Analysis of current testing practices

Clin Genet 2015: 87: 368 372 Printed in Singapore. All rights reserved Short Report Analysis of current testing practices for biallelic MUTYH mutations in MUTYH-associated polyposis 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12375 Landon M., Ceulemans S., Saraiya D.S., Strike B., Arnell C., Burbidge L.A., Moyes K., Theisen A., Fernandes P.H., Ji J.Q., Abbott B., Kaldate R.R., Roa B. Analysis of current testing practices for biallelic MUTYH mutations in MUTYH-associated polyposis. Clin Genet 2015: 87: 368 372. John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2014 MUTYH-associated polyposis (MAP) is an autosomal recessive syndrome caused by biallelic mutations in the base excision repair gene MUTYH. Owing to potential limitations in the MAP testing strategy and testing criteria, it is possible that MAP is being under-identified both genotypically and phenotypically. To determine whether full sequencing of MUTYH would increase clinical sensitivity over a founder mutation (FM) strategy, a retrospective analysis of two datasets from a commercial clinical laboratory was performed. The first cohort contained 1522 individuals who received MUTYH analysis for two FMs with subsequent full-gene sequencing. Eighty-five biallelic individuals were identified; 47 carried two FMs, 17 carried one FM and one mutation identified on full sequencing, and 21 carried biallelic mutations identified only on full sequencing. The second cohort contained 921 patients with colorectal cancer <50 years and <10 reported colorectal adenomas who had undergone MUTYH mutation testing. In this cohort, 19 of 921 (2.1%) individuals were identified as biallelic MUTYH carriers. Of these, 13 did not have a personal or family history of polyps and would not have met guidelines for MUTYH testing. These results suggest that individuals with biallelic MUTYH mutations are under-ascertained based on both genotype and phenotype under current standard testing practices. Conflict of interest All authors are employees of Myriad Genetic Laboratories, Inc. and receive salary and stock as compensation. M. Landon, S. Ceulemans, D.S. Saraiya, B. Strike, C. Arnell, L.A. Burbidge, K. Moyes, A. Theisen, P.H. Fernandes, J.Q. Ji, B. Abbott, R.R. Kaldate and B. Roa Myriad Genetic Laboratories, Inc., Salt Lake City, UT, USA Key words: autosomal recessive colorectal adenomatous polyposis genetic testing MUTYH MUTYH-associated polyposis Corresponding author: Michelle Landon, MS, LCGC, Myriad Genetic Laboratories, Inc., 320 Wakara Way, Salt Lake City, UT 84108, USA Tel: 801.584.1198; fax: 801-584-3099; e-mail: mlandon@myriad.com Received 10 December 2013, revised and accepted for publication 10 March 2014 Approximately 20% of patients with colorectal cancer (CRC) or adenomatous polyps have a family history of these neoplasms in a first-degree relative. However, pathogenic inherited genetic alterations have been identified in only 5% of patients with CRC (1). A subset of hereditary colon cancers has been attributed to MUTYH-associated polyposis (MAP). MAP is an autosomal recessive syndrome caused by biallelic mutations in MUTYH (2), a member of the base excision repair pathway that detects and protects against oxidative DNA damage. MAP has historically been characterized by the development of multiple colorectal adenomas (3 6). Individuals with MAP have a 50-fold increased risk for CRC in addition to predisposition to extracolonic cancers (7). The National Comprehensive Cancer Network (NCCN) 2013 guidelines recommend MUTYH gene testing in patients with a personal history of >10 adenomas or who meet the criteria for serrated polyposis syndrome with at least some adenomas (8). Current testing guidelines for individuals presenting with polyposis or CRC suggestive of autosomal recessive inheritance recommend testing for two common northern European MUTYH founder mutations (FMs), Y165C and G382D, 368

