeling. Analysis of sub-type specific mutation data from the BC cohort demonstrates an increased mutational load in ECM-related proteins, particularly the myosins, along with increased TP53 clonality in the non-LAR subtypes. CONCLUSIONS: Using TCGA RNASeq data, we have confirmed the presence of 5 major TNBC subtypes, including the LAR; which was negligible in basal composition by PAM50 intrinsic modeling. SPIA pathway analysis indicates a core set of pathways demonstrating altered expression across the TNBC sub-types and the identification of molecular targets within each subtype is ongoing. Citation Format: Kevin J. Thompson, Xiaojia Tang, Zhifu Sun, Jason P. Sinnwell, Hugues Sicotte, Douglas W. Mahoney, Steven Hart, Peter T. Vedell, Poulami Barman, Jeanette E. Eckel Passow, Eric D. Wieben, James N. Ingle, Judy C. Boughey, Liewei Wang, Richard Weinshilboum, Krishna R. Kalari, Matthew P. Goetz. Molecular classification of triple negative breast cancer via RNA-sequencing data. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5592. doi:10.1158/1538-7445.AM2014-5592

Abstract INTRODUCTION Next generation sequencing (NGS) of patients has significantly changed our ways to study cancer genomics as it provides precise estimates of gene expression, fusion transcripts, expressed single nucleotide variants (eSNVs), splice variants and copy number variants. The Breast Cancer Genome Guided Therapy (BEAUTY) is an ongoing clinical study in which RNA sequencing (RNAseq) and whole exome sequencing (WES) are performed prior to, during and after neoadjuvant chemotherapy. Here we report the use of these sequencing technologies to investigate gene expression levels and mutational profiles in a triple negative breast cancer (TNBC) patient enrolled in BEAUTY whose disease did not respond to neoadjuvant paclitaxel and anthracycline/cycphosphamide. METHODS Computational approaches were used to integrate WES and RNAseq data obtained before therapy (V1T), after 12 weekly paclitaxel treatments (V2T) and after anthracycline-based regimen at surgical resection (V3T) to study a single patient with persistent TNBC disease after neoadjuvant chemotherapy. RESULTS Using RNA-Seq data, we identified an inter-chromosomal fusion transcript between chromosome 20 and 22 (GNAS-TTC38) that was highly expressed at V1T, V2T and V3T. We also identified intra-chromosomal fusion transcripts that were expressed at two time points, such as fusion transcript (KANSL1-ARL17A) on chromosome 17 for V1T and V2T and fusion transcript (RBM12B-LINC00535) on chromosome 8 for V2T and V3T time points. Several gene expression changes were also observed. Gene expression analysis of V1T, V2T and V3T tumors was performed. Differential temporal gene expression profiles of 9884 genes that were significant and varying at different time points were obtained for pathway analysis. Pathway analysis of 9884 genes identified up regulation and down regulation of several transcription factors with a fold change of 2x or more. When compared to blood, DNA tumor and RNA-Seq data, we identified 81 common somatic eSNVs that were expressed in both V1T and V2T time points and we are in the process of investigating V3T data. We found alterations of key transporter domains (CD225, Coatamer_beta_C, DUF2435, Dynamitin, EI24, GLTP, LMF1, Porin_3, V-ATPase_C) in our V1T and V2T SNV data. Similar to gene expression analysis, we are in the process of obtaining the list of mutations at various time points to identify driver and passenger mutation candidate genes for this specific TNBC patient. CONCLUSIONS Our initial time-series analysis of eSNV, fusion transcripts and gene expression data demonstrate that intensive analysis for individual patients is feasible. Further investigation of drug transporters and transcription regulators may help develop personalized treatment strategies for patients with disease resistant to current regimens. Citation Format: Krishna R. Kalari, Xiaojia Tang, Kevin J. Thompson, Douglas W. Mahoney, Poulami Barman, Jason P. Sinnwell, Hugues Sicotte, Peter Vedell, Steven N. Hart, Travis J. Dockter, Katie N. Jones, Amy L. Conners, Ann M. Moyer, Daniel W. Visscher, Jia Yu, Bowen Gao, Sarah A. McLaughlin, John A. Copland, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard J. Gray, Vera J. Suman, Jeanette E. Eckel Passow, Jean-Pierre A. Kocher, Eric D. Wieben, Gianrico Farrugia, Cloann G. Schultz, James N. Ingle, Richard Weinshilboum, Matthew P. Goetz, Liewei Wang, Judy C. Boughey. Analysis of sequencing data to identify potential drug targets for an individual newly diagnosed with basal breast cancer who failed to respond to current standard neoadjuvant chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4185. doi:10.1158/1538-7445.AM2014-4185

