Familial Cancer (2010) 9:713–748 DOI 10.1007/s10689-010-9351-8

Abstracts 3rd Biennial Meeting of InSiGHT (International Society for Gastrointestinal Hereditary Tumours) Du¨sseldorf June 24–27 2009

Small bowel polypectomy by double balloon enteroscopy: advancing endoscopic therapy for patients with Peutz–Jegher’s syndrome

laparotomy and prolonged recovery, however experienced surgical cover should always be available for this complex group of patients in view of the risk of potential complications.

Edward Despott, Robin Phillips, Sue Clark, Aymer Postgate, Aine Fitzpatrick, Eric Tripoli, Krysia Konieczko, Chris Fraser

High cumulative risk of intussusceptions in patients with Peutz–Jeghers syndrome

The Wolfson Unit for Endoscopy & The Polyposis Registry, St Mark’s Hospital, Harrow, UK Introduction Small bowel (SB) obstruction and GI bleeding due to polyps are major causes of morbidity in patients with Peutz–Jegher’s Syndrome (PJS). Priorto double balloon enteroscopy (DBE), a technique that allows complete enteroscopy, the main therapeutic option available for PJS patients was laparotomy with intra-operative enteroscopy. We report our experience of the endoscopic management by DBE of SB polyps in the largest cohort of PJS patients from the UK. Aims & methods Data on PJS cases managed by DBE at St Mark’s since 2005 were prospectively collected. Patients deemed to have significant SB polyps ([15 mm) at capsule endoscopy or magnetic resonance enterography or both, were referred for elective DBE polypectomy. DBE procedures were performed under GA. In one case, an emergency DBE was performed in the setting of SB obstruction due to polyp-induced intussusception. All patients remain under follow up and surveillance. Results Nine patients (mean age 34 years, range 16–45 years) underwent 13 DBE procedures (3 patients had 2 DBEs). Although all patients had previous laparotomies (some multiple), adhesions did not cause significant hindrance to DBE. On average 3 polyps were removed per patient (mean size 18 mm, range 8–37 mm). Polyp stalks were injected with lifting solution (dilute adrenaline and methylene blue) with additional endo-looping on occasion priorto snaring to reduce bleeding risk. Sessile polyps were elevated with lifting solution to minimise perforation risk. One patient required 3 DBE procedures, one of which was laparoscopically assisted fora sessile distal duodenal polyp; 1 patient suffered a small post-polypectomy bleed which settled spontaneously and 1 patient in whom an emergency DBE was attempted, polypectomy of a sessile 30 mm polyp led to perforation with conversion to IOE through the defect. Conclusion We highlight the role of DBE as an emerging therapeutic option for SB polyps in PJS that avoids the need for

M. G. F. van Lier, A. Wagner, A. M. Westerman, J. H. P. Wilson, F. W. M. de Rooij, E. J. Kuipers, M. E. van Leerdam Erasmus MC, University Medical Center, Rotterdam, The Netherlands Peutz–Jeghers Syndrome (PJS) is an inherited disorder characterized by gastrointestinal hamartomas and mucocutaneous pigmentations. Germline mutations in the STK11-gene can be found in 70% of clinically affected patients. Hamartomas, mainly located in the small bowel, may cause intussusceptions. Since balloon-enteroscopy (BE) enables endoscopic removal of these polyps, we assessed the risk and onset of intussusception. Patients diagnosed with PJS based on clinical diagnostic criteria or proven STK11 mutation were included in this prospective cohort study (1995–2008). Clinical data were obtained by interview and chartreview. Genotype-phenotype correlations were evaluated. The cumulative risk of intussusception was calculated by Kaplan–Meier analyses. Forty-four PJS patients (57% males) were included from 18 PJS families; 32 patients still alive had a median age of 44 years (10–74 years) and 12 patients had deceased at a median age of 45 years (11–73 years). A germline STK11 mutation was detected in 32 patients (73%). Thirty-four patients (77%) had a history of one or more (1–6) episodes of intussusception due to small bowel polyps. The median age at the first intussusception was 13.5 years (3–50 years). Surgery was required in 33 patients (75%). There was no significant difference in intussusception incidence according to sex (p = 0.15) or mutation-status (p = 0.70). Kaplan–Meier analyses showed that intussusception had occurred in 50% of the cohort at a median age of 16 years (95% CI 11–21), increasing to 75% (95% CI 62–88) at the age of 35 years. The 10-year probability of intussusception was 25% (95% CI 12–38). PJS patients carry a high cumulative risk of intussusception caused by small bowel hamartomas (50% at 16 years), independent of STK11 mutation-status. These findings support the approach of

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714 enteroscopic surveillance with removal of small bowel hamartomas. The effect on the incidence of intussusception remains to be established and weighted against burden and complication-risk of the intervention.

Phenotype variability within CDH1 mutated families Laetitia Huiart1, Franc¸ois Eisinger1, Violaine Bourdon1, Bruno Buecher2, Martine Blayau2, Olivier Caron2, Jean-Franc¸ois Fle´jou2, Jean-Pierre Gendre2, Astrid Schielke2, Alain Se´zeur2 1

Sylviane Olschwang pour le re´seau PHRATries; Institut Paoli-Calmettes, Marseille, France

2

CDH1 mutations are associated with diffuse gastric carcinoma and lobular breast cancer. However, little is known on age dependent penetrance of CDH1 mutations and on relevance of current clinical management guidelines. Our objective was therefore to describe personal and familial history of cancer in families with a CDH1 mutation. We identified 16 families who tested positive for a deleterious CDH1 mutation between 1998 and 2008. All but one mutation were unique: c.1565+1del was found in 4 unrelated families. Index cases were affected with gastric cancer in all cases but 2. Nine out of the 14 index cases of gastric cancers were diffuse, 5 were unspecified. Ages at diagnosis ranged from 21 to 63 years (mean = 38). The 2 index cases free of gastric cancers were tested either because of a bilateral lobular breast cancer at 42, or because of a rectal linitis at 23 years of age. Overall 35 cases of gastric cancers were reported. No other family history of gastric cancer was found for 5 mutation carriers. Associated breast cancer was reported in 3 families, of which 2 were specified as lobular breast cancer. Interestingly in a family where 13 members were tested for the familial mutation (4 carriers, 9 non carriers), 2 mutation carriers were free of cancer at 65 and 49 respectively. In another family, among 10 mutation carriers, 6 underwent prophylactic gastrectomy, all showed invasive and in situ signet cell foci. However the oldest carrier who declined gastrectomy was clinically asymptomatic at 64 years of age. In total, 6 mutation carriers were clinically asymptomatic although older than the mean age at diagnosis observed in index cases. The intrafamilial phenotype variability observed in our series indicates the need for international consortium to provide reliable data on penetrance of CDH1 mutations before validate guidelines for clinical management.

Risk of pancreatic cancer in hereditary nonpolyposis colorectal cancer Jennifer E. Axilbund1, Alison P. Klein1,2,3, Judith A. Bacon2, Li Wang3, Daniel Edelstein4, Carolina E. Fasola2, Francis M. Giardiello1,4, Constance A. Griffin1,2 Department of Oncology, 2Department of Pathology, Sol Goldman Pancreatic Cancer Research Center, 4Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

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Background Hereditary Nonpolyposis Colorectal Cancer (HNPCC) is characterized by early-onset colorectal cancer. Other associated cancers include endometrial, ovarian, gastric, small bowel, urinary tract and biliary tract. It has been suggested that the risk of pancreatic cancer is also increased, though the precise magnitude is unknown. Pancreatic cancer is the 4th leading cause of cancer death in the United States, leading to an estimated 35,000 annual deaths. The aim

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Abstracts of this study was to assess the lifetime risk of pancreatic cancer in HNPCC families to guide screening recommendations. Methods In a retrospective, registry and clinic questionnaire-based study, we estimated the risk of pancreatic cancer in families that meet the Amsterdam-I criteria and/or have an identified mutation in an HNPCC-associated gene. Families were specifically queried regarding pancreatic cancer occurrence to avoid bias in reporting to a colon cancer registry and/or clinic. Person-years of follow-up were calculated for each individual from their birth until either the date health information was last updated, date of pancreatic cancer diagnosis or death. Standardized incidence ratios (SIR) were calculated by comparing the observed number of pancreatic cancer cases among individuals enrolled in the Johns Hopkins Hereditary Colorectal Cancer Registry and/or the Johns Hopkins Cancer Risk Assessment Program to those expected from SEER incidence rates. Results 1,045 individuals from 66 families were followed for a total of 63,045 person-years. 8 pancreatic cancers were reported. Overall risk of pancreatic cancer was 1.38 (CI = 0.63–2.63.) Risk of pancreatic cancer in individuals with a first-degree relative with colorectal or endometrial cancer was 1.51 (CI = 0.65–2.97). Conclusions These RESULTS: (a) demonstrate a small, not statistically significant increased risk for pancreatic cancer with HNPCC, and (b) suggest that pancreatic cancer screening is not currently warranted for the majority of HNPCC families. Funded by the Jennifer L. Brager Memorial Fund and CA62924.

Results of surveillance for hereditary pancreatic cancer using annual MRI (CP) Wouter H. de Vos1, Martin N. Wasser2, Bert A. Bonsing3, Anneke M. van Mil4, Daniel W. Hommes1, Johan A. Offerhaus6, Hans Morreau5, Hans F. Vasen1,7 Department of 1Gastroenterology and Hepatology, 2Radiology, 3 Surgery, 4Clinical Genetics, 5Pathology, Leiden University Medical Center, Leiden, The Netherlands; 6Pathology Utrecht University Medical Center, Utrecht, The Netherlands; 7Netherlands Foundation for the Detection of Hereditary Tumors, Leiden, The Netherlands Surveillance for pancreatic cancer (PC) in high risk groups may lead to early detection and may improve overall survival. We screened for early pancreatic neoplasia in individuals with a p16-germline mutation or with a strong family history of PC (FPC). Methods Since 2000, high risk individuals were offered annual surveillance by Magnetic Resonance Imaging (MRI(CP)). In case of suspected lesions surgery or additional follow up (FU) was provided. In case of doubt, the examination was repeated within 2–4 months. Results 71 (28 males) individuals with an average age of 56 years (range 40–72) were studied (64 from p16-and 7 from FPC families). The median FU was 3.4 years (range 0–7). IPMN (intraductal papillary mucinous neoplasm)-like lesions were identified in at least five p16 carriers (8%) and in four patients from FPC families (57%). Three patients (FPC n = 2 and p16 n = 1) underwent a prophylactic partial resection of the pancreas. Six patients (9%) were diagnosed with PC. Of them, 4 asymptomatic patients underwent a partial pancreatectomy. Two of 4 had no evidence of residual disease after surgery. The third patient had positive resection margins and nodes but survived 2 years. The fourth patient had both metastatic carcinoid

Abstracts and pancreatic cancer at surgery. The remaining two patients did not undergo surgery because of metastatic disease; one had pulmonary metastases of a melanoma, the other patient developed metastatic pancreatic cancer 3 months after the first screening examination on which a small pancreatic tumor was missed. Conclusion Small premalignant lesions can be identified by MRI(CP). In view of the substantial morbidity and mortality of pancreatic surgery, a major challenge is to decide at what stage and to what extent a patient should undergo prophylactic surgery. Close observation of patients with pancreatic lesions could add valuable information to this question.

Comparative yield of endosonography and magnetic resonance imaging in individuals at high-risk for pancreatic cancer F. Harinck1, I. Kluijt6, J.-W. Poley1, A. Cats7, C. M. Aalfs4, D. J. Gouma3, C. Y. Nio5, P. Fockens2, M. J. Bruno1,2 1 Department of Gastroenterology and Hepatology, Erasmus Medical Center Rotterdam, Netherlands; 2Department of Gastroenterology and Hepatology, Academic Medical Center Amsterdam, Netherlands; 3 Department of Surgery, Academic Medical Center Amsterdam, Netherlands; 4Department of Clinical Genetics, Academic Medical Center Amsterdam, Netherlands; 5Department of Radiology, Academic Medical Center, Amsterdam Netherlands; 6Department of Clinical Genetics, Netherlands Cancer Institute Amsterdam, Netherlands; 7Department of Gastroenterology and Hepatology, Netherlands Cancer Institute Amsterdam, Netherlands

Introduction Individuals at high-risk for pancreatic cancer (PC) are (1) mutation carriers of PC prone hereditary syndromes and (2) firstdegree relatives of patients with PC from familial PC kindreds. Noninvasive pre-cursor lesions of PC include pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). A surveillance program may improve the prognosis of highrisk individuals by detecting asymptomatic early cancers or precursor lesions. Endosonography (EUS) has shown to be a potentially valuable tool. Data for MRI are lacking. We present preliminary results of a comparative study between baseline EUS and MRI screening investigations in individuals entering a yearly surveillance program. Methods Asymptomatic high-risk individuals prospectively underwent EUS and MRI. Both investigations were carried out and scored according to predefined criteria. Investigators were blinded to the results of the alternative imaging modality. Results Thirty-three individuals underwent both EUS and MRI. In eight individuals (24%) focal lesions were detected, one with a mass lesion (11 mm) and seven with cystic lesions. The mass lesion, latter proven to be an adenocarcinoma, was only detected by EUS. The cystic lesions were detected by both techniques in four individuals (12%), by MRI only in two (6%) and by EUS only in two (6%). The overall number of cystic lesions detected varied between EUS and MRI (8 vs. 16). Communication between cysts and the pancreatic duct (PD) was more often reported by EUS than MRI (4 vs. 1). Conclusion Based on these preliminary results, EUS and MRI seem complementary techniques to detect (pre)malignant lesions in individuals at high-risk for developing PC. MRI detected more cystic lesions, but EUS detected communication between cyst and PD more often. The latter is valuable information since it differentiates a simple cyst from a side-branch IPMN. EUS detected one adenocarcinoma in these 33 patients, which was missed by MRI.

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In familial adenomatous polyposis: multiple targeted endoscopic biopsies are accurate in assessing severity of duodenal adenomatosis Musa Drini1, Anthony Speer2, Chris Dow3, Prithi Bhathal4, Neil Collier5, Finlay Macrae1 Colorectal Medicine and Genetics, 2Gastroenterology Department, Anatomical Pathology, 5Department of Surgery, The Royal Melbourne Hospital, Parkville Victoria 3050, Australia; 4Melbourne Pathology, Collingwood, Victoria 3066, Australia 1

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Background and study aims Duodenal polyps are common in FAP. The cumulative risk for development of duodenal cancer is reported to be 4.5% by the age of 57 year (95% CI 0.1–8.9%). Most upper gastrointestinal screening protocols are based on Spigelman’s classification. However the published experience on surveillance of duodenal adenomatosis in FAP, suggests that carcinomas identified in screening are advanced and often not curable. Patients and methods Review of FAP surveillance database between January 1999 and November 2008 is presented. Our surveillance protocol included multiple targeted endoscopic biopsies of selected lesions. Upper Gastrointestinal endoscopy is performed with side viewing instrument. Endoscopic findings were classified as: macroscopically normal or small polyps, benign macroscopically, suspicious for carcinoma macroscopically or confirmed carcinoma. Results Among 67 patients, 11 underwent surgical resection. Pancreas-preserving duodenectomy (PPD) was performed in four patients (five procedures), and Whipple’s operation in seven patients. The average size of polyps was 43 mm (range 17–65 mm), and the average number of targeted endoscopic biopsies per lesion was 7.5 (range 5–10). The section of surgical specimens most representative of the surface epithelium was evaluated for heterogeneity and from this we calculated the approximate percentage of dysplasia/carcinoma on the surface of the lesion, which would be amenable to endoscopic biopsy. Two intramucosal carcinomas were diagnosed on endoscopic biopsies, each understaged compared with the subsequent surgical specimen. All carcinomas identified were resectable with no evidence of local spread or distant metastasis. There was one postoperative death a patient with significant co morbidities, but no cancer related deaths. Conclusion Our screening program identified two early carcinomas and both underwent curative resections. Histology of resected polyps shows considerable heterogeneity of dysplasia and or carcinoma throughout polyps. Endoscopic biopsies understage some lesions. High-risk lesions are best identified in a screening program by multiple targeted endoscopic biopsies.

What happens when you remove the duodenum from a patient without a colon? Is bowel function or quality of life impacted? Yehuda Kariv, R. Kavir, A. da Luz Moreira, R. Mackay, Z. Kutalyli, R. M. Walsh, J. Church Cleveland Clinic Foundation, Cleveland, Ohio, USA Background Patients with familial adenomatous polyposis (FAP) and Spigelman IV duodenal adenomas need surgery to minimize cancer risk. Options for duodenectomy include pancreas sparing duodenectomy (PSD) and pancreaticoduodenectomy (PD). We studied

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716 the influence of duodenal surgery and its specific types on functional outcome and QOL in colectomized FAP patients. Methods FAP patients who had undergone colon resection were identified in a prospectively maintained polyposis registry. Long term ([1 year follow up from last surgery) function and QOL data were obtained using a periodic questionnaire and completed by a telephone survey. Outcomes in patients who had both colonic and duodenal surgery (CDS) were compared to patients who underwent colonic surgery without duodenal surgery (CS). CDS patients’ data was also analyzed according to the specific type of colonic or duodenal surgery. Results 51 FAP patients underwent CDS. 9 patients died during follow up (median, 192 (range 43–540) months from first surgery, 64 (range 19–250) months from last surgery). Long term functional and QOL data were available in 33 CDS and compared to 249 CS patients (Table). Median bowel movements per 24 h were 6 (CDS) and 5 (CS). Median quality of life (CCF Global Score) was 9/10 (CDS) and 9/10 (CS). Higher rates of diet (45% vs. 27%), social (19% vs. 5%), sexual (17% vs. 5%) and work restrictions (21% vs. 9%) were reported by CDS patients. CDS had lower quality of health scores. CDS patients with an ileoanal pouch had more frequent bowel movements but were similar in other functional and QOL parameters. Outcomes were comparable between different types of duodenal surgery. Conclusions FAP patients needing duodenectomy can be reassured that quality of life will be maintained.

Prophylactic pancreaticoduodenectomy for advanced duodenal adenomatosis in familial adenomatous polyposis J. Skipworth1, C. Morkane1, N. West3, M. Deheragoda2, D. Raptis1, C. Imber1, S. Olde-Damink1, M. Malago1, R. Phillips3, S. Clark3, A. Shankar1 1

Department of Hepatopancreaticobiliary Surgery, University College London Hospital NHS Trust, London; 2Department of Pathology, University College London Hospital NHS Trust, London; 3 The Polyposis Registry, St. Mark’s Hospital, Harrow Background Patients with familial adenomatous polyposis (FAP) develop duodenal polyps that may progress to malignancy, via the adenoma-carcinoma sequence. In 2002, this centre reported data on 16 FAP patients who underwent prophylactic duodenal resection for duodenal polyps. We now present the extended results, representing the largest series of pancreaticoduodenectomy for FAP reported. Methods We performed a retrospective case-notes review of all patients undergoing prophylactic duodenal resection for advanced duodenal adenomatosis, in a single centre. Data collected included demographics, resection type, Spigelman staging, morbidity and mortality. Results 40 (24F:16M) FAP patients underwent prophylactic resection for advanced duodenal adenomatosis (Spigelman stage III/IV or cancer) between January 1994 and November 2008. Data for 8 patients were incomplete. Median patient age was 48 years and median hospital stay 27 days. Available data revealed that 36 pancreaticoduodenectomies and 1 local excision + cholecystectomy were performed. Perioperative mortality was 2 (5%): both patients dying from multi-organ failure following re-operation for haemorrhage. Six further patients have subsequently died, four from metastatic disease, one following a

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Abstracts cerebrovascular accident and data is missing in one case. Complications occurred in 24 (60%) patients. Early complications included six anastomotic leaks, seven post-operative haemorrhages (three necessitating redo laparotomy, one requiring gastroduodenal artery embolisation and one needing embolisation of a jejunal vessel), three enterocutaneous fistulae, one pulmonary embolus, one lymphatic leak and 11 post-operative collections. Late complications included pancreatic insufficiency/steatorrhoea in 13 patients, diabetes in one and delayed gastric emptying in four patients. Postoperative histology revealed three (8%) pre-operatively undetected ampullary/duodenal cancers—two of these patients have subsequently died. Conclusion Prophylactic pancreaticoduodenectomy is associated with significant morbidity and mortality; however, FAP patients with advanced duodenal polyposis have a significant risk of malignant transformation and some have undetected malignancy. Thus, the identification of FAP patients requiring surgical intervention for highrisk of duodenal malignant transformation remains challenging.

Is there a genotype–phenotype correlation for ileal pouch polyposis in patients with familial adenomatous polyposis? Alexander C. von Roon, Olivia C. Will, Ripple F. Man, Kay F. Neale, R. John Nicholls, Robin K. S. Phillips, Paris P. Tekkis, Susan K. Clark The Polyposis Registry & Department of Surgery, St Mark’s Hospital and Department of Biosurgery and Surgical Technology, Imperial College, Harrow, Middlesex, UK Aim To examine whether in patients with familial adenomatous polyposis (FAP) who have undergone ileal pouch anal anastomosis (IPAA), the severity of pouch polyposis is associated with APC codon 1309 mutation or number of intact 20 amino acid beta-catenin binding and degradation sites (20aa repeats) on the mutant allele. Methods All pouch endoscopy reports for patients with FAP attending for annual surveillance after IPAA were reviewed. Only patients with more than 10 years follow-up after IPAA and a known APC mutation were included. The maximum incidence of pouch body neoplasms and mutation status were recorded. Significance was assessed with the Gamma statistic. Results Of 206 patients who underwent IPAA, 54 met the inclusion criteria. Fifteen patients had a codon 1309 mutation, 39 had other mutations. A larger proportion of patients with a codon 1309 mutation remained polyp-free (10/15 [67%] vs. 9/39 [23%]), a similar proportion had 1–10 pouch polyps (4/15 [27%] vs. 11/39 [28%]), and a lower proportion had over 10 pouch polyps (1/15 [7%] vs. 19/39 [49%]) after 10 years (p = 0.004). Of the 54 patients, 27 had mutations resulting in no 20aa repeats, 19 had one repeat, 6 had 2 repeats and 2 had 3 repeats. When compared with patients who had no 20aa repeat, a larger proportion of those with one repeat remained polypfree (10/19 [53%] vs. 6/27 [22%]), a similar proportion had 1–10 polyps (7/19 [37%] vs. 6/27 [22%]), and a lower proportion had more than 10 polyps (2/19 [11%] vs. 15/27 [56%]) after 10 years (p = 0.025). Conclusion The APC mutation at codon 1309 and mutations resulting in a single 20aa repeat on the mutant allele appear to be inversely correlated with the severity of pouch polyposis. This finding contrasts with the genotype–phenotype correlation observed in colonic polyposis. Larger studies are required to confirm these findings.

Abstracts

Children with familial adenomatous polyposis who underwent early colectomy: psychological, quality of life and pouch outcome C. Durno, K. Butler, T. Berk, N. Alingary, M. J. Esplen Dr. Zane Cohen Digestive Diseases Research Center, Familial Gastrointestinal Cancer Registry and Department of Surgery, Mount Sinai Hospital, Division of Gastroenterology, Hepatology and Nutrition, Department of Paediatrics, Hospital for Sick Children, University of Toronto, Toronto, Canada Background The impact of ileal pouch-anal anastomosis on quality of life in adolescents with FAP is favorable. There is a small group of children with FAP who develop polyps at a younger age and have a severe polyposis phenotype requiring earlier colectomy. The literature does not focus on this very young subgroup. Patients who undergo early colectomy may have a poor functional outcome and quality of life. Aim To investigate functional outcome and quality of life in patients with FAP who had colectomy \14 years of age. Methods A cross-sectional quantitative survey was used to assess patients with FAP recruited through a FAP Registry. Standardized instruments included: psychosocial functioning, quality of life and functional outcomes. Results Among 1337 patients with FAP from 409 kindreds 59 (4%) patients underwent colectomy at\14 years of age. Response rate was 84% (32/38). The mean age at the time of colectomy was 12 years (SD 2), with a current mean age of 24 years (SD 8.5). Time since colectomy was 12 years (SD 8.4, range 1–37 years). Bowel function: 78% of patients ‘‘rarely or never’’ have restrictions at work/school and 60% are ‘‘always to sometimes’’ embarrassed. Patients currently \18 years of age (9/32) have more restrictions. For the majority psychosocial functioning was in normal ranges. Self and body esteem were within normal ranges. Having a family member with FAP or having lost a family member to FAP (11/32) did not affect quality of life, self-esteem, or psychological outcome. Patients understand that their risk of bowel cancer is still increased compared to the general population. The majority of patients (n = 24, 75%) have had surveillance endoscopy within the last 2 years. Conclusions Patients who had early colectomies are functioning well psychologically and with bowel function. Health-related quality of life and compliance with endoscopic surveillance is very good. A subgroup who are currently under 18 years of age demonstrate emotional issues and difficulty adapting. There are some restrictions due to bowel and psychological symptoms. This subgroup would benefit with added psychological interventions to enhance coping.

717 nearly universal. We describe the development and initial evaluation of MyFAP, an Internet-based, multimedia psychosocial intervention for AYAs with FAP. The goal of MyFAP is to facilitate self-management and coping with the medical and psychological issues faced by young persons with FAP. Grounded in Social Cognitive Theory (SCT), MyFAP includes the following components: medical and genetic aspects of FAP; self-management (e.g., screening, self-care issues); emotional concerns and support; communication with peers, family and health care providers; and, preventive surgery. Consistent with SCT, the intervention includes multiple elements to facilitate the adoption of coping and problem-solving skills through cognitivebehavioral activities and interactive multimedia features. MyFAP includes a social networking component to promote social interaction and support with peers. Twenty-three AYAs age 13–24 years completed an initial evaluation of the intervention. The mean correct score on a baseline measure of FAP-related knowledge was 51%, indicating the need for improved education about clinical aspects of FAP. Greater than 90% of users rated the highest level of satisfaction with MyFAP’s attractiveness, control, efficiency, helpfulness, and learn ability, using a standardized measure. Findings showed that an Internet-based psychosocial intervention is a feasible and acceptable method for addressing the informational and supportive needs of AYAs with FAP.

Compliance with endoscopic surveillance advice for familial adenomatous polyposis (FAP): room for improvement Kirsten F. L. Douma, Eveline M. A. Bleiker, Neil K. Aaronson, Annemieke Cats, Miranda A. Gerritsma, Chad M. Gundy, Hans F. A. Vasen The Netherlands Cancer Institute, Antoni van Leeuwenhoek hospital, Amsterdam, The Netherlands Background & Aims Familial adenomatous polyposis (FAP) is characterized by the development of hundreds of adenomas in the colorectum that, without surgery, lead to colorectal cancer. The purpose of the study was to assess compliance with endoscopic surveillance advice before as well as after (prophylactic) surgery for FAP. Methods In this nationwide, cross-sectional study, individuals from families at high risk for FAP registered with the Netherlands Foundation for the Detection of Hereditary Tumours were invited to complete a questionnaire on psychosocial issues and endoscopic screening experiences. Compliance data were derived from medical records and via self-report.