Analysis of current testing practices for MUTYH-associated polyposis with subsequent full sequencing if a single FM is identified. However, several studies suggest there is both genotypic and phenotypic heterogeneity of MUTYH that would confound current common testing strategies. As initial studies of MAP were performed on APC-negative polyposis patients, the phenotype commonly associated with MAP is distinctly polyposis-like. However, some patients present with CRC in the absence of recognizable polyposis (3 6). In addition, a subset of MAP + patients in large-population cohorts lacks a northern European FM (9, 10). Because of potential limitations with the common testing strategy and restrictive testing criteria, it is possible that MAP is being under-identified both genotypically and phenotypically. To assess these potential ascertainment biases, a retrospective analysis of two datasets from a commercial clinical laboratory was performed to determine whether full sequencing of MUTYH as a reflex test for MAP, regardless of polyposis history or ethnicity, would increase clinical sensitivity. Materials and methods Study inclusion criteria A retrospective database analysis was conducted to identify potential MAP + patients who were missed because of genetic testing strategy or phenotypic characteristics. Cohort 1 contained individuals who underwent testing for APC full-gene analysis and testing for the two common MUTYH FMs, Y165C and G382D. Subsequent full MUTYH sequencing was performed for all patients, except for those biallellic for the two FMs and those positive for an APC mutation. Eight of these patients had reflex testing for full MUTYH sequencing even though they were positive for an APC mutation (n = 7) or had a MUTYH FM on both alleles (n = 1). For each sample, clinical history and ethnicity were extracted from test requisition forms (TRF) provided by the referring healthcare provider. Individually identifiable patient information was not extracted. Cohort 2 contained patients with CRC <50 years and <10 reported colorectal adenomas who had MUTYH mutation testing between July 2006 and May 2012. MUTYH analysis consisted of genotyping for Y165C and G382D with subsequent full-gene sequencing in patients with a heterozygous mutation or direct full-gene MUTYH analysis. In this cohort, 370 patients had initial or reflex testing for mismatch repair (MMR) gene mutations associated with Lynch syndrome. Demographics, clinical characteristics and family histories were reviewed based on data reported on test request forms submitted to a commercial testing laboratory. Mutational testing results were also reviewed. Biallelic MUTYH mutation carriers were assessed by personal and/or family history to determine whether they met NCCN guidelines (Amsterdam 1 and Bethesda criteria) for MUTYH testing (8). MUTYH analysis Sequencing analysis was performed on DNA extracted from blood or buccal mouthwash samples. Targeted MUTYH DNA sequencing analysis was performed for European MUTYH FMs Y165C and G382D. In some cases, full-gene sequencing of MUTYH was performed in both forward and reverse directions. Sequencing results were analyzed by a proprietary computer-based review system followed by visual inspection and confirmation of all clinically significant variants. Genetic variants were detected by comparison with a consensus wild-type sequence for each gene. All variants in the biallelic state in this manuscript were considered deleterious according to standard American College of Medical Genetics and Genomics (ACMG) guidelines (11, 12). Results Genotypic variability analysis In Cohort 1, 1522 individuals had full-gene MUTYH sequencing subsequent to full APC gene analysis and MUTYH analysis for Y165C and G382D. Of these, 85 had biallelic MUTYH mutations, with 47 carrying two FMs, 17 carrying one FM and 1 mutation identified on full sequencing, and 21 carrying biallelic mutations identified only on full sequencing. Table 1 shows the ethnicity and mutation spectrum of these biallelic mutation carriers. Phenotypic variability analysis Cohort 2 contained 921 patients with CRC diagnosed <50 years with <10 reported colorectal adenomas. Of these individuals, 214 had full MUTYH sequencing, and 707 had genotyping for Y165C and G382D (Table 2). Biallelic MUTYH mutations were identified in 19 of 921 (2.1%) of the individuals. Of the biallelic mutation carriers, 9 had no reported adenomas and 10 had 1 9 adenomas. Overall, 13 (68.4%) did not have a personal or family history meeting NCCN guidelines for MUTYH testing based upon polyp history. In addition, 11 of 921 patients were positive for mutations