Abstracts 3rd Biennial Meeting of InSiGHT(International Society for Gastrointestinal Hereditary Tumours)

Dusseldorf June 24–27 2009

Small bowel polypectomy by double balloon

enteroscopy: advancing endoscopic therapy for patients

with Peutz–Jegher’s syndrome

Edward Despott, Robin Phillips, Sue Clark, Aymer Postgate, Aine

Fitzpatrick, Eric Tripoli, Krysia Konieczko, Chris Fraser

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 tech-

nique that allows complete enteroscopy, the main therapeutic option

available for PJS patients was laparotomy with intra-operative enter-

oscopy. 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-poly-

pectomy 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 thera-

peutic option for SB polyps in PJS that avoids the need for

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.

High cumulative risk of intussusceptions in patients

with Peutz–Jeghers syndrome

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 chart-

review. Genotype-phenotype correlations were evaluated. The cumu-

lative 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 intussus-

ception 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

123

Familial Cancer (2010) 9:713–748

DOI 10.1007/s10689-010-9351-8

enteroscopic surveillance with removal of small bowel hamartomas.

The effect on the incidence of intussusception remains to be estab-

lished and weighted against burden and complication-risk of the

intervention.

Phenotype variability within CDH1 mutated families

Laetitia Huiart1, Francois Eisinger1, Violaine Bourdon1, Bruno

Buecher2, Martine Blayau2, Olivier Caron2, Jean-Francois Flejou2,

Jean-Pierre Gendre2, Astrid Schielke2, Alain Sezeur2

1Sylviane Olschwang pour le reseau PHRATries;2Institut Paoli-Calmettes, Marseille, France

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 int-

rafamilial phenotype variability observed in our series indicates the

need for international consortium to provide reliable data on pene-

trance 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

1Department of Oncology, 2Department of Pathology, Sol Goldman

Pancreatic Cancer Research Center, 4Department of Medicine, The

Johns Hopkins University School of Medicine, Baltimore, MD, USA

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

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 cal-

culated 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 pan-

creatic 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 statis-

tically 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,3Surgery, 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 muta-

tion 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 pro-

vided. 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 pap-

illary 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

714 Abstracts

123

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

1Department of Gastroenterology and Hepatology, Erasmus Medical

Center Rotterdam, Netherlands; 2Department of Gastroenterology and

Hepatology, Academic Medical Center Amsterdam, Netherlands;3Department 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) first-

degree relatives of patients with PC from familial PC kindreds. Non-

invasive pre-cursor lesions of PC include pancreatic intraepithelial

neoplasia (PanIN) and intraductal papillary mucinous neoplasia

(IPMN). A surveillance program may improve the prognosis of high-

risk individuals by detecting asymptomatic early cancers or precursor

lesions. Endosonography (EUS) has shown to be a potentially valu-

able 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 under-

went 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 indi-

viduals 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 adenocar-

cinoma in these 33 patients, which was missed by MRI.

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

1Colorectal Medicine and Genetics, 2Gastroenterology Department,3Anatomical Pathology, 5Department of Surgery, The Royal

Melbourne Hospital, Parkville Victoria 3050, Australia; 4Melbourne

Pathology, Collingwood, Victoria 3066, Australia

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 clas-

sification. 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 mac-

roscopically, suspicious for carcinoma macroscopically or confirmed

carcinoma.

Results Among 67 patients, 11 underwent surgical resection. Pan-

creas-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 mul-

tiple 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

Abstracts 715

123

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

1Department of Hepatopancreaticobiliary Surgery, University College

London Hospital NHS Trust, London; 2Department of Pathology,

University College London Hospital NHS Trust, London;3The 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 resec-

tion 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 pancreaticoduodenec-

tomies and 1 local excision + cholecystectomy were performed. Peri-

operative 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

cerebrovascular accident and data is missing in one case. Complica-

tions occurred in 24 (60%) patients. Early complications included six

anastomotic leaks, seven post-operative haemorrhages (three necessi-

tating 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 pan-

creatic 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 high-

risk 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 pro-

portion 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 muta-

tions 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 polyp-

free (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 colo-

nic polyposis. Larger studies are required to confirm these findings.

716 Abstracts

123

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 sur-

veillance 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.

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

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

Texas, USA

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 sup-

portive 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

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-man-

agement 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 cognitive-

behavioral 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 com-

pleted 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 Foun-

dation 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.

