M1929

Utility of MS-Mlpa Methylation Analysis in the Diagnosis of Lynch SyndromeRodrigo Jover, Lucía Pérez-Carbonell, Cristina Alenda, Artemio Payá, José-Luis Soto,Adela Castillejo, Victor Barberá, Carmen Guillén

Despite the advances in the selection of patients for genetic testing in Lynch syndrome atleast a third of patients with Bethesda criteria and lack of MLH1 expression have sporadictumours and should not receive genetic testing. The aim of this study is to investigate therole of the methylation analysis using MS-MLPA in the diagnosis of Lynch syndrome. MS-MLPA is a novel and methodologically easy technique for quantitative measure of genemethylation. Methods. Sixty colorectal cancer (CRC) specimens with loss of MLH1 expressionwere selected. DNA from paraffin-embedded tumours was tested for 2 quantitative methyl-ation detection methods: Methylight (Applied biosystems) and MS-MLPA (Methylation Spe-cific-Multiplex Ligation Probe Amplification) (MCR Holland) calculating Methylation Ratioin MLH1. Somatic mutation in BRAF V600E was investigated from tumour DNA. Germlinemutations in MLH1 were analyzed by sequentiation. Results: Correlation of quantitativeresults of the 2 methylation analysis techniques (Methylation and MS-MLPA) was 96.7% (p<0,001). Germline mutations in MLH1 were found in 8 out of 60 patients (13,3%). Noneof the tumours from these patients with Lynch syndrome showed significant methylation.Forty-two out of 52 (80%) tumours from patients without germline mutation in MLH1showed significant methylation using Methylight. Using MS-MLPA, methylation was foundin 44 out of 52 patients (85%). Both techniques had sensitivity and negative predictivevalue of 100 % to detect germline mutation patients. Specificity was 80 % for Methylightand 85 % for MS-MLPA, and positive predictive value (PPV) was 44 % for Methylight and50 % for MS-MLPA. These values were clearly better than the obtained using BRAF V600Emutation, with a specificity of 53,8%. Conclusion: Methylation analysis is a high sensitivityand specific method for detecting sporadic CRC unstable cases. These results highlight therole of MS-MLPA assays as a simple and reliable method for selection of patients for genetictesting in Lynch syndrome.

M1930

Identification of Genomic Variants in Hereditary Non-Polyposis ColorectalCancer Without Mismatch Repair Deficiency (MSS HNPCC)Rosa M. Xicola, Trinidad Caldes, Pilar Garre, Brian J. Doyle, Prathap Bandipalliam, SapnaSyngal, Jessica Grzybowski, Vanessa R. Sapoznik, Michael N. Grzybowski, Tamara I.Martinez, Sathayaraj Murugappan, Antoni Castells, Rodrigo Jover, Montserrat Andreu,Ajay Goel, Xavier Llor

Lynch syndrome, caused by germline mutations in mismatch repair (MMR) system genesand characterized by microsatellite instability (MSI), is the best-known form of hereditarynon-polyposis colorectal cancer (HNPCC). Recently we have learnt that a significant numberof families with HNPCC lack MMR gene mutations and MSI. We have previously describedthat, in a population-based cohort, over half of all families fulfilling Amsterdam criteria ofHNPCC belong to this second group and the genetic alterations responsible for these tumorsare yet to be identified. Aims: To identify genetic variants present in HNPCC families withoutMMRdeficiency (MSSHNPCC) and to ascertain the potential causative role of these variants inthis group of inherited CRCs. Methods: 46 MSSHNPCCCaucasian patients were investigated.Germ-line DNA was extracted and all coding sequences and intron-exon boundaries of 11candidate genes implicated in colorectal carcinogenesis (MLH1, MSH2, MSH6, MYH, OGG1,NUDT1, β-CATENIN, AXIN2, TGFBR1, TGFR2 and MGMT) were analyzed through directsequencing. Results: The entire genetic sequence of all genes was analyzed in a subgroupof 17 patients. We identified 8 synonymous variants and 8 missense variants in these tumors.None of these variants were novel except for a synonymous variant in exon 15 of β-CATENINfound in one patient. We also identified 26 different intronic variants described as singlenucleotide polymorphisms (SNP) and 10 new intronic variants. None of these 10 variantswere clearly affecting donor or acceptor splicing sites of the involved gene. A statisticalanalysis (two-sided X2 or Fisher test) was performed comparing frequencies of all thesevariants with the frequencies reported in control Caucasian populations available in publicdata-bases. Eight known variants were found to be significantly different from controls(P<0.05): MLH1 rs9876116, MGMT I143V and K178R, MSH2 rs17224822, β-CATENINrs4135385, MYH Q335H, MSH6 rs2020911 and NUDT1 D142D. These 8 variants, plusthe rest of the identified missense variants were analyzed in the entire cohort of 46 patients.Variants in MLH1, MGMT, MSH2 and NUDT1 remained significantly different from controls.28% of tumors presented novel variants. Conclusions: These results suggest that MSS HNPCCtumors do not arise as a consequence of pathogenic germline mutations in any of the 11studied genes that are commonly involved in CRC development. Further studies are currentlyin progress to determine whether these alterations could affect branch splicing sites or exonicsplicing enhancers, and thus may play a potential role in the evolution of MSS HNPCC cancers