The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA

Results A total of 328 individuals were eligible for the study, of whom 85 were at risk for FAP, 108 had an intact rectum after a colectomy with ileorectal anastomosis (IRA), and 135 had a pouch following a proctocolectomy with ileoanal anastomosis (IPAA). Based on medical record data, 20% of the at-risk group and 6% of the IRA group were found to be less than fully compliant with surveillance advice. Under-compliance in the at-risk group was associated significantly with perceived self-efficacy, use of sedatives during surveillance, pain after surveillance and low perceived benefits of surveillance (p \ .05).

Adolescents and young adults (AYAs) with familial adenomatous polyposis (FAP) face unique medical and psychosocial demands related to genetic counseling and testing, routine colorectal screening, and preventive colectomy. There are scant informational and supportive resources available for this population. The Internet is a promising medium for the delivery of psychosocial interventions to persons with rare conditions, and Internet use by young persons is

Conclusions One-fifth of individuals at-risk for FAP are undercompliant with screening advice. Low self-efficacy, non-use of sedatives during surveillance and pain after surveillance are negatively associated with compliance behavior. We recommend that sedatives be routinely offered to individuals undergoing colorectal cancer surveillance for FAP and that adequate pain medication or spasmolytica be provided after endoscopy.

MyFAP: An internet-based psychosocial intervention for adolescents and young adults with FAP Susan K. Peterson, Martha Askins, Alex Prokhorov, Devki Saraiya, Thuy Vu, Miguel Rodriguez-Bigas, Patrick Lynch

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Familial adenomatous polyposis (FAP): attitudes towards genetic testing in childhood and reproductive decision-making Kirsten F. L. Douma, Neil K. Aaronson, Hans F. A. Vasen, Senno Verhoef, Chad M. Gundy, Eveline M. A. Bleiker The Netherlands Cancer Institute—Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands Purpose Childhood DNA testing, prenatal diagnosis (PND) and preimplantation genetic diagnosis (PGD) are available for most hereditary cancer susceptibility syndromes. However, the use of PND and PGD for these syndromes is controversial. The purpose of this study is to investigate attitudes towards, and experiences with, childhood DNA testing, PND and PGD among members of families at high risk for familial adenomatous polyposis (FAP). Methods In this nationwide cross-sectional study, individuals from families at high risk for FAP, were invited to participate. Questionnaire data were collected on attitudes towards and experiences with childhood testing, PND and PGD, and on a range of sociodemographic, clinical and psychosocial variables. Results 525 FAP-family members participated (response rate = 64%). Forty percent of FAP-patients expressed that the disease has influenced their desire to have children. Only a small proportion (15%) considered termination of pregnancy for FAP acceptable. Approximately 30% of individuals with a FAP-diagnosis and their partners had a positive attitude towards PND and PGD. A positive attitude towards PND and PGD was associated with higher levels of guilt and a positive attitude towards termination of pregnancy. Five individuals reported direct personal experience with PND and one with PGD. Ninety-three individuals with a FAP-diagnosis and 43 partners had children who were minors (\18 years) during the DNA testing procedure. Most of these parents (82%) were satisfied with the procedure. One-third of the individuals wanted DNA testing for their children before age 12. Conclusion FAP family members’ experiences with childhood DNA testing are predominantly positive. Approximately one-third of those with a FAP diagnosis have a positive attitude towards PND and PGD. Few, however, have had direct experience with these procedures.

Extracolonic tumour spectrum and incidence in 276 patients affected by MUTYH-associated polyposis (MAP) Stefan Aretz, Daria Christian, Christoph Engel, Maartje Nielsen, Natalie Jones, Astrid Kaufmann, Verena Steinke, Hans F. Vasen, Peter Propping, Frederik J. Hes, Julian R. Sampson Stefanie Vogt Institute of Human Genetics, Bonn, Germany Background To date, no systematic evaluation of extracolonic MAP manifestations has been reported. Methods In a collaborative multicentre European study a large cohort of MAP patients (276 cases from 181 apparently unrelated families) was recruited. Based on medical records and anamnestic information the extracolonic tumour spectrum and incidence were evaluated to assess cumulative lifetime risks and compared with general population rates to obtain standardized incidence ratios (SIRs). Results The median age at evaluation was 54 years. Duodenal polyposis occurred in 17%; the relative risk of duodenal cancer was very high (SIR 130; 95% CI 16–470), while the lifetime risk was

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Abstracts 3.6%. Regarding extraintestinal tumours we found a low to moderate but significant increase in the incidence of breast cancer (SIR 3.0; 95% CI 1.5–5.4; mean age at diagnosis 60 years), bladder carcinomas (SIR 7.23; 95% CI 1.97–18.5; mean age at diagnosis 59 years), and skin cancer (SIR 2.8; 95% CI 1.5–4.8; mean age at diagnosis 52 years), other malignant lesions were not increased significantly. The incidence of extraintestinal malignancies as a whole was almost doubled (SIR 1.8; 95% CI 1.3–2.4), the lifetime risk was 40% (95% CI 24–55%). Interestingly, sebaceous gland tumours (SGT), that are a characteristic of Muir–Torre syndrome, occurred in five patients. No genotype–phenotype correlation was identified. Conclusions The relative risks of a few cancers and of extraintestinal malignancies as a whole were increased, however, no predominant lesion was observed. The spectrum of cancers and their advanced age at onset do not suggest that specific surveillance recommendations other than frequent gastrointestinal endoscopies can be made at present. Although not significant, a trend towards a slightly increased frequency of gynaecological tumours (endometrial and ovarian cancer) and SGT point to a phenotypic overlap with Lynch syndrome. SGTs might serve as diagnostic marker lesion in some cases. The study was supported by the Deutsche Krebshilfe, the Dutch Digestive Diseases Foundation, and the Wales Gene Park and Cancer Research Wales.

Analysis of MUTYH genotypes and colorectal phenotypes in patients with MUTYH associated polyposis Maartje Nielsen, Mirjam C. Joerink-van de Beld, Natalie Jones, Stefanie Vogt, Carli M. Tops, Hans F. A. Vasen, Julian R. Sampson, Stefan Aretz, Frederik J. Hes Department of Clinical Genetics, Leiden University Medical Center (MN, MCJB, CMT, FJH), Institute of Medical Genetics, School of Medicine, Cardiff University (NJ, JRS), Institute of Human Genetics, University of Bonn (SV, SA), Department of Gastroenterology & Medical Oncology, Leiden University Medical Center (HFAV) Background & Aims Functional studies have demonstrated significant differences in base recognition and glycosylase activity between various MUTYH mutations, notably for the two mutations most frequently reported in MAP patients: Y179C and G396D (previously annotated as Y165C and G382D). Our goal was to establish correlations between genotypes and colorectal phenotype of patients with MAP. Methods In this multicenter study, we analyzed genotype and phenotype data from 257 MAP patients. Data included age at presentation of MAP, polyp count, the occurrence, location and age at presentation of CRC. Results Patients with a homozygous G396D mutation or compound heterozygous G396D/Y179C mutations presented later with MAP and had a significantly lower hazard of developing CRC than patients with a homozygous Y179C mutation (P \ 0.001). The mean ages of CRC diagnosis in patients were 58 years (homozygous G396D) and 52 years (compound heterozygous G396D/Y179C) versus 46 years (homozygous Y179C; P = 0.001, linear regression). Conclusions Our study identified the phenotypic effects of Y179C as relatively severe and of G396D as relatively mild. These clinical data are in accord with findings from in vitro functional assays. Genotypic stratification may become useful in the development of guidelines for counseling, surveillance and management of families with MAP.

Abstracts 1. Harris M (2008) Why all young bowel cancer patients should be screened for Lynch syndrome. ANZ J Surg 78:531–532.

Renal cancer as part of the phenotype of MYH-associated polyposis; the evidence gets stronger Lisa LaGuardia, Margaret O’Malley, Carol Burke, James Church Department of Colorectal Surgery, The Sanford R. Weiss, M. D. Center for Hereditary Colorectal Neoplasia, Digestive Disease Institute, Cleveland Clinic, Cleveland Ohio Background The full phenotype of MYH-Associated Polyposis (MAP) is still not known as the syndrome is relatively new and the number of affected families relatively sparse. The syndrome often seems to mimic attenuated FAP except that the pattern of inheritance is recessive rather than dominant. In 2006 a preliminary study of the tumor spectrum that was seen in 6 MAP families suggested that renal cancer may be found to excess. Now we have 7 additional MAP families with information that more strongly implicates renal cancer as part of the syndrome. Methods The families of thirteen probands with MAP were characterized by extensive pedigree building. Data was entered into the Polyposis registry database. Where available, pathology reports were requested to document the reliability of the family history. All probands had biallelic mutations of MYH. Results Relatives under the age of eighteen and the spouses of at-risk relatives are excluded. There were 247 at-risk relatives in the 13 families. There were 67 affected (27%), some with multiple tumors. The cancer spectrum is 23 patients had colon cancer out of 8 families, 6 had prostate cancer out of 3, 6 had renal cancers out of 6, 2 had uterine cancer out of 1, 2 had pancreatic cancers out of 1, 4 had breast cancers out of 4, 1 had bone cancer out of 1, 1 had brain cancer out of 1, 5 had lung cancers out of 5, and 2 had leukemia out of 2. Discussion The colorectal phenotype of these MAP families is as expected. 13.3% of relatives had adenomas although less than half of the relatives had screening colonoscopy. Colon cancers were found in 23 individuals, usually with an older age of onset. The spectrum of extracolonic cancers seen in these 13 families is unusual. Renal cancer occurred in six different families. There were four cases of prostate cancer in one family and two with pancreatic cancer in another 78% of patients with a cancer had at least one Y165C mutation; 62% had at least one G382D mutation. Conclusion The phenotype of MAP is evolving. Detailed studies on phenotype in much large numbers of families are needed. Renal cancer was present 46% of these families and renal ultrasound is recommended as part of the surveillance program.

Changing causes of death in familial adenomatous polyposis: signs of progress but more work to do Lisa LaGuardia, Margaret O’Malley, James Church, Carol Burke, Matthew Kalady The Sanford R. Weiss, MD, Center for Hereditary Colorectal Neoplasia, Digestive Disease Institute, Cleveland Clinic, Cleveland, Ohio Introduction Patients with FAP are at risk of dying from multiple benign and malignant tumors, from surgical complications, comorbid

719 diseases and the rigors of life in the twenty-first century. Our last study on why patients with FAP die was in 1990. We have analyzed causes of death since then and compared the two groups to see if the significant advances in medicine and technology are reflected in different patterns. Methods Causes of death were extracted from the 1990 study via the manuscript. Causes of death since then were determined from the registry database and confirmed by chart review. Results In 1990 there were 178 FAP families in the registry. There are now 761 families. 212 patients have died (0.28 deaths perfamily), 110 before 1990 (0.62 deaths perfamily) and 102 since 1990 (0.17 deaths per family). Death from colorectal cancer before 1990 were 64 (58.2%), and after 1990 were 41 (40.2%). Deaths from desmoid disease were 12 (10.9%), and 9 (8.8%), from periampullary cancer were 9 (8.2%), and 4 (3.9%), from brain cancer were 8 (7.3%), and 2 (2.0%), Perioperative deaths were 5 (4.5%), and 3 (2.6%). Accidental deaths were 3 (2.7%), and 0 and death from other causes were 9 (8.2%), and 24 (23.5%). Unknown 0, and 19 (18.6%). Overall death from cancer (excluding desmoids) was 85 (77.3%) before 1990 and 57 (55.9%) since 1990. ‘‘Other’’ deaths include cancers of the thyroid, stomach, esophagus, pancreas, breast, ovary and lung. There were also 2 suicides and one death from pancreatitis. Overall there have been fewer deaths per family since 1990. Deaths from colorectal and periampullary/duodenal cancer have declined (even if the ‘‘unknown’’ category is excluded) while those for desmoid remain constant. Perioperative deaths are fewer. Conclusion Improvements in education and more access to genetic testing, screening and surgical techniques should further reduce the death rate from FAP. There is scope for doing so.

Interobserver variability in the interpretation of immunohistochemical mismatch repair protein staining Louise Klarskov1, Susanne Holck1, Karina Ro¨nlund2, Jan Lindebjerg2, Jacob Elebro3, Britta Halvarsson4, Inge Bernstein5, Jenny von Salome´e6, Mef Nilbert7 1 Department of Pathology, Faculty of Health Sciences, Copenhagen University, Hvidovre Hospital, Copenhagen, Denmark; 2Department of Pathology, Vejle Hospital, Vejle, Denmark; 3Department of Pathology, Lund University Hospital, Lund, Sweden; 4Department of Pathology, HNPCC-register, Department of Gastroenterology, Copenhagen; 5University, Hvidovre Hospital, Copenhagen, Denmark Helsingborg Hospital, Helsingborg, Sweden; 6Department of Clinical Genetics, Stockholm University, Karolinska Hospital, Stockholm, Sweden; 7Clinical Research Centre, Faculty of Health Sciences, Copenhagen University, Hvidovre Hospital, Copenhagen, Denmark

Introduction Immunohistochemical staining for mismatch repair (MMR) proteins is a widely implemented diagnostic tool in the work up of suspected hereditary colorectal cancer, but quality assurance studies are scarce. In order to validate routine staining with respect to interobserver variability, we reviewed MMR protein immunostainings from 225 colorectal cancers. Materials and methods All tumors were stained as part of routine examination linked to genetic counselling for suspected hereditary colorectal cancer. Immunohistochemical stainings for MLH1, PMS2, MSH2 and MSH6 were reviewed in a blinded fashion. In order to test the impact of experience in immunohistochemical evaluation, 3 pathologists specialized in gastrointestinal diagnostics and 2 residents in pathology evaluated all samples. Stainings were evaluated as normal, completely lost, weak or non-evaluable. Consensus was

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720 defined as all five observers agreeing on one of the four predetermined staining patterns. Results Consensus was reached for MLH1 in 52%, PMS2 in 60%, MSH2 in 81%, and MSH6 in 43% of the stainings. Related kappavalues of interobserver agreement, two by two, varied between 0.35–0.81, 0.45–0.91, 0.61–0.95, and 0.23–0.77, respectively. Kappavalues among the 3 specialists did not differ from the values between the 2 residents. Discrepancy was predominantly related to cases judged weak instead of normal or completely lost by at least one of the observers, whereas discrepancy between normal and completely lost occurred in 2–5% of the stainings. Discussion Improved HNPCC diagnostics is crucial since it allows identification of high-risk individuals who should be offered participation in surveillance programmes. Herein standardization and validation of MMR protein immunostaining evaluation is essential. Our findings suggest that differences in interpretations of the MMR protein immunohistochemical stainings are common, which underscores the need for quality assurance programmes.

Narrow-band imaging improves the detection of polyps in patients with hyperplastic polyposis syndrome: a prospective randomized study K. S. Boparai, F. J. C. van den Broek, S. van Eeden, P. Fockens, E. Dekker Academic Medical Centre, Amsterdam, The Netherlands Background Hyperplastic polyposis syndrome (HPS) is associated with colorectal cancer (CRC). HPS patients receive endoscopic surveillance to prevent malignant progression of polyps. Endoscopic detection and removal of potentially premalignant sessile serrated adenomas (SSAs) and conventional adenomas may be an important step in preventing CRC development in HPS. However, polyps in HPS can be difficult to detect due to their unremarkable color and flat shape. Objective To prospectively compare the value of narrow-band imaging (NBI) and high-resolution white light endoscopy (WLE) for the detection of polyps in HPS and to assess the value of NBI for the differentiation of these polyps. Patients and interventions During surveillance endoscopy in 22 HPS patients, each colonic segment was inspected twice, once with WLE and once with NBI, in random order by one experienced endoscopist. Of all detected polyps the size, shape and location was assessed. Kudo pit-pattern analysis was performed in all polyps. Main outcome measurements The sensitivity of WLE and NBI for the detection of polyps was assessed. The diagnostic accuracy of NBI in differentiating detected polyps was determined by using histology as a gold standard. Results A total of 116 HPs, 42 SSAs and 24 adenomas (range: 2–20 mm) were detected. Polyps were classified as flat (60%), sessile (38%) and pedunculated (2%). The sensitivities of WLE and NBI for overall polyp detection were 64 and 90% respectively (p \ 0.001). For flat polyps these were 51 and 87% (p \ 0.001) and for sessile/pedunculated polyps 81 and 96% (ns). The sensitivities of WLE and NBI for HPs was 67 and 90% respectively (p = 0.017). For SSAs this was 38 and 86% (p = 0.003) and for adenomas 70 and 100% (ns). The accuracy of NBI for discriminating SSAs from HPs was 63% and for differentiating adenomas from HPs this was 75%.

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Abstracts Conclusion NBI significantly improves the detection rate of SSAs and predominant flat polyps in HPS. Therefore, NBI seems of clinical value for the endoscopic management of HPS patients.

Family history of colorectal cancer is inversely related to polyp count in individuals with multiple serrated polyps Joanne Young, Daniel D. Buchanan, Kevin Sweet, Musa Drini, Mark A. Jenkins, Aung Ko Win, Michael Gattas, Michael D. Walsh, Diane McKeone, Aedan Roberts, Mark Clendenning, Rhiannon Walters, Sven Arnold, Alasdair Young, Heather Hampel, John L. Hopper, Jack Goldblatt, Jill George, Graeme K. Suthers, Kerry Phillips, Graeme P. Young, Elizabeth Chow, Susan Parry, Kathy Tucker, Amanda Muir, Michael Field, Sian Greening, Steven Gallinger, Jane Green, Michael O. Woods, Renee Spaetgens, Albert de la Chapelle, Finlay Macrae, Jeremy R. Jass Familial Cancer Laboratory, Queensland Institute of Medical Research, Herston, Australia Hyperplastic polyposis (HPS) is a colonic polyposis condition of unknown aetiology. An association with an increased risk of colorectal cancer (CRC) has previously been established. The purpose of this study was to examine the spectrum of phenotypic variation in HPS. One hundred and twenty-six patients with HPS were recruited to the study. Patients were assigned to three categories based on polyp numbers which were extracted from histology and colonoscopy reports. Ethnicity of paternal and maternal lines was self-reported. Family history of colorectal cancer data were derived from pedigrees. All patients were screened for the two most common mutations in MUTYH. One hundred and fourteen of 120 (95%) HPS probands were of white, northern European ethnicity. One of 126 patients (\1%) was homozygous for the Y165C variant in the MUTYH gene. The average minimum reported polyp number was 39; 28, 59 and 13% of the participants were in the polyp category 1 (5–20 polyps), category 2 (21–70 polyps) and category 3 ([70 polyps), respectively. CRC was identified in 49 of 119 patients (41%) and 28% of these patients had multiple CRC. CRC was significantly associated with the presence of adenomas (P = 0.03). Overall, 59% of probands had at least one-first-degree relative affected with CRC. Family history of CRC (especially a first-degree relative with CRC) was inversely associated with polyp number categories (P = 0.03). In addition, males were more likely to have higher numbers of serrated polyps (P = 0.04). We conclude that HPS is associated with an increased personal risk of CRC, and higher polyp numbers in males. The inverse relationship between polyp numbers and family history of CRC suggests heterogeneous modes of inheritance.

Serological markers in evaluation of individual risk of gastric cancer among mutation carriers in Lynch syndrome 39 Mecklin, A. Ristima¨ki, P. Sipponen, K. Nuorva, V. Ka¨rja¨, S. Sarna, L. Renkonen-Sinisalo, M. Aarnio, M. Heikkinen, K. Pylva¨na¨inen, H. J. Ja¨rvinen Jyva¨skyla¨ Central Hospital, Jyva¨skyla¨, Finland

Abstracts Gastric cancer (GC) is the second or third most common cancer in Lynch syndrome (LS), but its mortality often exceeds the mortality of colorectal or endometrial cancers. A chronic, active infection by high virulence H. pylori strains can transform normal gastric mucosa into atrophic gastritis. This type of response to the chronic infection may eventually lead to dysplastic changes and transformation to GC. Over 90% of GCs in LS seems to be histologically of intestinal type (IT). Therefore, H. pylori infection and atrophic gastritis can be considered as markers of increased risk of GC in individuals with LS and indicative for interventions. Gastroscopy has been considered the only screening method available, but its cost-benefit is low especially in most western countries. Our purpose was to find a cost-benefit non-endoscopic alternative to identify Lynch syndrome mutation carriers at risk for gastric cancer. Experimental design 89 Lynch syndrome mutation carriers over 50 years underwent upper-GI-endoscopy with biopsies and blood test panel including pepsinogen I and II, fasting gastrin-17 and immunoglobin G antibodies to H. pylori. Results Normal gastric mucosa was diagnosed in 71.6–77.3%, superficial gastritis in 21.6–18.2% and atrophic gastritis in 6.8–4.5% by serology and histology, respectively. The sensitivity of serology to identify atrophic gastritis was 0.750 (95% CI 0.301–0.954) and specificity 0.988 (95% CI 0.936–0.988). Positive predictive value was 0.750 (95% CI 0.301–0.954) and negative predictive value 0.988 (95% CI 0.936–0.998). The serological test panel overestimated normal mucosa into superficial gastritis in four and atrophy in one cases. Conclusion Serological biomarkers identify reliably atrophic gastritis and they can be used as non-endoscopic screening method in evaluating individual risk for gastric cancer and need for endoscopic surveillance in Lynch syndrome in countries with a low general incidence for gastric cancer.

Natural history of Amsterdam patients following colorectal cancer resection Matthew F. Kalady, James Church, Jon Vogel, Ellen McGannon, Susan Fay, Elena Manilich, Lori Arroyo, Kathy Toderick, Janet Shenal Department of Colorectal Surgery, Sanford R. Weiss Center for Hereditary Colorectal Neoplasia, Cleveland Clinic, Cleveland, OH, USA Introduction Colorectal cancer patients meeting Amsterdam criteria are offered total rather than segmental surgical resection to reduce the risk of metachronous colorectal malignancy. There is sparse natural history information, however, for patients who undergo a segmental colectomy. This study evaluates the natural history of surgically treated Amsterdam criteria colorectal cancer patients. Methods A single institution hereditary colorectal cancer database was reviewed for all patients meeting Amsterdam I, II or-like criteria or germline-confirmed Lynch patients who underwent surgical resection. Hereditary syndrome, patient demographics, type of surgery performed, tumor characteristics and subsequent follow-up were recorded. The primary endpoints for patients undergoing surgery less than a total proctocolectomy were subsequent adenoma formation and further resection for cancer. Results 208 patients were included, 67 treated primarily at our institution and 141 referred to our registry after undergoing index

721 resection. 49% were female and 173 cancers (83%) were rightsided. The median age at index surgery was 51.9 years. 176 patients underwent a partial colectomy, 23 underwent a total or subtotal colectomy with ileorectal or ileosigmoid anastomosis, respectively, and 9 patients underwent a total proctocolectomy. 66 of 199 patients (33%) underwent subsequent polypectomy during surveillance, with removal of 131 adenomas including 24 that were [10 mm and 30 with tubulovillous or villous architecture. 25 of 176 (14%) who underwent a partial colectomy subsequently developed a colorectal cancer requiring resection at mean time of 124 months from index surgery. The stages at second resection were I-9, II-11, III-4. Conclusions Amsterdam patients undergoing partial colectomy have a significant rate of metachronous high-risk adenoma formation and cancer development, some of which present at advanced stage. Therefore, total colectomy is still advocated as the index surgery. However, if circumstances result in a segmental colectomy, patients should undergo timely surveillance protocols and intervention to prevent future malignancies.

The outcome of longterm surveillance of Lynch syndrome families in the Netherlands H. F. A. Vasen1, J. Kleibeuker2, M. van Kouwen3, J. J. Koornstra2, A. Cats4, E. Dekker5, A. M. J. Langers1, S. Sanduleanu6, J.-W. Poley7, J. C. H. Hardwick1, W. H. de Vos tot Nederveen Cappel1, A. E. van der Meulen-deJong1, F. N. Nagengast3, The Dutch Lynch Syndrome Study Group8 Departments of Gastroenterology, University Medical Centre of Leiden1, Groningen2, Nijmegen3, Netherlands Cancer Institute Amsterdam4, AMC Amsterdam5, Maastricht6, Rotterdam7 and The Netherlands Foundation for the Detection of Hereditary Tumours8 Background Carriers of an MMR gene mutation have a high risk of developing colorectal cancer (CRC). Because of evidence of an accelerated colorectal carcinogenesis in Lynch syndrome, the surveillance protocol for these families has been changed in the mid Nineties. Since then, the recommended interval between examinations has been 1–2 years in stead of 2–3 years. The aim of the study was (1) to evaluate the risk of developing CRC while under surveillance and (2) to identify risk factors. Patients and methods The database of the Registry was used. Only carriers of an MMR mutation who had more than one surveillance examination were selected. MMR gene carriers who underwent a previous colectomy were excluded. The observation time was from 1-1-1995 until 1-1-2008. Endpoints of the study were CRC, death or the last colonoscopy. Kaplan–Meyer analysis was used to calculate the risk of CRC. Results A total of 721 mutation carriers were included. The mean follow up was 6.7 years. Thirty-three patients developed CRC. 85% were at stage I or II. The cumulative risk of developing CRC was 5% at 10 years of follow up. Male carriers had a (nonsignificant) higher risk than female carriers. Carriers of an MLH1 or MSH2 mutation had a significant higher risk than MSH6 carriers. There was no difference in risk between surveillance at university medical centres and peripheral hospitals. Conclusions The risk of developing CRC under surveillance has decreased since shortening of the surveillance interval. The risk was highest in carriers of an MSH2 and MLH1-gene mutation. In these groups annual colonoscopy and/or the use of chromoendoscopy may be considered.

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Modifiers of colorectal cancer risk in Lynch syndrome J. T. Wijnen, R. M. Brohet, S. Jagmohan-Changur, R. van Eijk, A. Middeldorp, C. M. Tops, M. van Puijenbroek, M. G. E. M. Ausems, E. Go´mez Garcı´a, F. J. Hes, N. Hoogerbrugge, F. H. Menko, T. A. M. van Os, R. H. Sijmons, S. Verhoef, A. Wagner, F. M. Nagengast, J. H. Kleibeuker, P. Devilee, H. Morreau, I. P. Tomlinson, R. S. Houlston, T. van Wezel, H. F. A. Vasen Leiden University medical Center, Leiden, The Netherlands Recent genome-wide association studies have identified common low risk variants for colorectal cancer (CRC). To assess whether these variants influence CRC risk in the Lynch syndrome (LS), we genotyped these variants in 675 proven MMR mutation carriers from 127 different LS families. We used univariate and multivariate analysis to analyse the association between the presence of a risk variant and CRC risk. In our initial analysis of six variants on 8q24.21, 8q23.3, 10p14, 11q23.1, 15q13.3 and 18q21.1 we found a significant association between CRC risk and rs16892766 (8q23.3) and rs3802842 (11q23.1). The C allele of rs16892766 was associated with an elevated risk of CRC in a dose dependent fashion, with homozygosity for CC conferring a 2.16-fold increased risk compared to the AA homozygotes. For rs3802842 the increased risk of CRC associated with the C-allele was only found among female carriers, while CRC risk was substantially higher among homozygous (HR 3.08) than among heterozygous carriers of the C-allele (HR 1.49). In an additive model of both variants, the CRC risk was significantly associated with the number of risk alleles (HR 1.60 for carriers of two or more risk alleles compared to carriers of none or one risk allel). The observed effects were stronger in female carriers than in male carriers. Results on genotyping of eight additional low risk variants will be presented at the meeting. Conclusion So far we have identified two loci which are significantly associated with CRC risk in Lynch syndrome families that may be helpful to identify high risk individuals that require more intensive surveillance.

