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Mismatch repair (MMR) gene sequence variants of uncertain clinical significance are often identified in suspected Lynch syndrome families, and this constitutes a challenge for both researchers and clinicians. Multifactorial likelihood model approaches provide a quantitative measure of MMR variant pathogenicity, but first require input of likelihood ratios (LRs) for different MMR variation-associated characteristics from appropriate, well-characterized reference datasets. Microsatellite instability (MSI) and somatic BRAF tumor data for unselected colorectal cancer probands of known pathogenic variant status were used to derive LRs for tumor characteristics using the Colon Cancer Family Registry (CFR) resource. These tumor LRs were combined with variant segregation within families, and estimates of prior probability of pathogenicity based on sequence conservation and position, to analyze 44 unclassified variants identified initially in Australasian Colon CFR families. In addition, in vitro splicing analyses were conducted on the subset of variants based on bioinformatic splicing predictions. The LR in favor of pathogenicity was estimated to be ~12-fold for a colorectal tumor with a BRAF mutation-negative MSI-H phenotype. For 31 of the 44 variants, the posterior probabilities of pathogenicity were such that altered clinical management would be indicated. Our findings provide a working multifactorial likelihood model for classification that carefully considers mode of ascertainment for gene testing.

Background How body size influences risk of molecular subtypes of colorectal cancer (CRC) is unclear. We investigated whether measures of anthropometry differentially influence risk of tumours according to BRAF c.1799T>A p.V600E mutation (BRAF) and microsatellite instability (MSI) status. Methods Data from The Netherlands Cohort Study (n = 120 852) and Melbourne Collaborative Cohort Study (n = 40 514) were pooled and included 734 and 717 colorectal cancer cases from each study, respectively. Hazard ratios (HRs) and 95% confidence intervals (CIs) for body mass index (BMI), waist measurement and height were calculated and compared for subtypes defined by BRAF mutation and MSI status, measured from archival tissue. Results Results were consistent between studies. When pooled, BMI modelled in 5 kg/m increments was positively associated with BRAF wild-type (HR: 1.16, 95% CI: 1.08-1.26) and MS-stable tumours (HR: 1.15, 95% CI: 1.06-1.24). Waist measurement was also associated with BRAF wild-type (highest vs lowest quartile, HR: 1.59, 95% CI: 1.33-1.90) and MS-stable tumours (highest vs lowest quartile HR: 1.68, 95% CI: 1.31-2.15). The HRs for BRAF mutation tumours and MSI tumours were smaller and non-significant, but differences between the HRs by tumour subtypes were not significant. Height, modelled per 5-cm increase, was positively associated with BRAF wild-type and BRAF mutation tumours, but the HR was greater for tumours with a BRAF mutation than BRAF wild-type (HR: 1.23, 95% CI: 1.11-1.37, P = 0.03). Similar associations were observed with respect to height and MSI tumours (HR: 1.26, 95% CI: 1.13-1.40, P = 0.02). Conclusions Generally, overweight increases the risk of CRC. Taller individuals have an increased risk of developing a tumour with a BRAF mutation or MSI. Published by Oxford University Press on behalf of the International Epidemiological Association

The association between ovarian endometrioid adenocarcinoma and endometriosis is well established. However, not all endometrioid adenocarcinomas are directly related to endometriosis, and it has been suggested that there may be clinicopathologic differences between endometriosis-positive and endometriosis-negative tumors. Molecular alterations in endometrioid adenocarcinoma include KRAS and BRAF mutations, but the incidence of these abnormalities in previous reports has been highly variable (0%-36% and 0%-24%, respectively). This may be explained by relatively small sample sizes in earlier studies but could also reflect difficulties in accurately classifying high-grade ovarian malignancies. In the current study, we investigated KRAS and BRAF mutations in 78 low-grade (FIGO grade 1 and 2) endometrioid adenocarcinomas and compared the results with the presence of endometriosis in the tumor-associated ovary and/or in other pelvic sites. KRAS mutations were identified in 12 (29%) of 42 endometriosis-associated endometrioid adenocarcinomas with satisfactory analysis but in only 1 (3%) of 29 tumors in which endometriosis was not identified. BRAF mutation was identified only in a single endometriosis-associated case. These findings support the hypothesis that endometriosis-associated and independent endometrioid adenocarcinoma may develop via different molecular pathways and that KRAS mutations have an important role only in the former tumors. In contrast, BRAF mutations do not appear to have a significant role in either endometrioid adenocarcinoma subgroup. This may be relevant to future targeted therapies in patients with high-stage or recurrent disease and indicate that histopathologists should carefully examine endometrioid adenocarcinoma specimens, including nonneoplastic tissues, for the presence of endometriosis.

Serrated polyposis syndrome (SPS), also known as hyperplastic polyposis, is a syndrome of unknown genetic basis defined by the occurrence of multiple serrated polyps in the large intestine and associated with an increased risk of colorectal cancer (CRC). There are a variety of SPS presentations, which may encompass a continuum of phenotypes modified by environmental and genetic factors. To explore the phenotype of SPS, we recorded the histologic and molecular characteristics of multiple colorectal polyps in patients with SPS recruited between 2000 and 2010 from genetics clinics in Australia, New Zealand, Canada, and the United States. Three specialist gastrointestinal pathologists reviewed the polyps, which they classified into conventional adenomas or serrated polyps, with various subtypes, according to the current World Health Organization criteria. Mutations in BRAF and KRAS and mismatch repair protein expression were determined in a subset of polyps. A total of 100 patients were selected for the study, of whom 58 were female and 42 were male. The total polyp count per patient ranged from 6 to 150 (median 30). The vast majority of patients (89%) had polyposis affecting the entire large intestine. From this cohort, 406 polyps were reviewed. Most of the polyps (83%) were serrated polyps: microvesicular hyperplastic polyps (HP) (n=156), goblet cell HP (n=25), sessile serrated adenoma/polyps (SSA/P) (n=110), SSA/P with cytologic dysplasia (n=28), and traditional serrated adenomas (n=18). A further 69 polyps were conventional adenomas. BRAF mutation was mainly detected in SSA/P with dysplasia (95%), SSA/P (85%), microvesicular HP (76%), and traditional serrated adenoma (54%), whereas KRAS mutation was present mainly in goblet cell HP (50%) and in tubulovillous adenoma (45%). Four of 6 SSA/Ps with high-grade dysplasia showed loss of MLH1/PMS2 expression. CRC was diagnosed in 39 patients who were more often found to have a conventional adenoma compared with patients without CRC (P=0.003). Patients with SPS referred to genetics clinics had a pancolonic disease with a high polyp burden and a high rate of BRAF mutation. The occurrence of CRC was associated with the presence of conventional adenoma.

OBJECTIVES: Serrated polyposis (hyperplastic polyposis) is characterized by multiple polyps with serrated architecture in the colorectum. Although patients with serrated polyposis are known to be at increased risk of colorectal cancer (CRC) and possibly extracolonic cancers, cancer risk for their relatives has not been widely explored. The aim of this study was to estimate the risks of CRC and extracolonic cancers for relatives of patients with serrated polyposis. METHODS: A cohort of the 1,639 first- and second-degree relatives of 100 index patients with serrated polyposis recruited regardless of a family history of polyps or cancer from genetic clinics in Australia, New Zealand, Canada, and the USA, were retrospectively analyzed to estimate the country-, age-, and sex-specific standardized incidence ratios (SIRs) for relatives compared with the general population. RESULTS: A total of 102 CRCs were observed in first- and second-relatives (SIR 2.25, 95% confidence interval (CI) 1.75-2.93; P

Purpose To determine whether cancer risks for carriers and noncarriers from families with a mismatch repair (MMR) gene mutation are increased above the risks of the general population. Patients and Methods We prospectively followed a cohort of 446 unaffected carriers of an MMR gene mutation (MLH1, n = 161; MSH2, n = 222; MSH6, n = 47; and PMS2, n = 16) and 1,029 their unaffected relatives who did not carry a mutation every 5 years at recruitment centers of the Colon Cancer Family Registry. For comparison of cancer risk with the general population, we estimated country-, age-, and sex-specific standardized incidence ratios (SIRs) of cancer for carriers and noncarriers. Results Over a median follow-up of 5 years, mutation carriers had an increased risk of colorectal cancer (CRC; SIR, 20.48; 95% CI, 11.71 to 33.27; P < .001), endometrial cancer (SIR, 30.62; 95% CI, 11.24 to 66.64; P < .001), ovarian cancer (SIR, 18.81; 95% CI, 3.88 to 54.95; P < .001), renal cancer (SIR, 11.22; 95% CI, 2.31 to 32.79; P < .001), pancreatic cancer (SIR, 10.68; 95% CI, 2.68 to 47.70; P = .001), gastric cancer (SIR, 9.78; 95% CI, 1.18 to 35.30; P = .009), urinary bladder cancer (SIR, 9.51; 95% CI, 1.15 to 34.37; P = .009), and female breast cancer (SIR, 3.95; 95% CI, 1.59 to 8.13; P = .001). We found no evidence of their noncarrier relatives having an increased risk of any cancer, including CRC (SIR, 1.02; 95% CI, 0.33 to 2.39; P = .97). Conclusion We confirmed that carriers of an MMR gene mutation were at increased risk of a wide variety of cancers, including some cancers not previously recognized as being a result of MMR mutations, and found no evidence of an increased risk of cancer for their noncarrier relatives.