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 surveil-

lance 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).

Conclusions One-fifth of individuals at-risk for FAP are under-

compliant with screening advice. Low self-efficacy, non-use of sed-

atives during surveillance and pain after surveillance are negatively

associated with compliance behavior. We recommend that sedatives

be routinely offered to individuals undergoing colorectal cancer sur-

veillance for FAP and that adequate pain medication or spasmolytica

be provided after endoscopy.

Abstracts 717

123

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 pre-

implantation genetic diagnosis (PGD) are available for most heredi-

tary 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. Question-

naire data were collected on attitudes towards and experiences with

childhood testing, PND and PGD, and on a range of sociodemo-

graphic, 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

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 can-

cer) 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 signifi-

cant differences in base recognition and glycosylase activity between

various MUTYH mutations, notably for the two mutations most fre-

quently 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 phe-

notype 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.

718 Abstracts

123

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 char-

acterized 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 pro-

bands 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

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. Peri-

operative 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 Ronlund2,

Jan Lindebjerg2, Jacob Elebro3, Britta Halvarsson4,

Inge Bernstein5, Jenny von Salomee6, Mef Nilbert7

1Department 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

Abstracts 719

123

defined as all five observers agreeing on one of the four predeter-

mined staining patterns.

Results Consensus was reached for MLH1 in 52%, PMS2 in 60%,

MSH2 in 81%, and MSH6 in 43% of the stainings. Related kappa-

values 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. Kappa-

values 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 partici-

pation 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 under-

scores 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 sur-

veillance 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 dis-

criminating SSAs from HPs was 63% and for differentiating

adenomas from HPs this was 75%.

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 colo-

rectal 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 var-

iant 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 rela-

tionship 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. Ristimaki, P. Sipponen, K. Nuorva, V. Karja,

S. Sarna, L. Renkonen-Sinisalo, M. Aarnio, M. Heikkinen,

K. Pylvanainen, H. J. Jarvinen

Jyvaskyla Central Hospital, Jyvaskyla, Finland

720 Abstracts

123

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 immu-

noglobin 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 gas-

tritis 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 resec-

tion. 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

resection. 49% were female and 173 cancers (83%) were right-

sided. 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 sur-

veillance protocol for these families has been changed in the mid

Nineties. Since then, the recommended interval between examina-

tions 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 sur-

veillance 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.

Abstracts 721

123

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. Gomez Garcıa, 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 geno-

typed 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 ele-

vated 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 signifi-

cantly 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 pheno-

type 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 homo-

zygotes and heterozygotes (hazard ratio = 2.93, p = 0.007).

Evidence for earlier CRC onset was also observed in H63D homo-

zygotes with a median age of onset 6 years earlier than wild type or

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 Balmana3, Rowena

Mercado4, Sapna Syngal5

1Herbert Irving Comprehensive Cancer Center, Columbia University

Medical Center, New York, New York, USA; 2Erasmus Medical

Center, Rotterdam, The Netherlands; 3Hospital Vall d’Hebron,

Universitat Autonoma 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 pre-

vious 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 car-

riers, 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 quanti-

tative way.

722 Abstracts

123

Identification of novel genes involved in colorectal

cancer predisposition

Ramprasath Venkatachalam1, Marjolijn J. L. Ligtenberg1,2, Eveline J.

Kamping1, Eveline Hoenselaar1, Marsha Voorendt1, Heike Gorgens3,

Hans K. Schackert3, Ad Geurts van Kessel1, Nicoline Hoogerbrugge1,

Roland P. Kuiper1

1Departments of Human Genetics,2Pathology, Radboud University

Nijmegen Medical Centre, Nijmegen Centre for Molecular Life

Sciences, Nijmegen, The Netherlands; 3Department of Surgical

Research, Universitatsklinikum Carl Gustav Carus, Technische

Universitat Dresden, Dresden, Germany

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 mis-

match 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 mod-

erate- 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 iden-

tified 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 mul-

tiplex 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

1Department 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 muta-

tions 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 dele-

tion of 4.9 kb encompassing the last exons of EPCAM (formerly

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 EP-

CAM was shown to extend into MSH2.

As antisense transcription of CpG islands may lead to methyla-

tion, 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 epi-

genetic 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 tumor-

specific 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-asso-

ciated 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-g-

ELISpot analysis, CD107a mobilization and in vitro killing assays

were performed. TiTc isolated from HNPCC-associated CRCs spe-

cifically 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 differ-

ently 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 rec-

ognizing 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 devel-

opment of FSP-based vaccination approaches, particularly for the

preventive application in HNPCC mutation carriers.

