Petersen, Gloria M.

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 Pancreatic cancer is projected to become the second leading cause of cancer-related death in the United States by 2020. A familial aggregation of pancreatic cancer has been established, but the cause of this aggregation in most families is unknown. To determine the genetic basis of susceptibility in these families, we sequenced the germline genomes of 638 patients with familial pancreatic cancer and the tumor exomes of 39 familial pancreatic adenocarcinomas. Our analyses support the role of previously identified familial pancreatic cancer susceptibility genes such as BRCA2, CDKN2A, and ATM, and identify novel candidate genes harboring rare, deleterious germline variants for further characterization. We also show how somatic point mutations that occur during hematopoiesis can affect the interpretation of genome-wide studies of hereditary traits. Our observations have important implications for the etiology of pancreatic cancer and for the identification of susceptibility genes in other common cancer types. Significance: The genetic basis of disease susceptibility in the majority of patients with familial pancreatic cancer is unknown. We whole genome sequenced 638 patients with familial pancreatic cancer and demonstrate that the genetic underpinning of inherited pancreatic cancer is highly heterogeneous. This has significant implications for the management of patients with familial pancreatic cancer. Cancer Discov; 6(2); 166–75. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 109

Abstract A genome-wide association study (GWAS) of pancreatic cancer conducted within the NCI Cohort Consortium (PanScan I and II) identified pancreatic cancer susceptibility loci on chromosomes 1q32.1/NR5A2, 5p15.33/CLPTM1L/TERT, 9q34.2/ABO, and 13q22.1. The most significant single-nucleotide polymorphism (SNP) identified on 13q22.1, rs9543325, lies in a ~600 kb gene desert; the nearest genes are KLF5, KLF12, PIBF1, DIS3, and BORA (265–586 kb away). Imputation using the 1000 Genomes and DCEG reference datasets did not improve the GWAS signal, but produced a set of highly correlated SNPs for functional follow-up. We performed eQTL analysis to test for association between the genotypes of these functional candidate variants and expression of nearby genes. Among 64 normal derived pancreatic tissue samples, DIS3 expression showed the strongest association with a 30 bp indel variant in the risk locus (P = 4.8 × 10-4), indicating risk alleles associate with reduced DIS3 expression. Mutations in DIS3 have previously been identified in acute myeloid leukemia and multiple myeloma, and its expression has been correlated with metastatic potential in colorectal cancer, suggesting DIS3 is relevant to cancer biology. Chromosome conformation capture identified a physical interaction between the indel-containing locus and a region near the DIS3 promoter. Luciferase assay for regulatory function of this indel-containing locus revealed allele-specific silencer activity for the insertion allele. Supershift electromobility shift assay (EMSA) demonstrated binding of LEF1 specifically to the insertion allele of the indel, which contains two in silico predicted LEF1 binding elements. Finally, through immunohistochemical analysis, high DIS3 protein levels associated with better survival for pancreatic cancer patients (hazard ratio = 2.87, 95% CI = 1.49–5.53, P = 0.001). Our results suggest that at least one target gene for the pancreatic cancer risk variants on chr13q22.1 may be DIS3, and that the underlying biology may be mediated by the novel indel through a long-range repressive effect on DIS3 expression. Citation Format: Jason W. Hoskins, Abdisamad Ibrahim, Mickey Emmanuel, Sarah Manmiller, Jinping Jia, Hemang Parikh, Irene Collins, Kris Ylaya, Sean F. Altekruse, Stephen M. Hewitt, Gloria M. Petersen, Laufey T. Amundadottir. Functional analysis of the chr13q22.1 pancreatic cancer risk locus suggests allele-specific effects on DIS3 expression with prognostic implications. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-09.

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.