Haemochromatosis HFE gene polymorphisms as potential modifiers of hereditary nonpolyposis colorectal cancer risk and onset age R. J. Scott, Z. Shi, D. Johnstone, B. A. Talseth-Palmer, T. Evans, A. D. Spigelman, C. Groombridge, E. A. Milward, J. K. Olynyk, J. Suchy, G. Kurzawski, J. Lubinski Hereditary nonpolyposis colorectal cancer (HNPCC) is characterised by germline mutations in DNA mismatch repair genes, however variation in disease expression suggests there are potential modifying factors. Polymorphisms of the HFE gene, which cause the iron overload disorder hereditary haemochromatosis, have been proposed as potential risk factors for the development of colorectal cancer (CRC). To understand the relationship between HNPCC disease phenotype and polymorphisms of the HFE gene a total of 362 individuals from Australia and Poland with confirmed causative MMR gene mutations were genotyped for the HFE C282Y and H63D polymorphisms. A significantly increased risk of developing CRC was observed for H63D homozygotes when compared to combined wild type homozygotes and heterozygotes (hazard ratio = 2.93, p = 0.007). Evidence for earlier CRC onset was also observed in H63D homozygotes with a median age of onset 6 years earlier than wild type or

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Abstracts heterozygous participants (44 vs. 50 years of age). This effect was significant by all tests used (log-rank test p = 0.026, Wilcoxon p = 0.044, Tarone–Ware p = 0.035). No association was identified for heterozygosity of either polymorphism and limitations on power prevented investigation of C282Y homozygosity or compound C282Y/H63D heterozygosity. In the Australian sample only, women had a significantly reduced risk of developing CRC when compared to men (hazard ratio = 0.58, p = 0.012) independent of HFE genotype for either SNP. In conclusion homozygosity for the HFE H63D polymorphism appears to be a genetic modifier of disease expression in HNPCC. Understanding the mechanisms by which HFE interrelates with colorectal malignancies could lead to reduction of disease risk in HNPCC.

Development and validation of a prediction model to estimate gene-specific risk in Lynch syndrome Fay Kastrinos1, Ewout W. Steyerberg2, Judith Balman˜a3, Rowena Mercado4, Sapna Syngal5 1

Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York, USA; 2Erasmus Medical Center, Rotterdam, The Netherlands; 3Hospital Vall d’Hebro´n, Universitat Auto`noma de Barcelona, Spain; 4Dana-Farber Cancer Institute, Boston, Massachusetts, USA; 5Dana-Farber Cancer Institute, Brigham and Women s Hospital, Boston, Massachusetts, USA Background and aims Lynch Syndrome is caused by mutations in the mismatch repair (MMR) system. Our aim was to expand a previous clinical model predicting the likelihood of finding a deleterious gene mutation in at-risk patients incorporating MSH6 prediction and gene-specific risk estimates. Methods We analyzed an unreported cohort of 4538 unrelated probands undergoing clinical genetic testing for MLH1, MSH2 and MSH6 mutations at a commercial laboratory. Personal and family history of cancer, cancer types and ages at diagnosis were compared by gene mutation for probands and their first- and second-degree relatives. A multivariable model using logistic regression was developed to predict the likelihood of finding a MMR gene mutation and provide a risk estimate for each individual gene. Validation was by bootstrap resampling and independent validation in one-third of the cohort. Results Twelve percent (525/4538) of subjects had pathogenic mutations (204 MLH1, 250 MSH2, 71 MSH6). Strong predictors of MLH1 and MSH2 mutations included CRC history in probands (OR: 5.1, 4.5 for MLH1 and MSH2, respectively) and relatives (OR: 3.3 for MLH1 and MSH2), whereas CRC was not as predictive of a MSH6 gene mutation. Endometrial cancer among probands was predictive of MSH2 and MSH6 mutations (OR: 7.2 for both genes) with younger age of diagnosis most notable for a MSH2 mutation. The model discriminated well at external validation for each gene, with area under receiver operating characteristic curve of 0.85 (95% CI 0.80– 0.89) for MLH1, 0.87 (95% CI 0.83–0.92) for MSH2, and 0.80 (95% CI 0.68–0.92) for MSH6. Conclusions From this large cohort of MMR gene mutation carriers, we expanded a clinical prediction model to evaluate an individual’s gene-specific probability of being a mutation carrier in MLH1, MSH2, and MSH6 genes. Our model’s capacity to estimate mutation probability by affected gene offers healthcare professionals ability to convey clinical information in an individualized quantitative way.

Abstracts

Identification of novel genes involved in colorectal cancer predisposition Ramprasath Venkatachalam1, Marjolijn J. L. Ligtenberg1,2, Eveline J. Kamping1, Eveline Hoenselaar1, Marsha Voorendt1, Heike Go¨rgens3, Hans K. Schackert3, Ad Geurts van Kessel1, Nicoline Hoogerbrugge1, Roland P. Kuiper1 Departments of Human Genetics,2Pathology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands; 3Department of Surgical Research, Universita¨tsklinikum Carl Gustav Carus, Technische Universita¨t Dresden, Dresden, Germany 1

Colorectal cancer (CRC) is the second most common cancer in the Western world in terms of both incidence and mortality rate. A positive family history of CRC is observed in about 25% of the cases. High-penetrant germline mutations in APC, MUTYH and the mismatch repair genes MLH1, MSH2, MSH6 and PMS2 account for less than 5% of hereditary cases whereas in the majority of these families the genetic defect is still unknown. In order to identify novel moderate- to high-risk mutations contributing to CRC predisposition we employed genome-wide copy number profiling using high-resolution SNP-based arrayCGH on normal tissue DNA from 32 independent patients with microsatellite-stable CRC without polyposis. All patients were suspected for hereditary CRC because of their young age at diagnosis or their positive family history for CRC. We identified small (100–160 kb) copy number anomalies in five independent families (16%), in all the cases affecting only a single gene. None of the genes had previously been described to be involved in colorectal cancer susceptibility. All genomic lesions were validated with multiplex ligation-dependent probe amplification (MLPA). In four cases we were able to establish that the aberrations were inherited from one of the parents. Two of the genomic lesions were deletions affecting a microRNA gene, illustrating that constitutional defects in these gene expression regulators might be common. Interestingly, at least two of the identified genes could be linked to pathways involved in CRC development. In an ongoing locus-specific validation screen of independent families with suspected familial CRC (currently * 250), we thus far found at least one of the genes to be recurrently affected, which strongly supports its role in CRC predisposition.

A novel cause of Lynch syndrome: heritable somatic methylation of MSH2 due to deletion of the 30 exons of the upstream gene M. J. L. Ligtenberg1,2, R. P. Kuiper1, T. L. Chan3,4, M. Goossens2, K. M. Hebeda2, M. Voorendt1, D. Bodmer1, E. Hoenselaar1, S. J. B. Hendriks-Cornelissen2, H. G. Brunner1, A. Geurts van Kessel1, J. H. J. M. van Krieken2, S. Y. Leung3,4, N. Hoogerbrugge1 1 Department of Human Genetics, 2Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 3Department of Pathology, The University of Hong Kong; 4Department of Pathology, St. Paul’s Hospital, Hong Kong

Lynch syndrome patients are susceptible to colorectal, endometrial and a range of other cancers due to heterozygous inactivating mutations in one of the mismatch repair genes, MLH1, PMS2, MSH2 or MSH6. During routine diagnostics for germline mismatch repair gene mutations, multiple patients with an MSH2-deficient tumor presented an aberrant MLPA result of a probe, which is located 16 kb upstream of MSH2. All these patients carried an heterozygous germline deletion of 4.9 kb encompassing the last exons of EPCAM (formerly

723 known as TACSTD1), a gene directly upstream of MSH2 encoding the epithelial cell adhesion molecule Ep-CAM. Due to the deletion the transcription termination signal is lost and transcription of EPCAM was shown to extend into MSH2. As antisense transcription of CpG islands may lead to methylation, we tested whether the transcription of the MSH2 promoter would lead to methylation of its CpG dinucleotides. Indeed, the MSH2 promoter in cis with the deletion is methylated in Ep-CAM positive, but not in Ep-CAM negative, normal tissues, thus revealing a correlation between transcriptional read-through of the mutated EPCAM allele and epigenetic inactivation of the corresponding MSH2 allele. This mechanism explains the mosaic pattern of epigenetic inactivation of MSH2, that we observed in successive generations (Ligtenberg et al., Nature Genetics, 41: 112–117 (2009)). Because EPCAM is expressed in the target tissues of Lynch syndrome, subjects with a 30 end deletion of EPCAM are offered the standard Lynch syndrome surveillance protocol. To optimize patient care clinical data of subjects with such a deletion that leads to loss of one functional EPCAM allele and mosaic inactivation of MSH2 are being collected.

T cell immune response against frameshift-induced neopeptides in HNPCC Yvette Garbe, Matthias Kloor, Lena Ehret, Susanne Eiermann, Peter Kienle, Hanns-Peter Knaebel Mirjam Tariverdian, Magnus von Knebel Doeberitz Institute of Pathology, University of Heidelberg, Heidelberg, Germany Hereditary non-polyposis colorectal cancer (HNPCC)-associated colorectal cancers (CRCs) display high level microsatellite instability (MSI-H) as a consequence of mismatch repair deficiency. Features of a pronounced immune response are a hallmark of HNPCC-associated CRCs. Previous reports suggested that MSI-induced novel tumorspecific frameshift peptides (FSPs) underly the high immunogenicity of MSI-H CRCs. In a recent study, we could show that FSP-specific peripheral T cells are frequent in patients affected by HNPCC-associated CRCs. However, the role of immune surveillance mechanisms and the clinical significance in HNPCC still remains unknown. To further investigate the efficacy of FSP-specific tumor-infiltrating and peripheral T cells in HNPCC patients with MSI-H CRC, IFN-gELISpot analysis, CD107a mobilization and in vitro killing assays were performed. TiTc isolated from HNPCC-associated CRCs specifically recognized MSI-induced FSPs and showed a strong CD107a degranulation which correlates with tumor cell lysis of HLA-matched MSI-H, but not microsatellite stable (MSS) CRC cells. Moreover, FSP-specific T cell responses were also present in the majority of peripheral blood samples from patients with MSI-H, but not MSS CRCs (p \ 0.001). FSP-specific T cell reactivity was already detectable in the peripheral blood of healthy HNPCC family members with MMR gene germline mutations, in contrast to healthy control individuals without evidence for HNPCC (7/14 FSP differently recognized, p \ 0.02). Our data strongly suggest that FSPs presented by MSI-H CRC cells are effectively recognized by the patient’s immune system. Our data suggest that, in vitro, FSP-specific T cells are capable of recognizing and killing MSI-H CRC cells. These results are compatible with a prominent role of immune surveillance contributing to the clinical course and eventually the limited penetrance of the HNPCC syndrome. These observations are of high relevance for the development of FSP-based vaccination approaches, particularly for the preventive application in HNPCC mutation carriers.

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MTHFR 677 C[T and 1298 A[C polymorphisms and the age of onset of colorectal cancer in HNPCC R. J. Scott, S. G. Reeves, C. Meldrum, C. Groombridge, A. D. Spigelman, J. Suchy, G. Kurzawski, J. Lubinski, P. McElduff University of Newcastle, Division of Genetics, Hunter Area Pathology Service, Newcastle, Australia HNPCC or Lynch Syndrome is associated with an increased risk of developing an epithelial malignancy. There is considerable variability in disease expression observed in this syndrome which is thought to be due to a combination of genetic and environmental factors. Alterations in the kinetics of MTHFR due to the presence of polymorphisms in the MTHFR gene have been associated with an increased risk of CRC. Two common single nucleotide polymorphisms (SNPs) located within the MTHFR gene, 677 C[T and 1298 A[C that alter the function of the encoded protein have been the focus of many studies into CRC risk outside of the context of an inherited predisposition to disease. A total of 417 HNPCC patients were genotyped for the 677 C[T and 1298 A[C SNPs to determine if there exists an association with the age of disease onset of colorectal cancer. Associations in disease risk were further investigated using Kaplan–Meier survival analysis and Cox hazard regression. The average ages of disease diagnosis were found to be different between individuals harbouring either one of the MTHFR polymorphisms. Both Kaplan–Meier and Cox hazard regression analysis revealed a more complex relationship between the two polymorphisms and the age of CRC onset. Kaplan–Meier survival analysis revealed that compound heterozygotes for the two SNPs developed CRC 10 years later than those carrying only the wild type alleles.

Abstracts therefore do not have HNPCC and 479 tested positive for mutations in one of the known genes (hMLH1, hMSH2, hMSH6 and PMS2). 29% of the families submitted for mutation analysis were found to harbour a causative change in one of the 4 genes. Missense mutations of unknown significance were found in 12% of the families and mutations were not identified in 59% of the families tested. Average age of diagnosis of CRC is lower in mutation positive individuals (42 years for hMLH1, 44 years for hMSH2 and 45 years for hMSH6 mutation carriers than in mutation negative individuals 52 years). Of the families with available information on cancer types, there were: 71 probands with a hMLH1 mutation, 84 with a hMSH2 mutation, 17 with a hMSH6 mutation, 3 with a PMS2 mutation, 68 with a missense mutation and 352 mutation negative probands. The incidence of extracolonic cancers was calculated to determine which cancers were over-represented in the Australian HNPCC population. Typically, endometrial cancer was over-represented (78 families with causative changes in one of the MMR genes). Other cancers were also observed at frequencies typical for HNPCC. However, our previous data indicating an over-representation of breast cancer in 95 families was confirmed in our updated dataset of 688 families. This updated dataset can now be used to better define disease penetrance and to accurately assess age specific disease incidence. This knowledge will help in providing accurate cancer risk assessments to patients harbouring causative mutations in MMR genes.

Base excision repair pathway in microsatellite stable tumors from hereditary non-polyposis colorectal cancer Trinidad Caldes, P. Garre1, V. Bricen˜o1, O. Valentin1, R. Xicola2, X. Llor2, M. de la Hoya1, T. Caldes1 Hospital Clinico San Carlos, Madrid, Spain

Hereditary nonpolyposis colorectal cancer in 688 families: mutations, age of diagnosis and cancer incidence Bente A. Talseth-Palmer1,2, Mary McPhillips3, Cliff Meldrum3 Claire Groombridge4, Allan D. Spigelman5 and Rodney J. Scott1,2,3 1 School of Biomedical Sciences, University of Newcastle, NSW, 2308, Australia; 2Hunter Medical Research Institute, John Hunter Hospital, Newcastle, NSW, 2305, Australia; 3Hunter Area Pathology Service, John Hunter Hospital, Newcastle, NSW, 2305, Australia; 4 Hunter Family Cancer Service, Hunter New England Health, NSW 2305, Australia; 5St Vincent’s Hospital Clinical School, Sydney, NSW 2010, Australia

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant inherited cancer syndrome associated with germline mutations in DNA mismatch repair (MMR) genes, with the majority of mutations affecting hMLH1 and hMSH2. The primary function of MMR genes is to eliminate base–base mismatches and insertion– deletion loops which arise as a consequence of DNA polymerase slippage during DNA replication. HNPCC is characterized by earlyonset epithelial cancers, with patients reporting to have an 80% lifetime risk of developing colorectal cancer (CRC), but are also at risk of developing cancer in a variety of other organs that include the endometrium, stomach, ovary, bladder, pancreas and the urinary tract. Disease penetrance estimates have not been accurately assessed and there is currently doubt about the true incidence of disease as well as age dependent probabilities of cancer expression. From 1997 to 2007, 1376 individuals (belonging to 688 families) have been tested for HNPCC at the Hunter Area Pathology Service (HAPS). Of these, 384 tested negative for a predictive test and

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Base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. MYH, OGG1 and MTH1 are members of BER, and MYH germline mutations were recently identified in patients with multiple adenomas and colorectal cancer. A subset of colorectal cancers (CRCs) arises in families that, despite fulfilling clinical criteria for Hereditary NonPolyposis Colorectal Cancer (HNPCC), do not show evidence of a mismatch repair (MMR) deficiency. Aims To identified genetic variants in MYH, OGG1 and MTH1 genes present in HNPCC families without MMR deficiency (HNPCCMSS). Methods A total of 45 DNAs from HNPCC-MSS index cases, were screened for all coding sequences of OGG1 and MTH1 by direct sequencing. S326C and IVS5-15C[G (OGG1) and D142D (MTH1) polymorphisms were also typed in 353 DNAs from control individuals by TaqMan assay technology. G382D and Y165D at the MYH gen were studied previously (Peterlongo et al. 2006). Results Three new variants were detected R46Q, A95A and G308G in OGG1 gene and two in MTH1, the V106M and D122D that was detected in two families. The frequencies of the SNPs, S326C and IVS5-15C[G in OGG1 and D142D in MTH1 were 38, 35 and 56% respectively in HNPCC-MSS index cases and 40, 35 and 36% in the control population. The reported variant R154H in OGG1 was not observed in Spanish population. A statistical analysis (two-sided X2 or Fisher test) was performed comparing frequencies between case and controls. The two variants in the OGG1 gene were not significantly different but the variant D142D in MTH1 gene was found to be significantly different between HNPCC-MSS cases and controls (OR: 12.37, p = 0.016). The genetic variants R46Q, A95A in the OGG1 gene, segregate with colorectal cancer in the family by the contrary

Abstracts variants D122D and V106M in MTH1 gene didn’t segregate. We couldn’t established the segregation for the variant G308G. Conclusions This study suggests that genetic variants in OGG1 and MTH1 may explain by different ways the susceptibility to CRC in HNPCC-MSS patients. Further studies are required to confirm the reported associations.

An important role for RNA based mutation scanning in the mismatch repair gene PMS2 H. M. van derKlift, C. M. Tops2, E. C. Bik2, M. W. Boogaard2, H. Morreau3, F. Hes2, P. Devilee1,3, J. T. Wijnen1,2 Heterozygous mutations in the Mismatch Repair gene PMS2 are involved in Lynch Syndrome while bi-allelic mutations are found in Constitutional Mismatch Repair Deficiency Syndrome patients. Mutation scanning in PMS2 is complicated by the presence of many pseudogenes in the Human Genome, with 14 copies involving PMS2 exon 1–5 on chromosome 7q and one copy (PMS2CL) of the 30 end (exon 9 and 11–15) about 700 kb centromeric from PMS2 on 7p. Recently it has been recognized that frequent recombination, either gene conversion or cross-over, between PMS2 and PMS2CL has occurred and is still ongoing. This makes mutation detection based on PMS2 specific nucleotides highly unreliable, especially, as we have shown, in the region encompassing exon 12–30 UTR. Here we propose an alternative strategy for mutation detection in PMS2 in which RT–PCR plays an important role either as initial scanning or as confirmation of mutations found in genomic DNA. As a reliable source of RNA we use short term cultured lymphocytes in which Nonsense Mediated RNA decay can be inhibited. The cDNA is amplified in two overlapping amplicons spanning the entire PMS2 gene, thereby circumventing pseudogene sequences and gene conversion events. With this strategy we found in 26 Lynch Syndrome families 19 different pathogenic mutations of which 15 were detectable with RT–PCR, the other 4 being large deletions that cross one or both borders of the gene. Two out of 19 mutations were missed with MLPA, exon-by-exon or Long Range PCR and could be detected with RT– PCR and Southern Blot only: a genomic deletion of exon 14 and a large 2 kb insertion of an SVA repeat in intron 7 resulting in an mRNA with a spliced-in fragment of 71 bp. We conclude that in our experience RT–PCR represents an effective tool for mutation scanning in PMS2.

Early detection of upper gastrointestinal cancers by endoscopy in patients with Lynch syndrome Masami Arai, Toshiharu Yamaguchi, Masatoshi Oya, Junko Fujisaki, Masahiro Igarashi, Kensuke Kuraoka, Hiroshi Takahashi, Tetsuichiro Muto The Cancer Institute Hospital of JFCR, Tokyo, Japan Background Colonoscopy is considered useful for cancer surveillance in patients with Lynch syndrome, but the diagnostic role of upper gastrointestinal endoscopy remains unclear. At our hospital, patients with Lynch syndrome are recommended to undergo annual screening by upper gastrointestinal endoscopy and colonoscopy (plus gynecological examination in women). We summarize the outcomes of surveillance by upper gastrointestinal endoscopy. Patients profiles and methods 41 patients (17 males, 24 females, average age at entry 49.3 years) with Lynch syndrome satisfying the Amsterdam criteria II or with confirmed pathogenic mutations (including 1 epimutation in the promoter region of hMLH1) in either

725 MMR gene were observed from 2005 to 2006. Upper gastrointestinal endoscopy was performed once every 1–2 years. Tumor incidence and outcomes were studied. We used the incidence of tumor detection on cancer screening by upper gastrointestinal endoscopy in our hospital as a control. Results Among 41 patients, 5 had a history of cancer (4 gastric cancer, 1 cancer of the ampulla of Vater). A total of 107 upper gastrointestinal examinations were performed within 4 years. 8 lesions (4 gastric cancers, 2 duodenal cancers, 1 SMT of the duodenum, and 1 severe dysplasia of the esophagus; incidence 7.5%) were detected. All tumors could be detected at curative stage, and 3 were resected endoscopically. The control tumor incidence was 0.82%. Conclusion Regular upper gastrointestinal endoscopy can facilitate early cancer detection in Lynch syndrome. The 2nd portion of the duodenum should be examined to avoid missing duodenal lesions. Adenomas of the stomach and duodenum as well as the colorectum may be precancerous in Lynch syndrome. Because some cancers develop rapidly, upper gastrointestinal endoscopy should be performed every 1–2 years in patients with Lynch syndrome.

Primary peritoneal cancer following bilateral salpingooophorectomy in two patients with Lynch syndrome Kathleen M. Schmeler, Molly S. Daniels, Pamela T. Soliman, Russell R. Broaddus, Michael T. Deavers, Thuy M. Vu, George J. Chang, Karen H. Lu MD Anderson Cancer Center, Houston, Texas, USA Background Women with Lynch syndrome have a 40–60% lifetime risk of endometrial cancer and a 7–12% lifetime risk of ovarian cancer. Risk-reducing surgery including hysterectomy and bilateral salpingo-oophorectomy is currently recommended once childbearing is complete. Unlike women with BRCA mutations, there have been no reported cases of primary peritoneal cancer following bilateral salpingo-oophorectomy (BSO) in women with Lynch syndrome. The objective of this study was to describe two cases of primary peritoneal cancer following BSO in women with Lynch syndrome. Cases The first patient was a 44 year-old woman who underwent hysterectomy with BSO for benign disease. She presented 12 years later with a pelvic mass and was diagnosed with a high-grade serous primary peritoneal cancer. Genetic testing showed a mutation in the MSH2 DNA mismatch repair gene. The second case was 58 year-old woman who had a hysterectomy and BSO for endometrial cancer. She developed a high-grade serous primary peritoneal cancer 8 years later and was found to have a mutation in the PMS2 DNA mismatch repair gene. Conclusion Women with Lynch syndrome should be counseled that they are at risk for developing primary peritoneal cancer despite undergoing gynecologic cancer risk-reducing surgery. The magnitude of this risk remains to be determined.

Clinical and genetic characteristics of Japanese HNPCC Yoichi Furukawa, Teruhiko Yoshida, Yusuke Nakamura, Yoshihiro Moriya Institute of Medical Science, The University of Tokyo, Tokyo, Japan To uncover the clinical and genetic characteristics of Japanese HNPCC, we performed a collaborative study of HNPCC registry and

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726 genetic testing project in Japan. Patients who fulfilled with modified Amsterdam criteria that included gastric cancer as an HNPCC-related tumor were enrolled in this study. A total of 101 patients were subjected to genetic testing after the informed consent was obtained. We analyzed genetic alterations in MSH2, MLH1, and MSH6, three major responsible genes by PCR and direct sequencing and MLPA. As a result, we identified 56 pathogenic mutations in the 101 cases. Among the 56 alterations, 31, 23, and 2 mutations were found in MLH1, MSH2, and MSH6, respectively. Although 47 mutations were detected in 70 patients who fulfilled with revised Amsterdam criteria, the remaining 9 patients were found in 31 subjects who might not be enrolled if gastric cancer was not taken as a related tumor. This result suggest that inclusion of gastric cancer increases the sensitivity of diagnosis but decreases specificity, leaving further studies for the development of systematic screening system. Expectedly, we observed that early onset and multiple HNPCCrelated tumors were associated with mutation. In addition, we found six subjects who harbored the same deletion of MLH1 exon 5. Further information on their family trees has uncovered that two of the five probands were relatives in a large pedigree. These data may be useful for the development of screening system to Japanese HNPCC.