Colorectal cancer (CRC) that demonstrates microsatellite instability (MSI) is caused by either germline mismatch repair (MMR) gene mutations, or 'sporadic' somatic tumour MLH1 promoter methylation. MLH1 promoter methylation is reportedly correlated with tumour BRAF V600E mutation status. No systematic review has been undertaken to assess the value of BRAF V600E mutation and MLH1 promoter methylation tumour markers as negative predictors of germline MMR mutation status. A literature review of CRC cohorts tested for MMR mutations, and tumour BRAF V600E mutation and/or MLH1 promoter methylation was conducted using PubMed. Studies were assessed for tumour features, stratified by tumour MMR status based on immunohistochemistry or MSI where possible. Pooled frequencies and 95% CIs were calculated using a random effects model. BRAF V600E results for 4562 tumours from 35 studies, and MLH1 promoter methylation results for 2975 tumours from 43 studies, were assessed. In 550 MMR mutation carriers, the BRAF V600E mutation frequency was 1.40% (95% CI 0.06% to 3%). In MMR mutation-negative cases, the BRAF V600E mutation frequency was 5.00% (95% CI 4% to 7%) in 1623 microsatellite stable (MSS) cases and 63.50% (95% CI 47% to 79%) in 332 cases demonstrating MLH1 methylation or MLH1 expression loss. MLH1 promoter methylation of the 'A region' was reported more frequently than the 'C region' in MSS CRCs (17% vs 0.06%, p

Benign serrated polyps are commonly found in the colorectum but have rarely been described in other parts of the gastrointestinal tract. We report a series of 9 serrated polyps arising in the duodenum with clinicopathologic features, immunohistochemical expression profile of mucins (MUC2, MUC5AC, MUC6), and molecular analysis for BRAF and KRAS. The polyps were diagnosed as incidental endoscopy findings in 9 different patients, comprising 3 male and 6 female patients, with a mean age of 52.2 years (range, 21-72 years). The second part of the duodenum was the most common site (n = 5), followed by the ampulla (n = 1) and the distal duodenum (n = 1), with the location of the 2 remaining polyps unspecified. Other upper gastrointestinal tract pathology features included Barrett esophagus for 5 patients, Helicobacter gastritis for 1 patient, and mild chronic gastritis for 1 patient. The histologic appearance of the polyps was similar to microvesicular hyperplastic polyp in the colorectum. Immunostaining for mucins showed MUC6 expression in the crypt bases of all polyps, MUC5AC expression in 8 cases (89%), and mucin 2 expression in 6 cases (67%). Molecular testing was successful in 6 polyps, showing BRAF mutation (V600E) in 2 polyps, KRAS mutation in 2 polyps, and no mutation for either gene in 2 polyps. Colonoscopy reports were available for 6 patients, of whom 4 were diagnosed with hyperplastic polyps or sessile serrated polyps in the colorectum. However, no patient met the criteria for serrated polyposis. Although probably rare and of uncertain malignant potential, hyperplastic polyp should be considered in the differential diagnosis of benign duodenal polyp.

Background Carriers of a germline mutation in a DNA mismatch repair (MMR) gene—that is, persons with Lynch syndrome—have substantially high risks of colorectal (CRC), endometrial, and several other cancers. The proportion of carriers who have de novo mutations (not inherited from either parent) is not known. This study reports a case series of de novo mutations in MMR genes and estimates the frequency of de novo mutation in MMR genes using the Colon Cancer Family Registry. Methods Screening for germline MLH1 , MSH2 , MSH6 , and PMS2 mutations was performed for all incident CRC cases recruited from cancer registries (population based probands) displaying microsatellite instability (MSI) or loss of expression of MMR genes by immunohistochemistry (IHC) and probands with CRC in multi-case families recruited from clinics (clinic based probands), regardless of MSI or IHC status. All relatives of probands with a pathogenic mutation who donated a blood sample underwent testing for the mutation identified in the proband. Results Of 261 probands (202 clinic based, 59 population based) with MMR gene mutations for whom it was possible to determine the origin of the mutation, six (2.3%, 95% CI 0.9% to 5.0%) were confirmed as de novo, and the remaining 255 (97.7%, 95% CI 95.0% to 99.1%) were inherited. Of the de novo mutation carriers, three were clinic based probands (1.5%, 95% CI 0.3% to 4.5%) and three were population based probands (5.1%, 95% CI 1.2% to 14.5%). Two were in MLH1 , three in MSH2 , and one in MSH6 . Conclusion De novo MMR gene mutations are uncommon causes of Lynch syndrome.

Serrated polyposis has only recently been accepted as a condition which carries an increased personal and familial risk of colorectal cancer. Described over four decades ago, it remains one of the most underrecognized and poorly understood of all the intestinal polyposes. With a variety of phenotypic presentations, it is likely that serrated polyposis represents a group of diseases rather than a single entity. Further, neoplastic progression in serrated polyposis may be associated with premature aging in the normal mucosa, typified by widespread gene promoter hypermethylation. From this epigenetically altered field, arise diverse polyps and cancers which show a range of molecular features. Despite a high serrated polyp count, only one-third of colorectal cancers demonstrate aBRAFV600E mutation, the molecular hallmark of the canonical serrated pathway, suggesting that though multiple serrated polyps act as a marker of an abnormal mucosa, the majority of CRC in these patients arise within lesions other than BRAF-mutated serrated polyps.

Lynch syndrome, historically known as hereditary non-polyposis colorectal cancer (Jass, 2006), refers to colorectal and other cancers caused by germline mutations in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 (Vasen et al, 1999). Approximately 1 in 3000 people in the general population carry a mutation in an MMR gene (Dunlop et al, 2000). These MMR gene mutation carriers are at substantially increased risk of colorectal cancer (CRC) with an estimated cumulative risk to age 70 years between 40% and 70% depending on the carrier's sex and the MMR gene that is mutated (Chen et al, 2006; Jenkins et al, 2006; Senter et al, 2008; Baglietto et al, 2010). Physical characteristics or environmental exposures of the mutation carriers could also modify their risks of developing CRC (Jenkins et al, 2007). Identifying modifiers of CRC risk for carriers of MMR gene mutations is important for understanding carcinogenesis. Identifying potentially protective or harmful and avoidable risk factors also creates opportunities for mutation carriers to reduce their risks of life-threatening diseases. Previous studies have provided evidence for the existence of modifiers of CRC risk for MMR gene mutation carriers. For example, CRC risk for carriers is positively associated with alcohol consumption (Watson et al, 2004) and negatively associated with fruit consumption and dietary fibre intake (Diergaarde et al, 2007). For smoking, CRC risk for carriers is positively associated with current smoking (Diergaarde et al, 2007; Pande et al, 2010), but negatively or not associated with former smoking (Diergaarde et al, 2007; Pande et al, 2010). However, the association between body size and CRC risk for MMR gene mutation carriers is yet to be established. One CRC risk factor for people in the general population is body size, a collective term for body mass index (BMI), waist circumference and height (Dai et al, 2007; Larsson and Wolk, 2007; Moghaddam et al, 2007; Renehan et al, 2008). Previous case–control studies of CRC cases with a family history (therefore, likely to be enriched for MMR gene mutation carriers) compared with population-based controls who were unselected for family history, have shown that current BMI is positively associated with CRC risk (Slattery et al, 2003; Campbell et al, 2007). However, studies that have examined the association between current BMI and the occurrence of tumours with microsatellite instability (MSI), a common characteristic of CRCs caused by MMR defects, have reported no statistically significant associations with this form of disease (Slattery et al, 2000; Campbell et al, 2010). One study that examined the association between BMI and colorectal adenoma risk for MMR gene mutation carriers observed a positive association for males but not females (Botma et al, 2010). Identifying an association of BMI in early adulthood on subsequent cancer risk would be important for MMR gene mutation carriers who learn their mutation status as adults in their 20s, an increasingly common occurrence because of increased systematic testing in the population, with the consequent opportunity to reduce their risk of disease. In this study, we estimated an association between BMI at age 20 years and CRC risk for MMR gene mutation carriers. As a comparison, we also estimated the association for non-carriers to investigate whether BMI has a differential effect on risk for CRC by mutation status.

