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This review explores the chief genetic and epigenetic events that promote pathological progression in colorectal carcinogenesis. This article discusses the molecular and pathological basis for classifying colorectal neoplasia into suppressor, mutator and methylator pathways. These differing mechanisms of genomic instability are associated with specific cancer characteristics, and may provide the opportunity for more effective prevention and surveillance strategies in the future. This is the first review in a series of five topics outlining important and developing aspects of colorectal cancer.

In this chapter, it is pointed out that colorectal cancer is a heterogeneous disease. The case is made for a ‘serrated pathway’ of neoplasia that would evolve relatively rapidly through the early acquisition of DNA instability. DNA hypermethylation is likely to be of critical importance in driving this pathway. Inhibition of apoptosis is conceived as the first step. Thereafter, methylation of one of several DNA repair genes would result in a state of tolerated hypermutability. It remains to be shown whether this model applies to a small subset of colorectal cancers or in fact explains the great majority given the overall low risk of progression for an individual adenoma initiated by mutation of APC.

The mutated in colorectal cancer (MCC) gene is in close linkage with the adenomatous polyposis coli (APC) gene on chromosome 5, in a region of frequent loss of heterozygosity in colorectal cancer. The role of MCC in carcinogenesis, however, has not been extensively analysed, and functional studies are emerging, which implicate it as a candidate tumor suppressor gene. The aim of this study was to examine loss of MCC expression due to promoter hypermethylation and its clinicopathologic significance in colorectal cancer. Correspondence of MCC methylation with gene silencing was demonstrated using bisulfite sequencing, reverse transcription–polymerase chain reaction and Western blotting. MCC methylation was detected in 45–52% of 187 primary colorectal cancers. There was a striking association with CDKN2A methylation (P

BACKGROUND. The Wnt signalling pathway is altered in the majority of colorectal cancers by APC mutation. Cancers arising via the serrated neoplasia pathway are hallmarked by BRAF mutation and microsatellite instability (MSI), but do not commonly mutate APC. RNF43 and ZNRF3 are transmembrane E3 ubiquitin ligases that are localised to intestinal stem cells where they cooperate to suppress Wnt signaling. Mutation of these genes may be an alternate mechanism to bypass the Wnt signal. RNF43 / ZNRF3 double knockout mice develop intestinal neoplasia and germline mutation of RNF43 has been reported in serrated polyposis. We therefore hypothesised that somatic mutation of RNF43 and/or ZNRF3 would be important in the development of serrated colorectal neoplasia. METHODS. RNF43 and ZNRF3 mutations were assessed in a pilot series of 24 BRAF mutant cancers representing the serrated neoplasia pathway and 10 control BRAF wild type cancers using Agilent V4+UTR whole exome capture followed by sequencing on an Illumina HiSeq 2000. An extended cohort of 80 BRAF mutant cancers (48 MSI, 32 MSS) and 67 BRAF wild type cancers was examined by Sanger sequencing. The frequency of a common hotspot mutation in RNF43 was validated in a second series of 42 BRAF mutant and 22 BRAF wild type colorectal cancers. Transcript expression for RNF43 and ZNRF3 was assayed using Illumina HT-12v4 BeadChip expression arrays in 29 BRAF mutant cancers, 187 BRAF wild type cancers and a subset of 32 matched normal mucosa samples. RESULTS. RNF43 mutations were detected in whole exome sequencing data in 11/24 BRAF mutant but no BRAF wild type cancers (P CONCLUSIONS. RNF43 is a frequent mutational target in serrated neoplasia. The high frequency of both substitution and frameshift mutations support the functional relevance of these alterations. We hypothesise that RNF43 is mutated as an alternative to APC in serrated lesions as a mechanism of bypassing control of the Wnt signal. These data support our current model of serrated neoplasia whereby BRAF is mutated early in serrated polyp development and RNF43 is subsequently mutated to remove constraints on the Wnt signal and allow progression to cancer. Citation Format: Vicki L. Whitehall, Catherine Bond, Diane McKeone, Jonathan Ellis, Mark Bettington, Sally Pearson, Barbara Leggett. RNF43 is a frequent mutational target in serrated colorectal neoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2076. doi:10.1158/1538-7445.AM2015-2076

Background SnoN is an important regulator of the transforming growth factor beta (TGFβ) signalling pathway and has been shown to exhibit both tumour promotion and suppression activity. Methods To further explore the role of this complex molecule in colorectal tumorigenesis, we examined 52 paired normal and tumour colorectal specimens stratified by level of microsatellite instability; 18 with high-level microsatellite instability (MSI-H) and 34 microsatellite stable (MSS). SnoN transcript expression was quantitated by real-time PCR and analysed with respect to clinical indicators of prognosis. Results Within the MSI-H subgroup, SnoN was commonly either up-regulated (6/18, 33%) or down-regulated (7/18, 39%). A significantly different distribution of SnoN expression was observed in MSS cancers compared with MSI-H (P ≤ 0.001). Whilst 17/34 (50%) of MSS tumours demonstrated up-regulation, none showed down-regulated expression. Within the MSI-H subgroup, up-regulation was significantly correlated with lack of repeat tract mutation in the TGFβRII gene (P ≤ 0.025), suggesting that SnoN is more frequently up-regulated in the presence of functional TGFβ signalling. Conclusion Together these data support the notion that SnoN has both oncogenic and tumour suppressive properties depending on other genetic changes within the tumour, and that the MSI-H pathway of colorectal tumorigenesis presents an excellent model for the study of these opposing functions.