Identification of the first Spanish founder mutations in the MLH1 gene M. Pineda1,*, E. Borra`s1,*, I. Blanco2, G. Llort3, T. Calde´s4, M. Urioste5, C. Martı´nez-Bouzas6, B. Gran˜a7, J. Balman˜a8, A. Torres9, T. Ramo´n y Cajal10, J. Sanz9,11, M. Dura´n11, S. Castellvı´-Bel12, S. Gonza´lez1, C. La´zaro1, G. Capella´1 1 Laboratori de Recerca Translacional, Institut Catala` d’Oncologia, IDIBELL, Hospitalet de Llobregat, Spain; 2Unitat de Consell Gene`tic, Institut Catala` d’Oncologia, IDIBELL, Hospital Duran i Reynals, Hospitalet de Llobregat, Spain; 3Unitat de Consell Gene`tic, Institut Catala` d’Oncologia, Hospital Germans Trias i Pujol, Badalona, Spain; 4 Laboratorio de Oncologı´a Molecular, Hospital Clı´nico Sant Carlos, Madrid, Spain; 5Departamento de Gene´tica Humana, Centro Nacional de Investigaciones Oncolo´gicas, Madrid, Spain; 6Laboratorio de Gene´tica Molecular, Hospital de Cruces, Bizkaia, Spain; 7Unitat d’Avaluacio´ del Risc de Ca`ncer i Consell Gene`tic, Institut Catala` d’Oncologia, Hospital Universitari Josep Trueta, Girona, Spain; 8 Servei d’Oncologia Me`dica, Hospital Vall d Hebron, Barcelona, Spain; 9Unitat de Consell Gene`tic, Hospital Universitari Sant Joan, Reus, Spain; 10Servei d’Oncologia Me`dica, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 11Institut d’Oncologia Corachan-Centre Me`dic, Barcelona, Spain; 12Servei de Gastroenterologia, Institut de Malalties Digestives i Metabo`liques, Hospital Clı´nic, Barcelona, Spain; 13Instituto de Biologı´a y Gene´tica Molecular, Valladolid, Spain

Lynch syndrome is an autosomal disorder caused by germline mutations in any of four DNA mismatch repair genes: MLH1, MSH2 MSH6 or PMS2. Clinically it is characterized by the development of early-onset colorectal cancers and an increased risk of other cancers. Lynch syndrome tumors are associated with microsatellite instability and loss of mismatch repair proteins expression. In some populations, founder mutations appear to explain a substantial fraction of Lynch syndrome cases. We report here the identification of two frequently occurring germline substitutions of the MLH1 gene in Spanish families: c.306+5G[A and C.1865T[A (p.Leu622His). The c.306+5G[A mutation was identified in 17 unrelated families from the north-east

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Abstracts of Spain, whereas the C.1865T[A mutation was found in 12 unrelated families coming from the region of Jae´n, at the south of Spain. Both mutations segregate with the disease, and tumors show microsatellite instability and loss of expression of MLH1 protein. We demonstrate that c.306+5G[A is a pathogenic mutation affecting the mRNA processing. Experimental approaches are being performed to elucidate the pathogenicity of the C.1865T[A (p.Leu622His) mutation. To determine possible founder effects on the identified Spanish MLH1 mutations, haplotype analysis was performed using three MLH1 SNPs and seven microsatellite markers, spanning 12 Mb on chromosome 3. The haplotype analysis demonstrated two common haplotypes associated to c.306+5G[A and C.1865T[A MLH1 mutations in the families. The estimation of the age of the c.306+5G[A and C.1865T[A mutations was of 36–70 and 5–65 generations ago, respectively. Mutations c.306+5G[A and C.1865T[A (p.Leu622His) of MLH1 gene are the first founder MLH1 mutations identified in Spain. This finding should be taken into consideration when designing molecular diagnostic strategies in the Spanish Lynch syndrome families.

Assessing BRAF V600E usefulness in the analytical algorithm of Lynch syndrome M. Gausachs1, M. Pineda1, M. Mene´ndez1, C. La´zaro1, I. Blanco2, S. Gonza´lez1, G. Capella´1 1 Laboratori de Recerca Translacional, 2Unitat de Consell Gene`tic, Institut Catala` d’Oncologia, IDIBELL, l’Hospitalet de Llobregat, Spain

BRAF is a kinase-encoding gene from the RAS/RAF/MAPK pathway. The hotspot BRAF V600E is present in about 10–15% of sporadic colorectal cancers (CRC) and strongly associates with microsatellite instability (MSI). In MSI tumors with loss of MLH1 expression BRAF mutation detection is used in the selection of candidates to have Lynch syndrome, since V600E is believed to be diagnostic of sporadic tumors except for PMS2 families. The aim of this study was to assess the usefulness of BRAF V600E in the analytical algorithm of tumors from candidates to mismatch repair (MMR) germline screening. A set of 157 tumors from individuals with family history of CRC attended at our Cancer Genetic Counseling Unit [109 MSI and 48 MSS (microsatellite stable)] were included. SNuPE test was optimized to detect BRAF V600E in DNA from paraffin-embedded tumors with an analytical sensitivity of 1:200. Assay was initially validated in a set of selected sporadic CRC [24 MSI and 49 MSS]. In these tumors BRAF mutation was identified in 5 of 24 (20%) MSI and 2 of 49 (4%) MSS tumors. BRAF mutation was assayed in 15 of 157 (9.5%) familial tumors [12 of 109 (11%) MSI; 3 of 48 (6%) MSS]. Restricting the analysis to MSI tumors, MMR germline mutations were found in 63 (43 MLH1 and 18 MSH2 and 2 MSH2) of the 109 MSI CRC. One BRAF V600E mutation was detected in a tumor from a germline MLH1 carrier and the remaining 11 mutations were found in the cases without detectable germline mutation. BRAF sensitivity was assessed in 51 cases with loss of MLH1 protein expression. All BRAF mutations associated with loss of MLH1 expression, showing a sensitivity of 39% and a specificity of 96% for depiction of sporadic tumors. This is the first report of the coexistence of somatic BRAF V600E and MLH1 germline mutation. Our findings question its role in the diagnostic algorithm of Lynch syndrome.

Abstracts

Diagnostic microsatellite instability testing in Lynch syndrome: different tumours, different markers Kenneth Porter, Nathan Curtis, Lisa Happerfield, Mark J. Arends, Ian M. Frayling University of Southampton School of Medicine, Southampton General Hospital, Southampton, UK Microsatellite instability (MSI) testing plays a key role in the molecular diagnosis of Lynch Syndrome (LS), together with mismatch repair protein expression by immunohistochemistry, and germline mutation detection. Because different microsatellite markers have different sensitivities to detect MSI in different tumours, we have examined marker sensitivities in a diagnostic clinical setting. MSI results from 285 tumours, from affected members of families attending two cancer genetics clinics, who either met the Amsterdam criteria or had a strong clinical suspicion of LS, were audited. A panel of 10 microsatellites was used: 3 mono- and 7 dinucleotide repeats: BAT25, BAT26, BAT40, D5S346, ACTC, D17S250, D13S153, D5S406, D5S107 & D2S123. A diagnosis of LS-associated MSI was made if [29% of markers were unstable, and 5 or more markers gave informative results. LS was subsequently diagnosed in 55/285 families, and the individual and combined sensitivities of the markers (mononucleotides alone; dinucleotides alone; all combined) in the LS and non-LS groups were thus determined, in colorectal and non-colorectal tumours. Because most (76%) of the tumours were colorectal in origin, the size of the non-colorectal group did not justify sub-division into endometrial and other non-colorectal tumours. In those LS-associated colorectal cancers in which all three mononucleotide markers worked, MSI could always have been diagnosed on the basis of the mononucleotides alone, i.e. the dinucleotides did not add extra information. However, in non-colorectal tumours the mononucleotide markers were not, in themselves, always sufficient to make a diagnosis of MSI. We conclude that when testing colorectal tumours the marker set could be rationalized to 5 mononucleotides, reducing costs without compromising diagnostic efficiency. Whereas, when testing noncolorectal tumours a set of both mono- and dinucleotide repeats remains necessary. Further work is necessary to determine the optimum marker sets to diagnose LS-associated MSI in the various types of LS-associated tumours.

Methylation specific multiplex ligation-dependent probe amplification in the analysis of MLH1 promoter hypermethylation Lars Henrik Jensen, Jan Lindebjerg, Lene Byriel, Steen Kølvraa, Dorthe Cru¨ger Danish Colorectal Cancer Group South, University of Southern Denmark and Vejle Hospital, Vejle, Denmark Background Promoter hypermethylation can lead to transcriptional silencing of certain genes. MLH1 is one of the main genes in the postreplicative mismatch repair, and promoter hypermethylation of this gene is found in sporadic mismatch deficient colorectal cancer. Lynch Syndrome is caused by germ-line mutations in for example MLH1. Thus, promoter hypermethylation of MLH1 can separate sporadic mismatch deficient tumours from possible hereditary cases. The aim of this study was to evaluate methylation specific multiplex ligationdependent probe amplification (MS-MLPA) in the analysis of MLH1 promoter hypermethylation and to determine the importance of the different regions of the promoter.

727 Methods Two-hundred-thirty-one patients with primary colorectal cancer were included in the study. Mismatch repair deficiency has previously been determined by microsatellite analysis and immunohistochemistry. A commercially available kit was used for MS-MLPA (ME011, MRC-Holland, Amsterdam, The Netherlands). Methylation was determined in four different regions of the MLH1 promoter (A through D) and in intron 1. Results Ninety tumours (40.0%) were in some degree methylated in one or more of the regions A–D and intron 1. All five regions were methylated in 32 tumours (13.9%), only A in 28 (12.1%), only D in 22 (9.5%), A + B in 3 (1.3%), A + B + C in 2 (0.9%), A + D in 2 (0.9%), and A + C + D in 1 (0.4%). In all 32 tumours with methylation in five regions, immunohistochemistry was negative for MLH1 and they had a high degree of microsatellite instability. Isolated methylation of region A, region D, or the combinations of A, B, C, and D was not associated with microsatellite instability or loss of MLH1 protein. Methylation in a region of intron 1 was strongest correlated with loss of protein expression and microsatellite instability. Conclusion MS-MLPA is an excellent tool for analysis of MLH1 promoter hypermethylation. The single most important region for methylation analysis may be in intron 1.

Recommendation for the current endoscopic technique in the surveillance of HNPCC gene carriers R. Hu¨neburg1, T. Sauerbruch1, C. Lamberti1 1

Department of Internal Medicine I

Introduction Due to the high risk of colorectal cancer in carriers of hereditary nonpolyposis colorectal cancer (HNPCC), and due to the shortened adenoma–carcinoma sequence, good colonoscopic surveillance is obligatory in these patients. Up to 25% of small and flat adenomas are missed by standard colonoscopy (SC). Therefore an endoscopic approach is needed which is able to detect small adenomas. Methods criteria:

All endoscopic studies that met the following inclusion

1. Screening of HNPCC patients 2. Comparison of different endoscopic techniques. Results To date, there are 4 studies available, 3 published, 1 in progress. In two studies, SC was compared to chromocolonoscopy (CC), in one SC was compared to narrow-band-imaging colonoscopy (NBI), in the last one, a two-armed study, SC was compared to CC as well as CC to NBI. A total of 105 patients were screened by comparing SC to CC, 62 patients by comparing SC to NBI and 62 patients by comparing CC to NBI. In all CC studies indigo carmine dye was used. The percentage of HNPCC gene mutation carriers differed a lot (84/50%/ 13/90%) as well as the rate of patients who had a colonoscopy been performed before inclusion into the study (32/56%/100/92%). Three studies showed a significant increase of adenoma detection while using CC (SC 25 adenomas/CC 56). One study showed a significant increase while using NBI (SC 25/NBI 21). One study revealed that CC detected significantly more adenomas than NBI (NBI 10/CC 38). In all four studies no randomized controlled trial was performed. Conclusion CC is superior to standard and NBI colonoscopy in detection of polypoid lesions in patients with HNPCC. Further studies are needed, preferably randomized controlled trials.

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Colorectal cancers show distinct mutational spectra in members of the WNT pathway according to anatomical localization and geneticinstability Cristina Albuquerque5, Ce´lia Baltazar1, Bruno Filipe1, Filipa Penha1, Teresa Pereira2, Ron Smits3, Marı´lia Cravo4, Pedro Lage4, Paulo Fidalgo4, Isabel Veiga5, Jose´ Silva Ramos6, Isabel Fonseca2, Carlos Nobre Leita˜o4, Riccardo Fodde3 Centro de Investigac¸a˜o de Patobiologia Molecular (CIPM), 2Servic¸o de Anatomia Patolo´gica e, 4Servic¸o de Gastrenterologia, Instituto Portugueˆs de Oncologia de Lisboa Francisco Gentil, EPE, Lisboa, Portugal; 3Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands; 5Servic¸o de Gene´tica, Instituto Portugueˆs de Oncologia do Porto Francisco Gentil, EPE, Porto, Portugal; 6Servic¸o de Gastrenterologia, Hospital dos Capuchos, Lisboa, Portugal 1

Constitutive activation of the canonical WNT signal transduction pathway plays a rate-limiting role in colorectal cancer onset and progression. However, it is yet unclear whether the mutation spectra in WNT genes vary depending on the sporadic or hereditary nature of the tumors, microsatellite instability (MSI) status, and localization along the gastro-intestinal tract. To clarify this, we have analyzed the APC (adenomatous polyposis coli), CTNNB1 (beta-catenin) and AXIN2 (conductin) genes for somatic mutations in 52 colorectal tumors and performed a meta-analysis of previous studies. In our cohort, mutations were detected in the majority of sporadic microsatellite stable (MSS; 80%) and MSI-High (MSI-H; 60%) carcinomas and HNPCC (hereditary non-polyposis colorectal cancer) colorectal tumors (74%). Notably, significant differences were found in mutation spectra among the different groups. Truncating APC mutations encompassing at least two beta-catenin down regulating motifs (20 a.a. repeats) were significantly more frequent among sporadic MSI-H and HNPCC cases than in MSS tumors (p = 0.027). In the latter, APC mutations encompassing only one or none of the 20 a.a. repeats were prevalent. Also, beta-catenin mutations were almost exclusively detected in MSI-H tumors, being more frequent in HNPCC than in sporadic lesions (40% vs. 10%). With respect to anatomical localization, APC mutations encompassing at least two 20 a.a. repeats were also significantly more frequent in proximal tumors than in tumors located in the descending/sigmoid colon (p = 0.047). The metaanalysis confirmed our results, leading to more significant data. In conclusion, the observed spectra of WNT gene mutations in HNPCC and sporadic MSI-H tumors are likely to result from selection of specific levels of beta-catenin signaling, optimal for tumor formation in specific anatomical locations and genetic backgrounds and may contribute for distinct clinical features and prognosis.

Familial colorectal cancer type X tumours present frequently chromosomal instability, APC allelic loss and specific KRAS mutations Ineˆs Francisco1, C. Albuquerque1, H. Belo1, B. Filipe1, I. Vitoriano1, J. Dinis1, P. Lage2, I. Claro2, S. Ferreira2, P. Rodrigues2, C. Nobre Leita˜o2 Centro de Investigac¸a˜o de Patobiologia Molecular (CIPM), 2Servic¸o de Gastroenterologia, Instituto Portugueˆs de Oncologia de Lisboa Francisco Gentil, EPE (IPOLFG, EPE), Portugal

1

In a fraction of families fulfilling the Amsterdam Criteria (AC) for hereditary non-polyposis colorectal cancer, DNA mismatch repair gene mutations are not found and colorectal cancers (CRC) are mostly

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Abstracts localized in the distal colon and frequently do not present microsatellite instability (MSI). These families have been designated as familial colorectal cancer type X (FCCX). We aimed at characterize a group of FCCX families for both clinical and molecular features. Twelve patients with CRC and/or adenomas, from 10 FCCX families were included and 8 CRC and 4 adenomas were analyzed. Chromosomal instability (CIN) was evaluated in the 8 CRC by the analysis of loss of heterozygosity (LOH) at 5q, 17p and 18q. APC (point mutation/LOH) and KRAS mutations were analyzed in the 12 tumours. When compared with Lynch syndrome, FCCX families presented a lower frequency of CRC and extra-colonic tumours from the Lynch spectrum but showed a higher frequency of adenomas. Among CRC, 5/8 were classified as presenting CIN. LOH of APC was detected in 7/11 tumours, including all CIN carcinomas. APC and KRAS mutations were identified in 4/11 and 6/12 tumours, respectively, mostly in tumours presenting CIN or LOH of APC. Considering the high frequency of LOH of APC and the higher prevalence of adenomas in FCCX, we searched for APC germline mutations and found the E1317Q mutation in one family. In conclusion, FCCX families present specific clinical and molecular features and may be divided in two distinct groups: one that present CIN tumours, with frequent LOH of APC and a specific KRAS mutation signature and a second group where tumours do not present CIN and where these alterations may be less frequent. The identification of the E1317Q mutation in one of the families from the former group suggests that some APC missense mutations may contribute for these cases.

Analysis of colonic and extra-colonic tumors in Chilean patients with Lynch syndrome ´ lvarez1, E. Pinto1, T. Claudio Heine1, M. De la Fuente1, K. A U. Kronberg1, A. M. Wielandt1, P. Orellana1,2, P. Carvallo2, F. Lo´pez-Ko¨stner1 1 Colorectal Surgery Unit, Oncology and Molecular Genetics Laboratory, Clı´nica las Condes, Santiago, Chile; 2Human Molecular Genetics Laboratory, Pontificia Universidad Cato´lica de Chile, Santiago, Chile

Background The Lynch Syndrome or Hereditary Non Polyposis Colorectal Cancer (HNPCC) represents up to 5% of all cases of colorectal cancer. Theses patients have extracolonic tumors also in various organs such as endometrial, small intestine, ureter and renal pelvis. All this tumors have been included in different clinical criteria for patient selection and diagnosis. Objective To describe and characterize colonic and extra-colonic tumors in Chilean families with Lynch syndrome. Materials and methods From hereditary intestinal tumor registry, we selected families that meet the clinical criteria of Amsterdam or Bethesda (with microsatellite instability). In all families, we evaluated the presence of colorectal and extracolonic tumors. Results We identified 14 families (83 patients) who meet the Amsterdam criteria and 7 families (23 patients) who meet the Bethesda criteria (with microsatellital instability). A total of 143 tumors were identified, 82 colorectal (57%) and 64 extracolonic (43%). The distribution of colorectal tumors showed a higher frequency of right colon (54%) followed by left colon (22%) and rectum (24%). Regarding extracolonic tumors, the most frequent was endometrial cancer (13%) followed by gastric cancer (12%) and breast cancer (10%). We also note that other cancers appeared before colorectal cancer, like endometrial cancer (75%) and breast cancer (67%). Twenty-eight patients had more than one tumor (26%) and 7 presents more than 3 tumors (7%) with a higher proportion in females (71%).

Abstracts Conclusions The results confirm the high incidence of colonic and extracolonic tumors in Chilean families with Lynch Syndrome. It also highlights the high incidence of gastric tumors, which are not considered within the Amsterdam criteria II, and breast tumors, which are not included within the Bethesda criteria.

Mutation analysis of the MMR genes in patients with suspicion of HNPCC from Castilla-Leon (Spain: Identification of a novel and recurrent deletion in MSH2) L. Pe´rez-Cabornero, E. Velasco, M. Infante, D. J. Sanz, A. Acedo, L. Hernandez, N. Martinez, E. Lastra, C. miner, M. Dura´n Instituto de Biologı´a y Gene´tica Molecular (IBGM), Valladolid, Spain Introduction Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in DNA mismatch repair genes (MMR genes), predominantly MLH1, MSH2 and MSH6. Defects in the MSH2 gene may account for about 40% of HNPCC cases. The aim of the study is to describe the mutational spectrum in 140 nonrelated families with suspicion of HNPCC from Catilla-Leon. Methods Point mutations in the MLH1, MSH2 and MSH6 genes were screened by heteroduplex analysis by capillary array electrophoresis (HA-CAE) followed by direct sequencing. Genomic rearrangements were assessed by multiplex ligation-dependent probe amplification (MLPA) and the positive results were confirmed by RT–PCR. Results Fourty-three different MMR gene alterations were detected. Among these, 9 mutations in 13 families are considered pathogenic and an additional 9 variants from 16 families are considered to represent variants of unknown pathogenicity. About 40% (7/18) of the pathogenic and UV mutations were novel: one complex frameshift mutation (MLH1 c.2284-2287delACCT; 2284-2313ins30), one splicing mutation (MSH2 C.1661G[A), three unknown missense variants (MLH1 C.1574G[A; MSH6: c.4003 A[C, c.3425C[T and c.100g[C) and a large genomic deletion in MSH2 removing exon 4–8. The different MMR genes contribute to 61% (MSH2), 31% (MLH1), and 8% (MSH6) of the mutations. About 50% of pathogenic mutations in MSH2 are large genomic deletions. Most of them were present in a single family. Interestingly, we found a novel and recurrent deletion exon 4–8 in MSH2, previously recognize, was causative in 31% (4/13) of the MMR mutant families. Conclusion These data indicate that: (1) the majority of germline mutations in HNPCC families in Castilla-Leon affect the MSH2 locus; (2) In our population we have identified a high proportion of novel mutation; (3) The identification of a new recurrent deletion is relevant to the molecular diagnosis of HNPCC in this region of Spain.

In silico analyses and experimental validation of MLH1 and MSH2 splice site and missense mutations Beate Betz1, Stephan Theiss2, Murat Aktas1,3, Carolin Konermann4, Timm O. Goecke1, Gabriela Mo¨slein5, Heiner Schaal4, Brigitte Royer-Pokora1 1

Institut fuer Humangenetik, Universitaetsklinikum Duesseldorf, RESULT GmbH, Duesseldorf, 3Institut fuer Transplantationsdiagnostik und Zelltherapeutika, Universitaetsklinikum Duesseldorf, 4 Institut fuer Virologie, Universitaetsklinikum Duesseldorf 5 Klinik fuer Chirurgie, St Josefs-Hospital Bochum-Linden 2

Hereditary non-polyposis colorectal cancer is caused by inactivating mutations of DNA mismatch repair genes, mainly MLH1 and MSH2.

729 Identification and characterization of those mutations is essential for genetic counseling. However the clinical effect of missense and splice site mutations remains unclear, until the pathogenic effect can be demonstrated. Missense mutations, lying in cis-acting sequences of splicing regulatory proteins (splicing enhancers/silencers) can cause aberrant splicing. We assessed ten splice site mutations and nine missense mutations outside splice sites in MLH1 and MSH2 and experimentally analyzed their effect on aberrant splicing at the RNA level. We additionally tested and compared the reliability of several web-based programs for the prediction of splicing outcome for these mutations. We used four well-established algorithms for computational scoring of 50 and/or 30 splice sites (Shapiro & Senapathy, MaxEnt score, HBond score, SD-score). For the prediction of putative exonic splicing enhancers/ silencers, we tested five common web-based resources representing different algorithms (ESE-Finder 3.0, Rescue-ESE, FAS-ESS, PESX, ESR-Search). Our study revealed current limits of the applied computational tools in the diagnostics of possibly pathogenic splicing mutations. Seven splice site mutations caused aberrant splicing: in six cases exon skipping and in one case activation of a cryptic splice site was observed. One 50 ss mutation caused the creation of a new overlapping 50 ss. Splice site strength assessments showed a high consistency both among the four different algorithms examined and compared to the RNA-based data. However, some programs exhibited exemplary weakness in specific positions and the type of aberrant splicing (exon skipping or activation of a cryptic splice site) can not yet be predicted by any of the examined algorithms. None of the experimentally tested missense mutations in the MLH1 and MSH2 genes actually influenced splicing in our RNAassay. In contrast, we found considerable divergence in the softwarepredicted splicing regulatory elements.

Integrated evaluation of unclassified variants in the mismatch repair genes Pastrello Chiara, Pin Elisa, Agostini Marco, Barana Daniela, Fornasarig Mara, Tibiletti Maria Grazia, Quaia Michele, Ponz de Leon Maurizio, Pucciarelli Salvatore Viel Alessandra Centro di Riferimento Oncologico, Aviano (PN), Italy Genetic testing of mismatch repair genes for the diagnosis of Lynch Syndrome is routinely carried out in our laboratory. The result of a 14 year-long screening revealed, beside the most informative nonsense, frameshift and large-deletion mutations, also several missense, silent and deep intronic variants with uncertain pathogenic meaning (unclassified variants). To clarify the role of these variants, we performed an integrated analysis evaluating: Correlation to clinical data Cosegregation with disease Presence of tumour molecular markers (MSI, IHC, LOH) Predictive value of multiple in silico analyses cDNA splicing alterations cDNA allelic imbalance compared to gDNA Presence of the variant in healthy controls An arbitrary ‘‘pathogenicity value’’ was assigned to every parameter and then summed to obtain a final score for each variant. These sums were subdivided in 3 value ranges: probably pathogenic, probably non pathogenic and intermediate.We analyzed 42 variants: 15 MLH1, 12 MSH2, 5 MSH6 and 10 PMS2; 26 were missense, 8 were silent and 8 were deep intronic.

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730 p.Pro349Arg, p.Met688Arg and p.Cys697Arg for MSH2, p.Gly67Arg, p.Thr82Ala, p.Arg265His and p.Lys618Ala for MLH1 and p.Ser46Ile for PMS2 resulted probably pathogenic. On the contrary, we had evidence of neutrality for a large number of variants, among which the common p.Gly322Asp and c.1077-10 T[C for MSH2, and p.Ser406Asn for MLH1 that resulted probably non pathogenic. This simple integrated analysis of the unclassified variants helped to clarify their pathogenic role, supporting a better estimation of the risk and more appropriate carrier surveillance.

Practical usefulness and problem of immunohistochemistry and microsatellite instability test for recruitment of hereditary nonpolyposis colorectal carcinoma patients Noriko Motoi, Yurika Mitsuhashi, Tomoyo Kakita, Mayumi Ogawa, Yuri Sato, Masami Arai, Yo Kato Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan Background It is important for diagnosis of HNPCC to recruit candidates at general hospital. Immunohistochemistry (IHC) and microsatellite instability (MSI) test are relatively common technique, so it might be good means of HNPCC-screening. We report our screening system and the summary of data. Materials and methods Since 2005, 625 specimens were examined by IHC and MSI. IHC includes three major DNA mismatch repair (MMR) system (MLH1, MSH2 and MSH6). MSI test includes at least five of seven major loci (NCI panel, TGF beta-RII and BAT40). Germline mutation of MMR genes were examined among the IHC/ MSI screened HNPCC-candidates who accepted informed consents. Results Any loss of MMR expression by IHC was revealed in 54 samples (8.6%). 18 showed vague results whether normal or lost. Loss of MLH1 is the most (n = 30), followed by MSH2 (19) and MSH6 (3). Eight samples showed double loss of MSH2 and MSH6. MSI were done in 604 cases. Three samples were omitted due to technical problems. 18 samples were examined by IHC alone. MSI was high in 55 samples (9.1%), vague in 9 (1.5%) and low in 540. The results between IHC and MSI showed good correlation (p \ 0.001). The incidence of germline mutation was 79% of IHC-lost/MSI-high cases (26/33). The most common mutation was found in MSH2 (n = 16), followed by MLH1 (7) and MSH6 (3). Discordant between IHC and genetic test were found in three cases. No mutation was detected in 7 IHC-lost/MSI-high cases (21%), especially in IHC-MLH1-lost cases. Discussion This data might be the largest series of HNPCC screening of single institution in Japan. Sensitivity of IHC/MSI was very high, although some cases showed non-specific IHC-reactivity. The latter might be affected by epigenetic status or other genetic abnormalities. Further improvement of screening methods to exclude such non-specificity would be expected.

A homozygote splice site PMS2 mutation as cause of TURCOT syndrome gives rise to two different abnormal transcripts Wenche Sjursen, Inga Bjørnevoll, Helge Myrvold, Lars F. Engebretsen, Tore Halvorsen Department of Pathology and Medical Genetics, St Olavs University Hospital, Trondheim, Norway

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Abstracts Turcot syndrome is a rare, inherited disease predisposing to tumours in the central nerve system and in the colorectal system. We here describe a family in which two siblings have Turcot syndrome, and where we have identified the underlying molecular genotype. The proband is still alive at the age of 43. The patient was operated at the age of 10 by brain tumour and at the age of 16 by colorectal cancer. She has since been treated for multiple cancers (gastrointestinal, endometrial, basal cell carcinomas), and removal of adenomatous polyps at several occasions. The aim of this work was to investigate if there was any specific genotype that explains the remarkable clinical history. DNA and RNA mutation analysis were performed for genes involved in polyposis and mismatch repair. cDNA analysis for the mismatch repair gene PMS2 showed that the patients genotype was a homozygous splice site mutation, c.989-1 G\T, which resulted in two abnormal transcripts, not one as expected. T he patient’s long time survival may in part be explained by meticulous follow up by health care professionals. The other importing factor is probably the nature of the genotype. cDNA analysis showed that the homozygous mutation led to two abnormal transcripts, of which one is perhaps less detrimental. We have detected the same mutation in heterozygote form in three other families. It has been a discussion whether a heterozygote PMS2 mutation may be the cause of HNPCC/Lynch syndrome. We will present these families and discuss the effect of having the variant in heterozygous form.