Abstracts 723

123

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 polymor-

phisms (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 polymor-

phisms. Both Kaplan–Meier and Cox hazard regression analysis

revealed a more complex relationship between the two polymor-

phisms 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.

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

1School 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;4Hunter 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 muta-

tions 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 early-

onset 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

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 muta-

tions 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 assess-

ments 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. Briceno1, O. Valentin1, R. Xicola2,

X. Llor2, M. de la Hoya1, T. Caldes1

Hospital Clinico San Carlos, Madrid, Spain

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 muta-

tions 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 Non-

Polyposis 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 (HNPCC-

MSS).

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 individ-

uals 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 signifi-

cantly 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

724 Abstracts

123

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 con-

version 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 surveil-

lance 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

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 hos-

pital 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 gas-

trointestinal 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 per-

formed every 1–2 years in patients with Lynch syndrome.

Primary peritoneal cancer following bilateral salpingo-

oophorectomy 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

Abstracts 725

123

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 HNPCC-

related 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. Borras1,*, I. Blanco2, G. Llort3, T. Caldes4,

M. Urioste5, C. Martınez-Bouzas6, B. Grana7, J. Balmana8,

A. Torres9, T. Ramon y Cajal10, J. Sanz9,11, M. Duran11,

S. Castellvı-Bel12, S. Gonzalez1, C. Lazaro1, G. Capella1

1Laboratori de Recerca Translacional, Institut Catala d’Oncologia,

IDIBELL, Hospitalet de Llobregat, Spain; 2Unitat de Consell Genetic,

Institut Catala d’Oncologia, IDIBELL, Hospital Duran i Reynals,

Hospitalet de Llobregat, Spain; 3Unitat de Consell Genetic, Institut

Catala d’Oncologia, Hospital Germans Trias i Pujol, Badalona, Spain;4Laboratorio de Oncologıa Molecular, Hospital Clınico Sant Carlos,

Madrid, Spain; 5Departamento de Genetica Humana, Centro Nacional

de Investigaciones Oncologicas, Madrid, Spain; 6Laboratorio de

Genetica Molecular, Hospital de Cruces, Bizkaia, Spain; 7Unitat

d’Avaluacio del Risc de Cancer i Consell Genetic, Institut Catala

d’Oncologia, Hospital Universitari Josep Trueta, Girona, Spain;8Servei d’Oncologia Medica, Hospital Vall d Hebron, Barcelona,

Spain; 9Unitat de Consell Genetic, Hospital Universitari Sant Joan,

Reus, Spain; 10Servei d’Oncologia Medica, Hospital de la Santa Creu

i Sant Pau, Barcelona, Spain; 11Institut d’Oncologia Corachan-Centre

Medic, Barcelona, Spain; 12Servei de Gastroenterologia, Institut de

Malalties Digestives i Metaboliques, Hospital Clınic, Barcelona,

Spain; 13Instituto de Biologıa y Genetica 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

of Spain, whereas the C.1865T[A mutation was found in 12 unre-

lated families coming from the region of Jaen, 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 affect-

ing 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 muta-

tions 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. Menendez1, C. Lazaro1, I. Blanco2,

S. Gonzalez1, G. Capella1

1Laboratori de Recerca Translacional, 2Unitat de Consell Genetic,

Institut Catala d’Oncologia, IDIBELL, l’Hospitalet de Llobregat,

Spain

BRAF is a kinase-encoding gene from the RAS/RAF/MAPK path-

way. 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 opti-

mized 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.

726 Abstracts

123

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 mis-

match 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 indi-

vidual 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 dinu-

cleotides 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 non-

colorectal tumours a set of both mono- and dinucleotide repeats

remains necessary. Further work is necessary to determine the opti-

mum 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 Cruger

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 post-

replicative 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 ligation-

dependent probe amplification (MS-MLPA) in the analysis of MLH1

promoter hypermethylation and to determine the importance of the

different regions of the promoter.

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 immuno-

histochemistry. 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 methyl-

ation 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. Huneburg1, T. Sauerbruch1, C. Lamberti1

1Department 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 surveil-

lance 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 All endoscopic studies that met the following inclusion

criteria:

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.

