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Hereditary colorectal cancer (CRC) syndromes attributable to high penetrance mutations represent 9–26% of young-onset CRC cases. The clinical significance of many of these mutations is understood well enough to be used in diagnostics and as an aid in patient care. However, despite the advances made in the field, a significant proportion of familial and early-onset cases remains molecularly uncharacterized and extensive work is still needed to fully understand the genetic nature of CRC susceptibility. With the emergence of next-generation sequencing and associated methods, several predisposition loci have been unraveled, but validation is incomplete. Individuals with cancer-predisposing mutations are currently enrolled in life-long surveillance, but with the development of new treatments, such as cancer vaccinations, this might change in the not so distant future for at least some individuals. For individuals without a known cause for their disease susceptibility, prevention and therapy options are less precise. Herein, we review the progress achieved in the last three decades with a focus on how CRC predisposition genes were discovered. Furthermore, we discuss the clinical implications of these discoveries and anticipate what to expect in the next decade.

International guidelines recommend universal screening of endometrial carcinoma (EC) patients for Lynch syndrome (LS). This screening is generally based on mismatch repair (MMR) protein immunohistochemistry followed by

Purpose: Some 10% of familial adenomatous polyposis (FAP) and 80% of attenuated polyposis (AFAP) cases remain molecularly unexplained. We scrutinized such cases by exome-wide and targeted methods to search for novel susceptibility genes. Methods: Exome sequencing was conducted on 40 unexplained (mainly sporadic) cases with FAP or AFAP from Finland. The DNA mismatch repair (MMR) gene MLH3 (MutL Homolog 3) was pinpointed and prompted a subsequent screen of similar to 1000 Swedish patients referred to clinical panel sequencing for colon tumor susceptibility. Results: Three homozygous carriers of a truncating variant in MLH3, c.3563C>G, p.Ser1188Ter, were identified among the index cases from the Finnish series. An additional biallelic carrier of the same variant was present in the Swedish series. All four patients shared a 0.8-Mb core haplotype around MLH3, suggesting a founder variant. Colorectal polyps from variant carriers showed no instability at mono-, di-, tri-, or tetranucleotide repeats, in agreement with previous findings of a minor role of MLH3 in MMR. Multiple loci were affected by loss of heterozygosity, suggesting chromosomal instability. Conclusion: Our results show that a biallelic nonsense variant of MLH3 underlies a novel syndrome with susceptibility to classical or attenuated adenomatous polyposis and possibly extracolonic tumors, including breast cancer.

Ulcerative colitis increases colorectal cancer risk by mechanisms that remain incompletely understood. We approached this question by determining the genetic and epigenetic profiles of colitis-associated colorectal carcinomas (CA-CRC). The findings were compared to Lynch syndrome (LS), a different form of cancer predisposition that shares the importance of immunological factors in tumorigenesis. CA-CRCs (n=27) were investigated for microsatellite instability, CpG island methylator phenotype, and somatic mutations of 999 cancer-relevant genes ("Pan-cancer" panel). A subpanel of "Pan-cancer" design (578 genes) was used for LS colorectal tumors (n=28). Mutational loads and signatures stratified CA-CRCs into three subgroups: hypermutated microsatellite-unstable (group 1, n=1), hypermutated microsatellite-stable (group 2, n=9), and non-hypermutated microsatellite-stable (group 3, n=17). The group 1 tumor was the only one with MLH1 promoter hypermethylation and exhibited the mismatch repair deficiency-associated signatures 21 and 15. Signatures 30 and 32 characterized group 2, whereas no prominent single signature existed in group 3. TP53, the most common mutational target in CA-CRC (16/27, 59%), was similarly affected in groups 2 and 3, but DNA repair genes and Wnt signaling genes were mutated significantly more often in group 2. In LS tumors, the degree of hypermutability exceeded that of the hypermutated CA-CRC groups 1 and 2, and somatic mutational profiles and signatures were different. In conclusion, groups 1 (4%) and 3 (63%) comply with published studies, whereas group 2 (33%) is novel. The existence of molecularly distinct subgroups within CA-CRC may guide clinical management, such as therapy options. This article is protected by copyright. All rights reserved.