Germline allele-specific expression of TGFBR1: promoter methylation and frequency in African Americans and in colorectal cancer with mismatch repair deficiency Laura Valle1,2, Tarsicio Serena-Acedo1, Sandya Liyanarachchi1, Heather Hampel1, Ilene R. Comeras1, Stephan M. Tanner1, Albert de la Chapelle1 1

Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, OH; 2Present Affiliation: Laboratori de Recerca Translacional, Institut Catala´ d’Oncologia, Barcelona, Spain Most of the genetic predisposition to colorectal cancer (CRC) has not yet been explained: While the proportion of all CRC that is associated with genetic predisposition is expected to be as high as 35% only around 5% has been explained molecularly so far. Up to now the classical methods used to search for susceptibility genes in families with high CRC aggregation (linkage analyses), have failed to identify the genetic cause of the excess of heritability in CRC, and case– control or cohort genome-wide association studies (GWAS) have started to identify variants associated with very low risks and usually located in genomic regions ‘‘empty’’ of genes. Recently, we showed that germline allele-specific expression (ASE) of the TGFBR1 gene occurs in 10–20% of all CRC patients and only in 1–3% of healthy controls in a mostly Caucasian population. If the preliminary findings are confirmed in larger studies, this places ASE of TGFBR1 among the high-risk biological markers of CRC predisposition with an odds ratio of at least 8.7. It is vital to identify the cause of this partial allelic expression reduction that, despite the efforts invested, has not been identified yet. Two major TGFBR1 haplotypes are predominant among ASE cases, suggesting that the genomic change is in cis, but causative germline changes have not been identified. Moreover, the haplotype findings suggest that the frequency of ASE might show differences between populations.

Abstracts

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The partial reduction of expression of one allele might well be explained by germline allele-specific methylation. Methylation of CpG sites in the promoters of genes can lead to loss of transcription and has been repeatedly implicated in the silencing of tumor suppressor genes. This hypermethylation is usually somatic, but if it plays a role in ASE of TGFBR1, it must be heritable and occur in the germline. Germline methylation of promoter CpG sites has been documented (e.g. in MLH1), but it is a very rare phenomenon and its heritability has not been convincingly proved. Here we study germline methylation status of the TGFBR1 CpG island in 22 ASE and 15 non-ASE individuals, not finding significant differences. Moreover we determined the frequency of TGFBR1 ASE and the associated haplotypes in African American (AA) CRC patients (n = 84) and controls (n = 168), and assessed its involvement in the predisposition to microsatellite unstable (MSI) CRC (n = 116). To sum up, ASE of TGFBR1 is not caused by allele-specific promoter hypermethylation, predisposes to CRC in AAs to approximately the same degree as in Caucasians, and it is not involved in the susceptibility to CRC associated with an inherited or acquired deficient mismatch repair. In AAs, the presence at lower frequency of two Caucasian ASE-associated haplotypes suggests admixture, and the existence of different genetic variants responsible for ASE.

Conclusion IHC as a primary molecular screening modality would fail to identify two missense mutations. MSI analysis could potentially identify a tumour that has defective DNA MMR but intact IHC staining caused by non-truncating missense mutations. In conjunction with a comprehensively developed family history, tumour IHC analysis of all four MMR proteins (hMSH2, hMLH1, hMSH6 & hPMS2) in conjunction with MSI testing is the optimum molecular strategy to screen for Lynch Syndrome.

Functional studies of unclassified variants of the human mismatch repair protein MLH1 in the yeast Saccharomyces cerevisiae

Michael P. Farrell, M. J. Kennedy, D. Flannery, T. J. Boyle, B. J. Mehigan, R. B. Stephens, S. A. White, C. B. Muldoon1, A. J. Green, S. T. Duke2

1

2

2

Karin Hardt , Sven Boris Heik , Johannes H. Hegemann , Brigitte Royer-Pokora1, The German HNPCC Consortium 1

Institut fuer Humangenetik, Heinrich Heine Universitaet, Duesseldorf; 2Institut fuer Funktionelle Genomforschung, Heinrich Heine Universitaet, Duesseldorf Genome stability is ensured by DNA-repair mechanism and is conserved from bacteria to men. HNPCC is caused by germ line mutations in the mismatch repair genes MLH1, MSH2, MSH6 and PMS2. 30% of all mutations in the mismatch repair gene hMLH1 are missense mutations. Functional analyses of proteins with amino acid substitutions are important for the classification of missense mutations into pathogenic or non-pathogenic variants. Due to the high homology of the Saccharomyces cerevisiae repair system this organism can be used as model for functional analyses of the human mismatch repair protein hMLH1. 8 missense mutations were inserted into the ATPase domain and 18 into the interaction domain of hMLH1. The interaction with the partner hPMS2 was analysed in a yeast-2-hybrid system. Additionally these mutations were analysed with the dominant negative mutator phenotype assay. In this system the interference of the human MLH1 protein with the yeast mismatch repair system reveals information about the protein function. To determine the mutation rate, an existing yeast LYS2A14 reporter system was used. The endogenous lysine gene contains a frameshift due to the insertion of an (A)14 run, and cells are lysine auxotroph. Reversion to prototrophy primarily occurs through a onenucleotide deletion, which is caused by a defective repair system during replication. For further characterisation of the missense mutations, the stability of the protein was determined by Western Blot analysis. In these studies 17 of 26 missense mutations in the hMLH1 gene were classified as pathogenic. The characterisation of 5 missense mutations in the ATPase domain revealed only slight effects on protein function and 4 missense mutations in the hPMS2-interaction domain were classified as non-pathogenic. Methods Molecular genetic studies undertaken in two large Irish kindreds that satisfy the stringent Amsterdam criteria will be described.

References 1. Lynch HT, de la Chapelle (2003) Hereditary colorectal cancer. N Engl J Med 348:919–932. 2. Boland CR (2007) Clinical uses of microsatellite instability testing in colorectal cancer: an ongoing challenge. J Clin Oncol 25:754–755.

Optimum clinical & molecular strategies to screen for Lynch syndrome

1 St. James s Hospital & Trinity College Dublin, 2National Centre for Medical Genetics, Dublin

Background The Lynch Syndrome is a highly penetrant, autosomal dominant, multi-system cancer disorder caused mainly by heritable defects in the highly conserved DNA mismatch-repair (MMR) genes hMSH2, hMLH1, hMSH6 & hPMS2 1. Identification of a pathogenic germline mutation is extremely important because it enables pre-symptomatic testing of family members and structured surveillance of mutation carriers. Due to the heterogeneity of the mutation spectrum of the MMR genes, mutation analysis is time-consuming and expensive, therefore, screening strategies are required to pre-select those families that are likely to harbour a deleterious mutation. Various criteria (Amsterdam & Bethesda) have not proved definitive for identifying patients who may harbour a mutation. In addition to family history, histological, immunohistochemical and molecular analysis can be utilised to identify patients eligible for mutation analysis. Most tumours from patients with Lynch Syndrome have a characteristic molecular signature resulting from the involvement of defective MMR, i.e., the presence of microsatellite instability (MSI) and/or the absence of MMR protein expression by immunohistochemistry (IHC) 2.

Preliminary findings from a qualitative study of the reasons individuals decline the offer of genetic testing for colorectal cancer Louise A. Keogh, Belinda Mcclaren, Judith Maskiell, Clara Gaff, John Hopper, Mark Jenkins Key Centre for Women’s health in Society, The University of Melbourne, Carlton, Australia Since 1997, the Australasian Colorectal Cancer Family Registry (ACCFR) has been studying a large number of individuals who have

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732 had bowel cancer and their relatives. When a genetic mutation is identified by the ACCFR, family members are offered the chance to have genetic testing through a family cancer clinic to learn of their result. We know from quantitative analysis that between 17% (before August 2003) and 49% (after August 2003) of those offered genetic testing decline the offer, but so far we have not known the reasons why. We are currently following up a small group of the 120 participants who have been offered genetic testing to explore genetic testing decision-making in more detail. Twenty to thirty participants will take part in a qualitative interview about genetic testing decisionmaking in the area of bowel cancer. This will include 10–15 who have declined genetic testing and 10–15 who have accepted an offer of genetic testing. Thematic analysis will be used to determine the reasons for accepting or declining the offer of genetic testing. Preliminary findings based on interviews with decliners will be presented. We will report why participants decline the offer of genetic testing. A common reason given by participants for declining was the fear of losing death and disability entitlements or becoming ineligible for life insurance if found to carry a mutation. Individual’s perception of their personal risk of colorectal cancer as well as their perception of the value of knowing their mutation status consistently played a role in decision-making.

Soft tissue sarcomas and hereditary non polyposis colorectal cancer (HNPCC) syndrome: formulation of an hypothesis E. Urso1, M. Agostini1, S. Pucciarelli1, I. Mammi1, A. Viel2, I. Maretto1, D. Nitti1 l

Clinica Chirurgica 2^, Dipartimento di Scienze Oncologiche e Chirurgiche, Azienda Ospedaliera-Universita` di Padova, Italy; 2 Oncologia Sperimentale 1^, Centro Regionale Oncologico, IRCCS, Aviano, Italy Background Hereditary non polyposis colorectal cancer (HNPCC) is an inherited disorder caused by mismatch repair (MMR) gene(s) deficiency. HNPCC predisposes to several malignances (particularly colorectal and endometrial cancer) which not yet include soft tissue sarcomas. Aim To sustain the hypothesis that sarcomas could be comprised in the spectrum of HNPCC related tumors. Methods We report on a HNPCC patient with leiomyosarcoma of deltoid muscle and review the literature, finding four other cases of soft tissue tumors involving HNPCC patients. Results Data for supporting our hypothesis are the follow: sarcomas are rare tumours and so there is a low probability of random clustering with colorectal and endometrial cancer; mean age at diagnosis of sarcomas in HNPCC patients was 34 years vs. 56 years of the general population; sporadic sarcomas are not associated to microsatellite instability neither to loss of MMR proteins expression at immunohistochemistry, while all the five cases collected of HNPCC patients with soft tissue sarcoma, presented loss of MSH2 at immunohistochemistry and/or germline MSH2 mutation. Moreover, an excess of sarcomas in MMR defective mice was also reported. Conclusions Sarcomas could be associated to colorectal cancer and other HNPCC related tumors as the expression of the same germline disease. This report would encourage the scientific community to revise the HNPCC family trees, with particular attention to soft tissue tumors. More extensive data could confirm our hypothesis and broad the spectrum of HNPCC related cancers.

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Abstracts

Adenomas in Lynch syndrome: diagnostically useful? Michael D. Walsh, Daniel D. Buchanan, Sven Arnold, Mark Clendenning, Rhiannon Walters, John L. Hopper, Mark A. Jenkins, Jeremy R. Jass, Joanne Young Queensland Institute of Medical Research, Brisbane, Australia Debate continues as to the usefulness of testing adenomas for loss of mismatch repair (MMR) proteins to identify individuals with suspected Lynch syndrome. Some studies have suggested that it is only worthwhile testing large, proximal adenomas exhibiting severe dysplasia while others report disappointing ‘‘hit rates’’ even from known mutation carriers. We tested 77 adenomas from 49 mutation carriers arising from 38 Lynch syndrome families (14 MLH1, 20 MSH2, 3 MSH6 and 1 PMS2 mutation respectively) enrolled in the Australasian Colorectal Cancer Family Study. The average age of diagnosis of first polyp studied was 49.6 ± 10.6 years (30.0–79.2 years).All polyps were tested by immunohistochemistry (IHC) for four MMR proteins MLH1, MSH2, MSH6 and PMS2. Sixteen polyps demonstrated normal MMR IHC (6/16 polyps came from individuals who had other polyps which demonstrated abnormal IHC), whilst 61/77 (79.2%) adenomas from mutation carriers showed loss of appropriate protein expression. A subset of polyps was also assessed for evidence of microsatellite instability (MSI) with concordance between IHC and MSI testing in 30/32 (94%) cases. Here was no significant difference between adenoma site with 20/23 (87%) proximal vs. 26/30 (87%) distal showing MMR deficiency. Similarly, size was not a predictor of MMR deficiency with 23/31 (74%) adenomas \5 mm maximum dimension, 18/22 (82%) 5–10 mm, and 14/17 (82%) [ 10 mm MMR deficient. Of the MMR deficient polyps, 32 showed mild dysplasia (75%), 32 showed moderate dysplasia (84%) and 10 showed severe dysplasia (90%). Whilst there was a trend to greater likelihood of loss of MMR protein with increasing dysplasia, this was not statistically significant. In conclusion, testing adenomas from patients from Lynch syndrome families is a useful alternative in cases where cancers are unavailable since 79.2% adenomas from carriers show appropriate loss of MMR proteins and a high level of concordant microsatellite instability.

Patient preferences for risk management strategies in women with Lynch syndrome Charlotte C. Sun, Susan K. Peterson, Kristin White, Beatty Watts, Molly Daniels, Stephanie Boyd-Rogers, Kathleen Schmeler, Diane C. Bodurka, Karen H. Lu University Texas M. D. Anderson Cancer Center, Houston, Texas, USA Background Women with Lynch syndrome must balance clinical benefits of cancer prevention and screening strategies against potential detriments to quality of life. We assessed women’s preferences for screening, chemoprevention, and prophylactic surgical strategies. Methods We used the visual analog scale (VAS) and standard gamble (SG) to assess preferences of women with Lynch syndrome. The VAS asked women to rank strategies from 0 = worst to 100 = best. The SG asked what percentage of lifetime risk of cancer they needed to accept each strategy. Strategies included: endometrial screening/biopsy (GYN); (2) colonoscopy (CLSCPY); (3) combined GYN + CLSCPY; (4) prophylactic hysterectomy/oophorectomy (TAH/BSO); (5) prophylactic subtotal colectomy (SUBTCOL); (6)

Abstracts combined TAH/BSO + SUBTCOL; (7) oral contraceptives (OCPs); (8) COX-2 inhibitors (COX2); (9) combined OCPs + COX2. Results 50 women participated (median age = 37.7 years, range 24.7–57.9). Using the VAS, the most favorable strategy was annual combined GYN + CLSCPY (VAS = 98), followed by annual GYN, CLSCPY, and OCPs (VAS = 90). The least preferred strategy was combined premenopausal TAH/BSO + SUBTCOL (VAS = 28). Postmenopausal TAH/BSO was the most favorable surgical intervention (VAS = 85). If lifetime risk of cancer exceeded 40%, women accepted postmenopausal TAH/BSO; if lifetime risk exceeded 70%, women accepted premenopausal TAH/BSO. Women accepted SUBTCOL if lifetime risk exceeded 75%. Compared to unaffected women, women with a personal history of colon cancer gave less favorable scores for COX2 (90 vs. 50, p = .001), OCP + COX2 (85 vs. 40, p = .02), CLSCPY (95 vs. 80, p = .06) but were more willing to accept annual and biannual GYN screening at lower lifetime risk of cancer (30% vs. 20%, p = .042; 30% vs. 10%, p = .019, respectively). Conclusions Our data indicate that women with Lynch syndrome had the highest preferences for combined GYN + CLSCPY screening. Women favored all screening strategies over surgery. Reproductive age influences preferences for TAH/BSO. Women with a personal history of colon cancer accept GYN screening more readily than unaffected women.

Identification of the MLH1 and MSH2 genes mutations in Greek colorectal cancer patients Georgia Thodi, Fostira Florentia, Sandaltzopoulos Raphael, Yannoukakos Drakoulis National Center of Scientific Research ‘‘Demokritos’’, Athens, Greece Germline mutations in the mismatch repair genes, MLH1, MSH2, MSH6 and PMS2, predispose to the Lynch syndrome. Approximately 90% of the deleterious alterations, reported in putative Lynch syndrome patients so far, are scattered throughout the coding regions of the MLH1 and MSH2 genes and involve not only small aberrations but also large genomic rearrangements. Thus, their identification requires full sequencing of the implicated genes, as well as techniques for the detection of gross aberrations. Our main goal is to study the nature and frequency of the MLH1 and MSH2 pathogenic mutations encountered in Greek colorectal cancer patients with family history indicative of Lynch syndrome in order to facilitate the DNA-testing procedure. We have screened twenty families selected upon modified Amsterdam criteria or revised Bethesda guidelines for the presence of pathogenic alterations in MLH1 and MSH2 genes using both sequencing and multiplex ligation dependent-probe amplification. Four point mutations, three nonsense in the exons five, seven, thirteen of the MSH2 gene and one splice site alteration in the intron nine of the MLH1 gene, as well as three genomic rearrangements, one deletion, one duplication involving exon six of the MLH1 gene and one duplication in the exon twelve of the MSH2 gene, have been detected in eight among our fifteen Amsterdam positive families. The deletion was characterized using long range PCR while the duplications have not been confirmed with an independent method, yet. The nonsense alterations located in exons five and seven of the MSH2 gene, respectively, as well as the two duplications have not been described elsewhere, to the best of our knowledge. In conclusion, the MLH1 and MSH2 mutational spectrum observed in our patients’ cohort is quite heterogeneous while genomic deletions/duplications seem to contribute significantly to its configuration.

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Microsatellite instability for Lynch syndrome diagnosis in Chile: a multicentric study Carolina A. Rı´os1, Jorge Mun˜oz1, Leonardo Espindola2, Juan Ignacio Vergara2, Mario Abedrapo3, Claudio Mun˜oz3, Yamile Corredoira4, Jaime Contreras5, Isabel Castro6, Mo´nica Acun˜a1, Lucı´a Cifuentes1 1 Laboratorio de Epidemiologı´a Gene´tica, Facultad de Medicina, Universidad de Chile; 2Servicio de Cirugı´a, Hospital Militar de Santiago; 3Servicio de Cirugı´a, Hospital Clı´nico Universidad de Chile; 4Departamento de Anatomı´a Patolo´gica, Hospital Clı´nico San Borja Arriara´n, Campus Centro, Facultad de Medicina, Uiversidad de Chile; 5Departamento de Cirugı´a, Hospital Clı´nico San Borja Arriara´n, Campus Centro, Facultad de Medicina, Universidad de Chile; 6Escuela de Tecnologı´a Me´dica, Facultad de Medicina, Universidad de Chile. Santiago, Chile

Lynch syndrome is the most common hereditary colorectal cancer syndrome. In Chile, few studies have been developed regarding this syndrome, although the colorectal cancer mortality rate has increased in the last few years. The aim of our research is to establish diagnostic criteria for Chilean patients, considering the population characteristics, the available data from the patients, molecular tests and the final costeffectiveness. To accomplish this, we have started a multicentric study involving several hospitals from the area of Santiago, Chile. The patients, who meet the Revised Bethesda Guidelines (RBG) for identifying individuals at risk for HNPCC, were selected and informed consent was obtained from all of them. Fixed and fresh samples were collected, according to the availability, and DNA was obtained with the proper methodology. At the moment, 25 patients have been recruited from 4 different hospitals. 56% of the patients meet the age criterion and only 24% fulfil the family history criterion, probably caused by the lack of information. The rest of the patients had synchronous or metachronous colorectal cancer or tumours with MSI-high histology. The microsatellite instability (MSI) was studied using 5 loci: Bat25, Bat-26, D2S123, D5S346 and D17S250. The results show 9 patients (36%) with MSI-high (with 2 or more out of 5 loci unstable). The immunohistochemistry (IHC) for hMSH2 and hMLH1 is being developed at the moment. All samples with MSI or altered IHC will eventually be subjected to a BRAF V600E mutation analysis and MSH2 and MLH1 mutation scan. (Grants: Universidad de Chile FEBA-157, Conicyt AT-24080077).

‘‘Minor’’ mismatch repair genes involvement in genetic predisposition to Lynch syndrome F. Duraturo, R. Liccardo, A. Cavallo, M. De Rosa, P. Izzo Department of Medical Biochemistry and Biotechnology, University of Naples Federico II, Naples, Italy Lynch syndrome is commonly associated to mutations in at least three mismatch repair (MMR) genes: MLH1, MSH2 and MSH6. However, mutations in these three genes do not account for all Lynch syndrome families. Recently, it has been shown that germline mutations in others MMR genes, PMS2 and MLH3, play a far more important role in Lynch Syndrome than initially thought. So far, MSH3 gene germline mutations have not been described. In this study, a set of 63 Lynch syndrome unrelated patients, negative for germline mutations within MSH2 and MLH1 genes, were screened for mutations in ‘‘minor’’ MMR genes: MSH6, PMS2, MLH3 and MSH3. Patients fulfilling the Amsterdam Criteria, showing early and late onset colorectal cancer and microsatellite instable tumors, were selected for this study. ‘‘Minor’’ MMR genes were

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734 screened by denaturing high performance liquid chromatography (DHPLC). All samples exhibiting abnormal DHPLC profiles were analyzed by directed sequencing. We have identified 2 frameshift mutations causing a premature stop codon, 5 missense mutations, 7 silent mutations and 4 intronic changes within MSH6 gene; 11 missense mutations, 4 silent mutations and 7 intronic changes in PMS2 gene; 6 missense mutations, 1 frameshift mutation mutations causing a premature stop codon, 2 silent mutations and 4 intronic changes within MLH3 gene. Finally, we have identified 4 missense mutations, 1 silent mutation and 7 intronic changes within MSH3 gene. Several patients were carriers of at least two genomic variants of the ‘‘minor’’ genes. Thirty-four of the 65 genomic variants identified are novel, not reported in the international mutational databases. This study gives the first evidence of MSH3 germline mutations in Lynch Syndrome patients. Moreover, the simultaneous presence of several mutations in different minor genes could suggest that these genes might work together in a cooperative manner resulting in an increased risk of cancer in Lynch Syndrome.

Prevention of endometrial tumours, a randomised control trial of the effect of the Mirena intrauterine system (IUS) with surveillance, versus surveillance alone, on the development of atypical endometrial hyperplasia (AEH) and carcinoma (EC) in women with Lynch syndrome AGED 35-65Y Shirley Hodgson1, Peter Sasieni2, Victoria Murday3, Yorkhill Hospital, Glasgow; Eamonn Sheridan, Leeds General Infirmary, Leeds 1

St Georges, University of London; 2Queen Mary University of London; 3Yorkhill Hospital, Glasgow; Eamonn Sheridan, Leeds General Infirmary, Leeds Introduction Women with Lynch syndrome have an increased risk of developing endometrial cancer (up to 65% lifetime risk, 20% before 50 years). The Mirena progestogen-releasing intra-uterine system (IUS) greatly reduces the thickness of the endometrium. Methods We hope to evaluate whether treatment with the Mirena for 4 years reduces the rate of detection of AEH and EC in women with Lynch Syndrome on surveillance by annual transvaginal ultrasound (TVS) and endometrial biopsy (EB). Secondary outcomes include determination of the sensitivity and specificity of surveillance, the agerelated incidence of AEH and EC, the premalignant pathway to carcinoma and any adverse effects of surveillance and use of the IUS. Results Unfortunately we have experienced great difficulty in ascertaining women for the study, largely because appropriate women within have often had hysterectomies or are eligible but unwilling to be randomised. Currently, 17 sites are active in the UK, with a further 7 going through the approvals process. 17 patients have been randomised, with a further 9 consented. Interest from non-UK sites could take the study internationally. Conclusion If the study demonstrates a significant reduction in the development of endometrial neoplasia change in women with the Mirena IUS, it will have a very substantial impact on the management of women with Lynch Syndrome, since many such women do not wish to have a hysterectomy, particularly during childbearing years, but are worried that surveillance may not detect endometrial cancer early enough for effective treatment.

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Abstracts The effectiveness of surveillance is still being evaluated and women may well prefer the option of Mirena IUS if shown to be effective. However, as the study may not be feasible, we are proposing to do an observational study on all women ascertained by the study to try and establish from this data what the diagnostic effectiveness of endometrial ultrasound and biopsy is, and the effect of any hormonal treatment.

Is uptake of genetic testing for colorectal cancer influenced by knowledge of insurance implications? Louise A. Keogh, David M. Studdert, Judith Maskiell, Finlay Macrae, D. James St John, Clara Gaff, Mary Anne Young, Melissa C. Southey, Graham G. Giles, Doreen Rosenthal, John L. Hopper, Mark A. Jenkins Key Centre for Women’s health in Society, The University of Melbourne, Carlton, Australia Objective To assess whether knowledge of insurance implications influenced uptake of genetic testing by participants in a research study of the causes of colorectal cancer. Design and setting From 1999 to 2003 participants in the population-based Victorian Colorectal Cancer Family Study were not informed of any potential effect of genetic testing on their eligibility for future insurance due to their participation. In 2003 the protocol was changed when the investigators became aware that offering participants the opportunity to learn of relevant genetic information might affect their ability to purchase some forms of future insurance. Since 2003 an added step was introduced to the consent procedure, and information on the potential effect on insurance eligibility was provided to participants. Main outcome measure Uptake of genetic testing for mutations in DNA mismatch repair (MMR) genes at a family cancer clinic. Results Compared with 1999–2003, after 2003 the proportion of participants that declined the offer of genetic testing more than doubled from 19 to 49% (p = 0.002). This decline in uptake of genetic testing could not be explained statistically by adjustment for measured putative predictors. Conclusion Knowing one has a mutation in a MMR gene is potentially of substantial clinical importance, so the identification and screening of people with a mutation might be a cost-effective way to reduce the burden of colorectal cancer in the community. If individuals are choosing not to get this genetic information because of how it will affect their eligibility for insurance, reforms to existing insurance practices are indicated.

Utility of the immunochemical expression profile in the identification of patients with Lynch’s syndrome E. Alcaraz, C. Alenda, A. Paya´, L. Pe´rez, R. Jover, E. Rojas, J. L. Soto, A. Castillejo, V. M. Barbera´ Hospital General Universitario Alicante, Alicante, Spain Background MLH1 inactivation may be observed in sporadic and Lynch’s syndrome colorectal carcinoma (CRC). Lynch syndrome is caused by germline mutations in the DNA mismatch repair (MMR) genes. Sporadic CRC is caused by MLH1 promoter methylation. The aim of this study is to evaluate the value of an immunohistochemistry (IHC) panel in the prediction of germline MLH1 mutation in patients with tumours that show loss of MLH1 expression.