Abstracts 727

123

Colorectal cancers show distinct mutational spectra

in members of the WNT pathway according

to anatomical localization and geneticinstability

Cristina Albuquerque5, Celia Baltazar1, Bruno Filipe1, Filipa Penha1,

Teresa Pereira2, Ron Smits3, Marılia Cravo4, Pedro Lage4, Paulo

Fidalgo4, Isabel Veiga5, Jose Silva Ramos6, Isabel Fonseca2,

Carlos Nobre Leitao4, Riccardo Fodde3

1Centro de Investigacao de Patobiologia Molecular (CIPM), 2Servico

de Anatomia Patologica e, 4Servico de Gastrenterologia, Instituto

Portugues de Oncologia de Lisboa Francisco Gentil, EPE, Lisboa,

Portugal; 3Department of Pathology, Josephine Nefkens Institute,

Erasmus MC, Rotterdam, The Netherlands; 5Servico de Genetica,

Instituto Portugues de Oncologia do Porto Francisco Gentil, EPE,

Porto, Portugal; 6Servico de Gastrenterologia, Hospital dos Capuchos,

Lisboa, Portugal

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 micro-

satellite 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 muta-

tion 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 locali-

zation, 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 meta-

analysis 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

Ines Francisco1, C. Albuquerque1, H. Belo1, B. Filipe1, I. Vitoriano1,

J. Dinis1, P. Lage2, I. Claro2, S. Ferreira2, P. Rodrigues2, C. Nobre

Leitao2

1Centro de Investigacao de Patobiologia Molecular (CIPM), 2Servico

de Gastroenterologia, Instituto Portugues de Oncologia de Lisboa

Francisco Gentil, EPE (IPOLFG, EPE), Portugal

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

localized in the distal colon and frequently do not present microsat-

ellite 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. Chromo-

somal instability (CIN) was evaluated in the 8 CRC by the analysis of

loss of heterozygosity (LOH) at 5q, 17p and 18q. APC (point muta-

tion/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 muta-

tions were identified in 4/11 and 6/12 tumours, respectively, mostly in

tumours presenting CIN or LOH of APC. Considering the high fre-

quency 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

T. Claudio Heine1, M. De la Fuente1, K. Alvarez1, E. Pinto1,

U. Kronberg1, A. M. Wielandt1, P. Orellana1,2, P. Carvallo2,

F. Lopez-Kostner1

1Colorectal Surgery Unit, Oncology and Molecular Genetics

Laboratory, Clınica las Condes, Santiago, Chile; 2Human Molecular

Genetics Laboratory, Pontificia Universidad Catolica 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 dis-

tribution 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 endo-

metrial 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%).

728 Abstracts

123

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 con-

sidered 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. Perez-Cabornero, E. Velasco, M. Infante, D. J. Sanz, A. Acedo,

L. Hernandez, N. Martinez, E. Lastra, C. miner, M. Duran

Instituto de Biologıa y Genetica 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 non-

related 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 con-

tribute to 61% (MSH2), 31% (MLH1), and 8% (MSH6) of the mutations.

About 50% of pathogenic mutations in MSH2 are large genomic dele-

tions. 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 Moslein5, Heiner Schaal4, Brigitte

Royer-Pokora1

1Institut fuer Humangenetik, Universitaetsklinikum Duesseldorf,2RESULT GmbH, Duesseldorf, 3Institut fuer Transplantations-

diagnostik und Zelltherapeutika, Universitaetsklinikum Duesseldorf,4Institut fuer Virologie, Universitaetsklinikum Duesseldorf5Klinik fuer Chirurgie, St Josefs-Hospital Bochum-Linden

Hereditary non-polyposis colorectal cancer is caused by inactivating

mutations of DNA mismatch repair genes, mainly MLH1 and MSH2.

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 50ss mutation caused the creation of a new overlapping

50ss. 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 RNA-

assay. In contrast, we found considerable divergence in the software-

predicted 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 non-

sense, 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 per-

formed 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.

Abstracts 729

123

p.Pro349Arg, p.Met688Arg and p.Cys697Arg for MSH2, p.Gly-

67Arg, 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

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 pol-

yposis 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

1Human 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 popu-

lation. 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.

730 Abstracts

123

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 sup-

pressor 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 approxi-

mately the same degree as in Caucasians, and it is not involved in the

susceptibility to CRC associated with an inherited or acquired defi-

cient 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.

