Prevalence and Characteristics of MUTYH-Associated Polyposis in Patients with Multiple Adenomatous and Serrated Polyps

Published OnlineFirst January 27, 2014; DOI: 10.1158/1078-0432.CCR-13-1490

Clinical Cancer Human Cancer Biology Research See related article by Borras et al., p. 1061

Carla Guarinos1, Miriam Juarez 1, Cecilia Egoavil2, Mara Rodrguez-Soler1,3, LucaPerez-Carbonell 1,19, Ramon Salas1, Joaqun Cubiella4, Francisco Rodrguez-Moranta5, Luisa de-Castro8, Luis Bujanda9, Anna Serradesanferm6, David Nicolas-P erez 10, Maite Herraiz 11, Fernando Fernandez-Ba nares~ 12, Alberto Herreros-de-Tejada13, Elena Aguirre14, Judith Balmana~ 7, Mara-Luisa Rincon 15, Angeles Pizarro16, Francisco Polo-Ortiz17, Adela Castillejo18, Cristina Alenda2, Artemio Paya2, Jose-Luis Soto18, and Rodrigo Jover1,3

Abstract Purpose: The present study aimed to determine the prevalence of MUTYH mutations in patients with multiple colonic polyps and to explore the best strategy for diagnosing MUTYH-associated polyposis (MAP) in these patients. Experimental Design: This study included 405 patients with at least 10 colonic polyps each. All cases were genetically tested for the three most frequent MUTYH mutations. Whole-gene analysis was performed in heterozygous patients and in 216 patients lacking the three most frequent mutations. Polyps from 56 patients were analyzed for the KRAS-Gly12Cys and BRAF V600E somatic mutations. Results: Twenty-seven (6.7%) patients were diagnosed with MAP, of which 40.8% showed serrated polyps. The sensitivity of studying only the three common variants was 74.1%. Of 216 patients without any monoallelic mutation in common variants, whole-gene analysis revealed biallelic pathogenic mutation in only one. G396D mutation was associated with serrated lesions and older age at diagnosis. There was a strong association between germinal MUTYH mutation and KRAS Gly12Cys somatic mutation in polyps. BRAF V600E mutation was found in 74% of serrated polyps in MUTYH-negative patients and in none of the polyps of MAP patients. Conclusions: We observed a low frequency of MUTYH mutations among patients with multiple adenomatous and serrated polyps. The MAP phenotype frequently included patients with serrated polyps, especially when G396D mutation was involved. Our results show that somatic molecular markers of polyps can be useful in identifying MAP cases and support the need for the complete MUTYH gene analysis only in patients heterozygous for recurrent variants. Clin Cancer Res; 20(5); 1158–68. 2014 AACR.

Introduction MUTYH gene. Patients with MAP exhibit a mean of 50 polyps MUTYH-associated polyposis (MAP; OMIM #608456) is (3), and this disease is responsible for 7% of attenuated an autosomal recessive disease that usually appears in adenomatous polyposis and 6.6% of classic polyposis cases patients with an attenuated polyposis phenotype (1, 2). This (4). MAP cases with colorectal cancer not showing a poly- syndrome is associated with biallelic mutations in the posis phenotype have also been described previously (5).

Authors' Afﬁliations: 1Unidad de Investigacion, Departments of 2Pathol- ment of Gastroenterology, Hospital Basurto, Bilbao; 18Department of ogy, and 3Gastroenterology, Hospital General Universitario, Alicante; Molecular Genetics, Hospital General Universitario, Elche, Spain; and 4Department of Gastroenterology, Complexo Hospitalario Universitario de 19Gastrointestinal Cancer Research Laboratory, Baylor Research Institute, Ourense, Ourense; 5Department of Gastroenterology, Bellvitge University Baylor University Medical Center, Dallas, Texas Hospital; 6Institut de Malaties Digestives i Metaboliques, CIBERehd, Hos- pital Clnic; 7Department of Oncology, Hospital Vall d'Hebron, Barcelona; 8Gastroenterology Department, Complexo Hospitalario de Vigo, Vigo; Note: Supplementary data for this article are available at Clinical Cancer 9Department of Gastroenterology, Hospital Donostia, CIBERehd, Univer- Research Online (http://clincancerres.aacrjournals.org/). sidad del Pas Vasco, San Sebastian; 10Department of Gastroenterology, Hospital Universitario de Canarias, La Laguna, Tenerife; 11Department Corresponding Author: Rodrigo Jover, Unidad de Gastroenterologa, of Gastroenterology, Clnica Universitaria de Navarra, Pamplona; 12Depart- Hospital General Universitario de Alicante, C/Pintor Baeza 12, 03010 ment of Gastroenterology, Hospital Mutua de Terrassa, Terrassa; Alicante, Spain. Phone: 34-965933468; Fax: 34-965933468; E-mail: 13Department of Gastroenterology, Hospital Puerta de Hierro, Madrid; [email protected] 14 15 Department of Oncology, Hospital Arnau de Vilanova, Lleida; Depart- doi: 10.1158/1078-0432.CCR-13-1490 ment of Gastroenterology, Hospital Bidasoa, Irun; 16Department of Gas- troenterology, Hospital Universitario Virgen del Roco, Sevilla; 17Depart- 2014 American Association for Cancer Research.

