Advances in Population Genetics and Hereditary Prediction Models for Pancreatic Cancer

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1 Advances in Population Genetics and Hereditary Prediction Models for Pancreatic Cancer Gloria M. Petersen, Ph.D. Department of Health Sciences Research Mayo Clinic Rochester, Minnesota Pancreasfest

2 Take Home Messages 1. Pancreatic cancer susceptibility gene discovery has uncovered new insights and has matured to translation. 2. Patients with and without positive family history can carry germline mutations in an array of genes. 3. Risk modeling for pancreatic cancer using genomic data remains a work in progress.

3 Complex Diseases: Genetic Variants of Different Effect Manolio, T et al. Finding the missing heritability of complex diseases. Nature 2009 October 8; 461(7265): McCarthy MI, et al. Genome-wide association studies for complex traits: consensus, uncertainty and challenges. Nature Rev. Genet. 2008;9:

4 Gene discovery in pancreatic cancer Subjects Design Genetic analysis technology Discovery goal Familial Cases Probands and/or family members Whole genome/exome sequencing Linkage marker genotyping Rare susceptibility genes with high effect (penetrance) Sporadic Cases Cases only Whole genome/exome sequencing Susceptibility genes, rare or moderate frequency with high effect Sporadic Cases & Healthy Controls Case-control association study (GWAS) Genome wide SNP panels Common variants with low effect

5 Genetics of Pancreatic Cancer: Families 10% of pancreatic cancer patients have a first degree relative with pancreatic cancer Positive Family History

6 Factors Influencing Pancreatic Cancer Risk Age older age Smoking Family History Race - Black High BMI, Obesity Hx Diabetes Hx Chronic Pancreatitis Heavy alcohol use Short telomeres Risk X X X X X X X X X Protective

7 Factors Influencing Pancreatic Cancer Risk Allergies Aspirin Use Diet Well-done, BBQ meat Fruits, vegetables, fiber Pesticides, benzene, asbestos, chlorinated hydrocarbons Risk X X Protective X X X

8 Pancreatic cancer risk in first degree relatives Case-Control studies Authors Cases Controls OR (95% C.I.) Falk et al. (1988) 362 1, ( ) Fernandez et al. (1994) 363 1, ( ) Silverman et al. (1999) 484 2, ( ) Schenk et al. (2001) ( ) Prospective Number of Affected First Degree Relatives SIR (95% CI) ( ) ( ) ( ) Klein AP, Brune KA, Petersen GM, et al.. Prospective risk of pancreatic cancer in familial pancreatic cancer kindreds. Cancer Res. 2004; 64: Axilbund,J, Wiley E. Genetic Testing by Cancer Site: Pancreas. Cancer Journal. 18(4): , 2012.

9 Inherited Susceptility to Pancreatic Cancer: Updated HBOC Peutz Jeghers Syndrome Hereditary pancreatitis Familial atypical mole and melanoma Hereditary nonpolyposis colorectal cancer (Lynch syndrome) HBOC Klein AP. Identifying people at a high risk of developing pancreatic cancer. Nat Rev Cancer Jan;13(1):66-74.

10 Zhen DB et al. Prevalence of BRCA1, BRCA2, PALB2, and CDKN2A Mutations in Familial Pancreatic Cancer (FPC): A PACGENE Study. Genetics in Medicine, 2015 Jul;17: Pancreatic Cancer Genetic Epidemiology (PACGENE) Consortium sites assembled 2,853 unrelated kindreds containing at least two family members affected with pancreatic cancer. 727 unrelated kindreds that contained at least two biologically related family members affected with pancreatic cancer and for which a proband DNA sample was available.

11 Table 3. Germline mutation prevalences stratified by deleterious/suspected deleterious and variants of uncertain significance among probands from Familial Pancreatic Cancer (FPC) kindreds, and probands from kindreds not meeting FPC definition. Results shown for probands who were tested for all four genes (total n=716). Gene Deleterious/Suspected Deleterious Mutations n (%) FPC (n=515) Non-FPC (n=201) Total (n=716) Variants of Uncertain Significance n (%) FPC (n=515) Non-FPC (n=201) Total (n=716) BRCA1 6 (1.2) 0 (0.0) 6 (0.8) 3 (0.6) 0 (0.0) 3 (0.4) BRCA2 19 (3.7) 6 (3.0) 25 (3.5) 2 (0.4) 1 (0.5) 3 (0.4) PALB2 3 (0.6) 1 (0.5) 4 (0.6) 11 (2.1) 5 (2.5) 16 (2.2) CDKN2A 13 (2.5) 0 (0.0) 13 (1.8) 10 (1.9) 3 (1.5) 13 (1.8) Total 41 (8.0) 7 (3.5) 48 (6.7) 26 (5.0) 9 (4.5) 35 (4.9) Zhen DB et al. Genetics in Medicine, 2015 Jul;17:569-77