Abstracts Methods The study was performed in 159 CRC with loss of MLH1 IHC expression from patients of the Genetic Counselling in Cancer Department of HGUE and from a series of non-selected CRC specimens from the EPICOLON study and the HGUA. IHC analysis for p53, p16, β-catenina, CK20, CDX-2, COX-2, CK7 and β2-microglobulina was performed on tissue microarray (TMA). The MLH1 methylation analysis was performed by real-time PCR assay Methylight. V600E BRAF mutation was detected using specific TaqMan probes by real time PCR. 48 patients were classified as familial, 79 were classified as sporadic and 33 had not enough information for classification. Results There was found statistically significant association between familiar status and IHC expression of p53 (familiar 2.6% vs. sporadic 15.9%, p \ 0.05), p16 (familiar 100% vs. sporadic 62.5%, p \ 0.001), CDX-2 (familiar 81.3% vs. sporadic 51.9%, p \ 0.01), COX-2 (familiar 34.2% vs. sporadic 55.4%, p \ 0.05) and β 2-microglobulin (familiar 60% vs. sporadic 81.8, p \ 0.05). In the regression model analysis p16 and β2-microglobulin resulted to be independents variables that help to detect the familiar status of CRC MLH1 negatives. Conclusions CRC with MLH1 inactivation show a different IHC profile according to hereditary or familiar nature. Differences founded with the markers used in this study allow create a prediction model for familiar or sporadic CRC which could be useful in the clinical practice.

No association between MUTYH AND MSH6 germline mutations in 64 HNPCC patients Verena Steinke1, Nils Rahner1, Monika Morak2, Gisela Keller3, Hans K. Schackert4, Heike Go¨rgens4, Wolff Schmiegel5, Brigitte Royer-Pokora6, Wolfgang Dietmaier7, Matthias Kloor8, Christoph Engel9, Peter Propping1, Stefan Aretz1, The German HNPCC Consortium 1

Institute of Human Genetics, University of Bonn, Bonn, Germany; Institute of Human Genetics, Ludwig-Maximilians-University, Munich, Germany, and Center of Medical Genetics, Munich, Germany; 3Department of Pathology, Munich University of Technology, Munich, Germany; 4Department of Surgical Research, Technische Universita¨t Dresden, Dresden, Germany; 5Department of Medicine, Knappschaftskrankenhaus, Ruhr-University Bochum, Bochum, Germany; 6Institute of Human Genetics, Heinrich Heine University, Duesseldorf, Germany; 7Department of Pathology, University of Regensburg, Regensburg, Germany; 8Institute of Molecular Pathology, University of Heidelberg, Heidelberg, Germany; 9Institute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany 2

Biallelic mutations in the base excision repair (BER) gene MUTYH are associated with multiple adenomas and an increased risk of colorectal cancer. The heterozygote carrier frequency in the general population is approximately 1–2%. Gu et al. (2002) reported on an interaction of MUTYH and mismatch repair (MMR) proteins. They showed that the MSH2/MSH6 complex enhances the binding affinity to the mismatched DNA substrate and the glycosylase activities of MUTYH. Mutations in MMR genes are known to cause hereditary nonpolyposis colorectal cancer (HNPCC/Lynch syndrome), an autosomal dominant tumor predisposition syndrome. Based on the reported interaction of the MMR complex and the base excision repair protein MUTYH, it was hypothesised that MUTYH mutations serve as phenotypical modifiers in HNPCC families. Recently, a significantly

735 higher frequency of heterozygosity for MUTYH mutations among MSH6 mutation carriers was reported. We examined 64 MSH6 mutation carriers (42 truncating mutations, 19 missense mutations and 3 silent mutations) of the German HNPCC Consortium for MUTYH mutations by sequencing the complete coding region of the MUTYH gene. We identified monoallelic MUTYH mutations in two of the 64 patients (3.1%): the truncating MUTYH mutation c.247C [ T;p.Arg83X was found in a patient harbouring a pathogenic MSH6 mutation and the missense mutation c.502C [ T;p.Arg168Cys was identified in a carrier of a MSH6 silent mutation of unknown functional relevance. No biallelic MUTYH mutation carrier was found. The frequency of MUTYH mutations was not significantly higher than in healthy controls, neither in the whole patient group (p = 0.30) nor in different subgroups regarding mutation type. Our results do not support an association between MSH6 and heterozygosity for MUTYH mutations. The study was supported by the Deutsche Krebshilfe.

Lynch syndrome in Rosario, Argentina: 3 years experience Enrique Spirandelli1, Sergio Chialina1, Florencia Spirandelli2, Fernando Serra2, Ariel Naves3, Claudio Settecase2, Juan Carlos Moreno2, Gustavo Castellani2, Gustavo Botti2, Ana Maria Moriena2, Luciana Spirandelli2 2 Integrantes del Servicio de Coloproctologia del Hospital Espan˜ol.1STEM SRL; 3Instituto de Histopatologia jefe del servicio de Coloproctologia Hospital Espan˜ol de Rosario

The Lynch Syndrome is characterized by the development of colorectal cancer, endometrial cancer and other associated cancers. It is caused by a mutation in one of the mismatch repair genes: mainly in MLH1 and MSH2 but also in other genes, such as MSH6 or PMS2. In Rosario city, Argentina, which has a population of 1.200.000 inhabitants we have established since 2005 a multidisciplinary group named ACETIEHR for the diagnosis and management of patients with hereditary cancer such as the Lynch Syndrome. Materials and methods Between September 2005 and December 2008 we have already identified 71 probands (male: 54%, female: 46%) fulfilling the revised Bethesda guidelines. Mean age at diagnosis 52.9 (range 17–81 years). We found extracolonic tumors in 57 probands and 86 family members (most frequent 31 in females and 55 in males). All the probands and their relatives received genetic counseling. We have already performed 29 microsatellite instability (MSI)tests using 5 markers (BAT-25, BAT-26, DS17250, D5S346, D2S123). We are also setting up immunohistochemistry which combined with MSI profiling is the current method of identifying the gene to be sequenced. Results We found 9 tumors with positive MSI. In 3 probands MSI(+)who underwent molecular sequencing, a pathologic mutation was found: 2 in MSH2 gene (c1864 C-[T exon 12 and c1224 T-[A exon 7) and 1 in MLH1 (c1852_1854 del AAG exon 16).Ten persons at risk were tested, and of these, 7 had the mutation and 3 did not. Discussion We estimate that in Rosario city there are 500–2000 mutation carriers in risk of developing colorectal cancer and other cancers associated with the Lynch Syndrome that could be early detected in order to adopt medical prevention measures. Because of the accumulating evidence that MSI is a predictive factor for response to 5FU-based chemotherapy, our group considers that MSI status will be a relevant prognostic factor for all colorectal cancers.

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A prospective single blinded randomized trial of polyethylene glycol-electrolyte solution vs. sodium phosphate as a bowel preparation for colonoscopy in Lynch syndrome gene carriers M. W. J. van Vugt-van Pinxteren, M. H. van Kouwen, M. G. H. van Oijen, F. M. Nagengast Department of Gastroenterology, UMC St. Radboud Nijmegen, The Netherlands Background Lynch syndrome gene carriers undergo regular surveillance colonoscopies. Polyethylene glycol-electrolyte solution (PEG) is routinely prescribed for bowel cleansing but often poorly tolerated by these patients. Sodium phosphate (NaP) may be an effective alternative. Aim To randomly compare the effects of bowel preparation on colonic cleansing and patients’ acceptance. Methods During a 1 year inclusion period Lynch syndrome patients, who previously underwent a colonoscopy were invited to participate. Patients were randomly assigned to either PEG or NaP and asked to fill in a questionnaire about preparation tolerability and future preferences prior to and 1 week after the preparation for colonoscopy. The endoscopist (blinded for the preparation) filled out a report about the colon cleansing. Results A total of 125 carriers were included in the study. Nine (7%) were excluded because of missing data. The remaining 116 patients (M/F 58/58, mean age 50 ± 30 years) were included in the statistical analysis. Of those, 53 used PEG and 63 NaP. Baseline characteristics did not differ between groups. Of the patients using PEG, 13% found the preparation almost intolerable, in contrast to 8% of those using NaP (p = 0.005). Future preparation preferences were 18% for PEG, 51% for NaP, 27% did not have a preference (of 4% no data). The colonoscopy was poorly tolerated in 28% of individuals using PEG and in 25% of the NaP participants (p = 0.963). The endoscopist reported a good to excellent clean colon in 83% of the patients on PEG and in 71% of those on NaP (p = 0.096). The proportion of colonoscopies with introduction into the cecum within 25 min did not differ between groups: 68% PEG and 72% NaP (p = 0.645). Conclusion Lynch syndrome carriers tolerated NaP better and preferred this formula for bowel preparation. Colon cleansing was suboptimal by both treatments with a tendency towards a cleaner colon with PEG.

Capsule endoscopy for the small bowel in juvenile polyposis syndrome O. Will, A. J. Postgate, C. H. Frase, A. Fitzpatrick, R. K. S. Phillips, S. K. Clark St. Mark’s Hospital, Middlesex, UK Introduction Juvenile polyposis syndrome (JPS) is one of the hamartomatous polyposis syndromes and demonstrates phenotypic heterogeneity. All patients with JPS develop colorectal polyps and are at risk of colorectal cancer. Small bowel involvement in JPS is variably described. Small intestinal cancer is reported but is rare and currently there is no evidence-based protocol for small intestinal surveillance. Aim This study investigates the utility of capsule endoscopy (CE) in patients with JPS.

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Abstracts Methods Ten patients with JPS underwent CE. Medical records were reviewed to characterise the genotype. Conventional upper and lower gastrointestinal endoscopy were used to characterise the phenotype of the rest of the bowel. Results Two patients had small bowel polyps beyond the range of oesophagogastroduodenoscopy (OGD) identified at CE: a 6 mm ileal polyp in one and 10 and 6 mm ileal polyps in the second. In addition duodenal polyps were detected in a third patient at CE. Three further patients had previously documented duodenal polyps at surveillance OGD. A SMAD4 mutation was identified in 7 patients and there was no obvious association with gastric/small bowel polyposis. Patients reported CE as comfortable and convenient. Conclusions CE provided information additional to conventional endoscopy and was well-tolerated. However no lesions requiring clinical intervention were identified and polyp numbers were small. CE may be used as a non-invasive method of investigating the small bowel JPS, but there is no evidence to support routine surveillance in colonic-confined JPS.

A novel mutation in MUTYH: associated polyposis (MAP) syndrome Florentia Fostira1, Christos Panopoulos2, Anna Efraimidis2, Drakoulis Yannoukakos1 1 N.C.S.R ‘‘Demokritos’’, Molecular Diagnostics Laboratory, R-RP Institute, Athens, Greece; 2Oncology Anticancer Hospital ‘Agios Sabbas’, 2nd Department of Medical Oncology, Athens, Greece

MUTYH-Associated Polyposis (MAP) syndrome is inherited as a recessive trait and is characterized by the intrinsic phenotypic feature of adenomas present at the individual’s colorectum. In the case of non-surgical removal of these polyps, adenoma will transform to carcinoma. Colorectal tumours from mutation carriers display an excess of somatic mutations, specifically G:C to T:A transversions. The risk of colorectal cancer among biallelic mutations carriers seems to be close to 100% by the age of 60. Four families meeting the MAP phenotypic criteria were studied. Genomic DNA was purified from peripheral blood leukocytes following standard chloroform extraction. The complete coding sequences of the MUTYHgene, including splice junctions, were amplified by Polymerase Chain Reaction (PCR) and electrophorized in an ABI Prism 310 Genetic Analyzer. Analysis of the MUTYH gene revealed five different germline mutations, one of which novel and never characterized in respects of its pathogenicity. A mutation has been identified on exon 7 of the MUTYH gene at a homozygous state, in an individual with less than one hundred colorectal polyps at the age of forty. Another patient diagnosed with colorectal cancer at the age of 34 carried two maternally and one paternally inherited mutation, located on exon 14. A milder MAP phenotype characterized an individual, who was diagnosed with less than one hundred polyps at the age of 69. Genetic testing revealed that the patient was compound heterozygote for two missense variants located on exons 8 and 12 of the MUTYH gene. A novel mutation located on intron 6 of the MUTYH gene has been identified in two families. Our findings confirm previous observations highlighting the necessity for genetic testing of MUTYH in patients with MAP phenotypic features, as well as his/her extended family, in order to determine the phenotyping expression of each mutation and propose the appropriate clinical surveillance, which includes biennial colonoscopy after the age of 30. In specialized cases surgical intervention might be essential.

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APC or MUTYH mutations account for the majority of clinically well characterized families with familial adenomatous polyposis and attenuated familial adenomatous polyposis phenotype and patients with more than 30 adenomas 1

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Bruno Filipe , Ce´lia Baltazar , Cristina Albuquerque , Sofia Fragoso1, Pedro Lage2, Ineˆs Vitoriano1, Susana Ma˜o de Ferro2, Isabel Claro2, Paula Rodrigues2, Paulo Fidalgo2, Marı´lia Cravo2, Carlos Nobre Leita˜o2 1

Centro de Investigac¸a˜o de Patobiologia Molecular (CIPM), Instituto Portugueˆs de Oncologia de Lisboa de Francisco Gentil, EPE, Lisbon, Portugal; 2Department of Gastroenterology, Instituto Portugueˆs de Oncologia de Lisboa de Francisco Gentil, EPE, Lisbon, Portugal Patients presenting familial adenomatous polyposis (FAP), attenuated FAP (AFAP) or multiple colorectal adenomas (MCRA) phenotype are clinically difficult to distinguish. We aimed to evaluate the contribution of APC and MUTYH germline mutations for 107 clinically well characterized patients with FAP related phenotype, classified according with the recent guidelines for the clinical management of FAP. Patients were stratified into 5 groups: FAP, AFAP, MCRA (10–99 colorectal adenomas) without familial history of colorectal cancer or few adenomas (FH), MCRA (10–99) with FH, MCRA (3–9) with FH. Most FAP patients presented APC mutations (38/43; 81%) and few showed MUTYH mutations (2/46; 4%), whereas AFAP patients presented a lower APC but a higher MUTYH mutation frequency (5/16; 31% vs. 8/20; 40%). MCRA patients did not present APC mutations but showed distinct MUTYH mutation frequencies: 0, 2/14 (14%) and 7/13 (54%), respectively for 3–9, 10–29 and 30–99 adenomas (P = 0.033). APC large deletions and expression deficiency in mutation negative patients suggest that both defects may account for a fraction of these cases. The remaining cases may represent a new clinical entity. We validate the recently proposed guidelines in our patient’s cohort and show that APC or MUTYH germline defects are responsible for the majority of clinically well characterized families with FAP, AFAP phenotype and patients with more than 30 colorectal adenomas. The different mutation frequencies among the five groups, specially according with the number of adenomas, points out for the importance of excellent clinical characterization and colonoscopy techniques for the management of FAP related phenotypes.

Adrenal incidentalomas in FAP patients A. Hansmann, O. C. C. Will, R. K. S. Phillips, F. F. Palazzo, K. Meeran, M. Marshall, S. K. Clark The Polyposis Registry, St. Mark’s Hospital, Harrow, United Kingdom Department of Radiology, St. Mark’s Hospital, Harrow, United Kingdom; Department of Endocrine Surgery, Hammersmith Hospital, London, United Kingdom; Department of Endocrinology, Hammersmith Hospital, London, United Kingdom Aim Adrenal incidentaloma (AI) is often diagnosed in patients with Familial Adenomatous Polyposis (FAP), since they frequently undergo cross-sectional imaging of the abdomen, and also have a higher incidence of AI than the general population. This study investigates the natural history of AI in FAP, and suggests a management protocol. Patients and methods An original cohort of 14 FAP patients with AI, identified 12 years ago in a prospective study, was followed up

clinically and radiologically. A further group of 16 FAP patients with AI was also identified. All had AI [ 1 cm in size. For both cohorts, characteristics of patients (genotype, age at diagnosis, concomitant diagnoses) and incidentaloma (size, laterality, rate of growth, outcome) are described. Results Overall, three of 30 patients underwent adrenalectomy; one had phaeochromocytoma and another an adenoma of borderline malignancy. A further three lesions were radiologically suspicious for malignancy at time of diagnosis, one in a patient who was unfit for surgery but died of non-adrenal causes after more than 9 years. None of the lesions radiologically benign at diagnosis showed an aggressive course, but one patient required referral for surgery after 12 years of follow-up due to slow size increase. There were no associations with genotype. Conclusions FAP-associated AI warrants long-term follow-up. While the natural history is similar to that of sporadically-occurring lesions, these patients have concomitant FAP-associated manifestations under regular radiological surveillance, and we suggest a management protocol tailored accordingly.

Audiology in familial adenomatous polyposis: do you hear what I hear? Margaret O’Malley, James Church, Lisa LaGuardia, Richard Naugle, Cynthia Gensur, Jeff Hammel, Carol A. Burke Digestive Disease Institute, Sanford R. Weiss, MD Center for Hereditary Colorectal Neoplasia, Cleveland Clinic, Department of Colorectal Surgery, Department of Psychology, Department of Audiology, Department of Gastroenterology, Cleveland, Ohio, USA Introduction The APC protein has an important role in maintaining function of microtubules in the ear that play a significant part in the mechanism of hearing. Preliminary data suggests that the APC protein is associated with an increased incidence of abnormal hearing which may affect intellectual function. We sought to assess the hearing among patients with FAP (Familial Adenomatous Polyposis). Methods Patients with FAP were recruited for an IRB-approved study assessing hearing and intelligence. Hearing was tested by pure tone air conduction audiometry, less than or equal to 30 db at 3/4 of the following frequencies (500, 1000, 2000 and 4000 Hz). Subjects were then administered the Kaufman Brief Intelligence Test (KBIT-2). We then analyzed the proportion of individuals with an abnormal audiometry as compared to age and gender adjusted normalized hearing standards. Results 44 patients were recruited from 42 families. Subjects included 22 men with a mean age of 42 years. When compared to normalized hearing standards, 19 (43.2%) of the 44 patients failed to meet the standard normal range. Audiologic abnormalities showed unilateral hearing impairment was documented in 6 patients, bilateral impairment in 13. Of these patients 63% were impaired at a single frequency; the other 37% were at multiple frequencies. 59% of patients showed right hearing impairment with the highest deficit (35%) at 2000 Hz. 71% of patients had left sided impairment with the greatest number (32%) at 4000 Hz. Conclusion A large subset of our sample of FAP patients (43.2%) had abnormal audiologic results when compared to the normalized standard. Differences in IQ scores for patients with and without audiologic abnormalities are not statistically significant, suggesting that these results do not reflect an association between hearing and intellectual functioning in our sample.

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Cognitive function in FAP: anyone out there listening? Margaret O’Malley, James Church, Lisa LaGuardia, Richard Naugle, Cynthia Gensur, Jeff Hammel, Carol A. Burke Digestive Disease Institute, Sanford R. Weiss, MD Center for Hereditary Colorectal Neoplasia, Cleveland Clinic, Department of Colorectal Surgery, Department of Psychology, Department of Audiology, Department of Gastroenterology, Cleveland, Ohio, USA Introduction Preliminary data suggests the APC protein is critical for microtubule dependent pathways in the cochlea and may be important in cognition. Abnormal audiometrics have been documented in FAP. We studied cognitive function among patients with FAP. Methods FAP patients were recruited fora study assessing intelligence using the Kaufman Brief Intelligence Test (KBIT-2), which provides Verbal, Nonverbal and Composite IQs. The KBIT-2 was administered and scored by individuals experienced in administration of psychometric measures. Mean scores were analyzed and compared to standard normal ranges. Results 44 Subjects from 42 families (22 men), mean age of 42 years were included. KBIT-2 Composite IQ score was 98.4 ± 12.4 (95% CI 94.5–102.3) which is within the average range of 90–109. 27% of patients scored below average (less than 90) and 15% scored above average (greater than 109), not a significant imbalance (sign test p = 0.33). Nonverbal IQ scores show no difference from average, mean = 100.5; 24% scored below and 27% scored above average. Verbal mean score was 95.5 ± 12.0 (95% CI 91.7–99.2) significantly lower than average (one-sample T-test P = 0.020). There is an imbalance among patients with 27% below and 7% above the average range and a tendency toward lower than average scores (sign test P = 0.06). The mean number of points by which Nonverbal IQ exceeded Verbal IQ was 5.0 ± 12.5 (95% CI 1.1–9.0) (one-sample T-test P = 0.013). The non-verbal score exceeded the verbal score for 27 patients (65.9%), while the verbal score was larger for only 10 patients (24.4%) (Sign test P = 0.008). Conclusion Composite IQ scores suggest that FAP patients do not have lower IQ than the general population. However the verbal scores of FAP patients which are dependent on hearing are significantly lower than average and may reflect abnormal audiometrics or other effects of the APC mutation on cognitive function.

Your patient information website: how good is it? Ramawad Soobrah, Anand Patel, Sue Clark St Mark’s Hospital, Harrow, Middlesex, UK Aim Although the Internet has greatly improved access to health information for patients, experts have raised concerns about the overall quality of those websites. This study was designed to evaluate the accessibility, quality and reliability of information about familial adenomatous polyposis (FAP) on the Web. The secondary aim was to evaluate the websites of InSiGHT member institutions (IMI) separately. Method We searched for the keywords ‘‘familial adenomatous polyposis’’ using the three most popular search engines (Google, Yahoo and MSN). The search was restricted using the ‘‘English language’’ and ‘‘exact phrase’’ settings and we looked at the first 50 websites only. The LIDA tool (an online validated instrument for health care websites) was used to assess their accessibility, usability and reliability. LIDA scores can be classified as being high [90%), medium (\90%, [50%) or low (\50%). The readability of each document was assessed using the Flesch reading ease score (FRES). A FRES score of 60–69 represents a standard readability level.

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Abstracts Result Of the 150 possible sites, only 55 were analysed because of repetitions (52), irrelevant content (21) or inaccessible links (22). The mean LIDA score was 61.9% (SD = 10) and mean FRES score was 35.2 (SD = 16). Only seven IMI websites were identified in the list and their mean LIDA and FRES scores were 67.9% (SD = 8.8) and 47.3 (SD = 7.8) respectively. The mean reliability scores of all websites compared to IMI websites were 37.4% (SD = 16.7) and 56.1% (SD = 9.2) respectively. Conclusion Overall, information available for patients about FAP on the internet is difficult to access, and of poor quality. Although the mean LIDA and FRES scores of IMI websites tend to be higher, the readability of their contents remain poor and they do not necessarily appear among the top search results. Hence the need to develop clear, easily accessible and authoritative resource for FAP patients and their relatives.

The role of common MUTYH gene mutations in breast cancer appears insignificant Astrid A. Out1, Ivonne J. H. M. van Minderhout1, Carli M. Tops1, Maartje Nielsen1, Marjan M. Weijs1, Martijn H. Breuning1, Hans F. A. Vasen2,4, Peter Devilee1,3, Frederik J. Hes1 1 Center for Human and Clinical Genetics, 2Department of Gastroenterology, 3Department of Pathology, Leiden University Medical Center, 4The Netherlands Foundation for the Detection of Hereditary Tumours, Leiden, The Netherlands

Previously, we described a significantly higher frequency of breast cancer in female bi-allelic MUTYH mutation carriers as compared to the Dutch population (4 out of 22, Standardized Morbidity Ratio = 3.75). Accordingly, we further investigated the possible role of constitutional MUTYH mutations in the development of breast cancer. A Dutch population based cohort of 1219 breast cancer patients, 476 familial breast cancer patients and 1148 controls were genotyped by Taqman assays for the 3 most common Dutch mutations, Y179C, G396D and P405L, the unclassified variant R309C and reported benign variant S515F (previously annotated as Y165C, G382D, P391L, R295C and S501F). Additionally, direct sequencing of the whole coding part of the MUTYH gene was performed in 306 patients. Genotyping showed 2.2, 2.5 and 1.7% heterozygote carriers for one of the three mutations among patients, familial patients and controls respectively, giving no significant difference. Variation in the frequencies of R309C and S515F was also not-significant between groups. Remarkable was a doubled frequency of G396D among familial patients as compared to controls. Sequencing revealed 3 coding and 6 intronic rare unclassified variants, without yet clues for pathogenicity. Overall, no bi-allelic mutation carriers were found. In conclusion, these findings could not confirm an association of MUTYH variants with breast cancer, although G396D may be very weakly associated.

Detecting somatic mosaicism of the APC gene with high resolution melting curve analysis A. A. Out1, I. J. H. M. van Minderhout1, A. C. Schaap1, R. Louiszoon1, E. C. Bik1, C. M. Tops1, R. H. A. M. Vossen1, N. van der Stoep1, E. Bakker1, H. F. A. Vasen2,4, H. Morreau3, P. Devilee1,3, F. J. Hes1 Center for Human and Clinical Genetics, 2Department of Gastroenterology, 3Department of Pathology, Leiden University Medical Center, 4The Netherlands Foundation for the Detection of Hereditary Tumours, Leiden, The Netherlands

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Abstracts Given the relatively high frequency of de novo APC mutations in polyposis patients, a substantial proportion of mosaic APC mutations can be expected. In a previous study 10 mosaic cases were identified among 242 APC mutation carriers (4%) using direct sequencing, PTT and DGGE. Scanning APC and MUTYH negative polyposis patients with a uniform sensitive method may reveal more mosaic cases. From 208 patients, leukocyte derived DNA was scanned with High Resolution Melting curve analysis (HR-MCA) for mutations in APC exons 4–6, 8–12, 14 and part of exon 5. To test the sensitivity for detection of mosaic mutations, DNA samples with different heterozygous mutations were diluted with wild-type DNA to create a range of allelic percentages. Samples with aberrant melting curves were analyzed by direct sequencing analysis and variants were additionally quantified by pyrosequencing. The median detectable level of mosaicism in diluted samples was 6%, which varied between different amplicons and mutations (2–25%). So far, one mosaic APC mutation carrier was detected. A C.1958+1G[T change in exon 14 was observed, showing a minimal elevated T-peak on direct sequencing, whereas HR-MCA and pyrosequencing analyses showed a 15–20% allele frequency. Furthermore, 6 previously undetected heterozygous pathogenic mutations were found. These results prove that HR-MCA is a promising and sensitive method for screening and quantification of mosaic mutations. However, the minimal detectable frequency is variable. Our aim is to further optimize and complete the analysis for the whole APC gene, to screen further polyposis patients and to set up mutation analysis in formalin fixed paraffin embedded (FFPE) polyp tissue.

The MUTYH gene variant database Astrid A. Out1, Carli M. J. Tops1, Maartje Nielsen1, Marjan M. Weiss1, Hans F. A. Vasen2,3, Johan T. den Dunnen1, Stefan Aretz4, Julian R. Sampson5, Peter Devilee1, Frederik J. Hes1. Center for Human and Clinical Genetics, 2Department of Gastroenterology, Leiden University Medical Center, 3 The Netherlands Foundation for the Detection of Hereditary Tumours, Leiden, The Netherlands, 4Institute of Human Genetics, University of Bonn, Bonn, Germany; 5Institute of Medical Genetics, School of Medicine, Cardiff University, Cardiff, Wales, United Kingdom 1

The MUTYH gene encodes a DNA glycosylase involved in base excision repair. A bi-allelic defect in this gene, leading to accumulating somatic G to T transversions in genes like TP53, APC and KRAS, is associated with colorectal polyposis and cancer. Whereas much is known about the two most frequent mutations Y179C and G396D (previously annotated as Y165C and G382D), the effect of uncommon variants is less well known. Especially, the very rare and unreported unclassified variants remain a holdup in molecular diagnostics. The open access MUTYH gene variant database (www. lovd.nl/MUTYH) aims to supply a role in internationally collecting and sharing of useful variant data combined with available phenotypic information. The current content consists of data extracted from published literature and an increasing number of unpublished variant records submitted by different international institutes. Submitted variants will become public after curation and annotation conform to current mutation nomenclature (reported nomenclature also listed). International submission could be especially helpful to catalog and exchange information on rare variants, which would be impossible through published literature. This locus-specific database (LSDB), in the Leiden Open (source) Variation Database (LOVD) format, is easy to consult and to submit information to. Furthermore, it is maintained following the recommendations of the Human Variome Project (HVP). With our own efforts, available and future collaborations, we

739 aim to maintain an up-to-date online resource of MUTYH data, valuable for researchers and clinicians in the field.