Abstract Pancreatic ductal adenocarcinoma (PDAC), as with all cancer, is driven by dysregulation of genetic programs due to accumulation of somatic mutations, epigenetic modifications and changes in the micro-environment. Holistic views of perturbations in gene expression networks could illuminate key regulators and pathways for this highly lethal cancer. Toward this end, we performed massively-parallel mRNA-sequencing in normal (n = 10) and tumor (n = 8) derived pancreatic tissue samples as well as pancreatic cancer cell lines (n = 9), and determined differential gene expression (DE) patterns. Sub-network enrichment analyses of all expressed genes based on magnitude of DE identified HNF1A as the regulator of the most significantly and consistently dysregulated expression sub-network in our pancreatic tumor samples (median P = 7.56 x 10-7, median rank = 1, range = 1-25). To explore the effect of HNF1A expression in pancreatic tumor-derived cells we generated stable HNF1A-inducible clones in two pancreatic cancer cell lines. Induction of HNF1A overexpression caused severe growth inhibition (5.3-fold, P = 4.5x10-5 for MIA PaCa-2 clones; 7.2-fold, P = 2.2x10-5 for PANC-1 clones), and G0/G1 cell cycle arrest. This was accompanied by down-regulation of 49 out of 84 assayed cell cycle genes, while only 1 displayed increased expression. These data, combined with HNF1A’s direct regulation of pancreatic development and homeostasis genes (i.e. PDX1, PTF1A and NR5A2), suggest it may be an important tumor suppressor in pancreatic cells. Citation Format: Jason W. Hoskins, Marta Flandez, Jinping Jia, Hemang Parikh, Irene Collins, Mickey Emmanuel, Abdisamad Ibrahim, Wenming Xiao, John Powell, Nuria Malats, Gloria M. Petersen, Fransisco X. Real, Laufey T. Amundadottir. Transcriptome analysis in pancreatic cancer reveals a tumor suppressor function for HNF1A. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B112.

Abstract A genome-wide association study (GWAS) of pancreatic cancer conducted within the NCI Cohort Consortium (PanScan I and II) identified pancreatic cancer susceptibility loci on chromosomes 1q32.1/NR5A2, 5p15.33/CLPTM1L/TERT, 9q34.2/ABO, and 13q22.1. The most significant single-nucleotide polymorphism (SNP) identified on 13q22.1, rs9543325, lies in a 600 kb gene desert; the nearest genes are KLF5, KLF12, PIBF1, DIS3, and BORA (265–586 kb). Imputation using the 1000 Genomes and DCEG reference datasets did not improve the GWAS signal, but produced a set of highly correlated SNPs for functional follow-up. We performed eQTL analysis to test for association between the genotypes of these functional candidate variants and expression of nearby genes. Among 100 normal derived pancreatic tissue samples, DIS3 expression showed the strongest association with a novel 30 bp indel variant in the risk locus (P = 4.0 × 104), indicating risk alleles associate with reduced DIS3 expression. Mutations in DIS3 have previously been identified in acute myeloid leukemia and multiple myeloma, and its expression has been correlated with metastatic potential in colorectal cancer, suggesting DIS3 is relevant to cancer biology. Chromosome conformation capture identified a physical interaction between the indel-containing locus and a region near the DIS3 promoter. Luciferase assay for regulatory function of this indel-containing locus revealed allele-specific silencer activity for the insertion allele. Supershift electromobility shift assay (EMSA) demonstrated binding of LEF1 specifically to the insertion allele of the indel, which contains two in silico predicted LEF1 binding elements. Finally, through immunohistochemical analysis, high DIS3 protein levels associated with better survival for pancreatic cancer patients (hazard ratio = 2.87, 95% CI = 1.49–5.53, P = 0.001). Our results suggest that at least one target gene for the pancreatic cancer risk variants on chr13q22.1 may be DIS3, and that the underlying biology may be mediated by the novel indel through a long-range repressive effect on DIS3 expression. Citation Format: Jason W. Hoskins, Abdisamad Ibrahim, Mickey Emmanuel, Hemang Parikh, Jinping Jia, Irene Collins, Kris Ylaya, Sean F. Altekruse, Stephen M. Hewitt, Gloria M. Petersen, Laufey T. Amundadottir. Functional analysis of the chr13q22.1 pancreatic cancer risk locus suggests allele-specific effects on DIS3 expression with prognostic implications. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B111.