The detection of defective mismatch repair (MMR), as assessed by the presence of tumor microsatellite instability (MSI) and/or loss of MMR protein expression by IHC, has been useful for risk assessment, prognosis, and prediction of treatment in patients with colorectal cancer. We analyzed tumors for the presence of defective MMR from 5927 Colorectal Cancer Family Registry patients recruited at six international consortium sites. We evaluated the appropriate percentage instability cutoff used to distinguish the three MSI phenotypes [ie, stable (MSS), low instability (MSI-L), and high instability (MSI-H)]; the sensitivity, specificity, and performance characteristics of individual markers; and the concordance between MSI and IHC phenotypes. Guided by the results of the IHC testing, our findings indicate that the distinction between an MSI-H phenotype from a low-instability or MSS phenotype can best be accomplished by using a cutoff of 30% or greater of the markers showing instability. The sensitivity and specificity of the mononucleotide markers were higher than those of the dinucleotide markers. Specifically, BAT26 and BAT25 had the highest sensitivity (94%) and specificity (98%), and the use of mononucleotide markers alone identified 97% of the MSI-H cases correctly. As expected, the presence of MSI-H correlated with an older age of diagnosis, the presence of tumor in the proximal colon, and female sex.

Cancer risks for a person who has inherited a MUTYH mutation from only one parent (monoallelic mutation carrier) are uncertain. Using the Colon Cancer Family Registry and Newfoundland Familial Colon Cancer Registry, we identified 2,179 first- and second-degree relatives of 144 incident colorectal cancer (CRC) cases who were monoallelic or biallelic mutation carriers ascertained by sampling population complete cancer registries in the United States, Canada and Australia. Using Cox regression weighted to adjust for sampling on family history, we estimated that the country-, age- and sex-specific standardized incidence ratios (SIRs) for monoallelic mutation carriers, compared to the general population, were: 2.04 (95% confidence interval, CI 1.56-2.70; p < 0.001) for CRC, 3.24 (95%CI 2.18-4.98; p < 0.001) for gastric cancer, 3.09 (95%CI 1.07-12.25; p = 0.07) for liver cancer and 2.33 (95%CI 1.18-5.08; p = 0.02) for endometrial cancer. Age-specific cumulative risks to age 70 years, estimated using the SIRs and US population incidences, were: for CRC, 6% (95%CI 5-8%) for men and 4% (95%CI 3-6%) for women; for gastric cancer, 2% (95%CI 1-3%) for men and 0.7% (95%CI 0.5-1%) for women; for liver cancer, 1% (95%CI 0.3-3%) for men and 0.3% (95%CI 0.1-1%) for women and for endometrial cancer, 4% (95%CI 2-8%). There was no evidence of increased risks for cancers of the brain, pancreas, kidney, lung, breast or prostate. Monoallelic MUTYH mutation carriers with a family history of CRC, such as those identified from screening multiple-case CRC families, are at increased risk of colorectal, gastric, endometrial and possibly liver cancers.

There is evidence that tamoxifen treatment ofBRCA1andBRCA2carriers for prior breast cancer increases risk of endometrioid subtype endometrial cancer (EC), and suggestive evidence thatBRCA1andBRCA2mutation carriers may be predisposed to EC in the absence of tamoxifen exposure. We assessed the association of EC withBRCA1orBRCA2mutation status in Australasian breast-ovarian families. Report of at least one case of EC was significantly greater inBRCA1-positive families (35/218 (16%); p = .03) and non-significantly greater inBRCA2-positive families (23/189 (12%); p = .6), compared to high-risk breast cancer families without aBRCA1/2mutation (86/796 (11%)). EC was the first/concurrent cancer for 41% of EC cases with multiple cancer diagnoses fromBRCA1/2families, and early onset for most of these diagnoses. Mutation status was imputed for ungeno-typed individuals from 57BRCA1/2pedigrees reporting EC using BRCAPRO. Effects of genotype on EC diagnosis age, and interaction with tamoxifen therapy, were assessed using Cox proportional hazards regression analysis. EC risk was non-significantly marginally greater forBRCA1carriers (hazard ratio = 1.25, 95%CI = 0.65–2.41), andBRCA2carriers (HR = 1.12, 95%CI = 0.51–2.45), compared to non-carrier family members. Tamoxifen therapy was highly significantly associated with EC (HR = 6.68, 95%CI = 3.12–15.15; p = 1.7 x 10-6) inBRCA1/2families, with no evidence for interaction between tamoxifen therapy andBRCA1/2genotype. Our family-based study supports a 7-fold increase in EC risk with tamoxifen exposure for female family members fromBRCA1/2families. Early onset EC in carriers without tamoxifen use suggests that further study is required to assess association of modest EC risk withBRCA1/2mutation status alone.

Surgical management of colon cancer for patients with Lynch syndrome who carry a mismatch repair (MMR) gene mutation is controversial. The decision to remove more or less of the colon involves the consideration of a relatively high risk of metachronous colorectal cancer (CRC) with the impact of more extensive surgery.To estimate and compare the risks of metachronous CRC for patients with Lynch syndrome undergoing either segmental or extensive (subtotal or total) resection for first colon cancer.Risk of metachronous CRC was estimated for 382 MMR gene mutation carriers (172 MLH1, 167 MSH2, 23 MSH6 and 20 PMS2) from the Colon Cancer Family Registry, who had surgery for their first colon cancer, using retrospective cohort analysis. Age-dependent cumulative risks of metachronous CRC were calculated using the Kaplan-Meier method. Risk factors for metachronous CRC were assessed by a Cox proportional hazards regression.None of 50 subjects who had extensive colectomy was diagnosed with metachronous CRC (incidence rate 0.0; 95% CI 0.0 to 7.2 per 1000 person-years). Of 332 subjects who had segmental resections, 74 (22%) were diagnosed with metachronous CRC (incidence rate 23.6; 95% CI 18.8 to 29.7 per 1000 person-years). For those who had segmental resections, incidence was statistically higher than for those who had extensive surgery (P0.001). Cumulative risk of metachronous CRC was 16% (95% CI 10% to 25%) at 10 years, 41% (95% CI 30% to 52%) at 20 years and 62% (95% CI 50% to 77%) at 30 years after segmental colectomy. Risk of metachronous CRC reduced by 31% (95% CI 12% to 46%; p=0.002) for every 10 cm of bowel removed.Patients with Lynch syndrome with first colon cancer treated with more extensive colonic resection have a lower risk of metachronous CRC than those receiving less extensive surgery. This finding will better inform decision-making about the extent of primary surgical resection.

Causative genetic variants have to date been identified for only a small proportion of familial colorectal cancer (CRC). While conditions such as Familial Adenomatous Polyposis and Lynch syndrome have well defined genetic causes, the search for variants underlying the remainder of familial CRC is plagued by genetic heterogeneity. The recent identification of families with a heritable predisposition to malignancies arising through the serrated pathway (familial serrated neoplasia or Jass syndrome) provides an opportunity to study a subset of familial CRC in which heterogeneity may be greatly reduced. A genome-wide linkage screen was performed on a large family displaying a dominantly-inherited predisposition to serrated neoplasia genotyped using the Affymetrix GeneChip Human Mapping 10 K SNP Array. Parametric and nonparametric analyses were performed and resulting regions of interest, as well as previously reported CRC susceptibility loci at 3q22, 7q31 and 9q22, were followed up by finemapping in 10 serrated neoplasia families. Genome-wide linkage analysis revealed regions of interest at 2p25.2-p25.1, 2q24.3-q37.1 and 8p21.2-q12.1. Finemapping linkage and haplotype analyses identified 2q32.2-q33.3 as the region most likely to harbour linkage, with heterogeneity logarithm of the odds (HLOD) 2.09 and nonparametric linkage (NPL) score 2.36 (P = 0.004). Five primary candidate genes (CFLAR, CASP10, CASP8, FZD7 and BMPR2) were sequenced and no segregating variants identified. There was no evidence of linkage to previously reported loci on chromosomes 3, 7 and 9. Electronic supplementary material The online version of this article (doi:10.1007/s10689-010-9408-8) contains supplementary material, which is available to authorized users.