Mucins are complex mucosal glycoproteins that can be highly expressed by adenocarcinomas, having diagnostic, therapeutic, and biological significance. MUC13 encodes a cell surface membrane-anchored mucin expressed in the normal gastrointestinal tract, trachea, and kidney as well as colorectal, esophageal, gastric, pancreatic, and lung cancers. MUC13 protein expression was determined immunohistochemically in 99 sporadic colorectal cancers, assessing proportion of tumor cells stained, stain intensity, and localization. In normal colon, intense apical membrane and variable cytoplasmic MUC13 staining was present in both goblet and columnar cells, with strongest reactivity in the upper crypts and surface epithelium. All cancers showed staining of most tumor cells, being most conspicuous in the apical membranes of gland spaces. Left-sided tumors had a higher overall proportion of MUC13-positive tumor cells than right-sided tumors (P < .05), and high staining intensity was more frequent in adenocarcinomas (81%) than mucinous tumors (50%) (P < .05). Poorly differentiated and late-stage tumors were more likely to have high-intensity cytoplasmic staining (P

Hepatocellular carcinoma (HCC) is associated with multiple risk factors and is believed to arise from pre-neoplastic lesions, usually in the background of cirrhosis. However, the genetic and epigenetic events of hepatocarcinogenesis are relatively poorly understood. HCC display gross genomic alterations, including chromosomal instability (CIN), CpG island methylation, DNA rearrangements associated with hepatitis B virus (HBV) DNA integration, DNA hypomethylation and, to a lesser degree, microsatellite instability. Various studies have reported CIN at chromosomal regions, 1p, 4q, 5q, 6q, 8p, 10q, 11p, 16p, 16q, 17p and 22q. Frequent promoter hypermethylation and subsequent loss of protein expression has also been demonstrated in HCC at tumor suppressor gene (TSG), p16, p14, p15, SOCS1, RIZ1, E-cadherin and 14-3-3 sigma. An interesting observation emerging from these studies is the presence of a methylator phenotype in hepatocarcinogenesis, although it does not seem advantageous to have high levels of microsatellite instability. Methylation also appears to be an early event, suggesting that this may precede cirrhosis. However, these genes have been studied in isolation and global studies of methylator phenotype are required to assess the significance of epigenetic silencing in hepatocarcinogenesis. Based on previous data there are obvious fundamental differences in the mechanisms of hepatic carcinogenesis, with at least two distinct mechanisms of malignant transformation in the liver, related to CIN and CpG island methylation. The reason for these differences and the relative importance of these mechanisms are not clear but likely relate to the etiopathogenesis of HCC. Defining these broad mechanisms is a necessary prelude to determine the timing of events in malignant transformation of the liver and to investigate the role of known risk factors for HCC.

We have recently reported that germline epimutations of the mismatch repair gene, MLH1 , produce a clinical phenotype consistent with hereditary non-polyposis colorectal cancer (HNPCC).1 MLH1 germline epimutations have now been reported in eight individuals clinically classified as having HNPCC and in whom no sequence mutations of the mismatch repair genes were identified.1–4 These manifest as soma-wide monoallelic hypermethylation of the promoter (in the absence of a sequence mutation within the gene) and cause transcriptional silencing of the epimutant allele.1 These cases provide evidence that germline epimutations represent a novel mechanism for disease, which can mimic that of a genetic mutation by conferring a similar phenotype. We hypothesised that germline epimutations may occur in genes other than MLH1 to cause other cancer prone syndromes apart from HNPCC. The APC gene represented a likely candidate as heterozygous inactivating mutations of APC are strongly associated with colorectal polyposis and cancer, but not all individuals harbour germline sequence mutations. Autosomal dominant familial adenomatous polyposis (FAP) is caused by heterozygous sequence mutations of the APC gene,5 and a recessive form is associated with biallelic mutations of MYH .6 However, no mutations of either gene are identifiable in approximately 20% of cases. Furthermore, somatic methylation of the CpG island encompassing …

Three studies report estimates of the cost and effectiveness of alternate strategies for screening the average-risk Australian population for colorectal cancer. The options considered are faecal occult blood testing, double contrast barium enema, sigmoidoscopy and colonoscopy. At present, there is no consensus over which screening method is optimal by the economic criterion. Also, the existing studies report a mixture of average and incremental cost-effectiveness ratios derived from data collected between 1994 and 2002. We suggest average cost-effectiveness ratios are not useful for decision-making and illustrate how they differ from the preferred incremental cost-effectiveness ratio. We then update the cost data reported in the three studies to 2002 prices and calculate incremental cost-effectiveness ratios where not previously available. Our re-analysis of one study contradicts the conclusions drawn by the authors, who had only calculated average cost-effectiveness ratios. In particular, we find their recommendation of population screening with colonoscopy would cause, annually, between 33 and 1,322 years of life to be lost and between $M17 and $M87 to be wasted. Based on updated cost data and the incremental analysis, our findings indicate that population screening using biennial faecal occult blood testing ($39,459 per life-year gained), annual faecal occult blood testing ($30,556 per life-year gained) and colonoscopy ($26,587 per life-year gained) are cost-effective. Hence, the decision over which method of screening is optimal remains ambiguous across the three studies. We recommend policy-makers choose the study they believe produces the most accurate estimates of cost and health effect, identify their willingness to pay for health benefits and consider other issues relevant to the decision.