Abstract Background Cancer genomic investigations have identified recurrent genomic aberrations critical for cancer initiation, progression, and metastases. However, these investigations are typically performed in isolation, and the effects of treatment on the clonal selection of tumor cells are mostly unknown. We hypothesized that molecular profiling of residual tumors after neoadjuvant chemotherapy (NAC) would identify new drug targets/pathways in patients at high risk for disease recurrence. To better identify clonal populations of resistant breast cancer cells, we utilized DNA content-based flow sorting of nuclei to identify and isolate clonal populations for aCGH and next generation sequencing (NGS) both before and after NAC. Methods The Breast Cancer Genome Guided Therapy Study (BEAUTY) (NCT 02022202) is a prospective study of patients with high-risk breast cancer treated with neoadjuvant 12 weekly paclitaxel (T) +/- trastuzumab followed by 4 cycles of anthracycline based chemotherapy. Tumor tissue from baseline, residual disease from surgery, distant metastases, and patient derived xenografts (PDX) are obtained for cell sorting by DNA ploidy, aCGH, RNA and exome sequencing. Results: 140 patients have been enrolled, 104 have completed surgery and 30 unique PDX have been established corresponding to 26 patients prior to chemotherapy and 4 from residual disease at surgery. Baseline exome and RNA sequencing is complete in 140. Currently, genomic analyses of flow sorted matched baseline, surgical, PDX, and distant disease samples are available in 6 patients. Substantial genomic variation was observed in the surgical sample compared to the primary tumor including gain of oncogenic drivers (EGFR) and loss of negative regulators (ATG5) (Table). The PDX recapitulated these events with excellent fidelity compared to the corresponding human tumor. In patients with TNBC, RNA seq obained from matched samples demonstrated changes in immune related pathways. Evaluation of drug targets/pathways identified in the resistant tumors are ongoing using the PDX and sequencing of the remaining matched baseline/surgical disease will be reported. Clonal changes occurring over time in patients with residual disease or disease recurrence after NACTumor SubtypeResidual Cancer BurdenDisease StatusClonal Aberration Changes (baseline and post NAC)TNBC (AR subtype)3Contralateral lymph node recurrence at 150 days2p25.2 - p25.1 amplicon lost at recurrence; 6p21.32 -p21.31 amplicon lost at recurrenceTNBC (BL 1)3Bone/liver/lymph node recurrence at 135 days5q11.2 amplicon gained at surgery; 12p13.33 - p13.2 amplicon gained at surgeryTNBC (BL 1)3Disease-free at day 1506q21 amplicon lost at sugeryTNBC (BL 2)0Brain recurrence at 390 daysChr 2 chromothripsis at surgeryLuminal B3Progression during chemotherapy7p12.1 - p11.2 amplicon gained at surgeryLuminal HER23Disease-free at day 3609q33.2 amplicon lost at surgery; 15q13.1 - q13.3 amplicon lost at surgery; 15q22.2 - q24.1 amplicon lost at surgery Conclusions: We observed substantial evolutionary changes in residual breast tumors remaining after NAC. Our findings suggest that a comprehensive assessment of the mutational landscape that has evolved during NAC can inform drug development in high risk breast cancer patients. Citation Format: Matthew P Goetz, Michael T Barrett, Krishna R Kalari, Vera J Suman, Sarah A McLaughlin, Alvaro Moreno-Aspitia, Ann M Moyer, Donald W Northfelt, Richard J Gray, Jason Sinnwell, Douglas Mahoney, Poulami Barman, Peter Vedell, Xiaojia Tang, Kevin Thompson, Travis Dockter, Katie Jones, Sara J Felten, Amy Conners, Jeanette Eckel-Passow, Hughes Sicotte, Steven N Hart, Jia Yu, Daniel W Visscher, Eric D Wieben, Cloann Schultz, Minetta C Liu, James N Ingle, Liewei Wang, Richard W Weinshilboum, Judy C Boughey. Impact of neoadjuvant chemotherapy on the clonal composition of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-3.