Functional studies of unclassified variants of the human

mismatch repair protein MLH1 in the yeast

Saccharomyces cerevisiae

Karin Hardt1, Sven Boris Heik2, Johannes H. Hegemann2,

Brigitte Royer-Pokora1, The German HNPCC Consortium

1Institut fuer Humangenetik, Heinrich Heine Universitaet,

Duesseldorf; 2Institut fuer Funktionelle Genomforschung,

Heinrich Heine Universitaet, Duesseldorf

Genome stability is ensured by DNA-repair mechanism and is con-

served 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 neg-

ative 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 one-

nucleotide 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.

Conclusion IHC as a primary molecular screening modality would

fail to identify two missense mutations. MSI analysis could poten-

tially 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.

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

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

1St. 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

Abstracts 731

123

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 par-

ticipants 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 decision-

making 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. Pre-

liminary 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

lClinica Chirurgica 2^, Dipartimento di Scienze Oncologiche

e Chirurgiche, Azienda Ospedaliera-Universita di Padova, Italy;2Oncologia 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 immuno-

histochemistry, while all the five cases collected of HNPCC patients

with soft tissue sarcoma, presented loss of MSH2 at immunohisto-

chemistry 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.

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 sus-

pected Lynch syndrome. Some studies have suggested that it is only

worthwhile testing large, proximal adenomas exhibiting severe dys-

plasia 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 defi-

ciency. 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 mod-

erate 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 syn-

drome 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 poten-

tial 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)

732 Abstracts

123

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 interven-

tion (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 syn-

drome 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 rear-

rangements, 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 config-

uration.

Microsatellite instability for Lynch syndrome diagnosis

in Chile: a multicentric study

Carolina A. Rıos1, Jorge Munoz1, Leonardo Espindola2, Juan Ignacio

Vergara2, Mario Abedrapo3, Claudio Munoz3, Yamile Corredoira4,

Jaime Contreras5, Isabel Castro6, Monica Acuna1, Lucıa Cifuentes1

1Laboratorio de Epidemiologıa Genetica, Facultad de Medicina,

Universidad de Chile; 2Servicio de Cirugıa, Hospital Militar

de Santiago; 3Servicio de Cirugıa, Hospital Clınico Universidad

de Chile; 4Departamento de Anatomıa Patologica, Hospital Clınico

San Borja Arriaran, Campus Centro, Facultad de Medicina,

Uiversidad de Chile; 5Departamento de Cirugıa, Hospital Clınico San

Borja Arriaran, Campus Centro, Facultad de Medicina, Universidad

de Chile; 6Escuela de Tecnologıa Medica, 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 cost-

effectiveness. 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 metachro-

nous colorectal cancer or tumours with MSI-high histology.

The microsatellite instability (MSI) was studied using 5 loci: Bat-

25, 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, show-

ing early and late onset colorectal cancer and microsatellite instable

tumors, were selected for this study. ‘‘Minor’’ MMR genes were

Abstracts 733

123

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 muta-

tions 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 inter-

national 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

1St 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 age-

related incidence of AEH and EC, the premalignant pathway to car-

cinoma 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.

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 pro-

posing to do an observational study on all women ascertained by the

study to try and establish from this data what the diagnostic effec-

tiveness 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 popula-

tion-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 indi-

viduals 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. Perez, 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.

734 Abstracts

123

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 speci-

mens 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 Gorgens4, Wolff Schmiegel5,

Brigitte Royer-Pokora6, Wolfgang Dietmaier7, Matthias Kloor8,

Christoph Engel9, Peter Propping1, Stefan Aretz1,

The German HNPCC Consortium

1Institute of Human Genetics, University of Bonn, Bonn, Germany;2Institute 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 Universitat 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

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 non-

polyposis 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 phe-

notypical modifiers in HNPCC families. Recently, a significantly

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 MU-

TYH 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 muta-

tion 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

2Integrantes del Servicio de Coloproctologia del Hospital Espanol-

.1STEM SRL; 3Instituto de Histopatologia jefe del servicio de

Coloproctologia Hospital Espanol de Rosario

The Lynch Syndrome is characterized by the development of colo-

rectal 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 inhab-

itants 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 diag-

nosis 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)t-

ests 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.

Abstracts 735

123

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 sur-

veillance 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 pref-

erences 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 pro-

portion 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 pre-

ferred 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 hamar-

tomatous polyposis syndromes and demonstrates phenotypic hetero-

geneity. 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.