Microsatellite instability in the insulin–like growth factor II receptor gene in gastrointestinal tumours

Colorectal cancers (CRCs) may be categorised according to the degree of microsatellite instability (MSI) exhibited, as MSI-high (MSI-H), MSI-low (MSI-L), or microsatellite stable (MSS). MSI-H status confers a survival advantage to patients with sporadic CRC.To determine if low levels of MSI are related to the clinicopathological features and prognosis of sporadic stage C CRC.A total of 255 patients who underwent resection for sporadic stage C CRC were studied. No patient received chemotherapy. Minimum follow up was five years.DNA extracted from archival malignant and non-malignant tissue was amplified by polymerase chain reaction using a panel of 11 microsatellites. MSI-H was defined as instability ator =40% of markers, MSS as no instability, and MSI-L as instability at0% but40% of markers. Patients with MSI-H CRC were excluded from analysis as they have previously been shown to have better survival.Thirty three MSI-L and 176 MSS CRCs were identified. There was no difference in biological characteristics or overall survival of MSI-L compared with MSS CRC but MSI-L was associated with poorer cancer specific survival (hazard ratio 2.0 (95% confidence interval 1.1-3.6)).Sporadic MSI-L and MSS CRCs have comparable clinicopathological features. Further studies are required to assess the impact of MSI-L on prognosis.

Background: Colorectal cancers resulting from defective DNA mismatch repair can occur in both hereditary non-polyposis colon cancer (HNPCC) and in the sporadic setting. They are characterised by a high level of microsatellite instability (MSI-H) and superficially resemble each other in that they are frequently located in the proximal colon and share features such as circumscribed tumour margins and tumour-infiltrating lymphocytes. However, significant differences can be demonstrated at the molecular level including widespread promoter hypermethylation and BRAF-activating mutations which occur significantly less often in HNPCC. Aims: In this study, we sought to determine whether the presence of widespread promoter hypermethylation and BRAF mutations would exclude HNPCC. Materials and methods: We investigated the methylation status of four methylated in tumour markers (MINTs 1,2,12 and 31), and the promoter regions of 5 genes hMLH1, HPP1, MGMT, p16 INK4A and p14 ARF , in 21 sporadic MSI-H colorectal cancers and compared these with 18 cancers from HNPCC patients. The methylation status of CpG islands were determined by either methylation specific PCR (MSP) or combined bisulfite restricton analysis (COBRA). In addition we considered the BRAF mutation status of 18 HNPCC tumours and 19 sporadic MSI-H cancers which had been previously determined by RFLP analysis and confirmatory sequencing. Results: Methylation of the promoter regions in target genes occurred less frequently within the HNPCC tumours (27% of analyses), compared with the sporadic MSI-H tumours (59% of analyses)(P < 0.001). Methylation of MINTs 1, 2, 12 and 31 occurred in 4% of analyses for HNPCC tumours contrasted with 73% for sporadic MSI-H tumours (P < 0.001). BRAF mutations were detected in 74% of sporadic tumours but none of the HNPCC cancers tested. Conclusions: The total number of genes and MINTs methylated in HNPCC was lower than in MSI-H colorectal tumours. No HNPCC tumour showed evidence of widespread promoter hypermethylation or BRAF mutation suggesting this feature could be used as a discriminator between familial and sporadic cases.

Background . Population-wide screening for people at average risk of colorectal cancer (CRC) by faecal occult blood test (FOBT) is under consideration in Australia. Methods . Qualitative methods were used to examine the views of community members who did ( n = 18) or did not ( n = 12) participate in a pilot program of FOBT screening. In addition, views were obtained from general practitioners (GPs) and specialist gastroenterologists directly involved in the implementation of the program. Two focus group sessions were conducted with screening participants and interviews were conducted with nonparticipants, GPs and gastroenterologists. Results . The findings suggest that CRC screening by FOBT distributed to households by mail was well accepted by the community and by the medical practitioners involved in its implementation. The trial had little negative effect on general practice. Both medical practitioners and consumers raised concerns about the efficacy of FOBT screening. Medical practitioners were also concerned about the potential burden mass screening could place on the public (government-funded) health care sector. Conclusions . It would seem that CRC screening using FOBT will not enjoy unqualified support from the community or from medical practitioners involved in the continuum of screening. Information about the objectives of screening programs, in general, and the efficacy of FOBT screening in particular, needs to be provided to the community to ensure informed individual choice.

Mutations in BRAF have been linked with colorectal cancers (CRC) showing high level microsatellite instability (MSI-H). However, the distribution of BRAF mutations in MSI-H cancers remains to be clarified with respect to precursor lesions and the CpG island methylator phenotype (CIMP).Forty three hyperplastic polyps (HP), nine mixed polyps (MP), five serrated adenomas (SA), 28 conventional adenomas (AD), 18 hereditary non-polyposis colorectal cancers (HNPCC), and 127 sporadic CRC (46 MSI-H and 81 non-MSI-H) were collected from patients undergoing colectomy for either CRC or hyperplastic polyposis. Twenty five of 57 serrated lesions were derived from four patients with hyperplastic polyposis. HP were further subdivided according to recently documented morphological criteria into 27 classical HP and 16 variant lesions described as "sessile serrated adenoma" (SSA). All tumours were screened for BRAF activating mutations.The BRAF mutation was more frequent in SSA (75%) and MP (89%) than in classical HP (19%), SA (20%), and AD (0%) (p0.0001), and also in sporadic MSI-H cancers (76%) compared with HNPCC (0%) and sporadic non-MSI-H cancers (9%) (p0.0001). The BRAF mutation was identified more often in CIMP-high serrated polyps (72%) and CIMP-high CRC (77%) than in CIMP-low (30%) and CIMP-negative (13%) polyps (p = 0.002) as well as CIMP-low (18%) and CIMP-negative (0%) CRC (p0.0001).The BRAF mutation was frequently seen in SSA and in sporadic MSI-H CRC, both of which were associated with DNA methylation. Sporadic MSI-H cancers may originate in SSA and not adenomas, and BRAF mutation and DNA methylation are early events in this "serrated" pathway.