Up to one third of colorectal cancers show familial clustering and 5% are hereditary single-gene disorders. Hereditary non-polyposis colorectal cancer comprises DNA mismatch repair-deficient and -proficient subsets, represented by Lynch syndrome (LS) and familial colorectal cancer type X (FCCTX), respectively. Accurate knowledge of molecular etiology and genotype-phenotype correlations are critical for tailored cancer prevention and treatment.The authors highlight advances in the molecular dissection of hereditary non-polyposis colorectal cancer, based on recent literature retrieved from PubMed. Future possibilities for novel gene discoveries are discussed.LS is molecularly well established, but new information is accumulating of the associated clinical and tumor phenotypes. FCCTX remains poorly defined, but several promising candidate genes have been discovered and share some preferential biological pathways. Multi-level characterization of specimens from large patient cohorts representing multiple populations, combined with proper bioinformatic and functional analyses, will be necessary to resolve the outstanding questions.

Some 10&ndash, 50% of Lynch-suspected cases with abnormal immunohistochemical (IHC) staining remain without any identifiable germline mutation of DNA mismatch repair (MMR) genes. MMR proteins form heterodimeric complexes, giving rise to distinct IHC patterns when mutant. Potential reasons for not finding a germline mutation include involvement of an MMR gene not predicted by the IHC pattern, epigenetic mechanism of predisposition, primary mutation in another DNA repair or replication-associated gene, and double somatic MMR gene mutations. We addressed these possibilities by germline and tumor studies in 60 Lynch-suspected cases ascertained through diagnostics (n = 55) or research (n = 5). All cases had abnormal MMR protein staining in tumors but no point mutation or large rearrangement of the suspected MMR genes in the germline. In diagnostic practice, MSH2/MSH6 (MutS Homolog 2/MutS Homolog 6) deficiency prompts MSH2 mutation screening, in our study, 3/11 index individuals (27%) with this IHC pattern revealed pathogenic germline mutations in MSH6. Individuals with isolated absence of MSH6 are routinely screened for MSH6 mutations alone, we found a predisposing mutation in MSH2 in 1/7 such cases (14%). Somatic deletion of the MSH2-MSH6 region, joint loss of MSH6 and MSH3 (MutS Homolog 3) proteins, and hindered MSH2/MSH6 dimerization offered explanations to misleading IHC patterns. Constitutional epimutation hypothesis was pursued in the MSH2 and/or MSH6-deficient cases plus 38 cases with MLH1 (MutL Homolog 1)-deficient tumors, a primary MLH1 epimutation was identified in one case with an MLH1-deficient tumor. We conclude that both MSH2 and MSH6 should be screened in MSH2/6- and MSH6-deficient cases. In MLH1-deficient cases, constitutional epimutations of MLH1 warrant consideration.

Background: Familial adenomatous polyposis (FAP) is a condition typically caused by pathogenic germline mutations in the APC gene. In addition to colon polyps, individuals with FAP have a substantially increased risk of developing papillary thyroid cancer (PTC). Little is known about the events underlying this association, and the prevalence of somatic "second-hit" mutations in APC is controversial. Methods: Whole-genome sequencing was performed on paired thyroid tumor and normal DNA from 12 FAP patients who developed PTC. Somatic mutation profiles were compared with clinical characteristics and previously sequenced sporadic PTC cases. Germline variant profiling was performed to assess the prevalence of variants in genes previously shown to have a role in PTC predisposition. Results: All 12 patients harbored germline mutations in APC, consistent with FAP. Seven patients also had somatic mutations in APC, and seven patients harbored somatic mutations in KMT2D, which encodes a lysine methyl transferase. Mutation of these genes is extremely rare in sporadic PTCs. Notably, only two of the tumors harbored the somatic BRAF p.V600E mutation, which is the most common driver mutation found in sporadic PTCs. Six tumors displayed a cribriform-morular variant of PTC (PTC-CMV) histology, and all six had somatic mutations in APC. Additionally, nine FAP-PTC patients had rare germline variants in genes that were previously associated with thyroid carcinoma. Conclusions: Our data indicate that FAP-associated PTCs typically have distinct mutations compared with sporadic PTCs. Roughly half of the thyroid cancers that arise in FAP patients have somatic "second-hits" in APC, which is associated with PTC-CMV histology. Somatic BRAF p.V600E variants also occur in some FAP patients, a novel finding. We speculate that in carriers of heterozygous pathogenic mutations of tumor suppressor genes such as APC, a cooperating second-hit somatic variant may occur in a different gene such as KTM2D or BRAF, leading to differences in phenotypes. The role of germline variance in genes other than APC (9 of the 12 patients in this series) needs further research.