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MUTYH-Associated Polyposis in Patients with Serrated Polyps

authors have proposed different diagnostic strategies, and Translational Relevance no consensus has been reached (4, 5, 17, 18). Our results could lead to advances in the characteri- The present study aimed to determine the prevalence of zation and genetic diagnosis of MUTYH-associated poly- MUTYH mutations in a population of patients with mul- posis (MAP), consequently improving colorectal cancer tiple colonic polyps, including both adenomatous and prevention and the management and surveillance of serrated polyps, and to explore the diagnostic yield of these patients. We demonstrated that almost half of different strategies for germline MUTYH testing in these MAP cases showed both adenomatous and serrated patients. Moreover, we investigated the usefulness of somat- polyps, especially when G396D mutation was involved, ic molecular markers in the polyps for MAP diagnosis. Our and even in cases with less than 10 adenomas. Therefore, results could lead to better characterization of the syndrome genetic testing for MUTYH should be offered not only in and enhanced strategies for the genetic diagnosis of the cases with the classically described attenuated adeno- disease, consequently improving colorectal cancer preven- matous polyposis phenotype, but also and especially in tion and the management and surveillance of these patients. cases presenting both adenomatous and serrated polyps. Moreover, our results support a MAP diagnostic strategy Materials and Methods that starts with analysis of the more common mutations, Patients including locally prevalent mutations, and includes The study included 405 patients from the EPIPOLIP study, whole-gene analysis only in cases showing a heterozy- a multicenter nationwide project that investigated causes of gous mutation in one of the gene hotspots. In addition, multiple colonic polyps and incorporated patients from 24 the majority of MUTYH mutation carriers showed the Spanish hospitals. Patients were retrospectively recruited somatic KRAS Gly12Cys mutation in their polyps. There- during the years 2009 to 2010 (19). Patients diagnosed with fore, this mutation may be useful not only as a pre- at least 10 polyps of any histology were included in the screening test for MAP diagnosis but also as a useful tool present study. Patients previously diagnosed with familial for classifying variants of uncertain significance at adenomatous polyposis, Lynch syndrome, or inflammatory MUTYH. bowel disease, and those who had only hyperplastic recto- sigmoid polyps were excluded. Informed written consent was obtained from all participants. The study was approved by the ethical committees from the participating hospitals. Colorectal cancer risk in patients with biallelic mutations is Demographic data about age, sex, and personal and about 80% by the age of 70 years without treatment, and familial history of polyposis, colorectal cancer, or other colorectal cancer is diagnosed simultaneously with the diag- neoplasia were collected. Endoscopy reports and the corre- nosis of polyposis in approximately 50% of patients (6). sponding histopathology reports were also reviewed to Patients with MAP can also present duodenal adenomatous collect information about the number, size, morphology, polyps and polyps in the fundus (6, 7). Extraintestinal distribution, and histology of colonic polyps. Polyps were neoplasias of breast, gastric, thyroid, testis, and hematologic classified as adenomatous or serrated. Polyps were consid- origin have been described in MAP syndrome (8, 9). ered proximal if located in the transverse or ascending colon > Two common mutations, c.536A G; p.Y179C and or cecum, and distal if located in the descending or sigmoid > c.1187G A; p.G396D, are reportedly responsible for colon or rectum. A central review of all specimens was approximately 80% of MAP cases in Caucasian populations performed by three experienced pathologists (A. Paya, (2, 10), although large deletions (11) and other low-fre- C. Egoavil, and C. Alenda). On the basis of the polyp quency mutations have also been identified in this gene histology, cases were classified as adenomatous polyposis, (12). The International Society for Gastrointestinal Hered- multiple adenomatous, and serrated polyps, or only mul- itary Tumors (InSiGHT) database presently includes 300 tiple serrated polyps (20). Serrated polyps included hyper- MUTYH unique DNA variants of the gene, some of which plastic polyps, traditional serrated adenomas, sessile serrat- are probable founder mutations in different populations. ed polyps, and mixed hyperplastic/adenomatous polyps. However, about 20% to 33% of the tested index patients of southern European populations do not carry one of these Samples most common mutations (13). DNA was extracted from peripheral blood samples of the It has been recently reported that the MAP phenotype 405 patients using the QIAamp DNA Kit (QIAGEN) fol- differs from other previously described polyposis syn- lowing the manufacturer’s instructions. dromes in that it may involve the coexistence of both A total of 605 polyps were collected from 56 patients. adenomatous and serrated polyps (14). However, to date, From each polyp, we prepared nine 5-mm paraffin-embed- MAP diagnosis has been based on the presence of adeno- ded sections containing only the representative biopsy. matous polyps (4, 15, 16). This differential phenotype Genomic DNA was extracted with the QIAamp DNA Inves- makes it unclear which patients should be tested for tigator Kit (QIAGEN) following the manufacturer’s instruc- MUTYH, and whether patients with serrated polyps or those tions. All samples were dissected macroscopically by a with multiple adenomatous and serrated polyps should pathologist (A. Paya, C. Alenda, and C. Egoavil) to ensure also be investigated for this disease. Moreover, different that they contained predominantly neoplastic tissue.

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Guarinos et al.

Germline mutation analysis Genetic Analyzer (Applied Biosystems). Sequencing results DNA sequencing. All cases were analyzed by PCR and were analyzed using Sequencing Analysis v. 5.1 and Variant sequencing of the MUTYH exons 7 and 13, where the two Reporter v. 1.1 software (Applied Biosystems). most frequent mutations are located (c.536A>G; p.Y179C Large genomic rearrangements. The 183 cases with more and c.1187G>A; p.G396D). All patients were also examined than 10 adenomas were tested for large insertion/deletion for the frameshift change c.1227_1228dup (Glu410- of the APC gene. Cases with biallelic or monoallelic path- GlyfX43), which was previously reported as a common ogenic mutations were analyzed for large rearrangements of mutation in different Mediterranean populations (21, the MUTYH gene. These analyses were performed by mul- 22). Patients who were heterozygous for any of these three tiplex ligation-dependent probe amplification (MLPA) mutations were sequenced for the whole coding region and using MLPA Kits P043 APC, and P378 MUTYH (MRC- intron–exon boundaries to determine the existence in trans, HOLLAND). MLPA assays were performed following the of a second mutational event. In addition, 216 patients who manufacturer’s instructions. PCR fragments were separated showed none of the three analyzed mutations were also and quantified by capillary electrophoresis on a 3500 investigated for germline mutations along the whole coding Genetic Analyzer (Applied Biosystems). Fragments were sequence of the MUTYH gene. This group was a represen- analyzed using GeneMapper v. 4.0 analysis software tative sample of the initial population, with no significant (Applied Biosystems). differences in age, gender, familial history, diagnosis of Interpretation of variants of uncertain significance. colorectal cancer, or in the number, size, type, and location Genetic analysis results were interpreted on the basis of the of polyps (Fig. 1). American College of Medical Genetics and Genomics Cases with more than 10 adenomas were also tested for (Bethesda, MD) recommendations for Standards for Inter- APC mutations (n ¼ 183). Direct amplicon sequencing was pretation of Sequence Variations and the InSiGHT database. performed using BigDye v3.1 terminators and a 3500 Bioinformatics tools were used to perform in silico analyses