Background and aim: E-cadherin binds to beta-catenin to form the cadherin/catenin complex required for strong cell adhesion. Inactivation of this complex in tumors facilitates invasion into surrounding tissues. Alterations of both proteins have been reported in hepatocellular carcinomas (HCC). However, the interactions between E-cadherin and beta-catenin in HCC from different geographical groups have not been explored. The aim of the present study was to assess the role of E-cadherin and beta-catenin in Australian and South African patients with HCC. Methods: DNA was extracted from malignant and non-malignant liver tissue from 37 Australian and 24 South African patients, and from histologically normal liver from 20 transplant donors. Chromosomal instability at 16q22, promoter methylation at E-cadherin, beta-catenin mutations and E-cadherin and beta-catenin protein expression was assessed using loss of heterozygosity, methylation-specific polymerase chain reaction, denaturing high-performance liquid chromatography and immunohistochemistry, respectively. Results: Loss of heterozygosity at 16q22 was prevalent in South African HCC patients (50%vs 11%; P < 0.05, chi(2)). In contrast, E-cadherin promoter hypermethylation was common in Australian cases in both malignant (30%vs 13%; P = not significant, chi(2)) and non-malignant liver (57%vs 8%, respectively, P < 0.001, chi(2)). Methylation of non-malignant liver was more likely to be detected in patients over the age of 50 years (P < 0.001, chi(2)), the overall mean age for our cohort of patients. Only one beta-catenin mutation was identified. E-cadherin protein expression was reduced in one HCC, while abnormalities in protein expression were absent in beta-catenin. Conclusion: Contrary to previous observations in HCC from other countries, neither E-cadherin nor beta-catenin appears to play a role in hepatocarcinogenesis in Australian and South African patients with HCC. (C) 2004 Blackwell Publishing Asia Pty Ltd.

Gene silencing through CpG island hypermethylation has been associated with genesis or progression of frequent microsatellite instability (MSI-H) cancers. To identify novel methylation sites unique to MSI-H colon cancers in an unbiased fashion, we conducted a global expression profiling-based methylation target search. We identified 81 genes selectively down-regulated in MSI-H cancers using cDNA microarray analysis of 41 primary colon cancers. Forty six of these 81 genes contained CpG islands overlapping their 5′untranslated regions. Initial screening of six genes in 57 primary colon cancers detected the following gene with MSI-H cancer-specific hypermethylation: RAB32, a ras family member and A-kinase-anchoring protein, was methylated in 14 of 25 (56%) MSI-H cancers but in none of 32 non-MSI-H cancers or 23 normal colonic specimens. RAB32 hypermethylation correlated with RAB32 mRNA down-regulation and with hMLH1 hypermethylation. In addition, the protein-tyrosine phosphatase receptor type Ogene, PTPRO, was frequently methylated in right-sided tumors. This methylation screening strategy should identify additional genes inactivated by epigenetic silencing in colorectal and other cancers.

Biallelic germ-line variants of the 8-hydroxyguanine repair gene MYH have been associated with multiple colorectal adenomas that display somatic G:CT:A transversions in APC. However, the effect of single germ-line variants has not been widely studied. To examine the relationship between monoallelic MYH variants and susceptibility to sporadic colorectal cancer (CRC), 92 cases of sporadic CRC, 19 cases of familial CRC not meeting the Bethesda guidelines, 17 cases with multiple adenomas, and 53 normal blood donors were screened for 8 potentially pathogenic germ-line MYH variants. Loss of heterozygosity (LOH) at 1p adjacent to the MYH locus, microsatellite instability (MSI) status, and somatic mutations in KRAS2 and APC were analyzed in sporadic cancers. Neither homozygote nor compound heterozygote MYH variants were observed in the germ-line of any subjects with sporadic CRC. There was no difference in the incidence of monoallelic variants between this group (20 of 92, 22%) and cancer-free controls (14 of 53, 26%). However, the presence of monoallelic germ-line MYH variants was negatively associated with an MSI-high (MSI-H) tumor phenotype, with an incidence of only 1 of 23 (4%) MSI-H CRCs as contrasted with 19 of 69 (28%) non-MSI-H (P = 0.02). Further, 4 of 5 tumors with 1p LOH contained monoallelic MYH variants compared with 15 of 53 without 1p LOH (P = 0.04) and the normal population (P = 0.03). The presence of G:CT:A transversions in KRAS2 or APC was significantly more common in single MYH variant tumors (9 of 12) than in MYH wild-type tumors (11 of 33; P = 0.02). These results suggest that single germ-line variants of MYH may influence genetic pathways in CRC. © 2004 Wiley-Liss, Inc.

Expression of membrane-bound Fas ligand (FasL) by colorectal cancer cells may allow the development of an immune-privileged site by eliminating incoming tumour-infiltrating lymphocytes (TILs) in a Fas-mediated counter-attack. Sporadic colorectal cancer can be subdivided into three groups based on the level of DNA microsatellite instability (NISI). High-level NISI (NISI-High) is characterized by the presence of TILs and a favourable prognosis, while microsatellite-stable (MSS) cancers are TIL-deficient and low-level MSI (MSI-Low) is associated with an intermediate TIL density. The purpose of this study was to establish the relationship between MSI status and FasL expression in primary colorectal adenocarcinoma. Using immunohistochemistry and a selected series of 101 cancers previously classified as 31 MSI-High, 30 NISI-Low, and 40 MISS, the present study sought to confirm the hypothesis that increased TIL density in MSI-High cancers is associated with low or absent membrane-bound FasL expression, while increased FasL in MSS cancers allows the killing of host TILs. TUNEL/CD3 double staining was also used to determine whether MSS cancers contain higher numbers of apoptotic TILs in vivo than MSI-High or MSI-Low cancers. Contrary to the initial hypothesis, it was found that MSI-High cancers were associated with higher FasL expression (p = 0.04) and a stronger intensity of FasL staining (p = 0.007). In addition, mucinous carcinomas were independently characterized by increased FasL expression (p = 0.03) and staining intensity (p = 0.0005). Higher FasL expression and staining intensity did not correlate with reduced TIL density or increased numbers of apoptotic TILs. However, consistent with the hypothesis that curtailment of the host anti-tumour immune response contributes to the poor prognosis in MSS cancers, it was found that apoptotic TILs were most abundant in MSS carcinomas and metastatic Dukes' stage C or D tumours (p = 0.004; p = 0.046 respectively). This study therefore suggests that MSS colorectal cancers are killing incoming TILs in an effective tumour counter-attack, but apparently not via membrane-bound FasL. Copyright (C) 2003 John Wiley Sons, Ltd.