12 Figure 1. Probability (%) that probands affected with pancreatic cancer (PC) will test positive for a deleterious or suspected deleterious mutation in BRCA1, BRCA2, PALB2, or CDKN2A, if from kindreds with various cancer family histories. Number of PC includes proband. Size of sample subsets from which probabilities were estimated are shown in parentheses. (727 probands tested) Zhen DB et al. Genetics in Medicine, 2015 Jul;17:569-77

13 Mayo Clinic study of mutations in 25 genes (ASCO 2016) Germline pathogenic variant prevalence by type of familial proband (FPC vs familial Non-FPC), categorized by genes associated and not associated with PDAC. Genes with no pathogenic variants included APC, BMPR1A, BRIP1, CDH1, CDK4, EPCAM/TACSTD1, MLH1, MSH2, MSH6, PTEN, RAD51C, RAD51D, SMAD4, STK11, and TP53. FPC (n=186) Non-FPC (n=117) Total (n=303) N % N % N % All Genes Genes Associated with PDAC ATM BRCA BRCA CDKN2A (p16) PALB PMS Sub-Total* Genes Not Associated with PDAC BARD CHEK MUTYH/MYH NBN Sub-Total * One FPC case carried two pathogenic variants: one in BRCA1 and one in BRCA2.

14 Smith AL Gallinger S, Zogopoulos G. Candidate DNA repair susceptibility genes identified by exome sequencing in high-risk pancreatic cancer. Cancer Lett Jan 28;370(2): Search for novel DNA repair susceptibility genes for pancreatic cancer using whole exome sequencing 109 high-risk PC cases from Ontario or Quebec PC research registries Young - dx d <50 years (N=8) Familial PC (N=101 in 85 kindreds) Known mutation carriers excluded 513 genes: putative DNA repair genes plus CDKN2A, SPINK1 and PRSS1 Private truncating variants (PTVs) in 41 novel genes among 36 kindreds.

15 Top Candidate Novel Susceptibility Genes AATF BLM CEP164 CHD1L FAN1 FANCG FANCL MCIR MGMT NEIL1 NEK1 NEK11 RHNO1 SPP1 TONSL WDR48 WRN Smith AL, et al. Candidate DNA repair susceptibility genes identified by exome sequencing in high-risk pancreatic cancer. Cancer Lett Nov 3.

16 A PACGENE study Sequenced whole genomes of 638 familial pancreatic cancer patients from 593 kindreds Sequenced exomes of pancreatic adenocarcinomas from 39 of these same patients. Strongly support the role of previously identified familial pancreatic cancer susceptibility genes such as BRCA2, p16/cdkn2a and ATM, and identify candidate genes harboring rare, deleterious germline variants for further characterization. Roberts NJ et al. Cancer Discovery 6:166-75, 2015

17 Genetics of Pancreatic Cancer: Sporadic Positive Family History

18 PanScan GWAS (Wave 1) Amundadottir L, Kraft P, Stolzenberg-Solomon RZ, et al. Genome-wide association study identifies variants in the ABO locus associated with susceptibility to pancreatic cancer. Nature Genetics 41:986-90, pairs Petersen GM, Amundadottir A, Fuchs CS, et al. A genomewide association study identifies pancreatic cancer susceptibility loci on chromosomes 13q22.1, 1q32.1 and 5p Nature Genetics, 42:224-8, pairs

19 GWAS Wave 2: Wolpin B et al. Genome-wide association study identifies multiple susceptibility loci for pancreatic cancer. Nature Genetics 46 (9): , PanScan3 7,683 pancreatic cancer 14,397 publicly available data on controls GWAS Wave 3. Childs EJ et al. (PANC4 study). Common variation at 2p13.3, 3q29, 7p13 and 17q25.1 associated with susceptibility to pancreatic cancer. Nature Genetics, 47:911-6, cases and 11,569 controls including 4,164 newly genotyped cases and 3,792 controls controls in 9 study populations from N. America, Central Europe and Australia. GWAS Wave 4. Childs EJ et al. Association of common susceptibility variants of pancreatic cancer in higher risk patients: A PACGENE Study. Cancer Epidemiol Biomarkers Prev Jul;25(7): cases (906 FPC; 79 early onset; 877 family controls) icogs array plus custom SNPs

20 Manhattan plot of Combined Stage 1 Association analysis. Novel loci associated with pancreatic cancer in Caucasians shown in red. (ABO) (Nongenic) (NR5A2) (CLPTM1L-TERT) (LINC00673) Childs EJ et al. (PANC4 study). Nature Genetics 47:911-6, 2015.