Figure 1. Flowchart of the study, indicating the number of patients selected for different genetic tests.

1160 Clin Cancer Res; 20(5) March 1, 2014 Clinical Cancer Research

MUTYH-Associated Polyposis in Patients with Serrated Polyps

for estimating the functional effects at the RNA and protein interquartile range, 13–36). A majority of polyps were <10 levels of the MUTYH base substitution variants with mm, with patients having a median of 15 polyps <10 mm unknown clinical significance. Polymorphism Phenotyping (25–75 interquartile range, 11–33) and a median of v2 (PolyPhen-2) and SNPs3D can predict the possible two polyps 10 mm (25–75, interquartile range, 0–4). impact of an amino acid substitution on the structure and Patients had a median of eight proximal polyps (25–75 function of a human protein using straightforward physical interquartile range, 4–14) and 10 distal polyps (25–75 and comparative considerations. A PolyPhen-2 variant pre- interquartile range, 5–20). Polyp histology revealed that diction score of less than 5% was considered benign, and a 135 cases were adenomatous polyposis (33.3%), 219 score of more than 95% was considered to be probably involved multiple adenomatous and serrated polyps damaging. SNPs3D predictions produced support vector (54.1%), and 51 (12.6%) only showed multiple serrated machine (SVM) values; values below 0.5 were associated polyps, 40 of which met the World Health Organization with deleterious variants, whereas those more than þ0.5 (WHO) criteria for serrated polyposis (9.9%; ref. 20). were associated with neutral mutations. Splicing was stud- Of the 405 total patients, 183 (45.2%) had more than 10 ied using SplicePort, which predicts losses or gains of donor adenomas. These patients were tested for APC mutation, and acceptor splice sites. When these bioinformatics tools and 6 cases (3.3%) showed a germline heterozygous path- made at least two concordant predictions of pathogenicity ogenic mutation in the APC gene. Each case with APC for a given variants of uncertain significance (VUS), it was mutation showed exclusively adenomatous polyps. classified as clinically significant. MUTYH mutational analysis KRAS Gly12Cys somatic mutation analysis All of the 405 patients were studied for MUTYH germline Cases with polyp DNA available were tested for somatic mutations. Eighteen (4.4%) had biallelic mutations of the KRAS n ¼ mutation ( 56). We analyzed a mean of 10 polyps Y179C and G396D variants (Fig. 1, group 1; and Table 1), per patient. Direct sequencing was performed to identify of which 4 (22.2%) had a homozygous p.Y179C mutation, KRAS mutation at exon 1, including codons 12 and 13. Both 6 (33.3%) had a homozygous p.G396D mutation, and 8 mutations were assessed by direct amplicon sequencing (44.5%) were compound heterozygous. No patient with BigDye v1.1 terminators and a 3500 Genetic Analyzer showed biallelic c.1227_1228dup mutation. Two patients (Applied Biosystems). were carriers of compound heterozygous Y179C and c.1227_1228dup mutations (Fig. 1, group 2; and Table 1). BRAF analysis Homozygotes carriers for each of the two common muta- BRAF The V600E mutation was detected by real-time PCR tions did not exhibit any differences in age at diagnosis, (ABI PRISM 7500, Applied Biosystems) using specific Taq- familial history, colorectal cancer predisposition, or in the Man probes and the allelic discrimination software number, location, or size of polyps. However, serrated (Applied Biosystems), as previously described by Benlloch polyps were more common in p.G396D homozygotes and colleagues (23). carriers (83.3%) than in p.Y179D homozygotes carriers (0%; x2; P ¼ 0.05). Four (50%) compound heterozygous Statistical analysis cases showed only adenomatous polyps, while the remain- Data were tested for statistical significance using SPSS ing four showed both adenomas and serrated polyps. The software (SPSS 19.0). Parametric continuous variables are mean ages at diagnosis were 47 (homozygous p.Y179C), 57 reported as mean SD, whereas nonparametric continuous (homozygous p.G396D), and 57 years (heterozygous p. variables are reported as median (Q2–Q3 interquartile Y179C/p.G396D), indicating that homozygous mutations range). Categorical variables are reported as frequency or in Y179C were associated with early onset of MAP syndrome x2 percentage. The test, followed by the Fisher exact test (x2, P ¼ 0.05). Nineteen monoallelic carriers of any of the where appropriate, was used to identify correlations three most common mutations were found, including seven t between categorical parameters. The Student test was used monoallelic carriers for the Y179C mutation, 10 for the for quantitative data, but the Kruskal–Wallis test was used G396D mutation, and two for the c.1227_1228dup P for comparisons between more than two groups. A value mutation. of less than 0.05 was considered significant. Whole MUTYH gene study Results We performed whole-gene analysis for the 19 patients Patient clinical characteristics who were heterozygous for any of the three common A total of 405 unrelated patients with at least 10 polyps mutations. Nine of these patients (47.4%) showed a second from the EPIPOLIP study were included in the present MUTYH mutation, including 3 that had been previously investigation. The mean age at diagnosis was 57.5 years described as pathogenic (the nonsense pathogenic muta- (SD, 12.55; range, 13–84). There were 286 males (70.6%). tion Gln338X, the known pathogenic changes c.389-1G>A Personal history of colorectal cancer was reported for 145 and c.721C>T; refs. 24, 25). Of the new six VUS, three were patients (35.8%), and 162 patients (40%) had a familial classified as pathogenic based on the predictions of in silico history of colorectal cancer or colonic polyps in first-degree studies (Table 2). Therefore, another 6 patients were genet- relatives. The median number of polyps was 20 (25–75 ically diagnosed with MAP (Fig. 1, group 2; and Table 1).