Laparoscopic surgery in patients with colonic polyposis syndromes Francisco Lo´pez-Ko¨stner, C. Heine, P. Medina, U. Kronberg, C. Wainstein Colorectal Surgery Unit, Clı´nica las Condes. Santiago, Chile Background Laparoscopic colorectal surgery has been established as a safe procedure for patients with cancer and diverticular disease of the colon. Patients with colonics polyposis syndromes have a special condition determined by the magnitude of the procedure (total colectomy or proctocolectomy) and because most of the time this is a profilactic surgery (preventing the development of cancer). Aim To analyze results of laparoscopic surgery in patients with colonic polyposis syndromes. Materials and methods Data where obtained from the prospective database of laparoscopic colorectal surgery. We selected all patients operated on by colon polyposic syndromes (Familial adenomatous polyposis, Familial attenuated adenomatous polyposis and Mixed polyposis) between years 1998 and 2008. Results In these period where operated on 25 patients with colonic polyposis syndromes. Sixteen patients had classical familial adenomatous polyposis (FAP), 9 had attenuated familial adenomatous polyposis (AFAP) and one had mixed polyposis. The mean age was 41 years presenting a bimodal distribution according the type of disease (average 33 years in PAF and 55 years in APAF). Fifty-six percent were woman. Total colectomy with ileorectal anastomosis was performed in 18 patients (72%), and restorative proctocolectomy (ileal pelvic reservoir) in seven (28%). The mean operating time was 270 min (r: 180–400). The conversion rate was 5% (one patient) due to anatomical difficulties. Liquid diet was started at 36 h and the average hospital stay was 5.7 days. Complication rate was 10% (two patients) and there was no operative mortality. Conclusions The laparoscopic access is a feasible and a safe alternative for patients with colonic polyposis syndromes.

Computed tomographic colonography to assess perioperative colorectal polyp burden in familial adenomatous polyposis Margaret O’Malley, James Church, Lisa LaGuardia, Jon D. Vogel, Grace Cheah, Mark Baker, Carol A. Burke Digestive Disease Institute, Sanford R. Weiss, MD Center for Hereditary Colorectal Neoplasia, Cleveland Clinic, Department of Colorectal Surgery, Department of Radiology, Department of Gastroenterology, Cleveland, Ohio, USA Background CT Colonography (CTC) efficacy to assess timing for colectomy in FAP is unknown. Methods FAP patients scheduled for primary colon surgery age [13 years were eligible. CTC and colonoscopy were performed day of surgery by operators blinded to results of the other study. Size and number of colorectal polyps on CTC and colonoscopy were compared. Pathologic review of the surgical specimen was performed. Findings documented include ranges of colon and rectal polyp number, size of largest polyp, and potential mass lesions.

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740 Results Ten patients, 7 male, were enrolled (mean age 32). All subjects had 100–1000 polyps with largest mean polyp size of 22 mm on pathology (17.5 mm on CTC and 18.7 mm on colonoscopy). One patient had a rectal and colon cancer detected on all 3 modalities. CTC and colonoscopy agreed on colon polyp number in 70% but in 20% CTC count was lower than colonoscopy. The respective agreement on rectal polyp number was 60%, while CTC underestimated rectal polyp count in 40%. The number of colon polyps on CTC agreed with pathology in 50%, but was lower in 50%. Colonoscopy agreed with pathology on 60% but was lower in 40%. 3 patients had proctocolectomy: colonoscopy agreed with pathology on rectal polyp burden in 75%, CTC suggested a lower burden in 75%. Extracolonic findings reported on CTC in 40%; one possible pancreatic tumor, 2 patients with pulmonary nodules, and one adrenal mass removed during surgery and confirmed adenoma. Follow up CT ruled out a pancreatic abnormality, and pulmonary nodules were found to be calcified granulomas. Conclusions CTC underestimates colon and rectal polyp burden up to 40% in FAP patients compared to colonoscopy. Both modalities underestimate colorectal polyp burden compared to pathology but don’t miss significant lesions. Extracolonic findings are common, none impacted FAP surgery.

APC alternative splicing as responsible for phenotypic variability in familial adenomatous polyposis S. Gonza´lez1, M. Mene´ndez1, I. Blanco2, A. Obrador-Hevia3, C. La´zaro1, G. Capella´1 1

Translational Research Laboratory, Catalan Institute of Oncology, Genetic Counselling Unit, Catalan Institute of Oncology, 3 Biology of Cancer Group, University of Balearic Island 2

Familial Adenomatous polyposis (FAP) is a dominantly inherited colorectal tumour predisposition syndrome that results from germ-line mutations in the Adenomatous Polyposis Coli (APC) gene. FAP shows substantial phenotypic variability: classical polyposis patients develop more than 100 colorectal adenomas, whereas those with attenuated polyposis (AFAP) have fewer than 100 adenomas usually associated with late onset. Abnormalities of pre-mRNA splicing are increasingly recognized as an important mechanism through which gene mutations cause disease and may affect disease expression, leading to phenotypic variability. The aim of the study was to elucidate the molecular basis of the phenotypic variability of one FAP and one AFAP families harbouring the same c.834+1G[A APC mutation. RNA-based studies were performed in six members of family A characterized by the presence of thyroid cancer in most cases, desmoids tumours and a classical FAP phenotype and in eight members of family B, characterized by three generations of affected members displaying AFAP phenotype. Eleven healthy controls were also included. Lymphocytes of two carriers per family were cultured and treated with puromycin, a translational inhibitor, before RNA extraction. Family A: In affected members RNA analyses of exons 5–9 showed the appearance of two APC isoforms (WT, and a 11 bp frameshift deletion that will generate a truncated protein (p.Asn276PhefsX8). Family B: Affected relatives harbouran additional intronic change c.730-29A[T in APC gene, that does not predict any splicing aberration. These relatives displayed a third transcript (321 bp) with an in-frame exon 7 skipping (p.Arg244_Gln278del). Surprisingly, this transcript also appears in family A after puromycin-treatment. The c.834+1G[A APC mutation generates a complex pattern of mRNA splicing. Our findings suggest that the presence of c.73029A[T in family B is associated with increased levels of transcripts

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Abstracts lacking exon 7. Differences in mRNA processing may modify phenotypes associates with APC gene mutations.

Haplotype analysis and age estimation of the Y165C and G382D mutations in German MAP patients Stefan Aretz, Stefanie Vogt, Daniela Tavian, Astrid Kaufmann, Dietlinde Stienen, Markus M. Noethen, Sven Cichon, Waltraut Friedl, and Roberto Colombo Institute of Human Genetics, Bonn, Germany Background In Caucasian MAP patients, the combined allele frequency of the mutations Y165C and G382D ranges between 50 and 82%, while these mutations have not been identified in Asian populations, supporting the hypothesis of a founder effect that occurred in the history of Caucasians. Methods We genotyped 5 intragenic SNPs and 10 gene-flanking STRPs in 32 unrelated German MAP patients who were homozygous or compound-heterozygous for the Y165C and G382D mutations. To investigate the natural history of the two common MUTYH alleles, we calculated their apparent age in generations (g) by a set of population-genetic algorithms and under different assumptions on the spread and molecular evolution of the founder haplotypes. Results. A fully conserved intragenic SNP haplotype and two highly conserved core STRP haplotypes were identified in 63% of mutation-bearing chromosomes. An ancestral haplotype could be identified which occurs at significantly increased frequency in the Y165C and G382D chromosomes. According to parametric and Bayesian analysis of linkage disequilibrium’s decay over time, the age of the most common recent ancestors of the two MUTYH mutations was estimated as 62–87 g (overall 95% CI 49–120 g) and 136–184 g (overall 95% CI 105–206 g) for Y165C and G382D, respectively. Conclusions Our results suggest that the Y165C and G382D chromosomes sampled in German MAP patients derive from two ancestors that lived between the sixth century B.C. and the third century A.D., and between the middle of the third millennium B.C. and sixteenth century B.C., respectively. Comparative haplotype studies on chromosomes bearing the same MUTYH mutations in different populations will refine our knowledge of their origin and spread into the European continent. The demonstration of founder effects has implications for MUTYH mutation analysis in polyposis patients of different ethnic origin. The study was supported by the German Cancer Aid (Deutsche Krebshilfe) and the CARIPLO Foundation.

‘‘High Risk’’ clinic for hereditary colorectal neoplasia: a focus for patient care and an opportunity for clinical research Lisa LaGuardia, Margaret O’Malley, Jon D. Vogel, Brandie Leach, Carol Burke, Matthew Kalady, James Church Patients with Hereditary Colorectal Neoplasia and their families need specialized care to plan for appropriate surveillance and to ensure that they receive the most favorable treatment. This involves coordinating multidisciplinary appointments on the same day to minimize inconvenience to patients. We have established a special ‘‘High Risk’’ clinic for these patients and their families. In this study we are reporting our activity for the last 5 years.

Abstracts Methods Initially the clinic ran one morning a month but has grown in the last 2 years adding another half day session. Requests for appointments were triaged by Registry Coordinators. Patients with syndromes of Hereditary Colorectal Neoplasia were eligible for this clinic if the necessary appointments included multiple physicians. The Clinic is staffed by one of three colorectal surgeons, one gastroenterologist, one genetic counselor, one hepatobiliary/upper GI surgeon and often by a clinical geneticist. Results From January 2004 to November 2008 there have been 440 patient visits, 68 colonoscopies, 180 flexible sigmoidoscopies, and 226 EGD’s. 44 consults to medical genetics were performed, and 9 to General surgery. Clinic activity generated 101 surgeries including 37 colectomies and 7 duodenectomies. If all the appointments were done separately this would mean at least 967 separate visits. Conclusion The High Risk Clinic is a valuable resource for patients, insurers and registry workers.

APC germline allele-specific expression in familial adenomatous polyposis E. Castellsague´1, S. Gonza´lez1, I. Blanco2, E. Guino´3, C. La´zaro1, S. Gruber4, G. Capella1 1 Laboratori de Recerca Translacional, Institut Catala` d’Oncologia, IDIBELL, Hospitalet de Llobregat, Spain; 2Unitat de Consell Gene`tic, Institut Catala` d’Oncologia, IDIBELL, Hospital Duran i Reynals, Hospitalet de Llobregat, Spain; 3Unitat de Bioestadı´stica i Bioinforma`tica, Institut Catala` d’Oncologia, IDIBELL, Hospitalet de Llobregat, Spain; 4University of Michigan, Ann Arbor, MI, United States

Background About 13% of Familial Adenomatous Polyposis (FAP) families and 70% of Attenuated FAP (AFAP) families remain with unknown molecular pathogenic cause after APC and MYH mutational analyses. Also, mutations can affect allele expression at the germline level. Aim The aim of the study was to determine the presence of germline allele-specific expression (ASE) in the APC gene in FAP and AFAP with and without detectable APC or MYH mutations.

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Is familial adenomatous polyposis a ciliopathy? Encarna B. Gomez Garcia, Nine V. A. M. Knoers Department of Genetics and Cell Biology, Maastricht, The Netherlands Familial adenomatous polyposis (FAP) is an autosomal dominant form of intestinal polyposis and colorectal cancer caused by germ-line mutations in the APC gene. The term Gardner’s syndrome is used to describe a subset of families with FAP in which the extra-colonic manifestations, such as osteomas, skin cysts, congenital hypertrophy of the retinal pigmented epithelium (CHRPE), and desmoid tumors (aggressive fibromatosis) are specially prominent. Our hypothesis is that a ciliary dysfunction is the underlying pathogenetic mechanism of the extra-intestinal manifestations observed in patients with FAP. This hypothesis is based on the presence of common clinical manifestations (cysts, retinal abnormalities, fibrosis) in Gardner’s syndrome and cilia-related disorders. Secondly, both APC and the cilia have degradation of b-catenin as common downstream target in the WNT-signalling pathway. Mutations in the APC gene causing Gardner’s syndrome are clustered in a region encoding a series of amino acid repeats responsible for the binding to b-catenin. Proofs of principle that b-catenin can be the key mediator of the ciliary disorder rely also in the observations that overexpression of b-catenin induces polycystic kidney disease, as well as CHRPE phenotypes in animal models. Other candidates to be the common link are the APC-binding proteins: EB1 and Kif3a, both of them are ciliary proteins involved in intraflagellar transport. Finally, pathogenetic similarities between some ciliopathies and the extra-intestinal tumors observed in FAP indicate that also the latter can have a cilia defect. In conclusion, from the perspective of FAP as a ciliary disorder, the presence of extra-colonic manifestations can now be understood, and it may add a whole new range of therapeutic options for those patients.

Adenomatous polyposis coli germline mosaicism in a patient with classical Judith Necker, Miche`le Attenhofer, Bruno Reichlin, Karl Heinimann

Methods Germline RNA from fresh frozen and/or cultured lymphocytes of 17 APC/MYH-negative Polyposis (7 FAP, 10 AFAP) families (21 individuals) and 26 APC-mutated Polyposis (21 FAP, 5 AFAP) families (45 individuals) was analysed. Fourteen controls were also studied. ASE was investigated by single nucleotide primer extension (SNuPE) of rs2229992 APC coding SNP. Results In controls ASE was 1.05 ± 0.05. We found that 17% (3 of 17) APC/MYH(-) FAP/AFAP families showed ASE (range = 1.17– 1.39) and ASE co-segregated with disease. ASE was more intense in short-cultured lymphocytes and reversed by puromycin treatment. Experimental approaches are being performed to elucidate the molecular mechanism leading to APC ASE in these families. Eleven of 26 (42%) APC-mutated FAP/AFAP harboured ASE (range = 1.20– 8.54) being the mutant allele the under expressed one. ASE was restricted to splicing, nonsense and frameshift mutations outside exon 15. Again, puromycin reversed ASE in all cases analysed. Conclusions APC ASE is present in a significant proportion (17%) of APC/MYH(-) FAP/AFAP. ASE, due to nonsense-mediated decay (NMD), is present in APC-mutated Polyposis and is associated with specific mutation location, similar to reports for other hereditary syndromes. Our results show that expression-based molecular tests could have an important role in the molecular diagnostics of Polyposis.

Research Group Human Genetics, Department of Biomedicine, Basel, Switzerland Familial adenomatous polyposis (FAP) is an autosomal dominant cancer predisposition syndrome characterized by multiple colorectal adenomas, which, unless removed, inevitably develop into colorectal cancer. FAP is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Somatic mosaicism has been reported to account for 11–20% of patients with apparently de novo APC mutation. Here, we present the rare instance of a FAP patient with APC germline mosaicism. The index patient was diagnosed of classical polyposis and rectal cancer at age 41. While his parents were reportedly healthy, his two daughters displayed profuse polyposis at age 18 and 23, respectively. Analysis of leukocyte-derived DNA from the index patient using the protein truncation test (PTT) revealed a barely visible additional band at 1.3 kb, indicative of a truncating mutation in the region of exon 15c–15e. By conventional sequencing of the entire coding region and gene dosage analysis, however, no pathogenic alteration could be identified. Subsequent APC mutation analysis of one of the patient’s daughters revealed a strong additional PTT band at 1.3 kb resulting from a frameshift mutation in APC exon 15c (c.2802_2805delTTAC, p.Thr934ThrfsX19).

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742 Detailed mutation analysis of DNA from epithelial and smooth muscle portions of normal mucosa and adenomas confirmed the presence of the c.2802_2805delTTAC alteration in the index patient. Preliminary data from quantification analyses indicate that the proportion of mutated alleles in these tissues varies between 13 and 46%; further investigations on tissues originating from all three germ layers are under way. In conclusion, our data on a FAP patient with APC germline mosaicism (a)

highlight the importance of applying several mutation detection methods instead of relying solely on DNA sequencing (b) emphasize the need for early medical surveillance of the offspring, since disease severity may increase in the next generation, as strikingly exemplified in this family.

c.891 + 3A [ C is an Italian recurrent MutYH mutation associated with production of aberrant mRNA transcripts Viel Alessandra, Pin Elisa, Pastrello Chiara, Fornasarig Mara, Urso Emanuele, Tricarico Rossella, Tibiletti Maria Grazia, Genuardi Maurizio

Abstracts Studies into the function of the Base Excision Repair pathway and its role in oxidative damage repair have been fuelled by the identification of MUTYH defects predisposing to colorectal neoplasia. Initial studies identified that bi-allelic germline MUTYH mutations were responsible for a proportion of attenuated FAP families, which led to the term MUTYH-associated polyposis (MAP) syndrome. However, colorectal cancer case–control studies have demonstrated strong evidence that bi-allelic defects of MUTYH also predispose to cancer in the absence of multiple polyps. The question remains as to whether mono-allelic defects impart an increased risk to disease. Various studies have provided contradictory evidence to the role of monoallelic defects but the rarity of the 2 most common mutations (Y176C MAF * 0.005; G393D MAF * 0.01) reduces the power to detect weaker effects. Hence in order to refine the risk of bi-allelic defects and understand the role of mono-allelic defects, in December of 2006 we invited world-wide groups, which had published data on MUTYH analysis of colorectal cancer cases and controls, to participate in a large meta-analysis of their data sets. We were also approached by a number of other groups who were in the process of screening for MUTYH defects giving a total of 10 participating groups. We are currently in the final stages of analysis and we would like to present our findings on the role of bi-allelic and mono-allelic MUTYH mutations on colorectal cancer risk after analysis of the whole dataset (consisting of 16482 cases and 14732 controls) and by age and sex stratification.

Quaia Michele Centro di Riferimento Oncologico, Aviano, Italy In addition to p.Tyr165Cys and p.Gly382Asp, specific mutations of the MutYH gene have been identified in different populations and diagnostic screening strategies could be optimized accordingly. In our mutational screening of patients with suspected MutYH-Associated Polyposis (MAP) living in the North East of Italy, we have also frequently identified the c.891+3A[C mutation in both homozygous and compound heterozygous conditions. This mutation was in fact detected 12 times in 11 probands/families and it represents the 19% of mutated alleles in our population. The clinical phenotype of the only homozygous c.891+3A[C/c.891+3A[C carrier, characterized by development of less than 30 adenomas at age 51 years, was consistent with the diagnosis of attenuated polyposis. By RNA analysis, we demonstrated that this variant, changing the third base of intron 10, completely disrupts the normal splicing process, producing out-of-frame transcripts lacking exon 10 (145 bp) and transcripts lacking exon 10 and retaining intron 11 (173 bp). Lymphoblastoid cell lines from one homozygous and 4 compound heterozygous patients have been obtained and analyzed at the protein level. Western blot analyses with a mcAb directed toward the a.a. 436–536 indicated that production of the full length MutYH protein is completely abrogated. Loss of the C-terminal domain gives rise to a defective protein probably unable to recognize 8-oxoG and bind DNA, thus resulting in severely impaired DNA repair. Despite frequent recurrence in our series of MAP patients, the c.891+3A[C mutation has been reported only rarely in other populations. A founder effect is suspected, since all patients carrying this splice variant live and/or originate from the same geographic area (North East of Italy). Allelotype analyses are in progress to verify this hypothesis.

Are there age and sex effects on the influence of MUTYH variants on colorectal cancer risk? S. M. Farrington, E. Theodoratou, H. Campbell, A. Tenesa, M. G. Dunlop and The MUTYH Meta-Analysis Collaboration University of Edinburgh, Colon Cancer Genetics Group, 4th Floor MRC Human Genetics Unit, Edinburgh, Scotland

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Allele-specific expression of the APC gene in mutationnegative patients with adenomatous polyposis Stefanie Vogt, Astrid Kaufmann, Dietlinde Stienen, Nils Rahner, Verena Steinke, Markus M. No¨then, Per Hoffmann, Stefan Aretz Institute of Human Genetics, Bonn, Germany Background In a substantial number of patients with colorectal adenomatous polyposis the genetic basis of the disease is unknown. Recent studies indicate that reduced expression of tumour suppressor genes may have pathogenic relevance. Methods We examined the allele-specific expression of the APC gene in unselected patients with [10 synchronous colorectal adenomas, in whom no APC or MUTYH germline mutation was identified, by use of a SNaPshot analysis, a primer extension method. In 31 unrelated patients and 10 normal controls who were informative for two APC polymorphisms the ratio of the peak areas of the different alleles (nucleotide ‘‘C’’/‘‘T’’ in codon 486 and ‘‘G’’/‘‘A’’ in codon 535) in cDNA relative to genomic DNA (gDNA) extracted from blood samples was determined. Results In controls, the median cDNA/gDNA peak ratio was 0.89 (SD ± 0.13). 8/31 patients (26%) showed reduced (peak ratio ≤0.6) allelic mRNA expression, the degree of reduction ranged from 40 to 72%. In 3 patients results were inconsistent; the remaining cases had balanced expression. 6/8 cases showing reduced expression had an attenuated, 2 had a classical colorectal phenotype. Conclusions These findings suggest that reduced mRNA expression may be causative for the development of a polyposis in a subset of mutation-negative patients. Unbalanced allelic mRNA expression point to cryptic germline mutations in the APC gene or associated regulatory regions that are not detectable by standard methods of mutation analysis. However, the underlying mechanisms remain to be uncovered, yet. The study was supported by the Deutsche Krebshilfe (Grant no. 108421)

Abstracts

Association between colorectal cancer and MYH mutations in sporadic tumors and in attenuated familial polyposis coli (AFAP) E. Urso1, M. Agostini1, S. Pucciarelli1, I. Mammi1, A. Viel2, I. Maretto1, D. Nitti1 l

Clinica Chirurgica 2^, Dipartimento di Scienze Oncologiche e Chirurgiche, Azienda Ospedaliera-Universita` di Padova, Italy; 2 Oncologia Sperimentale 1^, Centro Regionale Oncologico, IRCCS, Aviano, Italy Background While MYH biallelic mutations are clearly related to the Attenuated Familial Adenomatous Polyposis (AFAP), the association to an increase risk of colorectal cancer (CRC), and the phenotype of monoallelic MYH mutations are still unclear. Aim To Investigate in a prospective series of CRC and AFAP patients the association between MYH mutations and colonic tumors. Methods Study population consisted in a consecutive unselected series of CRC patients who underwent surgery in 2003 and a consecutive cohort of 37 AFAP patients observed from 2003 to 2008 at Padova Hospital. All patients were screened for the most frequent MYH germline mutations (Y165C, G382D, IVS10+3A[C, 1395-7delGGA).

Results Of 430 CRC patients [F = 170; median age 67 years], 33 were B50 years old (yo). Two biallelic and 2 monoallelic MYH mutations were found and 1 patient B50 yo had monoallelicmutation (3% of the early onset CRC). 37 AFAP patients [F = 11; median age 47 years] were observed; median (range) polyps number was 40 (10–100), and 3 APC mutations were found. Seventeen AFAP patients had CRC and 11 of them were B50 yo. MYH biallelic and monoallelic mutations were found in 6 and 3 patients of the whole series, respectively. The corresponding figure for AFAP patients with CRC were 3 and 2, respectively; and the corresponding figure for AFAP patients with CRC B 50 yo were 2 and 1, respectively. Incidence of MYH biallelic and monoallelic mutation is not statistically different in AFAP patient withor without cancer, even considering AFAP patients with CRC B 50 yo. Conclusions The Incidence of MYH mutations is low in unselected CRCs patients; however it is not so negligible in patients B50 years. In AFAP patients, the presence MYH mutation (either mono or biallelic) is not associated with an increase incidence of CRC, also considering patients B50 yo.

Changing trends in causes of mortality for patients with familial adenomatous polyposis Ashish Sinha, S. Rashis, R. K. S. Phillips, S. K. Clark St Mark’s Hospital, Polyposis Registry, St Mark’s Hospital, Harrow, London, UK Introduction Widespread use of prophylactic colectomy in familial adenomatous polyposis (FAP) has resulted in reduction in the number of deaths due to colorectal cancer. Now extracolonic manifestations of FAP are thought to be the leading cause of mortality in these patients. Methods We retrospectively examined the cause of death in patients with a confirmed diagnosis of FAP who had undergone primary surgery at our institution, using the Polyposis Registry database. Results Of the 559 patients who underwent primary surgery for FAP at our institution 146 (70 male) have since died. Information was available on 143 patients. The median age of death before 1990 was 45 years (interquartile range 35–54) and since 1990 was 52 years

743 (40–62); p = 0.022 (Mann–Whitney U-test). Metastatic colorectal cancer caused 53 (37.1%) deaths at a median age of 43 years (35–54). Eleven of these patients had known cancers at the time of surgery, 42 subsequently developed rectal cancer and metastatic disease. Desmoids caused mortality in 16 (11.2%) at a median age of 34 years (25–42). Upper gastrointestinal malignancies caused 26 (18.2%) deaths at a median age of 53 years (43–66). Other causes of mortality included 28 (19.6%) non-FAP related deaths at a median age of 54 years (45–66) and 7 (4.9%) secondary to non-FAP related malignancies; median age 52 years (49–60). There were 13 (9.1%) perioperative mortalities. Nine (11.5%) and 4 (6.2%), p = 0.03 (Mann–Whitney U-test) were pre and post 1990 respectively. These occurred at a median age of 34 (25–39) and 53 (42–55), p = 0.01 (Mann–Whitney U-test) pre and post 1990 respectively. Conclusion Metastatic colorectal cancer remains the leading cause of death in FAP. In recent decades more deaths are attributable to extracolonic manifestations. Cardio-respiratory disease is the leading cause of non-FAP related death. Post 1990, surgery appears to be safer with significantly fewer perioperative mortalities.

How a nurse practitioner specialising in pollyposis supports both patients and doctors Muditha Samarasinghe, Kay Neale, Pam Nye The Polyposis Registry, St Marks Hospital, Harrow, Middlesex, UK Medical staff come to our hospital to gain experience in colorectal conditions. They stay for a maximum of 1 year but often only 6 months. On arrival they have rarely encountered patients with polyposis and know little of these syndromes. When they leave, they take their valuable, newly acquired knowledge with them. The patients inevitably suffer from this fluctuating level of experience. In her clinic the Nurse Practitioner sees patients who are attending for routine follow up. She carries out a full physical examination, reviews the endoscopy and other investigative reports. The clinics run concurrently with the Consultants’ clinics in order to provide immediate expert advice if patients present with new findings. On average 500 patients a year will be seen by the Nurse Practitioner alone. In addition medical staff will request a joint consultation with the Nurse for a similar number. Advice is given on things such as: • • • • •

Screening intervals Familial tendencies Genetic variation Genetic testing Screening protocols for desmoid disease or adrenal adenomas

The Nurse will also take over from the doctor to provide psychological support when necessary releasing the doctor for the next patient. Between clinic visits these patients require easy access to specialist medical advice. In addition they have particular psychological, emotional and social needs requiring sensitive management. The Nurse Practitioner works closely with the Nurse Specialist and Administrative staff and: • • •

a telephone and email helpline are provided patients who miss an appointment are contacted; the importance of screening is discussed and another appointment offered customised clinic lists are produced to alert the nurses to specific points to be covered at individual patients’ visits

Conclusion Polyposis syndromes are complex; patients’ lives are enhanced by continuity of care and doctors are helped by a specially trained nurse.