Background The incidence of oesophageal adenocarcinoma (OA) isincreasing rapidly with nearly all OA arising from underlying Barrett’soesophagus (BO). The primary risk factors for BO are gastro-oesophagealrefl ux (GOR) and obesity. The risk of obesity remains after controlling forGOR, suggesting that non-refl ux obesity factors may be important. Themetabolic syndrome (MS) results from obesity related hormonal and systemicinfl ammatory changes and is associated with breast, prostate andcolon cancers. The association between MS and BO is unknown.Aim To conduct a case-control study to quantify the association betweenMS and BO.Methods We undertook a structured interview, clinical and anthropometricmeasures and fasting metabolic and hormonal studies within acase-control study conducted in Brisbane, Australia. We recruited 236cases with histologically confi rmed BO diagnosed 2003–6 and 237 controlsfrom the electoral roll, frequency matched by age and sex to cases.Odds ratios (OR) and 95% confi dence intervals (CI) were estimated usingmultivariable logistic regression analysis.Results The prevalence of MS was 63% in cases and 47% in controls (p= 0.0005). There was a signifi cantly increased risk of BO among thosewith MS (OR 1.91 95%CI 1.32–2.76). The risk was unaffected in a logisticregression model adjusting for age, sex, and use of aspirin, NSAIDs,alcohol and cigarettes (OR 1.91 95%CI 1.27–2.88). Further adjustment forBMI (OR 1.62 95%CI 1.03–2.54) and GOR (1.34 95%CI 0.80–2.23)modestly attenuated the association.Conclusions MS is associated with an increased risk of BO. This associationis not explained simply by higher BMI or GOR among cases. Themechanism of this association remains unknown but hormonal and systemicinfl ammatory changes associated with obesity are factors warrantingfurther study.

Genetic risk factors are important contributors to the development of colorectal cancer. Following the definition of a linkage signal at 9q22-31, we fine mapped this region in an independent collection of colon cancer families. We used a custom array of single-nucleotide polymorphisms (SNP) densely spaced across the candidate region, performing both single-SNP and moving-window association analyses to identify a colon neoplasia risk haplotype. Through this approach, we isolated the association effect to a five-SNP haplotype centered at 98.15 Mb on chromosome 9q. This haplotype is in strong linkage disequilibrium with the haplotype block containing HABP4 and may be a surrogate for the effect of this CD30 Ki-1 antigen. It is also in close proximity to GALNT12, also recently shown to be altered in colon tumors. We used a predictive modeling algorithm to show the contribution of this risk haplotype and surrounding candidate genes in distinguishing between colon cancer cases and healthy controls. The ability to replicate this finding, the strength of the haplotype association (odds ratio, 3.68), and the accuracy of our prediction model (∼60%) all strongly support the presence of a locus for familial colon cancer on chromosome 9q. Cancer Res; 70(13); 5409–18. ©2010 AACR.

e15510Background: Microsatellite instability (MSI) has been associated with better survival outcomes in early stage CRC. In mCRC MSI is a smaller subgroup (3-5%). There is evolving evidence that tr...

Background Germline mutations in MSH6 account for 10%-20% of Lynch syndrome colorectal cancers caused by hereditary DNA mismatch repair gene mutations. Because there have been only a few studies of mutation carriers, their cancer risks are uncertain.Methods We identified 113 families of MSH6 mutation carriers from five countries that we ascertained through family cancer clinics and population-based cancer registries. Mutation status, sex, age, and histories of cancer, polypectomy, and hysterectomy were sought from 3104 of their relatives. Age-specific cumulative risks for carriers and hazard ratios (HRs) for cancer risks of carriers, compared with those of the general population of the same country, were estimated by use of a modified segregation analysis with appropriate conditioning depending on ascertainment.Results For MSH6 mutation carriers, the estimated cumulative risks to ages 70 and 80 years, respectively, were as follows: for colorectal cancer, 22% (95% confidence interval [CI] = 14% to 32%) and 44% (95% CI = 28% to 62%) for men and 10% (95% CI = 5% to 17%) and 20% (95% CI = 11% to 35%) for women; for endometrial cancer, 26% (95% CI = 18% to 36%) and 44% (95% CI = 30% to 58%); and for any cancer associated with Lynch syndrome, 24% (95% CI = 16% to 37%) and 47% (95% CI = 32% to 66%) for men and 40% (95% CI = 32% to 52%) and 65% (95% CI = 53% to 78%) for women. Compared with incidence for the general population, MSH6 mutation carriers had an eightfold increased incidence of colorectal cancer (HR = 7.6, 95% CI = 5.4 to 10.8), which was independent of sex and age. Women who were MSH6 mutation carriers had a 26-fold increased incidence of endometrial cancer (HR = 25.5, 95% CI = 16.8 to 38.7) and a sixfold increased incidence of other cancers associated with Lynch syndrome (HR = 6.0, 95% CI = 3.4 to 10.7).Conclusion We have obtained precise and accurate estimates of both absolute and relative cancer risks for MSH6 mutation carriers.

Introduction: Both smoking and alcohol consumption have been associated with modestly increased risks of colorectal cancer (CRC). Reports have suggested that these associations may differ by tumor molecular subtype, with stronger associations for microsatellite unstable (MSI-H) tumors. Methods: We used a population-based case-unaffected sibling design including 2,248 sibships (2,253 cases; 4,486 siblings) recruited to the Colon Cancer Family Registry to evaluate the association between smoking, alcohol consumption, and CRC. Associations were assessed using conditional logistic regression, treating sibship as the matching factor. Results: Although there were no statistically significant associations between any smoking variable and CRC overall, smoking did confer an increased risk of certain types of CRC. We observed an association between pack-years of smoking and rectal cancer [odds ratio (OR), 1.85; 95% confidence interval (CI), 1.23-2.79 for >40 pack-years versus nonsmokers; Ptrend = 0.03], and there was an increased risk of MSI-H CRC with increasing duration of smoking (OR, 1.94; 95% CI, 1.09-3.46 for >30 years of smoking versus nonsmokers). Alcohol intake was associated with a modest increase in risk for CRC overall (OR, 1.21; 95% CI, 1.03-1.44 for 12+ drinks per week versus nondrinkers), with more marked increases in risk for MSI-L CRC (OR, 1.85; 95% CI, 1.06-3.24) and rectal cancer (OR, 1.48; 95% CI, 1.08-2.02). Conclusions: We found associations between cigarette smoking and increased risks of rectal cancer and MSI-H CRC. Alcohol intake was associated with increased risks of rectal cancer and MSI-L CRC. These results highlight the importance of considering tumor phenotype in studies of risk factors for CRC. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2745–50)

Colorectal cancer (CRC) can be classified as high-level microsatellite instability (MSI-H), low-level MSI (MSI-L) and microsatellite stable (MSS) depending on levels of MSI. MSI-H CRC relies on a distinct molecular pathway due to the mismatch repair (MMR) deficiency and shows methylation in multiple gene promoters. The genetic pathway leading to MSI-L is unknown, although higher levels of promoter methylation are observed in this group compared with MSS CRCs. This study explored how promoter methylation affects MSI phenotype, by analysing the methylation status of eight CRC-related promoters, MSI phenotype and KRAS/BRAF mutations in a series of 234 CRCs. Promoter methylation of p14ARF was significantly related to MSI-L CRC with KRAS mutation. The MSI-H phenotype was related to methylation of MLH1 as expected, while the MSS phenotype was related to methylation of p16INK4a and O6-methylguanine-DNA methyltransferase, although this was not statistically significant. Thus, promoter methylation of p14ARF could be a significant alteration leading to CRC with MSI-L.

Reliable methods for predicting functional consequences of variants in disease genes would be beneficial in the clinical setting. This study was undertaken to predict, and confirm in vitro, splicing aberrations associated with mismatch repair (MMR) variants identified in familial colon cancer patients. Six programs were used to predict the effect of 13 MLH1 and 6 MSH2 gene variants on pre-mRNA splicing. mRNA from cycloheximide-treated lymphoblastoid cell lines of variant carriers was screened for splicing aberrations. Tumors of variant carriers were tested for microsatellite instability and MMR protein expression. Variant segregation in families was assessed using Bayes factor causality analysis. Amino acid alterations were examined for evolutionary conservation and physicochemical properties. Splicing aberrations were detected for 10 variants, including a frameshift as a minor cDNA product, and altered ratio of known alternate splice products. Loss of splice sites was well predicted by splice-site prediction programs SpliceSiteFinder (90%) and NNSPLICE (90%), but consequence of splice site loss was less accurately predicted. No aberrations correlated with ESE predictions for the nine exonic variants studied. Seven of eight missense variants had normal splicing (88%), but only one was a substitution considered neutral from evolutionary/physicochemical analysis. Combined with information from tumor and segregation analysis, and literature review, 16 of 19 variants were considered clinically relevant. Bioinformatic tools for prediction of splicing aberrations need improvement before use without supporting studies to assess variant pathogenicity. Classification of mismatch repair gene variants is assisted by a comprehensive approach that includes in vitro, tumor pathology, clinical, and evolutionary conservation data.