Peutz-Jeghers syndrome is an autosomal dominant condition leading to gastrointestinal polyps which often causes bowel obstruction. This syndrome also predisposes to gastrointestinal, pancreatic, breast, uterine and other malignancies. Prognosis is likely to be improved by the early commencement of appropriate surveillance programs. Diagnostic and predictive genetic testing is now possible in many families due to identification of causative mutations in the serine/threonine kinase (STK)-11 (also known as the LKB1) gene. Such testing has now entered routine clinical practice and will allow early recognition of the condition in young, at-risk family members.

We have examined a series of human colorectal adenomas, carcinomas and cell lines derived from human colorectal cancer for loss of heterozygosity (LOH) on chromosome 11q22-23 by polymerase chain reaction (PCR) amplification of a microsatellite polymorphism of the dopamine D2 receptor (DRD2) locus. LOH was demonstrated in 5/30 (16.7%) adenomas and 23/68 (33.8%) carcinomas. Only 2/20 (10%) cell lines showed homozygosity which could potentially be as a consequence of LOH. This moderate level of loss in the tumour samples was probably not an underestimation as a result of excessive stromal contamination because high rates (68-77%) have been detected in the same samples on chromosomes 17 and 18. In contrast to a previous report, LOH in carcinomas at 11q22-23 occurred at a lower frequency and was not associated with Dukes' stage, degree of differentiation, mucin production or the location of the cancer. However, a significant association was found between LOH on chromosome 11 and chromosome 14. Thus, inactivation of any putative tumour-suppressor gene at 11q22-23 by LOH is not a very common event in the development of colorectal tumours, but may be biologically significant if accompanied by chromosome 14 deletions. Images Figure 1

Colorectal cancer (CRC) has traditionally been classified into two groups: microsatellite stable/low-level instability (MSS/MSI-L) and high-level MSI (MSI-H) groups on the basis of multiple molecular and clinicopathologic criteria. Using methylated in tumor (MINT) markers 1, 2, 12, and 31, we stratified 77 primary CRCs into three groups: MINT++ (2), MINT+ (1-2), and MINT- (0 markers methylated). The MSS/MSI-L/MINT++ group was indistinguishable from the MSI-H/MINT++ group with respect to methylation of p16(INK4a), p14(ARF), and RIZ1, and multiple morphological features. The only significant difference between MSI-H and non-MSI-H MINT++ cancers was the higher frequency of K-ras mutation (P0.004) and lower frequency of hMLH1 methylation (P0.001) in the latter. These data demonstrate that the separation of CRC into two nonoverlapping groups (MSI-H versus MSS/MSI-L) is a misleading oversimplification.

1. Sulphotransferases are a superfamily of enzymes involved in both detoxification and bioactivation of endogenous and exogenous compounds. The arylsulphotransferase SULT1A1 has been implicated in a decreased activity and thermostability when the wild-type arginine at position 213 of the coding sequence is substituted by a histidine. SULT1A1 is the isoform primarily associated with the conversion of dietary N-OH arylamines to DNA binding adducts and is therefore of interest to determine whether this polymorphism is linked to colorectal cancer. 2. Genotyping, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, was performed using DNA samples of healthy control subjects (n = 402) and patients with histologically proven colorectal cancer (n = 383). Both control and test populations possessed similar frequencies for the mutant allele (32.1 and 31%, respectively; P = 0.935). Results were not altered when age and gender were considered as potential confounders in a logistic regression analysis. 3. Examination of the sulphonating ability of the two allozymes with respect to the substrates p-nitrophenol and paracetamol showed that the affinity and rate of sulphonation was unaffected by substitution of arginine to histidine at position 213 of the amino acid sequence. 4. From this study, we conclude that the SULT1A1 R213H polymorphism is not linked with colorectal cancer in this elderly Australian population.

This review compiles evidence for an alternative to the classical adenoma-carcinoma sequence in the evolution of colorectal cancer. It is suggested that between 30 and 50 of colorectal cancers are not initiated by mutation of the tumor suppressor gene APC, but through the epigenetic silencing of genes implicated in the control of differentiation, cell cycle control and DNA repair proficiency. The precursor polyps are often characterized by a serrated architecture, and include hyperplastic polyps, admixed polyps and serrated adenomas. The alternative pathway is heterogeneous and may culminate in cancers showing low or high level DNA microsatellite instability (MSI-L and MSI-H, respectively), and in cancers that are microsatellite stable (MSS). Cancers showing DNA MSI may be characterized by an accelerated evolution. Cancers in hereditary non-polyposis colorectal cancer show features of both classical (adenoma and APC mutation) and alternative pathways (rapid evolution, MSI-H and lack of chromosomal instability).