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 phe-

notype 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

1N.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 fea-

ture 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 chlo-

roform extraction. The complete coding sequences of the MUTYH-

gene, 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 char-

acterized 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 com-

pound 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 fea-

tures, as well as his/her extended family, in order to determine the

phenotyping expression of each mutation and propose the appro-

priate clinical surveillance, which includes biennial colonoscopy

after the age of 30. In specialized cases surgical intervention might

be essential.

736 Abstracts

123

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

Bruno Filipe1, Celia Baltazar1, Cristina Albuquerque1,

Sofia Fragoso1, Pedro Lage2, Ines Vitoriano1, Susana Mao de Ferro2,

Isabel Claro2, Paula Rodrigues2, Paulo Fidalgo2, Marılia Cravo2,

Carlos Nobre Leitao2

1Centro de Investigacao de Patobiologia Molecular (CIPM), Instituto

Portugues de Oncologia de Lisboa de Francisco Gentil, EPE,

Lisbon, Portugal; 2Department of Gastroenterology, Instituto

Portugues de Oncologia de Lisboa de Francisco Gentil, EPE, Lisbon,

Portugal

Patients presenting familial adenomatous polyposis (FAP), attenu-

ated FAP (AFAP) or multiple colorectal adenomas (MCRA)

phenotype are clinically difficult to distinguish. We aimed to eval-

uate the contribution of APC and MUTYH germline mutations for

107 clinically well characterized patients with FAP related pheno-

type, 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 impor-

tance 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 inci-

dence 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, out-

come) 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 manifesta-

tions 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 audi-

ometry 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.

Abstracts 737

123

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 intelli-

gence 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 ade-

nomatous 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.

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

1Center 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

1Center 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

738 Abstracts

123

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 Reso-

lution 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 muta-

tions 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. Fur-

thermore, 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.

1Center for Human and Clinical Genetics, 2Department

of Gastroenterology, Leiden University Medical Center,3The 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

The MUTYH gene encodes a DNA glycosylase involved in base

excision repair. A bi-allelic defect in this gene, leading to accumu-

lating 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 diag-

nostics. 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

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 Lopez-Kostner, C. Heine, P. Medina, U. Kronberg,

C. Wainstein

Colorectal Surgery Unit, Clınica 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 ade-

nomatous 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 alter-

native 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 com-

pared. 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.

Abstracts 739

123

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 agree-

ment 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. Gonzalez1, M. Menendez1, I. Blanco2, A. Obrador-Hevia3,

C. Lazaro1, G. Capella1

1Translational Research Laboratory, Catalan Institute of Oncology,2Genetic Counselling Unit, Catalan Institute of Oncology,3Biology of Cancer Group, University of Balearic Island

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. Lym-

phocytes 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 aberra-

tion. 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.730-

29A[T in family B is associated with increased levels of transcripts

lacking exon 7. Differences in mRNA processing may modify phe-

notypes 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 fre-

quency of the mutations Y165C and G382D ranges between 50 and

82%, while these mutations have not been identified in Asian popu-

lations, 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 pop-

ulation-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 com-

mon 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 chro-

mosomes 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 incon-

venience 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.

740 Abstracts

123

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 gastroen-

terologist, 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. Castellsague1, S. Gonzalez1, I. Blanco2, E. Guino3, C. Lazaro1,

S. Gruber4, G. Capella1

1Laboratori de Recerca Translacional, Institut Catala d’Oncologia,

IDIBELL, Hospitalet de Llobregat, Spain; 2Unitat de Consell Genetic,

Institut Catala d’Oncologia, IDIBELL, Hospital Duran i Reynals,

Hospitalet de Llobregat, Spain; 3Unitat de Bioestadıstica

i Bioinformatica, 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 germ-

line allele-specific expression (ASE) in the APC gene in FAP and

AFAP with and without detectable APC or MYH mutations.

Methods Germline RNA from fresh frozen and/or cultured lym-

phocytes 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.

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 abnor-

malities, 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. Muta-

tions 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, Michele Attenhofer, Bruno Reichlin, Karl Heinimann

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).

Abstracts 741

123

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

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 pro-

cess, 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 popula-

tions. 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

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 mono-

allelic 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.