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Downloaded from clincancerres.aacrjournals.org on October 4, 2021. © 2014 American Association for Cancer Research. 1162 Downloaded from lnCne e;2()Mrh1 2014 1, March 20(5) Res; Cancer Clin urnse al. et Guarinos

clincancerres.aacrjournals.org Table 1. Characteristics of patients diagnosed with MAP Published OnlineFirstJanuary27,2014;DOI:10.1158/1078-0432.CCR-13-1490

ID MUTYH MUTYH Onset Type of Total Total Colorectal cancer Extracolonic Familial Somatic KRAS (group) ﬁrst event second event age polyposis polyps adenomas (location) cancer (age) history mutation 1 (1) Y179C Y179C 53 AD 70 70 Yes (right colon) No No Yes 2 (1) Y179C Y179C 45 AD 40 40 No No Yes NA 3 (1) Y179C Y179C 56 AD 53 41 Yes (right colon) No Yes NA 4 (1) Y179C Y179C 34 AD 50 50 No No No NA 5 (1) G396D G396D 65 AD 16 16 No No No NA 6 (1) G396D G396D 52 SP 21 0 Yes (left colon and sigmoid) No Yes Yes 7 (1) G396D G396D 69 AþS 103 72 Yes (right colon) No No Yes on October 4,2021. ©2014 American Association forCancer Research. 8 (1) G396D G396D 56 AþS 28 21 No Endometrial (51) No NA 9 (1) G396D G396D 54 AþS 63 52 Yes [right colon (2)] No Yes Yes 10 (1) G396D G396D 47 AþS 123 100 No No Yes NA 11 (1) Y179C G396D 68 AþS 35 29 Yes (transverse and right colon) No No No 12 (1) Y179C G396D 55 AþS3128No NoNoNA 13 (1) Y179C G396D 65 AD 41 41 Yes (transverse colon and rectum) No Yes NA 14 (1) Y179C G396D 43 AD 40 40 Yes No No NA 15 (1) Y179C G396D 53 AþS 16 10 Yes (sigmoid and rectum) No No Yes 16 (1) Y179C G396D 49 AþS 207 178 No No No Yes 17 (1) Y179C G396D 60 AD 78 78 No No Yes Yes 18 (1) Y179C G396D 63 AD 50 50 Yes (rectum) No Yes NA 19 (2) Y179C Glu410GlyfX43 34 AD 18 18 No No No NA 20 (2) Y179C Glu410GlyfX43 48 AD 134 134 Yes (right colon) No Yes Yes 21 (2) Y179C Thr219Ala 75 AD 31 31 Yes (sigmoid) Ovarian (74) No Yes 22 (2) Y179C Arg241Trp 40 AD 50 50 Yes (right colon) No Yes Yes 23 (2) G396D c.389-1G>A67AþS 35 25 Yes (right colon) Endometrial (55) Yes NA 24 (2) G396D Leu186Trp 44 AD 50 50 Yes (left colon) No Yes NA

lnclCne Research Cancer Clinical 25 (2) Glu410GlyfX43 Arg109Gln 42 AþS 19 15 No Testis (32) Yes Yes 26 (2) Glu410GlyfX43 Gln338X 47 AD 90 90 Yes No No No 27 (2) Leu111Val Ala385ProfX23 40 AD 27 27 No No Yes NA

Abbreviations: AD, adenomatous; AþS, multiple adenomatous and serrated polyps; NA, not available; SP, serrated. www.aacrjournals.org Downloaded from

Table 2. Characteristics of VUS identiﬁed in MUTYH

clincancerres.aacrjournals.org Reported SNPs3D Somatic Somatic Protein Number of insight Mainly Polyphen-2 prediction KRAS BRAF Published OnlineFirstJanuary27,2014;DOI:10.1158/1078-0432.CCR-13-1490 MUTYH VUS change patients (times) reported as prediction (SVM) SplicePort mutation mutation Interpretation c.389-1G>A14þ/? (4/4) ——Pathogenic c.367delG 1 Nonreported ——Pathogenic c.1012C>T Gln338X 1 8 þ/þ(5/8) No No Pathogenic c.1147delC Ala385ProfX23 1 40 þ/þ(38/40) ——Pathogenic c.655A>G Thr219Ala 1 Nonreported Probably Deleterious No effect Yes No Pathogenic damaging (2.9) c.721C>T Arg241Trp 1 9 þ/? (5/9) Probably Deleterious No effect Yes No Pathogenic damaging (3.93)

on October 4,2021. ©2014 American Association forCancer Research. c.556-557 CT>TG Leu186Trp 1 Nonreported Probably Deleterious AS (S ¼ 99.98%; ——Pathogenic damaging (3.27) FPR ¼ 48%; TH ¼2) c.326G>A Arg109Gln 1 Nonreported Probably Deleterious AS (S ¼ 100%; Yes No Pathogenic damaging (2.46) FPR ¼ 89.9%; TH ¼4) MUTYH c.331C>G Leu111Val 1 Nonreported Probably Deleterious No effect ——Pathogenic damaging (2.72)