M1931

Endoscopy As An Alternative to Surgery for the Treatment of Severe DuodenalPolyposis in Patients with Familial Adenomatous PolypsisDriffa Moussata, Bertrand Napoleon, Vincent Lepilliez, Marie-George Lapalus, StéphaneNANCEY, Jean-claude Cenni, Christian Partensky, Thierry Ponchon, Jean-alain Chayvialle,Jean-Christophe Saurin

Duodenal carcinoma is the second cause of mortality after colectomy in patients with familialadenomatous polyposis. The treatment of reference for severe duodenal polyposis in patientswith FAP(stage IV according to Spiegelman's score) is pancreaticoduodenectomy(PD, butthere is almost no published experience of therapeutic endoscopy in this situation. Aim:We evaluated the efficacy and the risks of an endoscopy-based approach, considering thehigh morbidity and mortality rates of PD in this indication (1). Material and methods: Cohortstudy in 2 endoscopic centers. All patients presented at inclusion a stage IV duodenalpolyposis and endoscopic treatment was shedulded. We reviewed treatment modalities,com-plications and the Spiegelman's score calculated at each endoscopy. Results: 36 patientswere treated (16 men, mean age 48 +/- 12 years, range 22-73). The polyposis had evolved

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into stage IV after a mean delay of endoscopic follow-up of 4.7 +/- 4.8 years (median 4,range 0-17) and the complete endoscopic follow-up was of 11 +/- 6 years (0,5 - 20). Themean number of endoscopies was of 11,9 +/- 6,7 (2 - 26). The mean endoscopic follow-up was of 6 +/- 3,8 years (0,5 - 17, median 6). At inclusion, the mean Spiegelman's scorewas of 9,9 +/- 1 points (9 - 12) versus 5,8 +/- 1,7 (0 - 8) after treatment (p = 0,002).Different endoscopic therapies were used during the successive treatment sessions (numberof patients that had at least once the therapeutic modality): Nd:YAG laser (2), photodynamictherapy (6), argon plasma coagulation (APC) (27), APC and mucosectomy (7) and mucosec-tomy alone (18). A treatment was performed for neoplasia on the papilla in 27 cases (adenomawith low grade syplasia in 12, high grade dysplasia in 13, intramucosal cancer in (1):Nd:YAG laser (2), sphincterotomy (4), ampullectomy (15). Six %(12/197) of the endoscopictreatment were complicated by: delayed hemorrhage 7, pancreatitis 4, duodenal perforation1 (necessitating surgery). Three patients had surgery for neoplasia treatment: 1 surgicalampullectomy for recurrent adenoma of the papilla, 2 distal duodeno-jejunal resections.During the follow-up, 4 patients died from non-duodenal cancers: cholangiocarcinoma (2),rectal carcinoma (1), lung cancer (1). No invasive duodenal adenocarcinoma was diagnosed.Conclusion: This long term cohort follow-up shows that endoscopic treatment is possiblewith a limited morbidity, allows dowstaging of the polyposis in 95 % of cases, withoutinvasive duodenal cancer. When confirmed by other series, this approach compares favorablywith surgery. (1) Gallagher MC et al.Br J Surg 2004.