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by mutation of the APC gene. It is characterised by the appearance of hundreds to thousands of colorectal adenomas in adolescence and the subsequent development of colorectal cancer. Various extracolonic malignancies are associated with FAP, including desmoids and neoplasms of the stomach, duodenum, pancreas, liver, and brain. We present a family affected by FAP with an exon 14 APC mutation displaying two rare extracolonic lesions, a hepatoblastoma and a myoepithelial carcinoma. The hepatoblastoma was found in a male patient aged 2 years. The second lesion, a myoepithelial carcinoma of the right cheek, was found in a female patient aged 14 years. Inactivation of the normal APC allele was demonstrated in this lesion by loss of heterozygosity analysis, thus implicating APC in the initiation or progression of this neoplasm. This is the first reported case of this lesion in a family affected by FAP.

PURPOSE: To compare microsatellite instability (MSI) testing with immunohistochemical (IHC) detection of hMLH1 and hMSH2 in colorectal cancer. PATIENTS AND METHODS: Colorectal cancers from 1,144 patients were assessed for DNA mismatch repair deficiency by two methods: MSI testing and IHC detection of hMLH1 and hMSH2 gene products. High-frequency MSI (MSI-H) was defined as more than 30% instability of at least five markers; low-level MSI (MSI-L) was defined as 1% to 29% of loci unstable. RESULTS: Of 1,144 tumors tested, 818 showed intact expression of hMLH1 and hMSH2. Of these, 680 were microsatellite stable (MSS), 27 were MSI-H, and 111 were MSI-L. In all, 228 tumors showed absence of hMLH1 expression and 98 showed absence of hMSH2 expression: all were MSI-H. CONCLUSION: IHC in colorectal tumors for protein products hMLH1 and hMSH2 provides a rapid, cost-effective, sensitive (92.3%), and extremely specific (100%) method for screening for DNA mismatch repair defects. The predictive value of normal IHC for an MSS/MSI-L phenotype was 96.7%, and the predictive value of abnormal IHC was 100% for an MSI-H phenotype. Testing strategies must take into account acceptability of missing some cases of MSI-H tumors if only IHC is performed.

1359 FIRST SVR DATA WITH THE NUCLEOSIDE ANALOGUE POLYMERASE INHIBITOR MERICITABINE (RG7128) COMBINED WITH PEGINTERFERON/RIBAVIRIN IN TREATMENT-NAIVE HCV G1/4 PATIENTS: INTERIM ANALYSIS FROM THE JUMP-C TRIAL P. Pockros, D. Jensen, N. Tsai, R.M. Taylor, A. Ramji, C. Cooper, R. Dickson, A. Tice, S. Stancic, D. Ipe, J.A. Thommes, J.M. Vierling. Scripps Clinic Research Center, La Jolla, CA, Center for Liver Diseases, University of Chicago Hospitals, Chicago, IL, Hawaii Medical Center, Honolulu, HI, The University of Kansas Hospital Medical Center, Kansas City, KS, USA; Division of Gastroenterology, University of British Columbia, Vancouver, BC, The Ottawa Hospital, Ottowa, ON, Canada; Dartmouth-Hitchcock Medical Center, Lebanon, NH, Infections Limited Hawaii, Honolulu, HI, Roche, Nutley, NJ, Genentech, South San Francisco, CA, Baylor College of Medicine, Houston, TX, USA E-mail: pockros.paul@scrippshealth.org Introduction: Mericitabine (RG7128, MCB) is a potent, selective nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase with activity across all HCV genotypes. MCB has demonstrated a high barrier to resistance without treatmentemergent resistance observed to date. Methods: JUMP-C is an ongoing randomised, double-blind, placebocontrolled phase 2b study in treatment naive patients infected with HCV G1/4. The trial objective was to compare a response guided therapy regimen of MCB plus peginterferon alfa-2a/ribavirin (P/R) with P/R alone. Patients randomised to arm A achieving an extended RVR (eRVR; HCVRNA

The BRAF (V600E) mutation in colorectal cancers that are microsatellite stable (MSS) confers a very poor patient prognosis, whereas BRAF mutant microsatellite-unstable (MSI) colorectal cancers have an excellent prognosis. BRAF wild type cancers are typically MSS and display chromosomal instability (CIN). CIN is associated with a poor prognosis but has not been extensively studied on a genome-wide basis in relation to BRAF mutational status in colorectal cancer. BRAF mutant/MSS (BRAFmut/MSS) cancers (n=33) and BRAF mutant/MSI (BRAFmut/MSI) cancers (n=30) were compared for presence of copy number aberrations (CNAs) indicative of CIN, with BRAF wild type/MSS (BRAFwt/MSS) cancers (n=18) using Illumina CytoSNP-12 arrays. BRAFmut/MSS and BRAFwt/MSS cancers showed comparable numbers of CNAs per cancer at 32.8 and 29.8 respectively. However there were distinct differences in patterns of CNA length between MSS cohorts, with BRAFmut/MSS cancers having a significantly greater proportion of focal CNAs compared to BRAFwt/MSS cancers (p These results indicate that although comparable rates of CIN occur between MSS subgroups, significant differences in their patterns of instability exist as BRAFmut/MSS cancers display a ‘focal pattern’ and BRAFwt/MSS cancers have more a ‘whole arm pattern' of CIN. This and the genomic loci more frequently affected in BRAFmut/MSS cancers may warrant extended studies to ascertain correlations with clinical data. Overall this investigation has identified that differing patterns of genomic instability exist between the two MSS colorectal subgroups and provides further evidence of the biological distinctions of the aggressive BRAFmut/MSS cancers. Citation Format: Catherine E. Bond, Derek J. Nancarrow, Leesa F. Wockner, Leanne Wallace, Grant W. Montgomery, Barbara A. Leggett, Vicki LJ Whitehall. Microsatellite stable colorectal cancers stratified by the BRAF V600E mutation show distinct patterns of chromosomal instability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2422. doi:10.1158/1538-7445.AM2014-2422