Microsatellite instability (MSI) occurs in 10% to 20% of colorectal cancers (CRC) and has been attributed to both MLH1 promoter hypermethylation and germline mutation in the mismatch repair (MMR) genes. We present results from a large population- and clinic-based study of MLH1 methylation, immunohistochemistry, and MMR germline mutations that enabled us to (a) estimate the prevalence of MMR germline mutations and MLH1 methylation among MSI-H cases and help us understand if all MSI-H CRC is explained by these mechanisms and (b) estimate the associations between MLH1 methylation and sex, age, and tumor location within the colon. MLH1 methylation was measured in 1,061 population-based and 172 clinic-based cases of CRC. Overall, we observed MLH1 methylation in 60% of population-based MSI-H cases and in 13% of clinic-based MSI-H cases. Within the population-based cases with MMR mutation screening and conclusive immunohistochemistry results, we identified a molecular event in MMR in 91% of MSI-H cases: 54% had MLH1 methylation, 14% had a germline mutation in a MMR gene, and 23% had immunohistochemistry evidence for loss of a MMR protein. We observed a striking age difference, with the prevalence of a MMR germline mutation more than 4-fold lower and the prevalence of MLH1 methylation more than 4-fold higher in cases diagnosed after the age of 50 years than in cases diagnosed before that age. We also determined that female sex is an independent predictor of MLH1 methylation within the MSI-H subgroup. These results reinforce the importance of distinguishing between the underlying causes of MSI in studies of etiology and prognosis. (Cancer Epidemiol Biomarkers Prev 2008;17(11):3208–15)

Purpose: A woman with early-onset endometrial cancer (EC) may represent the “sentinel” cancer event in a Lynch syndrome kindred. The aim of this study was to determine the incidence of Lynch syndrome in a series of young-onset EC, and to identify molecular, clinical, and pathologic features that may alert clinicians to the presence of this disorder. Experimental Design: Patients with EC, ages ≤50 years, were identified from the Queensland Centre for Gynaecological Cancer. Tumor sections underwent histopathology review and were immunostained for mismatch repair proteins. Tumor DNA was tested for microsatellite instability and methylation of MLH1. Patients were conservatively classified as presumptive Lynch syndrome if their tumors showed loss of at least one mismatch repair protein and were negative for methylation of MLH1. Personal and family history of cancer was reviewed where available. Results: Presumptive Lynch syndrome was seen in 26 of 146 (18%) tumors. These tumors were more likely to be poorly differentiated, International Federation of Gynecology and Obstetrics stage II and above, have tumor-infiltrating lymphocytes, have higher mitotic rate, and have deeper myometrial invasion (P < 0.05). Lynch syndrome cases were more likely to be associated with a positive family history when analyzed for Amsterdam criteria II, diagnosis of a Lynch syndrome spectrum cancer in at least one first-degree relative, and family history of any cancer (P < 0.05). Conclusion: Presumptive Lynch syndrome was identified in 18% of early-onset EC. A risk of this magnitude would argue for routine immunohistochemical testing of tumors in patients diagnosed with EC at or before the age of 50 years.

A significant proportion of colorectal cancer (CRC) develops through the serrated neoplasia pathway. Such tumours show a distinctive molecular phenotype of somatic BRAF mutations and widespread concordant methylation events in CpG islands (CIMP). These features are also observed in the polyps developing in individuals with hyperplastic polyposis syndrome (HPS). In HPS, multiple serrated polyps develop in the colorectum, and approximately 50% of cases present with at least one CRC. Observations of rare affected sibships including identical twins, suggest a recessive or co‐dominant mode of inheritance. In addition, up to 50% of individuals with HPS report a family history of CRC. At a population level, recent work has demonstrated that patients with serrated pathway cancers characterised by BRAF mutation are four times more likely to have a family history of CRC than patients with common CRC. These findings suggest an increased genetic predisposition for serrated pathway CRC in the wider population. We propose that HPS results from the inheritance of two putative co‐dominant alleles in approximately 1 in 2000 individuals. Therefore carriers of one co‐dominant allele may number up to 1 in 25, and it is likely that these carriers are at increased risk of CRC, accounting for, at least in part, the burden of serrated pathway CRC in the population. This proposition may have important implications for screening and prevention of CRC in individuals who have an increased risk for development of serrated pathway cancers, namely those with multiple, proximal, large or advanced serrated polyps, and their relatives.

The revised Bethesda guidelines for Lynch syndrome recommend microsatellite instability (MSI) testing all colorectal cancers in patients diagnosed before age 50 years and colorectal cancers diagnosed in patients between ages 50 and 59 years with particular pathology features. Our aim was to identify pathology and other features that independently predict high MSI (MSI-H).Archival tissue from 1098 population-based colorectal cancers diagnosed before age 60 years was tested for MSI. Pathology features, site, and age at diagnosis were obtained. Multiple logistic regression was performed to determine the predictive value of each feature, as measured by an odds ratio (OR), from which a scoring system (MsPath) was developed to estimate the probability a colorectal cancer is MSI-H.Fifteen percent of tumors (162) were MSI-H. Independent predictors were tumor-infiltrating lymphocytes (OR, 9.1; 95% confidence interval [CI], 5.9-14.1), proximal subsite (OR, 4.7; 95% CI, 3.1-7.3), mucinous histology (OR, 2.8; 95% CI, 1.7-4.8), poor differentiation (OR, 1.9; 95% CI, 1.2-3.1), Crohn's-like reaction (OR, 1.9; 95% CI, 1.2-2.9), and diagnosis before age 50 years (OR, 1.9; 95% CI, 1.3-2.9). MsPath scoreor=1.0 had a sensitivity of 93% and a specificity of 55% for MSI-H.The probability an individual colorectal cancer is MSI-H is predicted well by the MsPath score. There is little value in testing for DNA mismatch repair loss in tumors, or for germline mismatch repair mutations, for colorectal cancers diagnosed in patients before age 60 years with an MSPath score1 (approximately 50%). Pathology can identify almost all MSI-H colorectal cancers diagnosed before age 60 years.

Purpose: Microsatellite instability (MSI) testing of colorectal cancer tumors is used as a screening tool to identify patients most likely to be mismatch repair (MMR) gene mutation carriers. We wanted to examine which microsatellite markers currently used to detect MSI best predict early-onset colorectal cancer caused by germ-line mutations in MMR genes. Experimental Design: Invasive primary tumors from a population-based sample of 107 cases of colorectal cancer diagnosed before age 45 years and tested for germ-line mutations in MLH1, MSH2, MSH6, and PMS2 and MMR protein expression were screened for MSI using the National Cancer Institute panel and an expanded 10-microsatellite marker panel. Results: The National Cancer Institute five-marker panel system scored 31 (29%) as NCIMSI-High, 13 (12%) as NCIMSI-Low, and 63 (59%) as NCIMS-Stable. The 10-marker panel classified 18 (17%) as 10MSI-High, 17 (16%) as 10MSI-Low, and 72 (67%) as 10MS-Stable. Of the 26 cancers that lacked the expression of at least one MMR gene, 24 (92%) were positive for some level of MSI (using either microsatellite panel). The mononucleotide repeats Bat26, Bat40, and Myb were unstable in all 10MSI-High cancers and all MLH1 and MSH2 mutation carriers (100% sensitive). Bat40 and Bat25 were unstable in all tumors of MSH6 mutation carriers (100% sensitive). Bat40 was unstable in all MMR gene mutation carriers (100% sensitive). By incorporating seven mononucleotide repeats markers into the 10-marker panel, we were able to distinguish the carriers of MSH6 mutations (all scored 10MSI-Low) from the MLH1 and MSH2 mutation carriers (all scored 10MSI-High). Conclusions: In early-onset colorectal cancer, a microsatellite panel containing a high proportion of mononuclear repeats can distinguish between tumors caused by MLH1 and MSH2 mutations from those caused by MSH6 mutations.

