Kate Mc Namara MD CCCF Research Scholar University

Kate Mc. Namara, MD CCCF Research Scholar University of Toronto

CCCF Annual Research Scholar Award

Zane Cohen Center for Digestive Diseases Research Treatment Education Support Gastrointestinal Cancers Research Groups Familial Gastrointestinal Cancer Registry (FGICR) Ontario Pancreas Cancer Study (OPCS) Ontario Familial Colorectal Cancer Registry (OFCCR) Soft Tissue Sarcoma Research Program (STSRP) Diseases Lynch Syndrome (LS) Familial Adenomatous Polyposis (FAP) MYH - Associated Polyposis (MAP) Peutz-Jeghers Syndrome (PJS) Juvenile Polyposis (JP) Hereditary Hemorrhagic Telangiectasia (HHT) Hereditary Diffuse Gastric Cancer Syndrome (HDGC) Pancreatic Cancer

Genetics of Early Age Onset Colorectal Cancer (EAO-CRC)

CRC incidence and mortality in the US ● third leading cause of cancer incidence in both males and females ● second leading cause of cancer mortality in both males and females age-standardized incidence rate per 100, 000 43. 7 age-standardized mortality rate per 100, 000 15. 9 estimated new cases in 2014 % of all new cancer cases estimated deaths in 2014 % of all cancer deaths 136, 830 8. 2% 50, 310 8. 6% SEER 9 Incidence & U. S. Mortality 1975 -2011, All Races, Both Sexes.

EAO-CRC incidence and mortality in the US ● approximately 10% of colorectal cancers have early-onset Estimated Numbers of New Colorectal Cancer Cases and Deaths by Age and Sex, United States, 2014 NEW CASES Male Age (yrs) DEATHS Female Male Female Count % 0 -49 7, 270 10 6, 250 10 1, 840 7 1, 450 6 50 -64 22, 890 32 16, 570 25 6, 780 26 4, 590 19 65 -79 27, 950 39 23, 050 35 10, 100 38 7, 710 32 80+ 13, 720 19 19, 130 29 7, 550 29 10, 290 43 Siegel, R. , De. Santis, C. and Jemal, A. (2014), Colorectal cancer statistics, 2014. CA: A Cancer Journal for Clinicians, 64: 104– 117. doi: 10. 3322/caac. 21220

CRC incidence and mortality in Canada ● incidence and mortality rates are higher in Canada than in the US ● second leading cause of cancer incidence and mortality in males ● third leading cause of cancer incidence and mortality in females age-standardized incidence rate per 100, 000 age-standardized mortality rate per 100, 000 estimated new cases in 2014 % of all new cancer cases estimated deaths in 2014 % of all cancer deaths 48. 9 17. 9 24, 400 12. 8% 9, 300 11. 5% Canadian Cancer Society’s Advisory Committee on Cancer Statistics. Canadian Cancer Statistics 2014. Toronto, ON: Canadian Cancer Society; 2014.

EAO-CRC incidence and mortality in Canada ● approximately 6% of colorectal cancers have early-onset Estimated Numbers of New Colorectal Cancer Cases and Deaths by Age and Sex, Canada, 2011 NEW CASES DEATHS Male Female Age (yrs) Count % 0 -29 45 <1% 10 <1% 5 <1% 30 -49 720 5 660 6 175 3. 5 145 3. 5 50 -59 2, 100 15 1, 550 14 580 11. 5 390 10 60 -69 4, 000 29. 5 2, 500 23 1, 250 25 700 18 70 -79 4, 000 29. 5 2, 800 26 1, 500 30 1, 000 25. 5 80+ 2, 800 20. 5 3, 300 30. 5 1, 450 29 1, 650 42 Canadian Cancer Society’s Advisory Committee on Cancer Statistics. Canadian Cancer Statistics 2014. Toronto, ON: Canadian Cancer Society; 2014.

CRC incidence trends in the US ● CRC ASIR have been decreasing on average 3. 1% per year since 2002 SEER 9 Incidence & U. S. Mortality 1975 -2011, All Races, Both Sexes. Rates are Age-Adjusted.