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‘‘Peyer’s patch’’: Two cases in familial adenomatous polyposis patients after colectomy R. Man St. Mark’s Hospital, Harrow, Middlesex, UK Introduction Peyer’s Patches, gut-associated lymphoid tissue (GALT), are lymphoid follicles (Gullberg & Soderholm 2006). They are associated with Crohn’s disease (Shikuwa et al. 2007), malignant lymphoma (Rappaport et al. 1971), idiopathic intussusception (Hasegawa et al. 1998) and spongiform encephalopathy. They often described as polyps and may be mistaken for dysplastic lesions. Aim and method This study aimed to report and describes the macroscopic appearance of Peyer’s patch using various endoscopic imaging techniques in two FAP patients with ileoanal pouch. A retrospective search of our endoscopy database identified two FAP patients (26 years old and 46 years old respectively) both with an atypical ileal lesion found during surveillance endoscopy (15 years and 2 years after pouch surgery respectively). The macroscopic appearance and characterization of these lesions was examined with three different imaging techniques: conventional endoscopy with white light, chromoendoscopy with indigo carmine and electronic chromoendoscopy with narrow band imaging. Biopsy was taken to for histological assessment. Results Both lesions appeared to be elevated from the surface mucosa. Round lymphoid follicles were identified on close observation. Enhanced imaging with indigo carmine and narrow band imaging ascertain the absence of dysplastic features. No excision applied to any of these lesions. Histology later confirmed the present of lymphoid follicles only. Conclusion Recognition of different lesions is one of the core objectives in endoscopy training. Advances in endoscopy, such as video capsule endoscope and double balloon enteroscopy, increase the observation of these unusual lesions in the more accessible distal ileum; in particular in FAP after colectomy with easy access to the distal ileum. Unusual lesions may not be familiar to some endoscopists. Descriptions of macroscopic appearance of Peyer’s patch in endoscopy are limited in the literature (Hizawa et al. 1996, Fujimura et al. 1996, Fujimura & Owen 2000). Accurate and early identification and differentiation of the types of pathology may allow prompt treatment and management. Inability to recognize these lesions may lead to unnecessary excision and the associated risks and complications. Enhanced imaging techniques may allow detailed interpretation of mucosal lesions, and may help reducing this problem. Peyer’s patch can be found in normal healthy person. Implication of Peyer’s Patch in FAP patient is not clear.

Completion proctectomy: three case studies in FAP patients with ileo-rectal anastomosis R. Man, K. Neale, S. K. Clark St. Mark’s Hospital, Harrow, Middlesex, UK Introduction FAP patients with ileo-rectal anastomosis need regular surveillance of their retained rectum. Any detection of high risk dysplastic lesions requires prompt excision. The choice of management could vary individually and sometimes could be difficult. This study attempted to highlight the management of three cases with high risk rectal lesions. Aim and method This study aimed to explore the choice of management in patients with high risk rectal lesions detected during endoscopic surveillance. A retrospective search from the St. Mark’s

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Abstracts polyposis registry identified three cases. Their management choices and its implications were examined. Case 1 A 55 year old man with FAP had a rectal lesion removed during a surveillance endoscopy, 29 years after IRA. Histology confirmed invasive carcinoma. Screening imaging including endorectal ultrasound, CT scan and blood tests found no evidence of local invasion or metastasis after endoscopic polypectomy. Hence, proctectomy was not performed. The patient died 10 years later from a malignant kidney tumour. Case 2 A 65 year old women with FAP was found to have a severely dysplastic lesions in the rectum, 22 years after IRA. The rectal lesion was removed endoscopically. Completion proctectomy was declined by patient. No further high risk rectal lesion was detected with an endoscopic follow up of 4 years. Case 3 A 62 year old women with FAP had multiple severely dysplastic rectal lesions detected 2 years after the construction of IRA. Proctectomy was attempted but failed due to the presence of extensive desmoid disease. Learning difficulties also limited her choice for end-ileostomy. She settled with regular endoscopy and the use of chemoprevention as a result. Seven years later, repeated histological reviews of the lesion remain severely dysplastic. Conclusion Completion proctectomy is usually suggested when rectal cancer is detected in FAP patients after ileo-rectal anastomosis. Advances in therapeutic endoscopy allow safe and effective excision of mucosal lesions including polyp cancer. It is not unknown whether surgery can be delayed if the rectal lesion can be completely removed locally. High risk rectal lesion in IRA is the most common predicting factor and indication for surgery. Not all IRA patients with high risk rectal lesion proceed to proctectomy. When deciding the need for proctectomy in FAP patients with ileo-rectal anastomosis, the situation can be complicated especially in mentally and physically compromised patients. The long term risk in patients who choose not to or failed to convert from IRA to pouch or end-ileostomy is not known. Endoscopic monitoring and chemoprevention may be employed as comprising options. These should be considered with caution.

Peutz–Jeghers syndrome: screening and follow-up Shirley V. Hodgson, Andrew D. Beggs St Georges, University of London, London, UK Peutz Jeghers syndrome (PJS) is a rare autosomal dominant condition characterised by the development of multiple hamartomatous polyps throughout the bowel, and mucocutaneous pigmentation which is variable, develops during childhood and may fade in later adulthood. These polyps develop in early childhood, predominantly in the small intestine but also in the large bowel and stomach. Diagnosis is often made by obstruction from intussusception (43%) or obstruction (23%); abdominal pain from infarction (14%) or rectal bleeding from ulceration (7%). A recent review by Giardello et al. estimated lifetime risk of GI cancer as 48% for the small bowel, 24% gastric, 24% colon, 5% pancreas (up to 100-fold increase in risk reported) and female breast cancer 32% by 60 years age. There was also an increased risk of sex cord tumours of the ovary or testis, which can secrete estrogenic hormones, which may explain the increased risk of adenoma malignum of the cervix. The total risk of death from cancer in PJS has been estimated to be 48% by age 57 years. Mutations in the STK11 serine/threonine kinase gene on chromosome 19p13.3 are responsible for PJS, with no clear evidence for genetic heterogeneity, and only a minor suggestion of genotype/ phenotype correlations. Clinical management has been developed to detect early neoplastic lesions, and includes annual clinical evaluation,

Abstracts abdominal and pelvic ultrasound, cervical smears and breast examination, biennial ‘‘top and tail’’ endoscopy & small bowel series, and mammography for affected women from 25 years age. Surveillance for pancreatic cancer remains problematic, and screening protocols are being revised in the light of clinical evidence. We have set out to obtain follow-up data on patients with PJS ascertained from centres around Europe to try and gain further insight into the incidence of cancer and the effectiveness of surveillance in affected individuals, and preliminary data from this will be presented.

The mutational spectrum of APC in Greek FAP patients including a distinct mutation on the alternative splice site of exon 9 Florentia Fostira1, Georgia Thodi1, George Fountzilas2,3, Drakoulis Yannoukakos1 1

Molecular Diagnostics Laboratory, I/R-RP, National Center for Scientific Research ‘‘Demokritos’’, Athens, Greece; 2Department of Medical Oncology, Aristotle University of Thessaloniki, Papageorgiou Hospital, Thessaloniki, Greece; 3Hellenic Cooperative Oncology Group, Athens, Greece Familial Adenomatous Polyposis (FAP), an autosomal dominant inherited disease caused by germline mutations within the APC gene, is characterized by early onset colorectal cancer as a consequence of the intrinsic phenotypic feature of multiple colorectal adenomatic polyps. Analysis of the APC gene performed in a Greek kindred consisting of twenty-five FAP families, revealed eighteen different germline mutations in twenty (80%) families, of which five novel. Twelve mutations are located within exon 15, carriers of which are characterized by a clearly earlier mean age of colorectal polyposis (30.4 years) compared to the rest, which are scattered between exons 3 and 11 (39.4 years). Interestingly, a novel mutation located on the alternatively splice site of exon 9 was identified in a patient with attenuated FAP phenotypic features. This is the first reported mutation in the specific site. Transcripts characterization revealed disruption of splicing occurring within exon 9, resulting in the expression of a shorter mRNA transcript, which surprisingly does not affect the ratio between the two wild type transcripts. The latter, along with the ‘three hit theory’, highlights the need for a third hit for cancer to develop, most probable contributing to the mildness of disease. This is the first full report on the clinical characterization and the mutation spectrum in Greek adenomatous polyposis families. Using all available published data on Greek FAP families, it is concluded that the population is characterized by genetic heterogeneity, lack of founder mutation in FAP syndrome and low incidence of genomic rearrangements in APC.

Your insight membership Kay Neale, Tina Isherwood The Polyposis Registry, St Mark’s Hospital, Harrow, Middlesex, UK In Japan in 2007 we presented a Poster to tell people about InSiGHT. It was felt important that current members should be helped to understand how the Society is organised and why they should continue to pay an annual subscription. Also, non-members of the Society attending the Scientific Meeting would be encouraged to join and told how to apply for membership. It is proposed that the poster giving the details listed below should be updated and presented again in Germany in 2009:

745 1. Names of the members of Council 2. The way in which the Officers and members of Council are elected 3. How Council is funded 4. Number of current members by country 5. Subscription rates and methods of payment 6. Distribution of the Journal 7. The database 8. Website address 9. Request for suggestions

Bowel phenomena and symptom experience after 6 months of total colectomy with familial adenomatous polyposis Yoshie Murakami Dult Nursing, Graduate School of Human Health Sciences, Tokyo Metropolitan University, Tokyo, Japan Purpose The purpose of this study was to explore FAP patients’ bowel phenomena and symptom experience during the first 6 months after undergoing a prophylactic colectomy. Methods A convenience sample of three patients, one female and two males were interviewed, using a semi-structured interview guide, based on the UCSF Symptom Management Model. Results The data revealed that three patients had six common bowel phenomena; defecation patterns, sense for defecation, ability to discriminate between stool and flatus, soiling, awareness of abdominal content movement (peristalsis), and awareness of bowel sounds. The data also revealed that each patient had unique perception of their symptom experience; diarrhea, fear of bowel blockage, and night soiling. All patients had cope with symptom experience and thought value of life, because they could defect colon cancer and not enough to death. Conclusion Nurses, to provide quality nursing care to FAP patient post-colectomy, need to become aware of each FAP patient’s unique perception of their symptom experience, as well as the bowel phenomena common to all such FAP patients.

MUTYH variants in Austrian patients with familial adenomatous polyposis-like symptoms: a first report Brigitte Wolf, Silke Gruber, Judith Karner-Hanusch Medical University of Vienna, Department of Surgery, Research Laboratories, Wien, Austria Background In the diagnosis of Familial Adenomatous Polyposis (FAP) a number of patients present an atypical medical history characterized by less polyps and an older age of onset. Some of these patients have been shown to bear an inherited defect in the base excision repair gene MutYH. Materials and methods We analyzed a series of 64 unrelated Austrian families clinically diagnosed with FAP (17; 26.6%), AFAP (31; 48.4%) or multiple colorectal adenomas (16; 25%) for mutations in the MutYH gene. All 16 exons of the gene were sequenced starting from genomic DNA. An inherited defect in the APC gene was excluded by sequence analysis and MLPA. Results and conclusion In 15 of 64 (23.5%) patients a mutation in the MutYH gene was identified. In nine (14.1%) patients both alleles

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746 were affected. Among the mutation carriers three (20%) patients presented classical FAP symptoms, 10 (66.7%) patients AFAP and two (13.3%) multiple colorectal adenomas. Three of the mutation carriers had polyps in the upper intestine and three developed colorectal cancer before the age of 50 years. Only one of the putative pathogenic missense variants has not been listed in the MutYH database before, but was detected in both alleles of an AFAP patient. Furthermore we identified 5 frequently reported variants and one variant 127 bp upstream of the gene. This study is the first comprehensive report of MutYH gene mutations in Austrian FAP and FAP-like patients.

Abstracts

Oncology Institute of Vilnius University and Vilnius University Santariskiu Clinic Center Branch, Vilnius, Lithuania

opportunity to meet families over and over again at follow up visits, or when they attend with relatives, to get to know them well and earn their trust. Conversations starting about football, pets or music sometimes lead to things rarely discussed or forgotten such as illegitimacy, infantile death or relatives distanced by family feud. Another advantage of working within the hospital is the opportunity to visit patients in the ward where they have time to talk. This is a good time to update the family pedigree, as relatives with a more extended knowledge may be visiting, and also to re-enforce information given previously at clinic visits. In addition to face to face contact time is spent in the office where advice and support can be given by telephone and e-mail and efforts are made to trace at risk relatives. Information obtained in the clinical setting is entered onto the Registry database and family trees drawn. A poster would give the opportunity to present more information about the role, including a case history of a young foreign national with a fondness for vodka who presented in A&E with pancreatitis and anaemia. Fortunately, he was seen by a knowledgeable doctor and FAP was diagnosed. The nurse was instrumental in ensuring that this extremely anxious and reluctant young man attended for surgery and subsequent care.

Introduction The Lithuanian Polyposis Register has been found in 1995. Up till then, no attempts for systematic registration, screening, treatment or follow-up have been made. The support for the initiation of the register has been received from EUROFAP project, and register itself has been established in a close collaboration with the Leeds Castle Polyposis Group.

Developing a protocol for genetic evaluation of APC-negative patients with multiple colorectal adenomas

The Lithuanian polyposis register: progress during past 14 years N. E. Samalavicius, T. Poskus

Materials and methods During the period January 1995 to December 2008, a total of 106 familial adenomatous polyposis (FAP) patients from 47 unrelated families were registered. Among them, 22 (46.8%) were isolated cases. Data on screening, surgical treatment, extracolonic manifestations and outcome have been investigated. Results Out of 106 registered FAP patients, 29 (27.4%) underwent prophylactic surgery, 49 (46.2%) were operated in the presence of colorectal cancer, and 18 (26.4%) were not operated. Methods of prophylactic treatment included either subtotal colectomy with caecorectal/ ileorectal anastomosis, or reconstructive proctocolectomy. Most of the patients with colorectal cancer underwent segmental resections. FAP patients were examined for extracolonic manifestations: in 94.7% of CHRPE were detected, in 68.2%—mandibular osteomas, in only 14.3%—fundicgland polyposis, and in 66.7% duodenal adenomas. Desmoid tumours were present in 7.1% of FAP patients, and 14.3% had epidermoid cysts. No cases of duodenal cancer or other site cancers were recorded. Conclusions The Lithuanian polyposis register has been successfully growing since 1995. In a country like Lithuania with approximately 3 million inhabitants, a centralized register has been proven to be the optimal solution. However, proper molecular genetic services are yet to be established.

The role of the nurse specialist at St Mark’s Hospital

Guy Rosner1, Dani Bercovich2, Hana Strul1, Revital Kariv, Zamir Halpern1, Paul Rozen1 1 Department of Gastroenterology, Tel Aviv Medical Center, Tel Aviv, Israel; 2Human Molecular Genetics & Pharmacogenetics, Migal, Galilee Bio-Technology Center

Background Genetic evaluation of APC-negative patients with multiple colorectal adenomas (MAP) is a clinical & economic burden. In Israel, this is confounded as HMO-financed APC genetic evaluation sequences exon 16 only up to 3,000 base-pairs from 50 . Aims Perform comprehensive genetic evaluation of APC-negative MAP patients & draw conclusions for a future evaluation protocol. Methods Study population: 29 Amsterdam-criteria negative Jewish individuals, 52% males of mean age 53 years (range 19–76). There was dominant neoplasia inheritance in 7, recessive in 14; 12 reported CRC in a first-degree relative. Numbers of adenomas were: 6–10 (3 patients, all with colorectal cancer [CRC]), 11–19 (7 patients), multiple (19 cases); 7 had CRC (6 in left colon). Genetic analyses included: completion of exon 16 sequencing, MLPA analysis for deletions/duplications, MUTYH gene sequencing, MSI in CRCs having Bethesda criteria. Results Exon 16 sequencing revealed a weakly pathogenic polymorphism. MLPA results are pending. Pathogenic MUTYH mutations were found in 3 patients; 2 others had variants of unknown significance; polymorphisms occurred in 12, 5 having[1. 1 patient was MSI-H with MLH1 immunohistology, 5 more Bethesda-positive CRCs await MSIà.

Jo Rawlings, Kay Neale The Polyposis Registry, St Mark’s Hospital, London, UK The role is much the same as in Registries the world over but in this job the nurse attends surgical clinics where patients with polyposis are also being seen. There are six clinics a week and a paediatric clinic once a month. In the last year 1,165 patients attended, 497 of these were seen by the nurse, 115 of whom had genetic counselling. In addition to this and the routine of taking a family history, there is an

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Origin

Exon 16 APC sequencing MLPA

European 11

Normal

MYH mutation

Pending None

MYH variants

MSI-Hà IH+

None

N. African 9

Normal

Pending 3 (33.3%) 1 (11%)

MiddleEastern 9

Normal

Pending None

1 (11%)

1 (11%)

Abstracts

747

Discussion Based on this small, APC-negative, MAP cohort: Lynch syndrome needs exclusion if MAP is associated with CRC; otherwise MUTYH sequencing is probably the first evaluation especially for Jews of North-African descent. The significance of MUTYH variants needs further assessment; MUTYH polymorphisms might serve as MAP modifiers. Sequencing exon 16 is relatively unrewarding. We await MLPA & MSI results to draw final conclusions on a MAP evaluation protocol. Supported by the Israel Cancer Association & Sistopali Fund for Gastrointestinal Cancer Prevention.

Supported, in part, by the Sistopali Fund for Gastrointestinal Cancer Prevention.

Is the ‘mutated in colorectal cancer’ gene important in colon cancer after all? L. Pangon, N. Sigglekow, E. A. Musgrove, M. Kohonen-Corish Garvan Institute of Medical Research, Sydney, Australia

Ethnic variation of MUTYH gene mutations in an Israeli population sample Guy Rosner1,2, Sharon Simchoni2, Avi Orr-Urtreger2, Ruth Shomrat2, Dani Bercovich3, Hana Strul1, Revital Kariv1, Zamir Halpern1, Paul Rozen1 1

Department of Gastroenterology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 2Department of Genetics, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 3Human Molecular Genetics & Pharmacogenetics, Migal, Galilee Bio-Technology Center Background MUTYH gene mutations predispose to colorectal polyposis (MAP). Mutations Y179 C (exon 7) and G396D (exon 13), are the most frequent, however novel variants occur in defined populations. Aims Analyze our initial results evaluating MUTYH mutations in an Israeli population. Methods These were 65 Jewish patients of different origins, having [5 colorectal adenomatous polyps & negative for APC mutations. All had sequencing of MUTYH exons 7 and 13, & 29 also had full gene sequencing. Results 26 abnormal alleles were found, 22 known to be pathogenic. There were 8 biallelic mutations (6 G396D homozygotes, 2 Y179C homozygotes) & 1 compound heterozygote (G396D/Y179C); 4 monoallelic mutations (1 G396D, 2 Y179C, one 1186–1187 insGG). 4 others had MUTYH variants of unknown significance (L406V, L401V, L417M, S512F). 16 polymorphisms were found in 12 patients with 5 having [1 polymorphism. 20 of 26 abnormal alleles were in Jews of North-African origin. No Jews of European origin had either of the 2 common pathogenic mutations.

The ‘mutated in colorectal cancer’ (MCC) gene was discovered in 1991 due to its close linkage with APC, during the search for the FAP susceptibility gene. Although MCC mutations were found in sporadic cancers, early attempts failed to determine the significance of an MCC defect in colon cancer. We have recently shown that the MCC promoter is hypermethylated in *50% of colon cancers and therefore this gene defect is more common than previously thought (Kohonen-Corish et al. 2007; Oncogene 26:4435). MCC methylation is strongly associated with the BRAFV600E mutation, CIMP+ and MSI-H phenotypes. These findings were recently confirmed in an independent patient cohort (Fukuyama et al. 2008; Oncogene 27:6044). MCC methylation occurs early in precancerous lesions and is more common in serrated polyps than in traditional adenomas, suggesting a role in the serrated neoplasia pathway. MCC is methylated independent of the adjacent APC gene, and therefore it is not a bystander effect. Previous studies indicate that MCC has a potential role in regulating cell cycle progression and the NFkB and WNT pathways. All these cellular processes are relevant in carcinogenesis and defects of each one have been described in colon cancer. We have extended these initial observations and further dissected the effect of MCC on the cell cycle. Our results are consistent with a previously reported role at the G1/S checkpoint and a potential new role at the G2/M checkpoint. Our mass spectrometry experiments identify new MCC interacting partners that support its role in the cell cycle. We have further refined the subcellular localisation of MCC and determined that its activity may be regulated through phosphorylation of specific amino acids in a cell cycle dependent manner. Our data provide a potential cellular mechanism whereby an MCC defect promotes colon cancer.

Mucosectomy and stapled pouch-anal anastomosis Origin/No. pts.

G396D Y179C No. alleles/ biallelic

N. African/27

10/4

European/14

0

Middle East/24 4/2

Other path. Variants of Polymormutations unknown phisms No. No. alleles significance persons/no. with [1 7/2 1

2

3/1

0

0

0

6/3

0

0

2

3/1

Discussion Based on this pilot study, MUTYH gene mutations are not uncommon in Israeli Jewish APC-negative MAP patients. Published experience of MUTYH gene mutations in Jews refers mainly to those in North America & usually of European-origin. In this initial study the two most common mutations were found in Jews of nonEuropean origin. The significance of MUTYH variants & biallelic polymorphisms needs further assessment in our population. Further investigation for MUTYH mutations in Jews of various origins is warranted.

Steffen Bu¨low The Danish Polyposis Register, Copenhagen, Denmark Background In comparison with IRA the advantage of an ileoanal pouch is the total removal of all premalignant rectal mucosa. In FAP most surgeons prefer to perform a mucosectomy with a hand-sewn pouch-anal anastomosis, although the function is better after a stapled anastomosis. We have tried to combine the advantages of a mucosectomy and a stapled anastomosis. Procedure Proctocolectomy is performed including rectal transsection at the pelvic floor and construction of a stapled J-pouch with the anvil of a circular stapler 29 mm inserted in the bottom of the pouch. A Lone star retractor is applied and the rectal mucosa is incised 10 mm above the dentate line. Mucosectomy is performed, and a purse-string suture is applied to the resection line. The circular stapler is introduced transanally, connected to the anvil and the purse string suture is ligated around the center rod. The stapler is pressed firmly upwards, approximated, fired and removed, thereby leaving a stapled

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748 anastomosis at the level of the dentate line. A protective loop ileostomy is used routinely. Results Ten patients (7 males and 3 females, median age 37 years, range 12–50) were operated: 6 with a proctocolectomy and 4 with a proctectomy after a previous IRA. One patient developed a small abscess behind the pouch, which was drained successfully, and in two patients an anastomotic stricture necessitated one and two dilatations, respectively. Two patients have not yet had their ileostomy closed. After a median observation of 25 months (range 3–121) 8 patients are fully continent without any urge and have a median of 5 bowel movements per day (range 3–8). No anastomotic adenomas have been observed at regular endoscopical follow-up. Conclusion Mucosectomy with a stapled pouch-anal anastomosis seems to result in good function without any increased risk of anastomotic adenoma formation in the short term.

Mutation analysis of the APC gene in Taiwanese FAP families; low incidence of apc germline mutation in FAP families with mixed type of polyps Jy-Ming Chiang, P. S Hsieuh, C. R. Chang-chen Chang Gung Memorial Hospital Tao-Yuan, Taiwan Purpose Familial adenomatous polyposis (FAP) is an autosomal dominant disease caused by germline mutations in the adenomatous polyposis coli (APC) gene. Affected individuals develop colonic polyposis and various extra-colonic manifestations. This study is aimed to investigate the genetic and clinical characteristics of Taiwanese FAP families, and to analyze genotype–phenotype correlations. Materials and methods Blood samples were drawn from patients diagnosed with classic FAP registered in hereditary colorectal cancer database. Mutation analysis of APC gene of 66 FAP patients from 47 unrelated families was first screened. Negative cases were tested with Multiplex ligation-dependent probe amplification (MLPA) and SSCP of MYH exon 7 and 13. Results 79% (37/47) families had 28 APC mutations including 19 frameshift mutations, 4 nonsense mutations, 3 genomic deletion mutations, 1 missense mutation and 1 splice site mutation. We identified 15 novel mutations in 32% (15/47) families. Patients without identified APC mutation significantly displayed less profuse Polyposis (p = 0.034) and less gastroduodenal polyps (p = 0.027). Furthermore, FAP families with mixed types of polyposis are significantly associated with low incidence of APC germline mutation (p = 0.002).

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Abstracts Conclusions We added APC germline mutation data of Taiwanese FAP patients to the world and indicated that clinically defined FAP family may demonstrate distinct variant that is less frequently caused by gremlin mutation of APC gene.

Location in the large bowel influences the APC mutations observed in FAP adenomas O. Will, S. J. Leedham, G. Elia, R. K. S. Phillips, S. K. Clark, I. P. M. Tomlinson St Mark’s Hospital, Middlesex, UK Introduction The right colon differs from the left, in embryological origin, luminal environment, and function. In both sporadic colorectal cancer and Familial Adenomatous Polyposis (FAP), polyp density and cancer susceptibility vary markedly by colonic site. Adenomas in FAP have a different mutational spectrum in small intestine versus colon. This study aimed to investigate whether colonic location also influences the APC mutation spectrum in FAP. Methods 127 1–2 mm mildly dysplastic adenomas from 5 patients with a codon 1309 germline mutation, and 41 from 3 patients with mutations proximal to codon 1265, were analysed to assess the frequency of loss of heterozygosity (LOH). We chose polyps from different locations in the colon. Immunohistochemistry for betacatenin, caspase-3 and Ki-67 was performed to assess WNT pathway activation, apoptosis and proliferation. Results In polyps from patients with a 1309 mutation, the frequency of LOH showed a gradient from rectum (highest) to caecum/ ascending colon (lowest), but this was not present in patients with proximal germline APC mutations. Crypt-by-crypt analysis confirmed the LOH findings from whole polyps. Beta-catenin and caspase-3 expression showed no significant variation by colonic region, but Ki-67 expression decreased from ascending colon to rectum in tumours and normal tissue. Conclusions Colonic site alters the mutational spectrum of APC, and crypt cell proliferation. The higher frequency of LOH in rectal polyps from patients with codon 1309 mutations may help to explain their increased polyp burden at this site compared with patients who have other germline APC mutations.