Juvenile polyposis syndrome (JPS) is an inherited hamartomatous-polyposis syndrome with a risk for colon cancer. JPS is a clinical diagnosis by exclusion, and, before susceptibility genes were identified, JPS could easily be confused with other inherited hamartoma syndromes, such as Bannayan-Riley-Ruvalcaba syndrome (BRRS) and Cowden syndrome (CS). Germline mutations of MADH4 (SMAD4) have been described in a variable number of probands with JPS. A series of familial and isolated European probands without MADH4 mutations were analyzed for germline mutations in BMPR1A, a member of the transforming growth-factor beta-receptor superfamily, upstream from the SMAD pathway. Overall, 10 (38%) probands were found to have germline BMPR1A mutations, 8 of which resulted in truncated receptors and 2 of which resulted in missense alterations (C124R and C376Y). Almost all available component tumors from mutation-positive cases showed loss of heterozygosity (LOH) in the BMPR1A region, whereas those from mutation-negative cases did not. One proband with CS/CS-like phenotype was also found to have a germline BMPR1A missense mutation (A338D). Thus, germline BMPR1A mutations cause a significant proportion of cases of JPS and might define a small subset of cases of CS/BRRS with specific colonic phenotype.

AIM—Colorectal cancer has been described in association with hyperplastic polyposis but the mechanism underlying this observation is unknown. The aim of this study was to characterise foci of dysplasia developing in the polyps of subjects with hyperplastic polyposis on the basis of DNA microsatellite status and expression of the DNA mismatch repair proteins hMLH1, hMSH2, and hMSH6. MATERIALS AND METHODS—The material was derived from four patients with hyperplastic polyposis and between one and six synchronous colorectal cancers. Normal (four), hyperplastic (13), dysplastic (13), and malignant (11) samples were microdissected and a PCR based approach was used to identify mutations at 10 microsatellite loci, TGFβIIR, IGF2R, BAX, MSH3, and MSH6. Microsatellite instability-high (MSI-H) was diagnosed when 40% or more of the microsatellite loci showed mutational bandshifts. Serial sections were stained for hMLH1, hMSH2, and hMSH6. RESULTS—DNA microsatellite instability was found in 1/13 (8%) hyperplastic samples, in 7/13 (54%) dysplastic foci, and in 8/11 (73%) cancers. None of the MSI-low (MSI-L) samples (one hyperplastic, three dysplastic, two cancers) showed loss of hMLH1 expression. All four MSI-H dysplastic foci and six MSI-H cancers showed loss of hMLH1 expression. Loss of hMLH1 in MSI-H but not in MSI-L lesions showing dysplasia or cancer was significant (p< 0.001, Fisher's exact test). Loss of hMSH6 occurred in one MSI-H cancer and one MSS focus of dysplasia which also showed loss of hMLH1 staining. CONCLUSION—Neoplastic changes in hyperplastic polyposis may occur within a hyperplastic polyp. Neoplasia may be driven by DNA instability that is present to a low (MSI-L) or high (MSI-H) degree. MSI-H but not MSI-L dysplastic foci are associated with loss of hMLH1 expression. At least two mutator pathways drive neoplasia in hyperplastic polyposis. The role of the hyperplastic polyp in the histogenesis of sporadic DNA microsatellite unstable colorectal cancer should be examined. Keywords: colon; hyperplastic polyposis; DNA mismatch repair; microsatellite instability

BACKGROUND AND AIM—Hereditary non-polyposis colorectal cancer (HNPCC), as its name implies, is associated with few adenomas, and the early evolution of colorectal neoplasia is poorly understood. In this study our aim was to clarify the genetic profiles of benign polyps in subjects with HNPCC using a combined molecular and immunohistochemical approach. METHODS—Thirty adenomas and 17 hyperplastic polyps were obtained from 24 affected HNPCC subjects. DNA was extracted from paraffin embedded tissue by microdissection and analysed for the presence of microsatellite instability (MSI) and mutations in five genes known to be targets in mismatch repair deficiency (TGFβRII, IGF2R, BAX, hMSH3, and hMSH6). Serial sections were stained by immunohistochemistry for hMLH1 and hMSH2. RESULTS—Twenty four (80%) of 30 adenomas showed MSI. Of MSI positive adenomas, 66.7% showed MSI at more than 40% of markers (high level of MSI (MSI-H)). Two of 17 hyperplastic polyps revealed MSI at one marker (low level of MSI (MSI-L)). A significant association was found between MSI-H and high grade dysplasia in adenomas (p=0.004). Eight of nine adenomas with mutations of coding sequences revealed high grade dysplasia and all nine were MSI-H. Four of the nine ranged in size from 2 to 5 mm. The presence of the hMSH6 mutation was significantly correlated with high levels of MSI (80% of markers) (p

BACKGROUND—Juvenile polyposis syndrome (JPS) is characterised by gastrointestinal (GI) hamartomatous polyposis and an increased risk of GI malignancy. Juvenile polyps also occur in the Cowden (CS), Bannayan-Ruvalcaba-Riley (BRRS) and Gorlin (GS) syndromes. Diagnosing JPS can be problematic because it relies on exclusion of CS, BRRS, and GS. Germline mutations in the PTCH, PTEN and DPC4 (SMAD4) genes can cause GS, CS/BRRS, and JPS, respectively. AIMS—To examine the contribution of mutations in PTCH, PTEN, and DPC4 (SMAD4) to JPS. METHODS—Forty seven individuals from 15 families and nine apparently sporadic cases with JPS were screened for germline mutations in DPC4, PTEN, and PTCH. RESULTS—No patient had a mutation in PTEN or PTCH. Five different germline mutations were detected in DPC4; three of these were deletions, one a single base substitution creating a stop codon, and one a missense change. None of these patients had distinguishing clinical features. CONCLUSIONS—Mutations in PTEN and PTCH are unlikely to cause juvenile polyposis in the absence of clinical features indicative of CS, BRRS, or GS. A proportion of JPS patients harbour DPC4 mutations (21% in this study) but there remains uncharacterised genetic heterogeneity in JPS. Keywords: juvenile polyposis syndrome; germline mutations