21 Pancreatic Cancer and CDKN2A Methods: 1,537 pancreatic adenocarcinoma patients Consecutive, unselected white non-hispanic patients Lymphocyte DNA; exons and splice sites sequenced for CDKN2A Results: 9 (0.6%) carried germline mutations in CDKN2A 3 mutations previously unreported: missense mutation in p14 (A120P) and two frameshift mutations that affect both p16 and p14arf (R80fs/ P135fs and V95fs/ G150fs) CDKN2A mutation carriers were more likely to have: family hx pancreatic cancer (p= 0.003); carrier rate 3.3% family hx melanoma (p= 0.03); carrier rate 5.3% personal hx melanoma (p= 0.01) McWilliams RR et al. Eur J Hum Genetics, 2011 Apr;19(4):472-8.

22 Germline Mutations in CDKN2A Among 1,537 Unselected Unrelated Pancreatic Cancer Patients Pt Sex/ Age* Family history of pancreatic cancer Family history of melanoma Personal history of melanoma Exon Protein Genetic change Protein change Function 1 F 61 Yes No No 1A p16-34g>t N/A Initiation codon 2 M 74 No Yes Yes 1A p16 47T>G L16>R AAC p16 3 F 65 No No No 1A p16 71G>C R24>P AAC p16 4 F 58 No No No 2 p16 p14 5 M 66 No No No 2 p16 p14 192G>C 358G>C del del L64>L A120>P R80fs P135fs AAC p14arf makes a hybrid p16/p14 protein after frameshift 6 M 65 Yes No No 2 p16 p del 449 del V95fs G150fs frameshift 7 M 45 No No No 2 p16 p14 318G>A 484G>A V106>V A162>T AAC p14arf 8 M 67 No Yes Yes 2 p16 457G>T D153spl Affects splicing in p16/p14arf 9 M 57 Yes No No 2 p16 457G>T D153spl Affects splicing in p16/p14arf * M= male, F=female, age (years) at diagnosis of pancreatic cancer, AAC = amino acid change McWilliams RR et al. Eur J Hum Genetics, 2011 Apr;19(4):472-8.

23 Prevalence of Germline Mutations in Cancer Predisposition Genes in Patients With Pancreatic Cancer Grant R et al. Prevalence of Germline Mutations in Cancer Predisposition Genes in Patients With Pancreatic Cancer. Gastroenterology 2015;148:

24 Prevalence of pathogenic mutations in cancer predisposition genes among pancreatic cancer patients. Hu C, Hart SN, Bamlet WR, Moore RM, Nandakumar K, Eckloff BW, Lee YK, Petersen GM, McWilliams RR, Couch FJ. Cancer Epi Biomarkers and Prevention, 2016 Jan;25(1): patients unselected for a family history of cancer who were recruited to the Mayo Clinic Pancreatic Cancer patient registry over a 12 month period Panel of 21 cancer predisposition genes Next-generation sequencing

25 Prevalence of pathogenic mutations in cancer predisposition genes among pancreatic cancer patients. Hu C, Hart SN, Bamlet WR, Moore RM, Nandakumar K, Eckloff BW, Lee YK, Petersen GM, McWilliams RR, Couch FJ.Cancer Epi Biomarkers and Prevention, 2016 Jan;25(1): Fourteen pathogenic mutations in eight genes were identified in 13 patients (13.5%). One patient carried mutations in both NBN and CHEK2. Gene Nucleotide Age# Personal history of other cancer (age at diagnosis) Cancer in 1 st and 2 nd degree relatives b ATM c.2880delc 64 None 3 colorectal Meets NCCN Criteria for Cancer Syndrome? ATM c.5932g>t 83 Breast (67) 1 breast ATM c.6012_6013insa 66 Liver (66) None ATM c.7327c>t 67 Unknown (67) 1 pancreatic BARD1 c.1921c>t 71 None 1 pancreatic BRCA1 c.212+1g>a 59 None 2 breast, 1 pancreatic Yes (HBOC) BRCA2 c.5864c>a 69 None 1 breast Yes (HBOC) BRCA2 c.6373dupa 51 None 2 pancreatic, 2 breast, 1 endometrial Yes (HBOC) CHEK2 c.1100delc 79 None None FANCM c.2586_2589delaaaa 76 Melanoma 2 breast, 1 pancreatic MSH6 c.3804dupa 63 Endometrial (43) 2 breast, 2 colorectal Yes (Lynch) MSH6 c _ del25 75 None 1 breast No (Lynch) NBN/CHEK2 c.657_661del5/1100delc 80 None None