CRC incidence is increasing in young adults Race/ethnicity All races • Seigel et al. Young adults 20 -49 y, SEER 1992 -2005 Non-Hispanic White Non-Hispanic Black Hispanic Asian American/ Pacific Islander • O’Connell et al. Young adults 20 -40 y, SEER 1973 -1999 Colon Rectum n APC M 10, 913 1. 5** F 9, 733 1. 6** M 6, 748 2. 0** F 5, 626 2. 2** M 1, 409 − 0. 2 F 1, 456 − 0. 6 M 1, 307 2. 7** F 1, 250 1. 1 M 1, 284 1. 2 F 1, 239 0. 6 Age (y) 1973 Incidence 1999 Incidence EAPC 20 -40 1. 8 2. 1 0. 75** 60+ 179 204. 4 0. 26 20 -40 0. 8 1. 4 3. 15** 60+ 86 72. 1 -0. 73** • Meyer et al. Young adults <40 y, SEER 1973 -2005 Subsite APC Rectum 2. 6** Sigmoid 0. 4 Descending colon -1. 8** Colon excluding rectum -0. 2

CRC incidence is increasing in young adults Seigel et al. CRC incidence trends among young non-hispanic white adults (20 -49 yrs) by age and anatomic subsite, 1992 to 2005 • O’Connell et al. Young adults 20 -40 y, SEER 1973 -1999 • Meyer et al. Young adults <40 y, SEER 1973 -2005 Age M 20 -29 30 -39 40 -49 n 249 1, 419 5, 080 APC 5. 2* 3* 1. 5* F 20 -29 240 30 -39 1, 125 40 -49 4, 261 5. 6* 2* 2. 1* Subsite n M Proximal 2, 054 Distal 1, 609 Rectum 2, 609 APC 0 1. 5* 3. 5* F Proximal 1, 548 Distal 1, 619 Rectum 2, 065 0. 8 2. 3* 2. 9*

CRC incidence and mortality trends in Canada ● CRC ASIR has been decreasing on average 0. 8% per year in males, 2001 -2010 ● CRC ASIR has been decreasing on average 0. 6% per year in females, 2001 -2010 Canadian Cancer Society’s Advisory Committee on Cancer Statistics. Canadian Cancer Statistics 2014. Toronto, ON: Canadian Cancer Society; 2014.

CRC incidence is increasing in young adults

Hereditary CRC risk • familial cases comprise approximately 30% of all CRC • mutations in highly penetrant CRC susceptibility genes account for 5 -10% of all CRC • etiology of remaining 20 -25% of inherited CRC is not completely understood

Hereditary CRC syndromes POLYPOSIS adenomatous polyposis syndromes Familial Adenomatous polyposis (FAP) – AD, mutation in APC Attenuated FAP (AFAP) - AD, APC mutations at 5’ or 3’ ends of gene or in certain locations of exon 9 MUTYH-associated polyposis (MAP) – AR, biallelic mutations in MUTYH Polymerase-proofreading associated polyposis – AD, mutation in POLE or POLD 1 hamartomatous polyposis Peutz-Jeghers syndrome (PJS) - AD, mutations in STK 11 Juvenile Polyposis syndrome (JPS) - AD, mutation in SMAD 4 or BMPR 1 A Cowden syndrome (PTEN hamartoma tumor syndrome) - AD, germline mutation in PTEN hyperplastic/serrated polyposis Serrated polyposis syndrome – uncertain genetic etiology; unclear hereditary predisposition Hereditary mixed polyposis syndrome NONPOLYPOSIS Lynch syndrome (LS) – AD, mutation in DNA mismatch repair (MMR) gene (MLH 1, MSH 2, MSH 6, PMS 2) or EPCAM Biallelic Mismatch Repair deficiency Other (Li-Fraumeni, Bloom syndrome)

POLYPOSIS adenomatous polyposis syndromes Familial Adenomatous polyposis (FAP) – AD, germline mutation in APC Ø 100 s to 1000 s of synchronous adenomas beginning in adolescence Ø CRC risk >95% by age 50 if untreated Attenuated FAP (AFAP) - AD, germline APC mutations at 5’ or 3’ ends of gene or in certain locations of exon 9 Ø 10 to 99 synchronous adenomas Øemergence of adenomas and CRC development delayed 10 to 20 years ØCRC risk 70% by age 80 MUTYH-associated polyposis (MAP) - AR, biallelic mutations in MUTYH Ø variable phenotype, most commonly 20 to 99 polyps but may present with <10 or >500 Ø CRC risk >40% by age 60 Polymerase-proofreading associated polyposis (PPAS) – AD, mutation in POLE or POLD 1 Ø 10 to 100 adenomas, may present with other polyp histology Ø CRC risk undefined