Allele-specific expression of the APC gene in mutation-

negative patients with adenomatous polyposis

Stefanie Vogt, Astrid Kaufmann, Dietlinde Stienen, Nils Rahner,

Verena Steinke, Markus M. Nothen, 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 adeno-

mas, 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 ran-

ged 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)

742 Abstracts

123

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

lClinica Chirurgica 2^, Dipartimento di Scienze Oncologiche

e Chirurgiche, Azienda Ospedaliera-Universita di Padova, Italy;2Oncologia Sperimentale 1^, Centro Regionale Oncologico, IRCCS,

Aviano, Italy

Background While MYH biallelic mutations are clearly related to

the Attenuated Familial Adenomatous Polyposis (AFAP), the asso-

ciation 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 consec-

utive cohort of 37 AFAP patients observed from 2003 to 2008 at Padova

Hospital. All patients were screened for the most frequent MYHgermline 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. Inci-

dence 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

(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 psy-

chological support when necessary releasing the doctor for the next

patient.

Between clinic visits these patients require easy access to spe-

cialist 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.

Abstracts 743

123

‘‘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 mac-

roscopic 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 characteriza-

tion of these lesions was examined with three different imaging

techniques: conventional endoscopy with white light, chromoendos-

copy 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 observa-

tion. 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 endosco-

pists. 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 complica-

tions. 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 manage-

ment 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 man-

agement in patients with high risk rectal lesions detected during

endoscopic surveillance. A retrospective search from the St. Mark’s

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 con-

firmed invasive carcinoma. Screening imaging including endorectal

ultrasound, CT scan and blood tests found no evidence of local

invasion or metastasis after endoscopic polypectomy. Hence, proc-

tectomy 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 his-

tological 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 compro-

mised 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 chromo-

some 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,

744 Abstracts

123

abdominal and pelvic ultrasound, cervical smears and breast exami-

nation, 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

1Molecular 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 pub-

lished 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 con-

tinue 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:

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 dis-

criminate 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 phe-

nomena 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 Aus-

trian 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

Abstracts 745

123

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 colo-

rectal 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.

The Lithuanian polyposis register: progress during

past 14 years

N. E. Samalavicius, T. Poskus

Oncology Institute of Vilnius University and Vilnius University

Santariskiu Clinic Center Branch, Vilnius, Lithuania

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.

Materials and methods During the period January 1995 to Decem-

ber 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 pro-

phylactic 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 adeno-

mas. 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 success-

fully 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

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

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 ille-

gitimacy, infantile death or relatives distanced by family feud.

Another advantage of working within the hospital is the oppor-

tunity 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 infor-

mation 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.

Developing a protocol for genetic evaluation

of APC-negative patients with multiple colorectal

adenomas

Guy Rosner1, Dani Bercovich2, Hana Strul1, Revital Kariv,

Zamir Halpern1, Paul Rozen1

1Department 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 evalua-

tion 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 poly-

morphism. 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 MSI�.

Origin Exon 16

sequencing

APC

MLPA

MYH

mutation

MYH

variants

MSI-H�

IH+

European 11 Normal Pending None None

N. African 9 Normal Pending 3 (33.3%) 1 (11%)

Middle-

Eastern 9

Normal Pending None 1 (11%) 1 (11%)

746 Abstracts

123

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.

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

1Department 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 pol-

yposis (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 mono-

allelic 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.

Origin/No. pts. G396DY179CNo. alleles/biallelic

Other path.mutationsNo. alleles

Variants ofunknownsignificance

Polymor-phisms No.persons/no.with [1

N. African/27 10/4 7/2 1 2 3/1

European/14 0 0 0 0 6/3

Middle East/24 4/2 0 0 2 3/1

Discussion Based on this pilot study, MUTYH gene mutations are

not uncommon in Israeli Jewish APC-negative MAP patients. Pub-

lished 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 non-

European 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.

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

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 hypermethy-

lated 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 find-

ings 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 neo-

plasia 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 regu-

lating 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

Steffen Bulow

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 muco-

sectomy and a stapled anastomosis.

Procedure Proctocolectomy is performed including rectal transsec-

tion 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

Abstracts 747

123

anastomosis at the level of the dentate line. A protective loop ileos-

tomy 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 anas-

tomotic 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 pol-

yposis 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 iden-

tified 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). Further-

more, FAP families with mixed types of polyposis are significantly

associated with low incidence of APC germline mutation (p = 0.002).

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 fre-

quency of loss of heterozygosity (LOH). We chose polyps from

different locations in the colon. Immunohistochemistry for beta-

catenin, 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.

748 Abstracts

123