c.1000C>G Pro334Ala 1 Nonreported Benign NA No effect ——Inconclusive Polyps Serrated with Patients in Polyposis -Associated c.55>G Arg19Gly 1 Nonreported Possibly NA No effect ——Inconclusive damaging c.740G>A Arg247Gln 1 Nonreported Possibly Benign (0.39) No effect No No Inconclusive damaging c.1276C>T Arg426Cys 1 10 ?/? (8/10) Benign Benign (0.25) AS (S ¼ 99.9%; No No Inconclusive lnCne e;2()Mrh1 2014 1, March 20(5) Res; Cancer Clin FPR ¼ 48%; TH ¼2) c.577-5A>T 1 Nonreported NA NA No effect Yes No Likely benign c.1544C>T Ser515Phe 4 23 /?(13/23) Benign NA No effect No No Likely benign c.1258C>A Leu420Met 1 4 ?/?(4/4) Benign Benign (0.65) No effect ——Likely benign c.696C>T Thr232Thr 1 Nonreported Benign Benign (0.25) No effect ——Likely benign

Abbreviations: AS, aberrant splicing predicted; FPR, false-positive rate; NA, not available; S, sensibility; TH, threshold; þ/þ, pathogenic; þ/?, probably pathogenic; /?, probably no pathogenicity; ?/?, no known pathogenicity. 1163 Published OnlineFirst January 27, 2014; DOI: 10.1158/1078-0432.CCR-13-1490

Guarinos et al.

Whole-gene analysis was also performed in a represen- colorectal cancer and serrated polyps were found in 11 tative sample of 216 patients who did not show any of the (40.8%). One patient fulfilled the WHO criteria for serrated three recurrent mutations. Only one of these patients polyposis (Table 1, patient #6; ref. 20), which supposes a (0.5%) had a biallelic pathogenic MUTYH mutation. This 2.5% of patients with serrated polyposis (1 of 40 patients patient (Table 1, # 27) was diagnosed at 40 years of age, and with serrated polyposis included in the study). Four patients had one brother who was also diagnosed with polyposis at (14.8%) showed other extracolonic cancers (2 had endo- 40 years of age; no genotyping data were accessible for this metrial cancer, 1 ovarian cancer, and 1 testicular germ cell relative. tumor). In all MAP cases, family history was consistent with Another patient exhibited two concurrent VUS, both recessive inheritance with one or more affected siblings— considered to be likely benign. Whole-gene analysis also except for patients 3 and 23 (Table 1), who showed inher- identified 7 patients as heterozygous, but only one VUS itance consistent with vertical transmission. identified in these patients was classified as probably path- Comparing MAP cases with the patients with multiple ogenic based on the in silico results. No large rearrangements colonic polyps who did not show MUTYH mutation (Table were identified in these patients (Tables 1 and 2). 3), we found that patients with MAP had an earlier age of polyposis onset (52.7 vs. 60.2; P ¼ 0.005) and more polyps Clinical and pathologic features of MAP patients (P < 0.001), as expected. Patients with MAP also more Overall, a total of 27 patients were diagnosed with MAP commonly had a personal history of colorectal cancer due to biallelic pathogenic mutations (Tables 1 and 3), (59.3% vs. 35%; P ¼ 0.014) and high-grade dysplasia indicating an estimated 6.7% prevalence of MAP among adenomas (P ¼ 0.02). Disregarding those patients who APC mutation-negative cases with multiple polyps in the were diagnosed with colorectal cancer in the first colonos- colon (27 of 405). Of these 27 patients, 16 (59.3%) had copy, we observed that the incidence of new cases of

Table 3. Clinical and pathologic characteristics of patients with MAP and non-MUTYH patients with multiple colonic polyps

MAP patients Non-MUTYH (n ¼ 27) patients (n ¼ 228)a P Age at diagnosis in years, mean (SD) 52.7 (11.02) 60.2 (11.97) 0.005 Total cases of colorectal cancerb, n (%) 16 (59.3%) 79 (35%) 0.014 New cases of colorectal cancerb, n (%) 7 (43.7%) 15 (19%) 0.03 Personal history of any neoplasm, n (%) 4 (14.8%) 16 (7%) 0.15 Familial history of colorectal cancer or colonic polyps, n (%) 14 (51.9%) 85 (37.3%) 0.17 Polyp number, median (25–75 interquartile range) 35 (20–62) 15 (12–28) <0.001 % Polyps >1 cm, median (25–75 interquartile range) 5 (0–8) 9 (0–21) 0.03 % Proximal polyps, mean (SD) 49 (26) 45 (28) 0.46 Presence of serrated polyps, n (%) 11 (40.75%) 148 (64.9%) 0.02 – Type (%) SSA 0.36 HP 11 24.5 – Location (%) 89 75.5 Proximal colon 0.008 Distal colon 83.3 31.6 16.7 68.4 Dysplasia in polyps (%) HGD adenomas 7.4 0.8 0.02 Serrated polyps 0 6.5 0.3 KRAS Gly12Cys mutation (%) 84.6 0.4 <0.001 BRAF V600E mutation (%) 0 31.2 0.001 Adenomas 0 0.9 0.3 Serrated polyps 0 74.3 <0.001

NOTE: Bold values are statistically significant. Abbreviations: HGD, high-grade dysplasia; SSA, sessile serrated adenoma; HP, hyperplastic polyps. aPatients subjected to whole-gene analysis with no MUTYH biallelic mutation. bTotal cases of colorectal cancer refer to the total number of colorectal cancer cases before and after the first colonoscopy performed with diagnosis of colonic polyposis. New cases of colorectal cancer refer only to the colorectal cancer cases developed after this first colonoscopy.