POLYPOSIS hamartomatous polyposis Peutz-Jeghers syndrome (PJS) - AD, germline mutations in STK 11 Ø Ø GI polyps in 90 -100% of cases mucocutaneous pigmentation in >95% of cases most commonly perioral and buccal mucosa lifetime CRC risk 40% elevated risk for gastric, small bowel , breast, lung, pancreatic, and gynecologic cancers Juvenile Polyposis syndrome (JPS) - AD, mutation in SMAD 4 or BMPR 1 A Ø multiple juvenile polyps in colon, small bowel, and stomach Ø CRC risk 20% by age 35 and up to 70% by age 60 Cowden syndrome (PTEN hamartoma tumor syndrome) - AD, germline mutation in PTEN Ø range of phenotypes, colon polyps present in up to 95%, <10 to 100 s of polyps, various histology Ø lifetime CRC risk may be up to 15%

hyperplastic/serrated polyposis Serrated polyposis syndrome Ø uncertain genetic etiology; unclear hereditary predisposition Hyperplastic Mixed Polyposis Syndrome Ø newly described, poorly characterized Ø oligopolyposis, mixed poly histology including adenomatous, serrated, hyperplastic and mixed types

NONPOLYPOSIS Lynch syndrome (LS) – AD, mutation in DNA mismatch repair (MMR) gene (MLH 1, MSH 2, MSH 6, PMS 2) or EPCAM Ø usually few (<10) early-onset adenomas, accelerated adenoma-carcinoma sequence Ø lifetime CRC risk dependent on MMR gene mutation ranging 25 -75% for MLH 1/MSH 2 mutations and lower risks for other gene mutations Ø associated with extracolonic malignancies – endometrial, gastric, ovarian, urinary tract, small bowel, brain, hepatobiliary Ø pedigrees characterized by Amersterdam criteria (3 affected, 2 generations, first-degree relatives) Biallelic Mismatch Repair deficiency – AD, biallelic mutations in MMR genes Ø more commonly associated with brain and hematologic malignancies at very early-age Other (Li-Fraumeni, Bloom syndrome)

Hereditary CRC syndromes and EAO-CRC Ø hereditary CRC syndromes account for greater proportion of EAO-CRC ≤ 40 Chang et al. 2012 surgical patients, Stanford Univ hosp 75 total; 13(17%) IHC def, 2(3%) FAP, 1(1%) JPS, 1(1%) LFS, 3(4%) IBD Steinhager et al. 2012 <50 surgical patients, MSKCC 198 total; 17 (9%) LS and 7 (3. 5%) VUS Limburg et al 2011 <50 CCFR 155 total; 18 (12%) IHC def Perea et al. 2010 ≤ 45 surgical patients , 2 hospitals in Spain 43 total; 8(19%) LS and 1(2%)FAP, 26(60%) sporadic 86 total; 21(24%) LS, 1(1%) FAP, 1(1%) LFS 48(56%) sporadic Jasperson et al. 2010 ≤ 35 3 US CRC registries Losi et al. 2005 <45 surgical patients, 2 hospitals in Italy 71 total; 7 Terdiman et al. 2002 ≤ 35 high risk clinic, USCF 43 total; 13(30%) US population based registry 23 total; 0 (10%) LS (0%) LS LS

FGICR and EAO-CRC Lynch Syndrome n=87 1. MMR (or EPCAM) gene mutation 2. IHC deficient tumor • 71 germline mutations • 12 VUS Classic FAP n=84 Undefined conditions n=122 • • • 97 complete testing 5 partial testing 20 no testing 1. >100 adenomas 2. 100 adenomas + 1⁰ relative with FAP • 43 germline mutations: 41 APC, 2 biallelic MUTYH • 10 VUS AFAP n=8 • 8 germline mutations: 4 APC, 4 biallelic MUTYH JPS n=6 • • 2 germline mutations: 1 SMAD 4, 1 BMPR 1 A 1 VUS other Øhigh-risk familial ▪ FCCTX Øcommon familial risk Biallelic MMR deficiency n=4 • • 3 germline mutations 1 VUS Bloom syndrome n=1 • 1 germline mutation IBD n =11 Ønon-familial