Abstract Purpose: Methylated DNA markers can discriminate upper from lower GI neoplasms (Gastroenterology 2013;144(5):S84). It is unknown if organ-specific molecular prediction of GI tumor site can be achieved. Experimental design: An unbiased search for neoplasm markers at each major GI organ site was conducted by reduced representation bisulfite sequencing. Top marker candidates were then blindly validated by methylation specific PCR on independent tissue DNA samples from normal mucosa [17 esophagus (E), 13 stomach (S), 33 pancreas (P), 35 bile duct/liver (BD), 21 colorectal (CR)] and neoplasia [42 E, 43 S, 36 P, 48 BD, 97 CR]. Recursive partitioning (rPart) trees modeled all stepwise combinations of single markers to classify controls vs neoplasms and then neoplasms by site (CR, gastroesophageal or pancreaticobiliary). A 2nd model was designed from tissue data for blinded clinical piloting on 42 independent plasma DNA samples (14 normal and 28 cancers (14 P, 14 CR). Results: From the top 100 markers, 95 were validated and used for rPart modeling. A 3- marker panel (chr12.133484978-5047, BMP3, chr11.123301058-255) was selected for the universal detection of GI neoplasms, as it classified neoplasms and control tissues with 95% accuracy. Two markers (chr7.25896389-501, QKI) then assigned lower vs upper GI neoplasms with 94% accuracy. Finally, 3 markers (PDGFD, ELOVL2, PCBP3) called pancreaticobiliary vs gastroesophageal neoplasms with 94% accuracy. All 8 markers applied in a single model (Table) accurately predicted control vs tumor by site [88% (p <0.0001)]. In plasma, a 2-marker panel (BMP3, QKI) was 78% accurate in assigning patients to cancer or control groups (p<0.0009) and 83% accurate in assigning cancer origin to CR or P sites (p<0.0001). Predicted by ModelTissue SourceControlsColorectal neoplasiaPancreatico-biliary cancerGastro-esophageal neoplasiaControls1169102Colorectal neoplasia18617Pancreatico-biliary cancer10654Gastro-esophageal neoplasia12872 Conclusion: Panels of methylated DNA markers were identified for both universal detection of GI neoplasia and accurate prediction of tumor site. Pilot testing of these markers in plasma supports potential clinical feasibility. Citation Format: John B. Kisiel, William R. Taylor, Tracy C. Yab, Hassan M. Ghoz, Patrick H. Foote, Mary E. Devens, Douglas W. Mahoney, Thomas C. Smyrk, Lisa A. Boardman, Gloria M. Petersen, Navtej S. Buttar, Lewis R. Roberts, Graham P. Lidgard, David A. Ahlquist. Accurate site prediction of gastrointestinal cancer by novel methylated DNA markers: Discovery & validation. [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 4252. doi:10.1158/1538-7445.AM2015-4252

Abstract Background: Epidemiologic studies have suggested that metformin use is associated with cancer death reduction. The results for pancreatic ductal adenocarcinoma (PDAC) are inconsistent. Retrospective studies of cancer survival may potentially have unintended biases; due to complexities of diabetes management, metformin use exposure is commonly categorized as simply “yes/no.” Nevertheless, in part based on epidemiologic studies, there are at least 20 open cancer clinical trials that include metformin in a treatment arm. We aimed to study metformin use and survival of patients with PDAC, addressing potential inherent biases. Methods: A retrospective cohort study for the years 2000-2011 was conducted using the patient database of the Mayo Clinic Specialized Programs of Research Excellence (SPORE) in Pancreatic Cancer. 1,360 PDAC patients (59% male; mean age 67) with diabetes were studied; overall survival was the primary outcome. Initiation and duration of metformin use was carefully abstracted from medical records. Patients were categorized into 5 groups based on metformin initiation: (A) Never used, n = 908 (reference group); (B) Metformin started >1 year prior to PDAC diagnosis, n = 84; (C) Metformin started within 1 year prior to PDAC diagnosis, n = 212; (D) Metformin started <30 days post-PDAC diagnosis, n = 104; (E) Metformin started >30 days post PDAC diagnosis, n = 34. Hazard ratios (HR) and 95% confidence intervals (C.I.) for survival differences between groups were computed using Cox proportional hazards models, adjusting for age, sex, disease stage, body mass index, and diagnosis year group. Results: Median survival of patients in Groups B-E was 292 days compared to 308 for Group A; this was not significantly different. However, among 413 resectable patients, metformin users appeared to survive longer than non-users (782 vs. 612 days, p = 0.07). Survival in each group was 308, 245, 249, 243 and 818 days, respectively. Because Group E had already survived >30 days, a bias existed. With Group E excluded, there was no survival difference among the other 4 groups (p = 0.18). Compared to Group A, HR and 95% C.I. for metformin use were 1.08 (0.85-1.37), 0.99 (0.85-1.17), 1.04 (0.83-1.31) and 0.49 (0.33-0.74), for Group B-E, respectively. Conclusions: Our thorough analysis suggests that metformin use does not improve survival of patients with PDAC. The benefit of metformin was observed only in patients who started metformin post-PDAC diagnosis; however, there is an inherent survival bias in this group of patients. Studies of medication exposure and cancer survival warrant very careful and detailed data collection. Researchers should exercise caution when initiating clinical trials based on retrospective epidemiologic studies. Citation Format: Roongruedee Chaiteerakij, David B. Zhen, Patrick A. Burch, Kari G. Chaffee, William R. Bamlet, Ann L. Oberg, Lewis R. Roberts, Gloria M. Petersen. Metformin use does not increase survival of pancreatic cancer patients: A cautionary lesson. [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 LB-183. doi:10.1158/1538-7445.AM2015-LB-183