26 Assessment of germline cancer predisposition genes in 392 unselected pancreatic cancer patients. McKay S, Humphris J, Johns A, Pinese M, Patch A, Nones K, APGI, Grimmond S, Biankin A. Abstract A02, AACR Conference on Pancreatic Cancer: Advances in Science and Clinical Care, May 12-15, Patients 13 genes analyzed among data from WES (n=208)and WGS (n=184) 23 (5.9%) patients classified as pathogenic or likely pathogenic carriers 3/23 were familial cases No mutations found in: APC, MLH1, MSH2, MSH6, PRSS1, or TP53

27 RISK MODELING THAT INCORPORATES GENETIC INFORMATION Klein AP et al. An absolute risk model to identify individuals at elevated risk for pancreatic cancer in the general population. PLoS One Sep 13;8(9):e Data on 3,349 cases and 3,654 controls from the PanScan Consortium Non-genetic and genetic risk factors for pancreatic cancer Absolute risks estimated based on relative risks and population incidence rates: Variable Multivariable OR [95% CI] Current smoking 2.20 [ ] Family history of pancreatic cancer 1.60 [ ] Obesity (body mass index >30 kg/m 2 ) 1.26 [ ] Non-O ABO genotype AO vs. OO genotype BB vs. OO genotype ABO genetic SNPs rs (chr1q32.1) rs401681(5p15.33) rs (13q22.1) ( Diabetes >3 years Nested case-control Case-control 1.23 [ ] 1.58 [ ] 1.29 [ [ ] 1.27 [ ] 1.57 [ ] 1.80 [ ] Heavy alcohol use (>3 drinks/day) 1.45 [ ]

28 Klein AP et al. An absolute risk model to identify individuals at elevated risk for pancreatic cancer in the general population. PLoS One Sep 13;8(9):e AUC for risk models: Only non-genetic factors: 58% Only genetic factors: 57% Both non-genetic and genetic factors: 61% Fewer than 3/1,000 U.S. non- Hispanic whites have more than a 5% predicted lifetime absolute risk. immediate clinical utility of model is limited. The genetic factors did not add substantively to a risk model based on life-style factors only, as most individuals remained in the same risk strata.

29 Klein AP et al. An absolute risk model to identify individuals at elevated risk for pancreatic cancer in the general population. PLoS One Sep 13;8(9):e72311.

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31 Take Home Messages 1. Pancreatic cancer susceptibility gene discovery has uncovered new insights and has matured to translation. 2. Patients with and without positive family history can carry germline mutations in an array of genes. 3. Risk modeling for pancreatic cancer using genomic data remains a work in progress.

32 Collaborators & Acknowledgments Mayo Clinic Rob McWilliams, MD Kari Chaffee, M.S. Suresh Chari, M.D. Hugues Sicotte, Ph.D. Fergus Couch, Ph.D. Julie Cunningham, Ph.D. Thomas Smyrk, M.D. Bill Bamlet, M.S. Lizhi Zhang, M.D. Martin Fernandez-Zapico, MD PanC4 GWAS Leadership Team: Harvey Risch, Ph.D., Yale Sara Olson, Ph.D., MSKCC Donghui Li, Ph.D., MDACC Steve Gallinger, MD, OICR Federico Canzian, PhD, DKFZ Paige Bracci, UCSF Rachel Neale, Queensland RO1 CA97075 (to GMP) R01 CA (to APK) SPORE (P50 CA ) R01 CA (to GMP, BK, SW) PanCAN CIDR PanScan Project Leadership Team: Patricia Hartge, Sc.D, NCI/DCEG Robert Hoover, M.D., Sc.D, NCI/DCEG Stephen Chanock, M.D., NCI/DCEG/CGF Laufey Amundadottir, Ph.D., NCI/CGF/DCEG Brian Wolpin, M.D., Harvard University Charles Fuchs, M.D., M.P.H, Harvard University Charles Kooperberg, Fred Hutchinson CR Peter Kraft, Ph.D., Harvard University Rachael Stolzenberg-Solomon, M.P.H., Ph.D., NCI Kai Yu, Ph.D., NCI Johns Hopkins University Ralph Hruban, M.D. Alison Klein, Ph.D. Mike Goggins, M.D. Mimi Canto, MD Nick Roberts, DVM, PhD PACGENE collaborators Melissa Bondy, Ph.D. MD Anderson/Baylor Steve Gallinger, M.D. Univ Toronto Ann Schwartz, Ph.D. - Wayne State Univ Sapna Syngal, M.D. Dana Farber Cancer Inst Daniela Seminara, Ph.D. NCI