Br J Surg. 2013 Dec; 100(13): 1719 -31. doi: 10. 1002/bjs. 9316. Systematic review of the impact of registration and screening on colorectal cancer incidence and mortality in familial adenomatous polyposis and Lynch syndrome. Barrow P 1, Khan M, Lalloo F, Evans DG, Hill J. METHODS • English-language studies describing CRC incidence and/or mortality in patients with FAP or LS • comparison of : screened and unscreened patients OR time periods before and after establishment of the registry RESULTS Ø FAP – 33/33 studies report reduction of CRC incidence and mortality with registration and screening Ø LS – 9/10 studies report reduction of CRC incidence and mortality with registration and screening

J Exp Clin Cancer Res. 2014 Jan 2; 33: 1. doi: 10. 1186/1756 -9966 -33 -1. Early-onset colorectal cancer patients without family history are "at very low risk" for lynch syndrome. Stigliano V 1, Sanchez-Mete L, Martayan A, Diodoro M, Casini B, Sperduti I, Anti M.

Clinicopathologic features of non-familial EAO-CRC ● ● more frequent location in distal colon and rectum later stage at presentation ● more frequent aggressive histologic features § § venous invasion perineural invasion mucinous histology signet ring histology Early-onset (≤ 40 years) colorectal adenocarcinoma (%) (N=55) Control (>40 years of age) colorectal adenocarcinoma (%) (N=73) P value  Right colon 11 (20) 31 (42) 0. 007  Left colon and rectum 44 (80) 42 (58) 24 (44) 20 (36) 33 (45) 9 (12) 6 (11) 14 (26) 20 (36) 15 (27) 14 (19) 21 (29) 28 (38) 10 (14) 0. 21 40 (73) 15 (27) 63 (86) 10 (14) 0. 06 Perineural invasion 16 (29) 8 (11) 0. 009 Venous invasion 12 (22) 4 (6) 0. 006 11 (15) 62 (85) 0. 22 1 (1) 72 (99) 0. 021 Tumor location  Sigmoid  Rectum Tumor stage I  III  IV Tumor grade  Low  High Mucinous histology  present 42 (76)  absent 13 (24) Signet ring histology  present 7 (13)  absent 48 (87) Chang DT, Pai RK, Rybicki LA, et al. Clinicopathologic and molecular features of sporadic early-onset colorectal adenocarcinoma: an adenocarcinoma with frequent signet ring cell differentiation, rectal and sigmoid involvement, and adverse morphologic features. Mod Pathol 2012 Aug; 25(8): 1128– 1139

PLo. S One. 2014 Aug 1; 9(8): e 103159. Sporadic early-onset colorectal cancer is a specific sub-type of cancer: a morphological, molecular and genetics study. Kirzin S 1, Marisa L 2, Guimbaud R 3, De Reynies A 2, Legrain M 4, Laurent-Puig P 5, Cordelier P 6, Pradère B 7, Bonnet D 8, Meggetto F 6, Portier G 7, Brousset P 9, Selves J 9. METHODS • surgical patients at university hospital in France 1999 -2005 ▪ 39 MSS tumor <45 y cases ▪ 9 MSI tumor <45 y cases ▪ 36 MSS tumor >60 y cases ▪ 14 MSI tumor >60 y cases RESULTS • absence of BRAF mutations and methylator phenotype in EAO-CRC MSS young MSS old MSI young MSI old mut 14 (37%) 16 (44%) 3 (33%) 4 (29%) WT 24 (63%) 20 (56%) 6 (67%) 10 (71%) BRAF V 600 E mut 0 (0%) 5 (14%) 0 (0%) 5 (36%) WT 39 (100%) 31 (86%) 9 (100%) 9 (64%) TP 53 mut 17 (44%) 17 (47%) 0 (0%) 5 (36%) WT 22 (56%) 19 (53%) 9 (100%) 9 (64%) PIK 3 CA mut 5 (21%) 2 (6%) 4 (80%) 3 (27%) WT 19 (79%) 32 (94%) 1 (20%) 8 (73%) CIMP unmethylated 38 (100%) 29 (81%) 9 (100%) 5 (38%) methylated 0 (0%) 7 (19%) 0 (0%) 8 (62%) KRAS • 49 signaling pathways upregulated in EAO-CRC CONCLUSIONS Ø EAO-CRC is distinct clinico-molecular entity