Apart from hysterectomy, there is no consensus recommendation for reducing endometrial cancer risk for women with a mismatch repair gene mutation (Lynch syndrome).To investigate the association between hormonal factors and endometrial cancer risk in Lynch syndrome.A retrospective cohort study included 1128 women with a mismatch repair gene mutation identified from the Colon Cancer Family Registry. Data were analyzed with a weighted cohort approach. Participants were recruited between 1997 and 2012 from centers across the United States, Australia, Canada, and New Zealand.Age at menarche, first and last live birth, and menopause; number of live births; hormonal contraceptive use; and postmenopausal hormone use.Self-reported diagnosis of endometrial cancer.Endometrial cancer was diagnosed in 133 women (incidence rate per 100 person-years, 0.29; 95% CI, 0.24 to 0.34). Endometrial cancer was diagnosed in 11% (n = 70) of women with age at menarche greater than or equal to 13 years compared with 12.6% (n = 57) of women with age at menarche less than 13 years (incidence rate per 100 person-years, 0.27 vs 0.31; rate difference, -0.04 [95% CI, -0.15 to 0.05]; hazard ratio per year, 0.85 [95% CI, 0.73 to 0.99]; P = .04). Endometrial cancer was diagnosed in 10.8% (n = 88) of parous women compared with 14.4% (n = 40) of nulliparous women (incidence rate per 100 person-years, 0.25 vs 0.43; rate difference, -0.18 [95% CI, -0.32 to -0.04]; hazard ratio, 0.21 [95% CI, 0.10 to 0.42]; P .001). Endometrial cancer was diagnosed in 8.7% (n = 70) of women who used hormonal contraceptives greater than or equal to 1 year compared with 19.2% (n = 57) of women who used contraceptives less than 1 year (incidence rate per 100 person-years, 0.22 vs 0.45; rate difference, -0.23 [95% CI, -0.36 to -0.11]; hazard ratio, 0.39 [95% CI, 0.23 to 0.64]; P .001). There was no statistically significant association between endometrial cancer and age at first and last live birth, age at menopause, and postmenopausal hormone use.For women with a mismatch repair gene mutation, some endogenous and exogenous hormonal factors were associated with a lower risk of endometrial cancer. These directions and strengths of associations were similar to those for the general population. If replicated, these findings suggest that women with a mismatch repair gene mutation may be counseled like the general population in regard to hormonal influences on endometrial cancer risk.

Sporadic colorectal cancers with a high degree of microsatellite instability are a clinically distinct subgroup with a high incidence of BRAF mutation and are widely considered to develop from serrated polyps. Previous studies of serrated polyps have been highly selected and largely retrospective. This prospective study examined the prevalence of sessile serrated adenomas and determined the incidence of BRAF and K-ras mutations in different types of polyps.An unselected consecutive series of 190 patients underwent magnifying chromoendoscopy. Polyp location, size, and histologic classification were recorded. All polyps were screened for BRAF V600E and K-ras codon 12 and 13 mutations.Polyps were detected in 72% of patients. Most (60%) were adenomas (tubular adenomas, tubulovillous adenomas), followed by hyperplastic polyps (29%), sessile serrated adenomas (SSAs; 9%), traditional serrated adenomas (0.7%), and mixed polyps (1.7%). Adenomas were more prevalent in the proximal colon (73%), as were SSAs (75%), which tended to be large (64%5 mm). The presence of at least one SSA was associated with increased polyp burden (5.0 vs 2.5; P.0001) and female sex (P.05). BRAF mutation was rare in adenomas (1/248 [0.4%]) but common in SSAs (78%), traditional serrated adenomas (66%), mixed polyps (57%), and microvesicular hyperplastic polyps (70%). K-ras mutations were significantly associated with goblet cell hyperplastic polyps and tubulovillous adenomas (P.001).The prevalence of SSAs is approximately 9% in patients undergoing colonoscopy. They are associated with BRAF mutation, proximal location, female sex, and presence of multiple polyps. These findings emphasize the importance of identifying and removing these lesions for endoscopic prevention of colorectal cancer.

In recent years, an alternative pathway of colorectal cancer development has been described in which serrated polyps replace the traditional adenoma as the precursor lesion. Importantly, serrated polyps and a subset of colorectal cancer show largely nonoverlapping mutation profiles to those found in adenomas and the majority of colorectal cancer. These genetic alterations include activating mutation of the BRAF proto-oncogene and widespread gene promoter hypermethylation (CpG island methylator phenotype or CIMP). Up to 15% of colorectal cancer is likely to develop on the basis of a strong genetic predisposition. The two most well-characterized syndromes, familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer (Lynch syndrome), both develop via the adenoma-carcinoma pathway and together account for approximately one third of familial colorectal cancer. We have recently described 11 families in which there is evidence that the genetic predisposition to autosomal dominant colorectal cancer is linked to the serrated pathway. This condition, serrated pathway syndrome, and the related condition, hyperplastic polyposis, the presentation of which suggests a recessive mode of inheritance, represent two syndromes in which BRAF mutation and methylation co-occur within serrated precursor lesions. Further, CIMP is observed in the normal colonic mucosa of individuals with hyperplastic polyposis consistent with a field defect in epigenetic regulation. The spectrum of serrated neoplasia may also implicate the apparently sporadic and later onset subset of colorectal cancer with high levels of microsatellite instability. The tendency for these lesions to be multiple, associated with smoking, and to show frequent BRAF mutation and CIMP points to a defect that may result from interactions between the environment and a weakly penetrant genetic alteration. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1778–84)

Background & Aims: Hyperplastic polyposis syndrome (HPS) is defined phenotypically with multiple, large and/or proximal hyperplastic polyps. There is no known germ-line predisposition. We aimed to characterize the clinicopathologic features of 38 patients with HPS and explore the role of germ-line mutations in the base excision repair genes MBD4 and MYH. Methods: Utilizing clinical databases of The Royal Melbourne Hospital Bowel Cancer Surveillance Service and the Familial Cancer Clinic, 38 patients with HPS were recruited. The patients were analyzed for age at first diagnosis, features of hyperplastic polyposis, family histories of polyposis and colorectal cancer (CRC), coexisting adenomas, serrated adenomas, incidence of CRC, and microsatellite instability in the tumours. Mutation analysis of MBD4 and MYH were performed. Results: Serrated adenomas were common (26%), and 19 (50%) of the 38 patients had a first-degree relative with CRC. Family history of HPS was uncommon, with only 2 cases found. Ten patients developed CRC, and 3 required surgery for polyposis. No pathogenic mutations in MBD4 were detected in the 27 patients tested, but 6 single nucleotide polymorphisms of uncertain functional significance were identified. Pathogenic biallelic MYH mutations were detected in 1 patient. Conclusions: Mutations in MBD4 are unlikely to be implicated in HPS; MYH mutations should be studied, especially when adenomas occur in the same patient. The clinical, histopathologic, and molecular findings of this study should contribute to our understanding of HPS and its relationship to the serrated neoplasia pathway.

Somatic mutation of K-ras is known to be a common event in colorectal cancer tumourigenesis however its association with age at onset has not been widely explored. In this study, we have analyzed tumours from a population-based study of colorectal cancer diagnosed before the age of 45 years, in which cases had been previously screened for germ-line mismatch repair gene mutations and for microsatellite instability. We used a micro-dissection and sequencing approach to search for somatic K-ras mutations in codons 12, 13 and 61 in 101 early-onset colorectal cancers. Six (6%) somatic K-ras mutations were detected; five in codon 12 (4 G>T transitions and 1 G>A) and one in codon 13 (G>A transition). All codon 12 mutations were identified in microsatellite stable tumours and the codon 13 mutation was identified in a MSI-high tumour. Four cases with K-ras mutations had no reported family history of colorectal cancer and two had some family history of colorectal cancer. None were known to carry a germ-line mutation in hMSH2, hMLH1, hMSH6 or hPMS2. The role of somatic K-ras mutations in early-onset colorectal cancer carcinogenesis appears to be minor, in contrast to its significant role in colorectal cancer of later age of onset.