// Noora K. Porkka 1 , Alisa Olkinuora 1 , Teijo Kuopio 2 , 3 , Maarit Ahtiainen 4 , Samuli Eldfors 5 , Henrikki Almusa 5 , Jukka-Pekka Mecklin 6 , 7 , 8 and Paivi Peltomaki 1 1 Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland 2 Department of Pathology, Jyvaskyla Central Hospital, Jyvaskyla, Finland 3 Department of Biological and Environmental Science, University of Jyvaskyla, Jyvaskyla, Finland 4 Department of Education and Research, Jyvaskyla Central Hospital and University of Eastern Finland, Jyvaskyla, Finland 5 Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland 6 Faculty of Sport and Health Sciences, University of Jyvaskyla, Jyvaskyla, Finland 7 Department of Surgery, Jyvaskyla Central Hospital, Jyvaskyla, Finland 8 Department of Education & Science, Jyvaskyla Central Hospital, Jyvaskyla, Finland Correspondence to: Noora K. Porkka, email: noora.porkka@helsinki.fi Keywords: Lynch syndrome; breast carcinoma; MSI; DNA mismatch repair; somatic mutation Received: February 13, 2020 Accepted: March 14, 2020 Published: April 07, 2020 ABSTRACT Inherited DNA mismatch repair (MMR) defects cause predisposition to colorectal, endometrial, ovarian, and other cancers occurring in Lynch syndrome (LS). It is unsettled whether breast carcinoma belongs to the LS tumor spectrum. We approached this question through somatic mutational analysis of breast carcinomas from LS families, using established LS-spectrum tumors for comparison. Somatic mutational profiles of 578 cancer-relevant genes were determined for LS-breast cancer (LS-BC, n = 20), non-carrier breast cancer (NC-BC, n = 10), LS-ovarian cancer (LS-OC, n = 16), and LS-colorectal cancer (LS-CRC, n = 18) from the National LS Registry of Finland. Microsatellite and MMR protein analysis stratified LS-BCs into MMR-deficient (dMMR, n = 11) and MMR-proficient (pMMR, n = 9) subgroups. All NC-BCs were pMMR and all LS-OCs and LS-CRCs dMMR. All but one dMMR LS-BCs were hypermutated (> 10 non-synonymous mutations/Mb; average 174/Mb per tumor) and the frequency of MMR-deficiency-associated signatures 6, 20, and 26 was comparable to that in LS-OC and LS-CRC. LS-BCs that were pMMR resembled NC-BCs with respect to somatic mutational loads (4/9, 44%, hypermutated with average mutation count 33/Mb vs. 3/10, 30%, hypermutated with average 88 mutations/Mb), whereas mutational signatures shared features of dMMR LS-BC, LS-OC, and LS-CRC. Epigenetic regulatory genes were significantly enriched as mutational targets in LS-BC, LS-OC, and LS-CRC. Many top mutant genes of our LS-BCs have previously been identified as drivers of unselected breast carcinomas. In conclusion, somatic mutational signatures suggest that conventional MMR status of tumor tissues is likely to underestimate the significance of the predisposing MMR defects as contributors to breast tumorigenesis in LS.