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colorectal cancer in patients with MAP was also higher than in the non-MUTYH patients (P ¼ 0.03). 21) Serrated polyps were more frequently located in the ¼ n right colon in patients diagnosed with MAP (P ¼ 0.008). Larger polyps (>1 cm) were more frequently found in cases without MUTYH mutation (P ¼ 0.03). No signiﬁ- 65.4 (25.4) cant differences were found regarding the presence of No truncative mutation ( family history of polyps and colorectal cancer or extra- 0.01 colonic cancer (Table 3). P

Comparing patients with biallelic mutations in the two 6) common MUTYH hotspots (group 1) with those carrying ¼ other pathogenic mutations (group 2) revealed no signif- n icant differences in age at diagnosis; total number, size, and location of polyps; presence of serrated polyps; familial

history; extracolonic cancer; or colorectal cancer predispo- Truncative mutation ( sition (Table 4). However, there was a trend for patients of group 2 to have an earlier age at diagnosis (48.5 vs. 54.8 11) 7 (63.6) 2 (33.3) 1 9 (42.9)

years). ¼ n 46.8 (21) 36 (7.3) Patients with MAP who carried the G396D mutation No Y179C ( showed older age at diagnosis, and this mutation was 0.04 0.06 signiﬁcantly associated with the presence of serrated lesions P (P ¼ 0.008). Carriers of the Y179C mutation more frequently showed proximal polyps (Table 4). 16) ¼

KRAS > n

c.34G T and BRAF V600E somatic mutation Y179C ( analysis A total of 605 polyps from 56 patients included in this study were analyzed for the KRAS Gly12Cys and BRAF 11) 1 (9.1) 4 (25) ¼

V600E somatic mutations. According to histology, 50.2% n 46.7 (11.6) 52.6 (11.9) 0.9 53 (10.3) 46.3 (11) 0.1 54.6 (10.5) No G396D ( of the polyps were classiﬁed as tubular adenomas, 9.5% as

tubulovillous adenomas, and 40.3% as serrated polyps. P 0.01 Among patients with MAP, 10.3% of polyps were serrat- 0.008 ed, whereas 58.7% were serrated in MUTYH-negative patients 16)

Polyps from 13 patients with MAP were analyzed for the ¼ n KRAS Gly12Cys somatic mutation. Eleven of these patients G396D ( (84.6%) showed this mutation in at least 10% of their polyps (mean, 29%; range, 10%–50%; Tables 1 and 2). MUTYH 9)

One of the ﬁve carriers of heterozygous mutations ¼

KRAS n showed the Gly12Cys mutation in 50% of polyps. Group 2 ( This mutation was not observed in any of the 373 polyps from 36 MUTYH-negative patients, or in the 2 patients P who carried benign VUS. Comparing MAP cases with those MUTYH cant. without mutation revealed a strong association 18) ﬁ 8 (44.4) 0.4 6 (66.7) 8 (50) 0.82 6 (54.5) 7 (43.8) 0.3 7 (63.6) 4 (66.7) 0.6 10 (47.6) 1 (5.6) 0.2 3 (33.3) 2 (12.5) 0.4 2 (18.2) 1 (6.2) 0.4 3 (27.3) 2 (33.3) 0.2 2 (9.5) MUTYH KRAS ¼ 54 (9.3) 0.2 48 (13.5) 56.9 (8.6) 10 (55.6) 0.7 6 (66.7) 10 (62.5) 0.7 6 (54.5) 10 (62.5) 0.7 6 (54.5) 3 (50) 0.7 13 (61.9) between germinal mutation and Gly12Cys 59 (46.7) 0.6 50 (38.3) 58.6 (49.8) 0.7 52.9 (34.6) 59 (48.3) 0.7 52 (37.5) 53.8 (47) 0.9 56.9 (43.6) n somatic mutation in polyps (P < 0.001; Table 3). Group 1 ( KRAS Gly12Cys somatic mutation was found more (%)

frequently in serrated polyps and tubulovillous adeno- n mas (61.5% and 63.6%, respectively) than in tubular adenomas (14%; P < 0.001). Gly12Cys mutation was more frequent in larger polyps (>5 mm): 38.1% versus 19.3% (P ¼ 0.02). No differences were found relating to (%) polyp location. n (%) Correlation between the different types of mutations found and phenotypical characteristics of patients with MAP 1 cm, mean (SD) 7.5 (14.3) 0.7 10 (10.4) 10 (15.3) 0.4 59.9 (28.5) 9.1 (15.7) 0.8 7.4 (8.4) 9.9 (11.2) 0.8 8 (13.6) > On the other hand, BRAF V600E mutation was not n detected in any of the 178 analyzed polyps from patients with MAP, and was detected in 31.2% (116 of 373) of mean (SD) cancer or colonic polyps, MUTYH BRAF cancer, neoplasm, Age at diagnosis in years, Polyp number, mean (SD) % Polyps % Proximal polyps, mean (SD) 64.6NOTE: (26) Bold values are statistically signi 0.1 47.1 (22.8) 57.2 (24.7) 0.8 59.9 (28.5) 67.4 (25.7) Familial history of colorectal Personal history of colorectal Personal history of any polyps from -negative patients. mutation was Presence of hyperplasic polyps 9 (50) 0.2 2 (22%) 10 (62.5) Table 4. predominantly found in serrated polyps (Table 3).

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Guarinos et al.