Am J Surg Pathol. 2009 Apr; 33(4): 572 -82. doi: 10. 1097/PAS. 0 b 013 e 31818 afd 6 b. Clinical, pathologic, and molecular features of early-onset colorectal carcinoma. Yantiss RK 1, Goodarzi M, Zhou XK, Rennert H, Pirog EC, Banner BF, Chen YT. METHODS • surgical patients UMass Memorial Healthcare and Weill Cornell Medical College 2000 -2007 § 24 patients <40 y § 15 patients 40 -40 y § 30 patients >50 y RESULTS • more frequent AMACR expression in patients <40 y • 4 micro-RNA species significantly overexpressed in patients <40 y (mi. R-21, mi. R-20 a, mi. R-181 b, mi-203) CONCLUSIONS Ø posttranscriptional regulation of m. RNA may be particularly important for the development of CRC in young patients

Exploring the genetics of EAO-CRC Next-generation sequencing (NGS)

Ø rare variant hypothesis ▪ undiscovered moderately penetrant genetic variants cause increased CRC risk v de novo dominant v recessive v biallelic somatic

Whole genome sequencing vs Exome sequencing Whole genome ü expensive ü IT issues ü complete sequence Exome ü ü cheaper more manageable coding regions incomplete sequence

Exome sequencing and cancer genetics Science. 2009 Apr 10; 324(5924): 217 Exomic sequencing identifies PALB 2 as a pancreatic cancer susceptibility gene. Jones S 1, Hruban RH, Kamiyama M, Borges M, Zhang X, Parsons DW, Lin JC, Palmisano E, Brune K, Jaffee EM, Iacobuzio-Donahue CA, Maitra A, Parmigiani G, Kern SE, Velculescu VE, Kinzler KW, Vogelstein B, Eshleman JR, Goggins M, Klein AP. Nat Genet. 2011 Jun 19; 43(7): 663 -7. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Comino-Méndez I 1, Gracia-Aznárez FJ, Schiavi F, Landa I, Leandro-García LJ, Letón R, Honrado E, Ramos-Medina R, Caronia D, Pita G, Gómez-Graña A, de Cubas AA, Inglada-Pérez L, Maliszewska A, Taschin E, Bobisse S, Pica G, Loli P, Hernández-Lavado R, Díaz JA, Gómez-Morales M, González-Neira A, Roncador G, Rodríguez-Antona C, Benítez J, Mannelli M, Opocher G, Robledo M, Cascón A. Nat Genet. 2011 May; 43(5): 442– 446. Exome sequencing identifies GRIN 2 A as frequently mutated in melanoma Xiaomu Wei, 1 Vijay Walia, 1, 12 Jimmy C Lin, 2, 12 Jamie K Teer, 3 Todd D Prickett, 1 Jared Gartner, 1 Sean Davis, 4 NISC Comparative Sequencing Program, 5 Katherine Stemke. Hale, 6 Michael A Davies, 6, 7 Jeffrey E Gershenwald, 8, 9 William Robinson, 10 Steven A Rosenberg, 11 and Yardena Samuels 1 Nature. 2011 Jan 27; 469(7331): 539– 542. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM 1 in renal carcinoma Ignacio Varela, 1 Patrick Tarpey, 1 Keiran Raine, 1 Dachuan Huang, 2 Choon Kiat Ong, 2 Philip Stephens, 1 Helen Davies, 1 David Jones, 1 Meng-Lay Lin, 1 Jon Teague, 1 Graham Bignell, 1 Adam Butler, 1 Juok Cho, 1 Gillian L. Dalgliesh, 1 Danushka Galappaththige, 1 Chris Greenman, 1 Claire Hardy, 1 Mingming Jia, 1 Calli Latimer, 1 King Wai Lau, 1 John Marshall, 1 Stuart Mc. Laren, 1 Andrew Menzies, 1 Laura Mudie, 1 Lucy Stebbings, 1 David A. Largaespada, 3 L. F. A. Wessels, 4 Stephane Richard, 5, 6 Richard J Kahnoski, 7 John Anema, 7 David A. Tuveson, 8 Pedro A. Perez-Mancera, 8 Ville Mustonen, 8 Andrej Fischer, 9, 10 David J. Adams, 11 Alistair Rust, 11 Waraporn Chan-on, 2 Chutima Subimerb, 2 Karl Dykema, 12 Kyle Furge, 12 Peter J. Campbell, 1 Bin Tean Teh, 2, 14 Michael R. Stratton, 1, 15 and P. Andrew Futreal 1