Some 10% of familial adenomatous polyposis (FAP) and 80% of attenuated polyposis (AFAP) cases remain molecularly unexplained. We scrutinized such cases by exome-wide and targeted methods to search for novel susceptibility genes.Exome sequencing was conducted on 40 unexplained (mainly sporadic) cases with FAP or AFAP from Finland. The DNA mismatch repair (MMR) gene MLH3 (MutL Homolog 3) was pinpointed and prompted a subsequent screen of ~1000 Swedish patients referred to clinical panel sequencing for colon tumor susceptibility.Three homozygous carriers of a truncating variant in MLH3, c.3563CG, p.Ser1188Ter, were identified among the index cases from the Finnish series. An additional biallelic carrier of the same variant was present in the Swedish series. All four patients shared a 0.8-Mb core haplotype around MLH3, suggesting a founder variant. Colorectal polyps from variant carriers showed no instability at mono-, di-, tri-, or tetranucleotide repeats, in agreement with previous findings of a minor role of MLH3 in MMR. Multiple loci were affected by loss of heterozygosity, suggesting chromosomal instability.Our results show that a biallelic nonsense variant of MLH3 underlies a novel syndrome with susceptibility to classical or attenuated adenomatous polyposis and possibly extracolonic tumors, including breast cancer.

Lynch syndrome (LS) is the most prevalent cancer predisposition syndrome in which germline mutation in one of four DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 causes significantly increased lifetime risks of colorectal, endometrial, ovarian, and other cancers. Whether or not breast cancer is part of LS tumor spectrum is currently debated. Moreover, the reasons for organ selectivity in germline mutation carriers are unknown. We have reported that breast carcinoma from LS individuals resembles common breast carcinoma in many respects, but differs with respect to genomic instability (Lotsari et al. 2012, Cancer Res.). To extend these preliminary observations, we are now carrying out an in-depth investigation into the spectra of somatic mutations in LS and sporadic breast carcinomas. All available breast carcinomas among 300 LS families from the nation-wide Hereditary Colon Cancer Registry of Finland have been identified and the tumors investigated for MMR status, DNA methylator phenotype, mechanisms of two-allele inactivation for MMR genes, and somatic mutation status. For the latter, we use massive parallel sequencing as described in our recent publication (Porkka et al. 2017, Oncotarget). To define the unique vs. shared molecular features of LS breast tumorigenesis, profiles of somatic alterations in LS breast carcinomas are compared to those of established LS spectrum carcinomas (colorectal and ovarian) as well as sporadic breast carcinomas with sequence information available from large electronic data-sets. Of 14 LS breast carcinoma samples, 9 (64 %) were MMR-deficient and 5 (36 %) MMR-proficient. This contrasts with LS colorectal and ovarian carcinomas, all of which were MMR-deficient (p=0.0012). MMR-deficient LS breast carcinomas harbored an average of 783 non-synonymous mutations and MMR-proficient tumors 555 mutations (statistically non-significant). In contrast to colorectal and ovarian carcinomas, in which loss of heterozygosity (LOH) or somatic MMR gene mutation provided a second hit consistent with two-hit inactivation in 89 % (25/28) of the tumors, a detectable second hit (mainly LOH) was present in only 43 % (6/14) of breast carcinomas (p=0.0025). Genes harboring high-frequency mutations in at least one-third of tumors were regarded candidates for driver genes. There were 133 such genes in LS colorectal carcinomas, 10 in ovarian carcinomas, and 18 in breast carcinomas. Genes involved in epigenetic regulation were significantly enriched in all three tumor types. In addition, a significant enrichment of NOTCH signaling associated genes characterized LS breast carcinomas. This research is likely to shed light to the mechanisms of organ-specific cancer susceptibility in germline carriers of MMR gene mutations. Our results are expected to guide the surveillance and other clinical management of LS individuals with breast carcinoma. Citation Format: Noora Porkka, Alisa Olkinuora, Satu Mäki-Nevala, Samuli Eldfors, Päivi Peltomäki. Tumorigenesis in Lynch syndrome: Somatic mutation profiles compared to sporadic counterparts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5369.