Table 5. Performance characteristics of selected strategies for identiﬁcation of patients with MAPa

Sensitivity Speciﬁcity Positive predictive Negative predictive (95% CI) (95% CI) value (95% CI) value (95% CI) Genetic testing Only two common variants 66.7 (46.0–83.4) 100 (98.4–100) 100 (81.3–100) 96.2 (92.9–98.2) Two common variants þ local variant 74.1 (53.7–88.8) 100 (98.4–100) 100 (83.1–100) 97.0 (93.9–98.8) Whole-gene heterozygous 96.3 (81.0–99.4) 100 (98.4–100) 100 (88.6–100) 99.6 (97.6–99.9) Polyposis phenotype Only adenomas 59.3 (38.8–77.6) 68.4 (61.9–74.4) 18.2 (10.8–27.8) 93.4 (88.5–96.7) >30 Adenomas 59.3 (38.8–77.6) 93.4 (89.4–96.3) 51.6 (33.1–69.8) 95.1 (91.4–97.5) >10 Adenomas 96.3 (81.0–99.4) 63.6 (56.7–69.8) 23.8 (16.2–33.0) 99.3 (96.2–99.9) Patient characteristics <60 y 70.4 (49.8–86.2) 61.8 (55.2–68.2) 17.9 (11.1–26.6) 94.6 (89.7–97.6)

aPerformance characteristics were calculated on the basis of the 255 patients subjected to whole-gene analysis (216 lacking the three most common mutations, 13 with only monoallelic mutation, and 20 with biallelic mutation in common variants).

Performance of different strategies for MAP diagnosis Biallelic germline mutations in MUTYH accounted for We found that only 18 of 27 (66.6%) MAP cases could be 6.7% of the analyzed cases that had an attenuated polyposis explained by biallelic mutations in the two common phenotype. This mutation detection rate was lower than MUTYH hotspots. Analysis of the two hotspots and the found in previous reports (2, 22, 26, 27), but similar to that third common variant in our population resulted in a described in more recent studies of cases with multiple sensitivity of 74.1% (20 of 27 cases). Whole-gene analysis colorectal adenomas (4). The frequencies of the Y179C and of cases heterozygous for any of the three common variants G396D alleles were consistent with those in previous stud- achieved the best performance, diagnosing 96.3% of ies (10, 22, 28), although the present population showed a cases. Table 5 shows the performance characteristics of these higher heterogeneity of MUTYH mutations—including 10 strategies. new variants, five of which were considered pathogenic. Considering the characteristics of our patients with MAP, Our study confirms previous reports indicating the presence investigation of the MUTYH gene in cases with only ade- of both adenomatous and serrated polyps in almost half of nomatous polyps would achieve a sensitivity of only 59.3%. the MAP cases. These results support the notion that MAP If we had performed the MUTYH study in only patients with should be particularly suspected in patients with multiple more than 30 adenomas, 10 patients with MAP (37%) adenomas coexisting with serrated polyps throughout the would not have been identified. However, by including colon, especially when familial history suggests a recessive patients with multiple adenomatous and serrated polyps hereditary pattern, and in patients who are young or who and more than 10 adenomas, only 1 patient would remain have a high number of polyps. We found one MAP patient undiagnosed (Table 5). Regarding age at diagnosis, limiting fulfilling the WHO criteria for serrated polyposis (20), MUTYH analysis to patients younger than 60 years would which represents a 2.5% of patients with serrated polyposis reduce the sensitivity to 70.4% (Table 5). included in the study, supporting the need for studies of MUTYH in patients with this disease (14). Discussion Massive parallel sequencing methodologies are undoubt- The present report describes several notable results. First, edly going to change the analysis strategy in cancer genetics; almost half of the identified MAP cases showed both ade- however, until next-generation sequencing becomes broad- nomatous and serrated polyps, especially when G396D ly available in diagnostic laboratories, Sanger sequencing mutation was involved. There were even cases showing less remains essential. Our present findings have important than 10 adenomas, raising the question of whether there is implications for the design of mutation detection strategies. an adenoma cutoff for MAP diagnosis in cases with both We recommend a sequential analysis strategy using the adenomatous and serrated polyps. Second, we found that direct sequencing of exons 7 and 13 of MUTYH for MAP the MAP diagnostic strategy should start with analysis of the diagnosis, starting with examination of the most locally most common mutations, taking into account locally prev- prevalent mutations. In our study, more than 95% of alent mutations, and it should include whole-gene analysis diagnosed MAP cases had at least one of the three most only in cases that present a heterozygous mutation in a gene common mutations, which were all located in these exons. hotspot. Third, in our population, the majority of MUTYH Correct MAP diagnosis requires identification of the locally mutation carriers showed the somatic KRAS Gly12Cys prevalent mutations that exist in a large proportion of cases mutation in their polyps, but BRAF V600E mutation was with MUTYH germline mutation by analyzing the whole not found in serrated polyps of patients with MAP. gene in a number of cases. Our results support the need for