M1932

Increased Colorectal Cancer Risk During Follow-Up in Patients withHyperplastic Polyposis Syndrome: A Multicenter Cohort StudyKaram S. Boparai, Elisabeth Mathus-Vliegen, Jan J. Koornstra, Fokko Nagengast, MoniqueE. van Leerdam, Carel J. van Noesel, Martin H. Houben, Annemieke Cats, Liselot vanHest, Paul Fockens, Evelien Dekker

Background: Hyperplastic polyposis syndrome (HPS) is believed to be associated with anincreased colorectal cancer (CRC) risk. However, the risk of developing CRC in a large HPScohort during a clearly described follow-up period of multiple years has not been reported.Hence, a causal relationship between HPS and CRC cannot be made by default. Patientsand methods: The aims of this study were to assess the cumulative risk of developing CRCduring follow-up in the largest HPS cohort described thus far and to examine a possibleassociation with frequency and interval of surveillance endoscopies. Databases of 7 medicalcenters were searched for patients satisfying the criteria for HPS (WHO) who were retrospect-ively analysed in this study. Endoscopy reports and corresponding histopathology reportswere collected to evaluate the length, interval and frequency of endoscopic surveillanceand to derive information regarding polyp characteristics. Information regarding CRC wasexamined by evaluating endoscopy and histopathology reports made at the time of CRCdiagnosis and histopathology reports of colon resection specimens. Results: In this study,77 patients with HPS with a median follow-up period of 2.8 years (range: 0.2-20.8) wereincluded. During surveillance, 1984 polyps were identified: 1407(71%) hyperplastic polyps(HPs), 302(15%) serrated adenomas (SAs), 273(14%) adenomas and 2 admixed polyps. SAsand adenomas were identified in 52% and 69% of patients, respectively. In 27(35%) patients,CRC was detected of which 22 at initial endoscopy. Five (6.5%) patients developed CRCafter the diagnosis HPS was made during a median follow-up time of 1.3 years (0.4-6.7)and a median of 3 previous surveillance endoscopies (range: 2-4). In these patients with aninterval CRC, the median interval between the last surveillance endoscopy and CRC detectionwas 11 months (range: 4-43). The cumulative risk of developing an interval CRC undersurveillance was 7% at five years and 16% at ten years. In 4/5 patients with interval carcinomasCRC was detected within a HP (3/4) or SA (1/4). In patients with interval carcinomas, 4/5(80%) had SAs and multiple polyps (≥30) compared to only 15/72(21%) patients withoutan interval carcinoma (p=0.01). Conclusions: HPS is causally related with CRC development.Moreover, despite regular surveillance endoscopies, 6.5% of HPS patients in this studydeveloped CRC. The presence of SAs and multiple (≥30) polyps seem to be associated withCRC development in HPS. Future prospective studies are required to assess the risk of polypprogression in HPS and to determine the optimal surveillance and treatment protocol forthese patients.

M1933

A Comprehensive Analysis of the Phenotypic Manifestations of MismatchRepair Gene Mutations: Comparing MSH6 with MLH1 and MSH2 MutationCarriersFay Kastrinos, Ewout W Steyerberg, Judith Balmaña, Sapna Syngal

Background: Lynch Syndrome is caused by germline mismatch repair (MMR) gene mutations.Previous reports of phenotypic differences between MMR mutation carriers present limiteddata pertaining to individuals with MSH6 alterations due to small sample sizes. We comparedphenotypic expression of MLH1, MSH2, and MSH6 gene mutation carriers in a large cohortof individuals undergoing clinical genetic testing. Methods: We analyzed new data from apreviously unreported cohort of 4187 unrelated probands undergoing clinical genetic testingfor MLH1, MSH2 and MSH6 mutations at a commercial laboratory. Personal and familyhistory of cancer, cancer types and ages at diagnosis were compared by mutation status forcarriers and their first- and second-degree relatives. Results: Thirteen percent (540/4187)of subjects had pathogenic mutations (211 MLH1, 255 MSH2, 74 MSH6). MLH1 carriershad a higher prevalence of colorectal cancer (CRC) (77% v 69% v 54%, p=0.004) thanMSH2 and MSH6 carriers respectively. Personal history of ≥2 CRC diagnoses was similaramong MLH1 and MSH2 carriers (12% v 12%) and less likely in MSH6 carriers (7%). MLH1and MSH2 carriers had a younger mean age at CRC diagnosis (41, 43 years) than MSH6carriers (46 years) with a significant difference between MLH1 and MSH6 carriers (p=0.03).Forty-two percent of female carriers had endometrial cancer and prevalence was highestamong MSH6 carriers (56% v 32% MLH1 and 45% MSH2, p=0.007) who were older atdiagnosis (51 years, p=0.02) than other carriers. MSH6 carriers had multiple family FDRaffected with endometrial cancer more often than MLH1 and MSH2 carriers (p=0.005). Othernonendometrial, extracolonic cancers were more frequent among MSH2 carriers (23% v 9%MLH1 and 12% MSH6, p=0.0001) with no difference in mean age of diagnosis between

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