The KIAA0101/p15(PAF)/OEATC-1 protein was initially isolated in a yeast two-hybrid screen for proliferating cell nuclear antigen (PCNA) binding partners, and was shown to bind PCNA competitively with the cell cycle regulator p21(WAF). PCNA is involved in DNA replication and damage repair. Using polyclonal antisera raised against a p15(PAF) fusion protein, we have shown that in a range of mammalian tumor and non-tumor cell lines the endogenous p15(PAF) protein localises to the nucleus and the mitochondria. Under normal conditions no co-localisation with PCNA could be detected, however following exposure to UV it was possible to co-immunoprecipitate p15(PAF) and PCNA from a number of cell lines, suggesting a UV-enhanced association of the two proteins. Overexpression of p15(PAF) in mammalian cells was also found to protect cells from UV-induced cell death. Based on similarities between the behaviour of p15(PAF) and the potential tumor suppressor product p33ING1b, we have further shown that these two proteins interact in the same complex in cell cultures. This suggests that p15(PAF) forms part of a larger protein complex potentially involved in the regulation of DNA repair, apoptosis and cell cycle progression. (c) 2005 Elsevier Inc. All rights reserved.

Colorectal adenomas have traditionally been regarded as homogeneous. The aim of our study was to identify molecular features that may differentiate sporadic adenomas front familial adenomas such as Familial Adenomatous Polyposis (FAP) and Multiple Adenoma patients. DNA methylation was tested at Methylated IN Tumor (MINT) loci (1,2,12,31) and the CpG promoter region of genes MLH1, HPP1, MGMT, p14(ARF) and p16(INK4a) in FAP-associated adenomas (33) from 5 patients with a known APC mutation (Group 1, FAP), adenomas (29) from 4 Multiple Adenoma patients (Group 2 Multiple), adenomas (14) from 3 patients with sporadic colorectal cancers showing high microsatellite instability (Group 3, MSI-H) and adenomas (16) from 7 patients, with sporadic colorectal cancers showing microsatellite stable or low level instability (Group 4, MSS/MSI-L). Aberrant Crypt Foci (ACFs), Hyperplastic Polyps (HPs) and cancers were also examined for methylation status as well as K-ras mutation. Multiple Adenoma patients were examined for germline polymorphisms in the base excision repair gene, MYH. The familial syndrome, FAP -associated adenomas showed a significantly low frequency of MINT methylation (15.5%) compared to sporadic MSS/MSI-L-associated adenomas (35.5%). Group 3 (MSI-H) adenomas were different in that many showed serration and a high level of methylation (57.1%). Group 2, Multiple Adenoma cases, resembled sporadic MSS/MSI-L-associated adenomas. However the promoter regions of key genes, MGMT, p14(ARF) and p16(INK4a) were methylated to a greater extent than MINTs in both sporadic and familial adenomas. Genetic profiling of adenomas supports the concept that adenomas belonging to familial syndromes pursue a different pathway to tumorigenesis than their sporadic counterpar\ts from their earliest formation. (c) 2005 Wiley-Liss, Inc.

Background & Aims: Recently, an alternative pathway of tumorigenesis has been identified in the colorectum associated with serrated precursor lesions, variable levels of microsatellite instability (MSI-V), and driven in part by activating mutations in the BRAF proto-oncogene (V599E). Somatic BRAF mutations in hereditary nonpolyposis colon cancer (HNPCC) are rarely observed. Here, we discuss their role in the development of other familial colorectal cancers (CRC). We studied non-FAP, non-HNPCC CRC families characterized by tumors that varied in their level of MSI between individual members. Methods: A subset of tumors from a total of 55 collected (25 polyp and 30 cancers) from 43 individuals across 11 families underwent pathology review, examination for V599E using allele-specific polymerase chain reaction, and for methylation of the MINT31 CpG island. Results: All MSI-V families met the current revised Bethesda Guidelines and 6 of 11 (55%) met the Amsterdam I criteria. V599E was observed in 12 of 19 (63%) polyps and 14 of 20 (70%) cancers (4 of 4 high MSI, 2 of 4 low MSI, and 8 of 12 stable MSI), a significant increase over HNPCC (0 of 15 or 0%), and unselected CRC (30 of 197 or 15.2%) (P < .05). Eight of the 10 (80%) cancers that underwent analysis showed hypermethylation of MINT31. CRCs showed early age at onset and were more likely to show a serrated architecture than unselected CRCs (P < .05). Conclusion: These data provide evidence that the families described here represent a syndrome of familial CRC that is distinct from HNPCC. High levels of BRAF mutation and MINT31 hypermethylation suggest an origin in the serrated pathway of CRC development.

Aim: Hyperplastic polyps (HP) of the colorectum have traditionally been regarded as non-neoplastic lesions. Recent data implicate HP in the pathogenesis of colorectal cancers (CRC) characterised by extensive DNA methylation and microsatellite instability. The aim of this study was to identify morphological and molecular features that may characterise subtypes of HP with potential for neoplastic progression. Materials and methods: HP (22) clustering around distal CRC (group I) were compared with HP (58) in subjects with hyperplastic polyposis (group II). DNA methylation was tested in methylated in tumour (MINT) loci (1, 2, 12, 31) and genes HPP1 , MGMT , p14 ARF , p16 INK4a , and hMLH1 . Results: Group II HP showed significantly more methylation than group I HP at all loci except MINT1 and MGMT . Group I showed the lowest frequency of DNA methylation but the highest frequency of K- ras mutation. Group II HP (termed HP variant) had the morphological features of the recently described “sessile serrated adenomas”. Methylation of hMLH1 was found most frequently in group II polyps that included foci of dysplasia (7/10) and in no group I lesions. Conclusion: The findings support the existence of morphological and molecular heterogeneity among HP and highlight a subset that is likely to have significant malignant potential.

Loss of heterozygosity (LOH) on 8p occurs at high frequencies in many tumor types, including colorectal carcinoma (CRC). We previously used microcell-mediated chromosome transfer (MMCT) into the CRC cell line SW620 to map a ∼7.7-Mb colorectal cancer–suppressor region (CRCSR) at 8p22–23.1. In the current study, we transferred small fragments of this CRCSR into SW620 to refine the region further. Two microcell hybrids containing a 321- to 898-kb region around the D8S552 marker at 8p23.1 showed suppression of soft agar clonicity and tumorigenicity in athymic mice when compared to control cell lines. These data suggest that the putative colorectal tumor–suppressor gene is within this small region. We analyzed two candidate genes within this region: FLJ23749 and KIAA1456. Expression of both genes was detected in normal colonic crypt cells and in mucosa. Quantitative reverse transcriptase polymerase chain reaction showed downregulation of KIAA1456 in 9 of 12 primary colorectal tumors compared to matching normal mucosa, but normal or increased expression of FLJ23749. FLJ23749 was expressed in all CRC cell lines tested; however, KIAA1456 was downregulated in three cell lines, including SW620, and was restored in the suppressed microcell hybrids. 5′aza-2′Deoxycytidine treatment of the downregulated cell lines restored expression of KIAA1456, but bisulfite genomic sequencing did not show a correlation between promoter methylation and expression. Forty percent of the primary tumors showed LOH at this CRCSR locus, and mutation analysis revealed somatic mutations in 1 of 88 primary colorectal tumors for both KIAA1456 and FLJ23749. Despite the rarity of somatic mutations, the expression data suggest that KIAA1456 is still a candidate for the putative 8p colorectal cancer tumor-suppressor gene. © 2004 Wiley-Liss, Inc.

To the Editor: Lynch syndrome (LS) (MIM#120435) is a cancer predisposition condition resulting from mutations within the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 or PMS2). In a suspected LS family, tumors are assessed for microsatellite instability (MSI) and/or immunohistochemical (IHC) absence of MMR proteins, both of which are hallmarks of LS. To enable appropriate diagnosis, counseling and surveillance, the gene indicated by the IHC profile is screened for mutations via techniques such as denaturing high pressure liquid chromatography (DHPLC), sequencing and multiplex ligation-dependent probe amplification (MLPA). Using these approaches, mutations are identified in the majority of cases suspected of LS; however, there remains a substantial proportion of families for which mutations cannot be identified...

Attempts to classify colorectal cancer into subtypes based upon molecular characterisation are overshadowed by the classical stepwise model in which the adenoma-carcinoma sequence serves as the morphological counterpart. Clarity is achieved when cancers showing DNA microsatellite instability (MSI) are distinguished as sporadic MSI-low (MSI-L), sporadic MSI-high (MSI-H) and hereditary non-polyposis colorectal cancer (HNPCC). Divergence of the 'methylator' pathway into MSI-L and MSI-H is at least partly determined by the respective silencing of MGMT and hMLH1. Multiple differences can be demonstrated between sporadic and familial (HNPCC) MSI-H colorectal cancer with respect to early mechanisms, evolution, molecular characterisation, demographics and morphology. By acknowledging the existence of multiple pathways, rapid advances in the fields of basic and translational research will occur and this will lead to improved strategies for the prevention, early detection and treatment of colorectal cancer.