Abstract Genome wide association studies (GWAS) have mapped multiple independent cancer risk loci (n = 6) to a small region on chr5p15.33 for at least ten distinct cancers, including bladder, breast, glioma, lung, melanoma, non-melanoma skin, ovarian, pancreas, prostate, and testicular germ cell cancer. This region harbors two plausible target genes, TERT which encodes the catalytic subunit of telomerase reverse transcriptase which maintains chromosome ends by adding telomeres repeats, and CLPTM1L which encodes the cleft lip and palate transmembrane protein 1-like protein which promotes cancer cell growth, protect cells from apoptosis, and can induce abnormal cytokinesis. The most significant pancreatic cancer GWAS SNP on chr5p15.33 was rs401681 (P = 3.7×10−7, OR = 1.19), located in the 13th intron of the CLPTM1L gene and within the independent multi-cancer risk locus in this region most distant from TERT. Imputation and fine mapping in pancreatic cancer resolved this signal by three orders of magnitude (P = 1.4×10−10, OR = 1.30) to a set of seven highly correlated SNPs. Electrophoretic mobility shift assays (EMSA) and luciferase assays across these SNPs led us to a single SNP, rs36115365, that showed allele-specific effects in both assays and overlapped with prominent ENCODE marks indicating regulatory potential. Examining this SNP across cell lines derived from multiple types of cancer linked to this locus, specifically pancreatic, testicular, lung, and melanoma, established a consistent pattern of a specific protein binding and enhanced regulatory activity for the risk allele across eight cancer cell lines (two cell lines per cancer type). siRNA-based targeting of the putative gene regulatory-region harboring rs36115365 resulted in a strong inhibition of TERT expression (average 60%, range 45-85%) but no effect on CLPTM1L expression. This inhibition of TERT expression was stronger from the pancreatic cancer risk as compared to the protective allele. Quantitative mass spectrometry identified Zinc finger protein 148 (ZNF148) as the protein specifically binding the risk allele, further supported by direct and allele-specific binding of purified recombinant ZNF148 in EMSA assays. Lastly, siRNA-mediated knockdown of ZNF148 resulted in significant reduction of TERT but not CLPTM1L expression across multiple cancer cell lines. Our results indicate that the pancreatic cancer GWAS signal at the single independent chr5p15.33 multi-cancer risk locus most distant from TERT may be explained by a single SNP that influences TERT expression via ZNF148. Citation Format: Kevin M. Brown, Jun Fang, Jinping Jia, Zhaoming Wang, Matthew Makowski, Tongwu Zhang, Jason Hoskins, Jiyeon Choi, Younghun Han, Mingfeng Zhang, Mai Xu, Peter Kanetsky, Andresson Thorkell, Gloria M. Petersen, Katherine L. Nathanson, Christopher I. Amos, Maria T. Landi, Stephen J. Chanock, Michiel Vermeulen, Laufey T. Amundadottir. Functional characterization of a multicancer risk locus on chr5p15.33 reveals regulation of TERT by ZNF148. [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 4610. doi:10.1158/1538-7445.AM2015-4610

Abstract Purpose: Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets. Experimental Design: At a referral center, we conducted four sequential case–control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated. Results: Sequencing identified >500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS). Conclusions: We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls. Clin Cancer Res; 21(19); 4473–81. ©2015 AACR.