Nat Genet. 2013 Feb; 45(2): 136 -44. Germline mutations affecting the proofreading domains of POLE and POLD 1 predispose to colorectal adenomas and carcinomas. Palles C 1, Cazier JB, Howarth KM, Domingo E, Jones AM, Broderick P, Kemp Z, Spain SL, Guarino E, Salguero I, Sherborne A, Chubb D, Carvajal-Carmona LG, Ma Y, Kaur K, Dobbins S, Barclay E, Gorman M, Martin L, Kovac MB, Humphray S; CORGI Consortium; WGS 500 Consortium, Lucassen A, Holmes CC, Bentley D, Donnelly P, Taylor J, Petridis C, Roylance R, Sawyer EJ, Kerr DJ, Clark S, Grimes J, Kearsey SE, Thomas HJ, Mc. Vean G, Houlston RS, Tomlinson I. • whole genome sequencing and linkage analysis • COlore. Rectal Gene Identification (CORGI) study; recruits from UK Clinical Genetics Departments • 20 discovery phase samples: § 15 unrelated probands diagnosed with ≥ 10 colorectal adenomas before age 60 § relative with >5 colorectal adenomas for 3 probands, and 2 affected relatives for 1 proband • 3, 805 white UK validation phase samples enriched for family history of CRC and early-onset • 6, 721 white UK control samples without personal history of CRC

POLE L 424 V variant • pedigree SM 2702, only variant shared by all 3 affected individuals • present in 12 additional unrelated cases from validation phase and no controls • additional genotyping in 12 families showed all carriers developed colorectal tumors POLD 1 S 478 N variant • pedigree SM 1645, shared by both of the 2 affected individuals • present in additional proband plus additional unrelated case from validation phase and no controls • additional genotyping in SM 1645 and proband family showed all carriers developed colorectal tumors Ø dominantly-inherited high penetrance susceptibility for colorectal adenoma and carcinoma Ø mutations map to proof-reading (exonuclease) domain of DNA polymerases ε and δ; predicted to impair correction of mispaired bases inserted during DNA replication

Hum Mutat. 2013 Jul; 34(7): 1026 -34. doi: 10. 1002/humu. 22333. Epub 2013 May 20. Exome resequencing identifies potential tumor-suppressor genes that predispose to colorectal cancer. Smith CG 1, Naven M, Harris R, Colley J, West H, Li N, Liu Y, Adams R, Maughan TS, Nichols L, Kaplan R, Wagner MJ, Mc. Leod HL, Cheadle JP. METHODS • exome sequencing of 50 germline DNA samples • unrelated sporadic CRC patients from UK national multicenter randomized controlled trials COIN and COIN-B

analysis strategy 1 § § § search for protein-truncating variants in subset of 1, 138 genes from pathways implicated in CRC oncogenes excluded search for LOH by Sanger sequencing of matched tumor DNA when available Ø 32 variants in 31 genes Ø 5 variants with somatic biallelic inactivation FANCM, LAMB 4, PTCHD 3, LAMC 3, TREX 2 analysis strategy 2 § § § search for protein-truncating variants in all genes initially limited search to 18 EAO-CRC cases in sample set expanded to include all cases Ø 173 variants in 159 genes in EAO-CRC Ø additional 331 variants in additional 305 genes in remainder

Cancer Genetics 208 (2015) 35 -40 Systematic search for rare variants in Finnish early-onset colorectal cancer patients Tomas Tanskanen a, Alexandra E. Gylfe a, Riku Katainen a, Minna Taipale b, e, Laura Renkonen-Sinisalo a, c, Heikki J€arvinen c, Jukka-Pekka Mecklin d, Jan B€ohm d, Outi Kilpivaara a, Esa Pitk €anen a, Kimmo Palin a, Pia Vahteristo a, Sari Tuupanen a, Lauri A. Aaltonen a, * METHODS • exome sequencing • unselected surgical patients from 9 Finnish hospitals 1994 -1998 • discovery set of 22 non-familial EAO-CRC cases <40 y • validation set of 95 familial CRC cases