in MMR genes. All individuals with biallelic MUTYH mutations had at least one FM; this is a result of ascertainment bias due to the testing strategy performed. Discussion These results suggest that individuals with biallelic MUTYH mutations are under-ascertained based on both genotype and phenotype under current standard testing practices. These results are in concordance with previous studies that have suggested screening of MUTYH should be considered in patients with multiple polyps, and in patients with early-onset CRC. In Cohort 1, FM testing with reflex to full MUTYH sequencing if positive for one of the two FMs identified 64 of 85 (75%) biallelic MUTYH mutation carriers. Full MUTYH sequencing identified an additional 21 biallelic MUTYH mutation carriers. Thus, analysis of only the 369

Landon et al. Table 1. Demographics of patients in Cohort 1. Biallelic MUTYH carriers (n = 85) No FM (n = 21) 1 FM (n = 17) 2 FM (n = 47) Non-biallelic carriers (n = 1437) % Female 9 (42.9%) 8 (47.1%) 23 (48.9%) 653 (45.4%) Mean age of polyp diagnosis a 41.2 years 45.7 years 49.3 years 47.7 years Range of reported polyps 100+ 9 (42.9%) 3 (17.6%) 9 (19.1%) 198 (13.8%) 10 99 12 (57.1%) 13 (76.5%) 34 (72.3%) 923 (64.2%) 1 9 0 (0.0%) 0 (0.0%) 2 (4.3%) 158 (11.0%) No reported polyps 0 (0.0%) 1 (5.9%) 2 (4.3%) 132 (9.2%) No polyp range specified 0 (0.0%) 0 (0.0%) 0 (0.0%) 26 (1.8%) Ethnicity African 0 (0.0%) 0 (0.0%) 0 (0.0%) 92 (6.4%) Ashkenazi 0 (0.0%) 0 (0.0%) 0 (0.0%) 51 (3.5%) Asian 5 (23.8%) 0 (0.0%) 0 (0.0%) 50 (3.5%) Central/Eastern Europe 0 (0.0%) 0 (0.0%) 1 (2.1%) 56 (3.9%) Latin American Caribbean 2 (9.5%) 1 (5.9%) 9 (19.1%) 99 (6.9%) Native American 0 (0.0%) 0 (0.0%) 0 (0.0%) 7 (0.5%) Near east/mideast 5 (23.8%) 0 (0.0%) 1 (2.1%) 16 (1.1%) Western/Northern Europe 6 (28.6%) 14 (82.4%) 28 (59.6%) 704 (49.0%) Multiple Ethnicities Indicated 1 (4.8%) 0 (0.0%) 3 (6.4%) 154 (10.7%) None Specified 2 (9.5%) 2 (11.8%) 5 (10.6%) 208 (14.5%) FM, founder mutation. a When provided. Table 2. Demographics of patients in Cohort 2 Biallelic MUTYH carriers (n = 19) Non-biallelic carriers (n = 902) % Female 12 (63.2%) 499 (55.3%) Mean age at time of colon 39.7 years 37.9 years cancer diagnosis Range of reported polyps 1 9 10 (52.6%) 396 (43.9%) No reported polyps 9 (47.4%) 506 (56.1%) Ethnicity African 0 (0.0%) 36 (4.0%) Ashkenazi 0 (0.0%) 19 (2.1%) Asian 0 (0.0%) 35 (3.9%) Central/Eastern Europe 1 (5.3%) 51 (5.7%) Latin American/Caribbean 3 (15.8%) 63 (7.0%) Native American 0 (0.0%) 7 (0.8%) Near east/mideast 0 (0.0%) 11 (1.2%) Western/Northern Europe 11 (57.9%) 468 (51.9%) Multiple Ethnicities Indicated 2 (10.5%) 98 (10.9%) None specified 2 (10.5%) 114 (12.6%) two MUTYH FMs would have missed 25% (21/85) of biallelic individuals in Cohort 1; 20% (17/85) of biallelic individuals would have been misidentified as monoallelic MUTYH carriers. Full-gene sequencing of MUTYH in patients with a suspected polyposis syndrome is likely to increase clinical sensitivity. Owing to the ethnicity distribution of mutations, it is difficult to predict who would be missed by FM testing with reflex to sequencing only if positive for one mutation. Almost half of biallelic FM carriers (40%) did not report Western/Northern European ancestry. However, 8.3 22% of polyposis patients with MAP identified through full sequencing do not have either FM (13 15). In addition, common mutations exist in other ethnic populations, including E466X (East Indian), Y90X (Pakistani), R245C and IVS10-2a > g (Japanese), c.1145delc (Italian), A473D (Finnish), and E383fxX451 (Portuguese) (9, 16). Interestingly, in Cohort 1, 6 of the 21 (28.6%) patients with no FMs were of Western/Northern European ethnicity (Table 1). While a higher proportion of patients with no FMs were identified to be of Asian and Near/Middle Eastern ethnicity, this is consistent with the expected outcome since as the tested FMs are seen at a higher frequency in the Western/Northern European population. In Cohort 2, all subjects with biallelic MUTYH mutations had at least one FM. However, this is a result of the testing strategy; any mutation carriers in the study would have first been identified by the presence of an FM. The analysis of patients in Cohort 1 had standard limitations common to any retrospective analysis. Given that many of these patients were tested in conjunction with APC analysis, there is an ascertainment bias for individuals whose personal and/or family history is suggestive of an adenomatous polyposis syndrome. Therefore, the reported phenotypic trends probably do not represent those of the overall biallelic MUTYH population. In addition, the accuracy of demographic, personal, and family cancer histories reported on the TRF was not verified through an independent medical record review. 370