1166 Clin Cancer Res; 20(5) March 1, 2014 Clinical Cancer Research

MUTYH-Associated Polyposis in Patients with Serrated Polyps

complete MUTYH gene analysis only in patients who are fying VUS at MUTYH; analyzing the presence of the somatic carriers of prevalent mutations (Y179C, G396D, and mutation Gly12Cys in polyps or colorectal cancer could Glu410GlyfX43), even if recessive inheritance is not con- support the pathogenicity of the VUS. This technique may firmed. We found a very low diagnostic yield from whole- be limited by the need for testing multiple polyps, as well as gene analysis in patients with multiple adenomatous and cost or technical problems in obtaining polyp tissue. Fur- serrated polyps. No MUTYH rearrangements were identified ther research should be performed to study possible uses of in this study. Not having analyzed the whole gene in every this promising molecular marker of MAP. Moreover, in this case was a limitation of our study, which precludes the study, we found no polyp showing the somatic BRAF V600E possibility of having more accurate values for the sensitivity mutation in patients with MAP. We believe this to be a very and specificity of the different strategies. However, given the striking fact, given the high proportion of BRAF mutation extremely low rate of biallelic pathogenic mutation found that we found in the serrated polyps of MUTYH-negative in the 216 studied cases, we decided not to complete the patients. This finding suggests the possibility of using BRAF whole-gene testing in all cases. mutation in serrated polyps as a negative molecular marker It has been suggested that there is a genotype–phenotype for MAP. association for MUTYH mutations, with worse pathogenic- In summary, our present results support the existence of ity and earlier age of onset in carriers of the Y179C allele different polyp phenotypes in MAP. Genetic testing should (29). Our results offer new insight into this association. We be offered, not only in cases with attenuated adenomatous report that the G396D variant was strongly associated with polyposis as has been classically described, but also and the presence of serrated polyps, even in heterozygosis. We especially in cases showing multiple adenomatous and also found 2 patients who developed endometrial cancer; serrated polyps. The determination of clinical and molec- one had a sister with MUTYH biallelic mutation who also ular hallmarks of MAP will enable more reliable correct had both endometrial and colorectal cancer. Endometrial identification of possible carriers of pathogenic MUTYH cancer has been proposed as an extracolonic neoplasia in mutations. MAP syndrome (30), and the present findings might support this partnership. However, the number of MAP cases Disclosure of Potential Conflicts of Interest that we included precludes our ability to provide solid No potential conflicts of interest were disclosed. evidence about the association between MAP and extracolonic neoplasms. APC MUTYH Authors' Contributions In our population, germline mutations in or Conception and design: C. Guarinos, J. Cubiella, F. Rodrguez-Moranta, accounted for only a small portion of cases with multiple A. Castillejo, R. Jover colonic polyps. Additional related mutations have been Development of methodology: C. Guarinos, C. Egoavil, R. Salas, F. Rodrguez-Moranta, A. Castillejo, C. Alenda, J.-L. Soto reported in genes involved in the base excision repair system Acquisition of data (provided animals, acquired and managed patients, or the Wnt pathway (31–35). Other mechanisms, such as provided facilities, etc.): C. Egoavil, M. Rodrguez-Soler, L. Perez-Carbo- APC nell, J. Cubiella, F. Rodrguez-Moranta, L. de-Castro, L. Bujanda, A. Serra- mosaicism and deep intronic gene mutations, are also desanferm, D. Nicolas-Perez, M. Herraiz, F. Fernandez-Banares,~ A. Herreros- reportedly associated with attenuated polyposis, and could de-Tejada, E. Aguirre, J. Balmana,~ M.-L. Rincon, A. Pizarro, F. Polo-Ortiz, explain a small proportion of cases (36, 37). The existence of A. Castillejo, C. Alenda, A. Paya, R. Jover Analysis and interpretation of data (e.g., statistical analysis, biosta- other yet unknown genes involved in cases showing mul- tistics, computational analysis): C. Guarinos, M. Rodrguez-Soler, R. Salas, tiple colonic polyps with incomplete penetrance, as well as J. Cubiella, A. Castillejo, C. Alenda, J.-L. Soto, R. Jover complex interactions between environmental factors and Writing, review, and/or revision of the manuscript: C. Guarinos, C. Egoavil, J. Cubiella, L. de-Castro, M. Herraiz, F. Fernandez-Banares,~ modifier genes might further account for the unexplained E. Aguirre, J. Balmana,~ A. Castillejo, J.-L. Soto, R. Jover cases. Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): C. Guarinos, C. Egoavil, R. Salas, One proposed molecular hallmark of carcinomas caused A. Herreros-de-Tejada, E. Aguirre, R. Jover by MUTYH deficiency is the presence of the c.34G>T (p. Study supervision: J. Cubiella, A. Castillejo, J.-L. Soto, R. Jover Gly12Cys) KRAS mutation in MAP colorectal cancers. Pre- Performance of the genetic testing of patients: M. Juarez vious reports have indicated that this mutation occurs at a high frequency in tumors of patients with MAP, and it has Grant Support been suggested that this analysis could be implemented as a This work was supported by the Instituto de Salud Carlos III (PI08/0726, MUTYH INT-09/208, PI11/2630, and INT-12/078). C. Guarinos received a predoc- prescreening test to help select persons for genetic toral grant from Conselleria d’Educacio de la Generalitat Valenciana testing (38, 39). Our results confirmed the existence of this (VALiþd. EXP ACIF/2010/018). M. Rodrguez-Soler received a grant from KRAS Gly12Cys somatic mutation in the polyps of patients Fundacion de la Comunidad Valenciana para la Investigacion en el Hospital General Universitario de Alicante and Instituto de Salud Carlos III (Rio- with MAP, supporting its possible role as a MAP diagnostic Hortega grant CM11/00066) and L. Perez-Carbonell received grants from tool, especially in atypical cases with young onset colorectal Instituto de Salud Carlos III (FI07-00303) and Fundacion Alfonso Martn cancer or recessive colorectal cancer familial history. This Escudero. The costs of publication of this article were defrayed in part by the molecular marker is more frequently found in serrated and payment of page charges. This article must therefore be hereby marked tubulovillous adenomas and, interestingly, it shows a high advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate specificity, having only been found in the polyps of patients this fact. with MAP and not in other cases with multiple colonic Received May 30, 2013; revised November 21, 2013; accepted November polyps. This marker could also be a useful tool for classi- 21, 2013; published OnlineFirst January 27, 2014.

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1168 Clin Cancer Res; 20(5) March 1, 2014 Clinical Cancer Research

Prevalence and Characteristics of MUTYH-Associated Polyposis in Patients with Multiple Adenomatous and Serrated Polyps

Carla Guarinos, Miriam Juárez, Cecilia Egoavil, et al.

Clin Cancer Res 2014;20:1158-1168. Published OnlineFirst January 27, 2014.

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