Background: Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends. The expression of telomerase is thought to be required for cellular immortality and oncogenesis. Methods: To investigate the role of telomerase in the pathogenesis of colorectal cancer, we analysed telomerase activity in biopsy samples of colorectal cancer and colonic adenomas. Using a polymerase chain reaction-based assay we examined telomerase activity in 52 samples of colorectal cancer, 12 colonic adenomas and 30 normal colonic mucosa samples obtained by endoscopic biopsy. Results: Telomerase activity was detectable in 88.5% (46/52) of colorectal carcinomas, in 50% (6/12) of colonic adenomas but not in normal colorectal mucosa. There was no correlation between telomerase activity and tumour location, type, size and differentiation (P > 0.05). Conclusions: It was concluded that telomerase activation plays a role in the evolution of colorectal cancer, and that measurement of telomerase activity in biopsied colorectal mucosa samples may provide information both as a diagnostic marker to detect small numbers of cancer cells, and as a screening method for patients at high risk for colorectal carcinoma.

Background The aim of this systematic review was to compare the safety and efficacy of dynamic graciloplasty with colostomy for the treatment of faecal incontinence. Methods Two search strategies were devised to retrieve literature from the Medline, Current Contents, Embase and Cochrane Library databases up until November 1999. Inclusion of papers depended on a predetermined protocol, independent assessments by two reviewers and a final consensus decision. English language papers were selected. Acceptable study designs included randomized controlled trials, controlled clinical trials and case series. Forty papers met the inclusion criteria. They were tabulated and critically appraised in terms of methodology and design, outcomes, and the possible influence of bias, confounding and chance. Results No high-level evidence was available and there were no comparative studies. Mortality rates were around 2 per cent for both graciloplasty and colostomy. Morbidity rates reported for graciloplasty appear to be higher than those for colostomy. Dynamic graciloplasty was clearly effective at restoring continence in between 42 and 85 per cent of patients, whereas colostomy is, by its design, incapable of restoring continence. However, dynamic graciloplasty is associated with a significant risk of reoperation. Conclusion While dynamic graciloplasty appears to be associated with a higher rate of complications than colostomy, it is clearly a superior intervention for restoring continence in some patients. It is recommended that a comparative, but non-randomized, study be undertaken to evaluate the safety of dynamic graciloplasty in comparison to colostomy, and that the procedure should be performed only in centres where it is carried out routinely.

Aims: In hereditary colorectal cancer (CRC) disorders such as familial adenomatous polyposis and hereditary non-polyposis colon cancer, the identification of germline mutations greatly assists in the clinical management of families. In addition, study of somatic mutations in the cancers themselves (both hereditary and sporadic) has been fundamental in the elucidation of the initiation and progression of CRC. Many of the genes underlying CRC development are large; hence mutation screening is a time-consuming and labour-intensive process requiring a rapid and accurate alternative to gel-based systems such as single-strand confirmational polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE). Here we report our progress using denaturing gradient high-pressure liquid chromatography (DHPLC) in the screening of the mismatch repair genes MLH1 and MSH2 and in screening the APC and HPP1 tumour suppressor genes for mutations. Methods: Genomic DNA was amplified using intronic primer sets spanning individual exons in the gene(s) under study. PCR products were subjected to DHPLC and the resultant chromatographs were compared with those of normal controls and aberrant peaks identified. Amplified products with aberrant peaks in the study samples underwent manual sequencing to confirm the presence of sequence variants. Results: The proportion of amplified fragments showing aberrant peaks (hits) ranged from 18 to 30% and in the case of every gene, more than 80% of these could be confirmed as a sequence variant by manual sequencing. The highest rate was found in HPP1, where all hits were found to be sequence variants, and the lowest rate was found in MSH2, where manual sequencing failed to find a sequence variant in 17% of the hits attained. Mutations varied in their nature from directly truncating through splice variants to missense and deletion mutations. Traces for each mutation displayed unique shapes and both deletions and single base changes were equally dramatic. During the mutation scanning many polymorphisms presented as aberrant peaks, as would be expected. Importantly, the same polymorphism gave an identical chromatographic tracing between individuals, opening the possibility to identify common polymorphisms on pattern recognition alone. There remains, though, the possibility that rare pathogenic variants may assume an identical shape. Conclusions: The results indicate that DHPLC is a sensitive and efficient technique for screening of DNA for sequence variants. Given that polymorphisms comprised the largest proportion of variants found in each gene (66-100%), excluding these by pattern recognition would markedly reduce the amount of sequencing required.

The average age at diagnosis for colorectal cancer (CRC) in Australia is 69, and the age-specific incidence rises rapidly after age 50 years. The incidence has stabilized or is declining in older age groups in Australia during recent decades, possibly related to the increased uptake of screening and high-risk surveillance. In the same time frame, a rising incidence of CRC in younger adults has been well-documented in the United States. This rise in incidence in the young has not been reported from other countries that share long-term exposure to westernised urban lifestyles. Using data from the Australian Institute of Health and Welfare, we examined trends in national incidence rates for CRC under age 50 years and observed that rates in people under age 40 years have been rising for the last two decades. We further performed a review of the literature regarding CRC in young adults to outline the extent of current understanding, explore potential risk factors such as obesity, alcohol, and sedentary lifestyles, and to identify the questions remaining to be addressed. Although absolute numbers might not justify a population screening approach, the dispersal of young adults with CRC across the primary health-care system decreases probability of their recognition. Patient and physician awareness, aided by stool and emerging blood-screening tests and risk profiling tools, have the potential to aid in identification of those young adults who would most benefit from a colonoscopy through early detection of CRCs or by removal of advanced polyps.

Background: Inheritance of a germline mutation in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 causes a high risk of colorectal and other cancers (Lynch Syndrome). Use of aspirin has been shown to be associated with a reduced risk of colorectal cancer for the general population as well as for MMR gene mutation carriers. The aim of this study was to determine whether use of aspirin and ibuprofen in a nontrial setting is associated with the risk of colorectal cancer risk for MMR gene mutation carriers. Methods: We included 1858 participants in the Colon Cancer Family Registry who had been found to have a pathogenic germline mutation in a MMR gene (carriers). We used weighted Cox proportional hazards regression to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). All statistical tests were two-sided. Results: A total of 714 carriers (38%) were diagnosed with colorectal cancer at a mean age of 42.4 (standard deviation 10.6) years. A reduced risk of colorectal cancer was associated with aspirin use (for 1 month to 4.9 years: HR = 0.49, 95% CI = 0.27 to 0.90, P =. 02; for 5 years: HR = 0.25, 95% CI = 0.10 to 0.62, P =. 003) and ibuprofen use (for 1 month to 4.9 years: HR = 0.38, 95% CI = 0.18 to 0.79, P =. 009; for 5 years: HR = 0.26, 95% CI = 0.10 to 0.69, P =. 007), compared with less than one month of use. Conclusion: Our results provide additional evidence that, for MMR gene mutation carriers, use of aspirin and ibuprofen might be effective in reducing their high risk of colorectal cancer.

The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.

Objective To estimate risk of colorectal cancer (CRC) for first-degree relatives of CRC cases based on CRC molecular subtypes and tumour pathology features. Design We studied a cohort of 33 496 first-degree relatives of 4853 incident invasive CRC cases (probands) who were recruited to the Colon Cancer Family Registry through population cancer registries in the USA, Canada and Australia. We categorised the first-degree relatives into four groups: 28 156 of 4095 mismatch repair (MMR)-proficient probands, 2302 of 301 MMR-deficient non-Lynch syndrome probands, 1799 of 271 suspected Lynch syndrome probands and 1239 of 186 Lynch syndrome probands. We compared CRC risk for first-degree relatives stratified by the absence or presence of specific tumour molecular pathology features in probands across each of these four groups and for all groups combined. Results Compared with first-degree relatives of MMR-proficient CRC cases, a higher risk of CRC was estimated for first-degree relatives of CRC cases with suspected Lynch syndrome (HR 2.06, 95% CI 1.59 to 2.67) and with Lynch syndrome (HR 5.37, 95% CI 4.16 to 6.94), but not with MMR-deficient non-Lynch syndrome (HR 1.04, 95% CI 0.82 to 1.31). A greater risk of CRC was estimated for first-degree relatives if CRC cases were diagnosed before age 50 years, had proximal colon cancer or if their tumours had any of the following: expanding tumour margin, peritumoral lymphocytes, tumour-infiltrating lymphocytes or synchronous CRC. Conclusions Molecular pathology features are potentially useful to refine screening recommendations for first-degree relatives of CRC cases and to identify which cases are more likely to be caused by genetic or other familial factors.