Analysis of current testing practices for MUTYH-associated polyposis Table 3. Syndromic characteristics of MAP positive patients in Cohort 2 (n = 19) Patients meeting criteria Personal history of polyps No 9/19 (47.4%) Yes 10/19 (52.6%) Lynch syndrome criteria Non-age related Bethesda Family history 7/19 (36.8%) Multiple a cancers 2/19 (10.5%) Family history and multiple cancers 2/19 (10.5%) Amsterdam I 1/19 (5.3%) a Two or more cancers. This could have resulted in an over/under representation of MUTYH biallelic + patients based on colon polyp number and cancer diagnosis in Cohort 2 but would have a minimal impact on the outcomes for Cohort 1. These data do not include TRFs missing personal and family history information (2/85 biallelic MUTYH carriers). In Cohort 2, 13 of 19 (68.4%) patients with biallelic MUTYH mutations lacked a personal or family history meeting guidelines for MUTYH testing based on polyp history. These patients displayed a phenotype more closely resembling Lynch syndrome with 9 of 19 biallelic MUTYH mutation carriers having no adenomas. These nine MAP patients would not have been identified if having multiple adenomas was the sole criterion for germline MUTYH testing. Most of the 19 patients with biallelic MUTYH mutations reported personal and family histories consistent with Lynch syndrome with 12 of the 19 meeting NCCN guidelines (Amsterdam I and Non-Age Related Bethesda criteria) for Lynch syndrome testing (Table 3). All patients met the Bethesda criteria for age. This phenotypic overlap is evidenced by the fact that 370 patients in this cohort were also tested for Lynch syndrome. These results are comparable to previous studies that show family histories of MAP patients consistent with Lynch syndrome (6). These results also support previous findings that MAP can contribute to CRC in the absence of colon polyps (4 6, 17). Conversely, 94% (80/85) of individuals in Cohort 1 reported 10 or more adenomas, meeting clinical diagnostic criteria for familial adenomatous polyposis (FAP). This was expected as most individuals underwent genetic testing for an adenomatous polyposis indication, which is reflective of a possible ascertainment bias. The phenotypic variability within and between Cohorts 1 and 2 shows the phenotypic overlap between inherited polyposis syndromes. Full-gene MUTYH sequencing as part of testing for inherited CRC syndromes regardless of polyp history may increase clinical sensitivity. MUTYH biallelic mutations are found at a similar incidence in CRC patients <50 years as mutations in individual MMR genes. The contribution of MAP to early-onset CRC may be comparable to that of each MMR gene (MLH1, MSH2, MSH6, PMS2) i.e. 1 2% (18). Full-gene MUTYH sequencing should be considered in patients <50 who meet Lynch syndrome criteria in the absence of MMR mutations. Although the prevalence of biallelic MUTYH mutations is higher in early-onset CRC, the overall prevalence relative to that of each MMR gene suggests testing would increase clinical sensitivity regardless of age. The phenotypic and genotypic under-ascertainment of MAP patients described in these datasets suggest that a possible revision of medical practice to include full MUTYH testing for patients with young-onset CRC in the absence of polyposis may be appropriate. One possible solution to the issue of phenotypic overlap is to consider the use of multi-gene panels that are becoming widely available. Such a testing strategy may help identify additional patients with a hereditary cancer syndrome and enable clinicians to better manage individuals and families who are affected or at risk for these inherited disorders with specific genetic and clinical counseling, screening, and treatment recommendations. 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