Abstract Background: Guidelines recommend germline mutation testing of breast cancer predisposition genes in triple negative (TN) breast cancer cases with a family history of breast or ovarian cancer or when diagnosed under age 60. However, the prevalence of mutations in these genes among TN cases unselected for family history of breast or ovarian cancer is not known. Methods: To assess the frequency of mutations in 16 predisposition genes in TN cases we screened a large cohort of TN patients (n=1824) unselected for family history of breast or ovarian cancer from 12 centers and 824 study matched unaffected controls for mutations using a panel-based sequencing approach. Results: Deleterious mutations were identified in 15% of TN patients: 8.5% had BRCA1, 2.7% had BRCA2, and 3.6% had mutations in 12 other genes. Mutations in non-BRCA1/2 genes encoding proteins implicated in homologous recombination repair of DNA double strand breaks were detected at the same frequency as in breast cancer families. TN cases with mutations had high-grade tumors and were diagnosed at an earlier age than non-mutated cases. However, 10% of TN cases diagnosed at ≥60 years and 5% with no family history of cancer were also found to carry mutations. Inactivating mutations in non-BRCA1/2 predisposition genes were associated with moderate to high risks of TN breast cancer. Conclusions: National Comprehensive Cancer Network (NCCN) guidelines support clinical genetic testing of breast cancer predisposition genes in 95% of TN breast cancer patients carrying mutations in susceptibility genes. In contrast, National Institute of Health and Care Excellence (NICE) guidelines in the U.K. do not support genetic testing of a substantial proportion of TN patients with predisposing alleles. Frequency tables for inherited mutations in known predisposition genes based on age of diagnosis and family history of cancer will allow for selection of TN patients most likely to carry mutations in the predisposition genes. Citation Format: Fergus J Couch, Steven N Hart, Priyanka Sharma, Amanda Ewart Toland, Penelope Miron, Janet E Olson, Andrew Godwin, Vernon S Pankratz, Curtis Olswold, Seth Slettedahl, Lucia Guidugli, Matthias W Beckmann, Brigitte Rack, Arif B Ekici, Irene Konstantopoulou, Florentia Fostira, George Fountzilas, Liisa M Pelttari, Song Yao, Judy Garber, Angela Cox, Hiltrud Brauch, Christine Ambrosone, Heli Nevanlinna, Drakoulis Yannoukakos, Susan L Slager, Celine M Vachon, Diana M Eccles, Peter A Fasching. Triple-negative breast cancer: Frequency of inherited mutations in breast cancer susceptibility genes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-03.

Abstract Germline mutations of BRCA1 and BRCA2 underlie joint susceptibility to breast and ovarian cancers, but do not account for all cases. Genome wide association studies show little overlap between common variants associated with breast and ovarian cancer susceptibility, whereas rare variants in genes initially identified as breast cancer susceptibility genes also confer susceptibility to ovarian cancer cases, unselected for breast cancer. Ovarian and breast tumors also are observed in Peutz-Jeghers and possibly in Lynch syndrome. To further elucidate rare germline susceptibilities for breast and ovarian cancer, we have ascertained a unique cohort of 90 BRCA1/2-negative probands with a personal history of breast cancer and ovarian/fallopian tube/peritoneal cancer, unselected for family history. Cases are from the clinical genetics services of Memorial Sloan-Kettering Cancer Center and University of Pennsylvania. Analysis is ongoing using both targeted and exome sequencing approaches. Germline DNA of 69 cases will be subjected to whole exome sequencing. A total of 21 additional cases will undergo targeted sequencing for 28 genes (8 cases) and an extended panel of 54 genes (13 cases). The majority of ovarian cancers are high-grade serous epithelial ovarian carcinoma (n=51), in combination with invasive ductal or DCIS (n=36), invasive lobular (n=4), or unspecified (n=11) breast cancers. The remaining ovarian subgroups comprise other epithelial subtypes; endometrioid (n=9), clear cell (n=3), mucinous and low-grade serous (n=3) and non-epithelial (n=4) or unclassified (n=20), in combination with invasive ductal or DCIS (n=21), invasive lobular (n=5) or unspecified (n=13) breast cancers. Preliminary analysis of the targeted set of genes in 69 cases, reveals 43 predicted pathogenic coding or splice site variants; 17 of which are within PALB2, MSH2, MSH6, BARD1, FANCE, CDKN2A, TP53, SETD2, SIRT1, BRIP1, RAD50, and RAD51D and are not seen in dbSNP138, 1000 Genomes or Exome Variant Server ESP6500 and 26 variants with reported frequencies in MUTYH, MSH6, MLH1, FAM175A, RAD50, UIMC1, JARID2, PHF3, SIRT1, MRE11A, ATM, BRCA2, TP53BP1, CDH1, RAD51D, BRCA1, BABAM1 of less than 1% allele frequency in public databases. Analysis of these variants across and within histologic subtypes is being undertaken to look for specific genotype-phenotype correlations. Analysis of novel candidate genes and pathways by co-segregation, functional analysis, as well as external replication is underway to determine whether shared rare variants, mutated genes, or altered pathways confer cancer susceptibility in this cohort. Germline susceptibility to breast and ovarian cancer is heterogenous and remains a critical research and clinical question. Improved understanding of the susceptibility genetics will allow further examination of the utility of targeted cancer prevention strategies in those who are found to be at risk. Citation Format: Kasmintan A. Schrader, Kara N. Maxwell, Joseph Vijai, Steven Hart, Tinu Thomas, Bradley Wubbenhorst, Lucia Guidugli, Robert Klein, Marina Corines, Liying Zhang, Susan Neuhausen, Jeffrey Weitzel, Namrata Gupta, Larry Norton, Clifford Hudis, Gad Getz, Mark Daly, Steven Lipkin, David Altshuler, Fergus Couch, Katherine Nathanson, Kenneth Offit. Determination of cancer susceptibility in probands with breast and ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3282. doi:10.1158/1538-7445.AM2014-3282