RESULTS § no genes with rare loss-of-function (Lo. F) variants present in more than one EOA-CRC patient § ADAMTS 4, CYTL 1, SYNE 1 Lo. F variants in one EAO-CRC patient and familial CRC cases § ACSL 5 p. Pro 71 Leu, INTS 5 p. Pro 922 Leu missense variants in 2 EAO-CRC patients and no familial CRC cases § MCTP 2, ARHGAP 12, ATM, DONSON, ROS 1 each with homozygous variants in one EOA-CRC patient § no compound heterozygous Lo. F variants CONCLUSIONS Ø results suggest genetic heterogeneity in unexplained EAO-CRC

The Toronto EAO-CRC cohort FGICR Undefined conditions n=122 • • • 97 complete testing 5 partial testing 20 no testing • complete testing • no polyposis • no significant family history: ▪ pedigree does not meet Amsterdam or Revised Bethesda criteria ▪ no relative with EAO-CRC (≤ 35 y) ▪ no first degree relative with CRC • germline and tumor DNA available Ø 30 EAO-CRC germline and tumor exomes

Ø rare variant hypothesis ▪ undiscovered moderately penetrant genetic variants cause increased CRC risk perhaps not one single variant but unique combinations ; OR the interaction of certain variant(s) with environmental exposures and/or epigenetic alterations, etc….

References Ferlay J, Soerjomataram I, Ervik M, et al. GLOBOCAN 2012 v 1. 0, Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer; 2013. Available from: http: //globocan. iarc. fr, accessed November 15, 2014. American Cancer Society. Colorectal Cancer Facts and Figures 2014 -2016. Atlanta: American Cancer Society; 2014. Canadian Cancer Society's Advisory Committee on Cancer Statistics (2014). Canadian Cancer Statistics 2014. Toronto, ON: Canadian Cancer Society. Siegel R, Desantis C, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014 Mar-Apr 64(2): 104 -17. Edwards BK, Ward E, Kohler BA, et al. Annual report to the nation on the status of cancer, 1975 -2006, featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates. Cancer. 2010 Feb 1; 116(3): 544 -73. Cheng L, Eng C, Neiman LX, Kapadia AS, Du XL. Trends in colorectal cancer incidence by anatomic site and disease stage in the United States from 1976 -2005. Am J Clin Oncol. 2011 Dec; 34(6): 573 -80 Surveillance Epidemiology and End Results. Fast Stats, http: // seer. cancer. gov/faststats. Accessed November 15, 2014. Bailey CE, Hu CY, You YN, et al. Increasing Disparities in the Age-Related Incidences of Colon and Rectal Cancers in the United States, 1975 -2010. JAMA Surg. 2014 Nov 5: 1 -6. Siegel RL, Jemal A, Ward EM. Increase in incidence of colorectal cancer among young men and women in the United States. Cancer Epidemiol Biomarkers Prev. 2009 Jun; 18(6): 1695 -8. You YN, Xing Y, Feig BW, Chang GJ, Cormier JN. Young-onset colorectal cancer: is it time to pay attention? Arch Intern Med. 2012 Feb 13; 172(3): 287 -9. Pignone M, Rich M, Teutsch SM, et al. Screening for colorectal cancer in adults at average risk: a summary of the evidence for the US Preventive Services Task Force. Ann Intern Med. 2002 Jul 16; 137(2): 132 -41. Whitlock EP, Lin JS, Liles E, Beil TL, Fu R. Screening for colorectal cancer: a targeted, updated systematic review for the U. S. Preventive Services Task Force. Ann Intern Med. 2008 Nov 4; 149(9): 638 -58. Myers EA, Feingold DL, Forde KA, et al. Colorectal cancer in patients under 50 years of age: A retrospective analysis of two institutions' experience. World J Gastroenterol. 2013 Sep 14; 19(34): 5651 -7. Kirzin S, Marisa L, Guimbaud R, et al. Sporadic early-onset colorectal cancer is a specific sub-type of cancer: a morphological, molecular and genetics study. PLo. S One. 2014 Aug 1; 9(8): e 103159. Chang DT, Pai RK, Rybicki LA, et al. Clinicopathologic and molecular features of sporadic early-onset colorectal adenocarcinoma: an adenocarcinoma with frequent signet ring cell differentiation, rectal and sigmoid involvement, and adverse morphologic features. Mod Pathol 2012 Aug; 25(8): 1128– 1139 Yantiss RK, Goodarzi M, Zhou XK, et al. Clinical, pathologic, and molecular features of early-onset colorectal carcinoma. Am J Surg Pathol 2009 Apr; 33(4): 572– 582 Fante R, Benatti P, di Gregorio C, et al. Colorectal carcinoma in different age groups: a population-based investigation. Am J Gastroenterol. 1997; 92: 1505– 1509. Minardi AJ, Sittig KM, Zibari GB, Mc. Donald JC. Colorectal cancer in the young patient. Am Surg. 1998; 64: 849– 853. Domergue J, Ismail M, Astre C, et al. Colorectal carcinoma in patients younger than 40 years of age. Montpellier Cancer Institute experience with 78 patients. Cancer. 1988; 61: 835– 840. Palmer ML, Herrera L, Petrelli NJ. Colorectal adenocarcinoma in patients less than 40 years of age. Dis Colon Rectum. 1991; 34: 343– 346. Chiang JM, Chen MC, Changchien CR, et al. Favorable influence of age on tumor characteristics of sporadic colorectal adenocarcinoma: patients 30 years of age or younger may be a distinct patient group. Dis Colon Rectum. 2003 Jul; 46(7): 904 -10. Parramore JB, Wei JP, Yeh KA (1998) Colorectal cancer in patients under forty: presentation and outcome. Am Surg 64: 563– 567 discussion 567– 568 Liang JT, Huang KC, Cheng AL, Jeng YM, Wu MS, et al. (2003) Clinicopathological and molecular biological features of colorectal cancer in patients less than 40 years of age. Br J Surg 90: 205– 214 Berg M, Danielsen SA, Ahlquist T, Merok MA, Agesen TH, et al. (2010) DNA sequence profiles of the colorectal cancer critical gene set KRAS-BRAF-PIK 3 CA-PTEN-TP 53 related to age at disease onset. PLo. S One. 2010 Nov 12; 5(11): e 13978. Alsop K, Mead L, Smith LD, Royce SG, Tesoriero AA, et al. (2006) Low somatic K-ras mutation frequency in colorectal cancer diagnosed under the age of 45 years. Eur J Cancer 42: 1357– 1361 Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990; 61: 759– 67. Reich DE, Lander ES. On the allelic spectrum of human disease. Trends Genet. 2001; 17: 502– 10. Wang WY, Barratt BJ, Clayton DG, Todd JA. Genome-wide association studies: theoretical and practical concerns. Nat Rev Genet. 2005; 6: 109– 118. Bodmer W 1, Bonilla C. Common and rare variants in multifactorial susceptibility to common diseases. Nat Genet. 2008 Jun; 40(6): 695 -701. doi: 10. 1038/ng. f. 136. Frayling I, Beck NE, Ilyas M, Dove-Edwin I, Goodman P, Beck JA, et al. The APC variants I 1307 K and E 1317 Q are associated with colorectal tumors, but not always with a family history. Proc Natl Acad Sci USA. 1998 Sep 1; 95(18): 10722– 7. Fearnhead N, Wilding JL, Winney B, Tonks S, Bartlett S, Bicknell DC, et al. Multiple rare variants in different genes account for multifactorial inherited susceptibility to colorectal adenomas. Proc Natl Acad Sci USA. 2004 Nov 9; 101(45): 15992– 7. Comino-Mendez I, Gracia-Aznarez FJ, Schiavi F, Landa I, Leandro-Garcia LJ, Leton R, et al. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nat Genet 2011 Jun 19; 43(7): 663– 7. Jones S, Hruban RH, Kamiyama M, Borges M, Zhang X, Parsons DW, et al. Exomic sequencing identifies PALB 2 as a pancreatic cancer susceptibility gene. 2009 Science Apr 10; 324(5924): 217 Palles C, Cazier JB, Howarth KM, Domingo E, Jones AM, Broderick P, et al. Germline mutations affecting the proofreading domains of POLE and POLD 1 predispose to colorectal adenomas and carcinomas. Nat Genet. 2013 Feb; 45(2): 136 -44. Smith CG, Naven M, Harris R, Colley J, West H, Li N, et al. Exome resequencing identifies potential tumor-suppressor genes that predispose to colorectal cancer. Hum Mutat. 2013 Jul; 34(7): 1026 -34. Newcomb PA, Baron J, Cotterchio M, Gallinger S, Grove J, Haile R, et al. Colon Cancer Family Registry: an international resource for studies of the genetic epidemiology of colon cancer. Cancer Epidemiol Biomarkers Prev. 2007 Nov; 16(11): 2331 -43.

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