Mucins are high molecular weight glycoproteins with a variety of postulated biological functions, including physicochemical protection from toxins and mutagens, adhesion modulation, signal transduction, and regulation of cell growth. Mucins are widely and differentially expressed in the gastrointestinal tract. To date, studies of cellular expression have relied on light microscopy using in situ hybridization and immunohistochemistry. Although informative, it has been difficult with these techniques to ascertain exactly which cell types are producing a given mucin. We studied expression of MUC1, MUC2, and MUC4 apomucins in a series of normal colon biopsies using a combination of immunoelectron microscopy and light microscopy. MUC1 mucin was localized to both goblet and columnar cells, where it was seen in secretory vesicles, microvilli, and in cytoplasmic remnants in goblet cell thecae. MUC2 expression was restricted to goblet cells, in which reactivity was concentrated in the rough endoplasmic reticulum (RER). MUC4 expression was seen in both columnar and goblet cells, localized to the RER. The inability to detect MUC2 and MUC4 apomucins in the Golgi complex and the mature mucous gel probably represents masking of peptide epitopes following O-glycosylation. This study has helped clarify lineage-specific mucin synthesis in the normal colon. (J Histochem Cytochem 47:1063–1074, 1999)

AIM: To investigate the distribution of DNA microsatellite instability (MSI) in a series of hyperplastic polyps, serrated adenomas, and mixed polyps of the colorectum. METHODS: DNA was extracted from samples of 73 colorectal polyps comprising tubular adenomas (23), hyperplastic polyps (21), serrated adenomas (17), and mixed polyps (12). The presence of MSI was investigated at six loci: MYCL, D2S123, F13B, BAT-40, BAT-26, and c-myb T22, using polymerase chain reaction based methodology. MSI cases were classified as MSI-Low (MSI-L) and MSI-High (MSI-H), based on the number of affected loci. RESULTS: The frequency of MSI increased in tubular adenomas (13%), hyperplastic polyps (29%), serrated adenomas (53%), and mixed polyps (83%) (Wilcoxon rank sum statistic, p < 0.001). Hyperplastic epithelium was present in nine of 12 mixed polyps and showed MSI in eight of these. MSI was mostly MSI-L. MSI-H occurred in two serrated adenomas and three mixed polyps. Clonal relations were demonstrated between hyperplastic and dysplastic epithelium in four of eight informative mixed polyps. CONCLUSIONS: The findings support the view that hyperplastic polyps may be fundamentally neoplastic rather than hyperplastic. A proportion of hyperplastic polyps may serve as a precursor of a subset (10%) of colorectal cancers showing the MSI-L phenotype, albeit through the intermediate step of serrated dysplasia. This represents a novel and distinct morphogenetic pathway for colorectal cancer.

Juvenile polyps are present in a number of Mendelian disorders, sometimes in association only with gastrointestinal cancer [juvenile polyposis syndrome (JPS)] and sometimes as part of known syndromes (Cowden, Gorlin and Banayan-Zonana) in association with developmental abnormalities, dysmorphic features or extra-intestinal tumours. Recently, a gene for JPS was mapped to 18q21.1 and the candidate gene DPC4 (SMAD4) was shown to carry frameshift mutations in some JPS families. We have analysed eight JPS families for linkage to DPC4. Overall, there was no evidence for linkage to DPC4; linkage could be excluded in two of the eight pedigrees and was unlikely in two others. We then tested these eight families and a further 13 familial and sporadic JPS cases for germline mutations in DPC4. Just one germline DPC4 mutation was found (in a familial JPS patient from a pedigree unsuitable for linkage analysis). Like all three previously reported germline mutations, this variant occurred towards the C-terminus of the DPC4 protein. However, our patient's mutation is a missense change (R361C); somatic missense mutations in DPC4 have been reported previously in tumours. We therefore confirm DPC4 as a cause of JPS, but show that there is considerable remaining, uncharacterized genetic heterogeneity in this disease.

Background—Up to 15% of colorectal cancers are characterised by DNA microsatellite instability (MIN), shown by the presence of DNA replication errors (RERs).Aims—To identify pathological features that are discriminating for colorectal cancer (CRC) showing extensive MIN.Subjects—A prospective series of 303 patients with CRC and no family history of either familial adenomatous polyposis or hereditary non-polyposis colorectal cancer.Methods—DNA was extracted from fresh tissue samples and the presence of MIN was studied at nine loci that included TGFβRII, IGFIIR, and BAX. The 61 cases showing RERs were compared with 63 RER negative cases with respect to a comprehensive set of clinical and pathological variables. Predictive utility of the variables was tested by decision tree analysis.Results—Twenty seven patients with CRC showed extensive RERs (three loci or more) (RER+) and 34 had limited RERs only (28 = one locus; 6 = two loci) (RER+/−), yielding a bimodal distribution. RER+ cancers differed from RER− and RER+/− cases. Tumour type (adenocarcinoma, mucinous carcinoma, and undifferentiated carcinoma) (p=0.001), tumour infiltrating lymphocytes (p=0.001), and anatomical site (p=0.001) were the most significant of the discriminating variables. Algorithms developed by decision tree analysis allowed cases to be assigned to RER+ versus RER− and +/− status with a global sensitivity of 81.5%, specificity of 96%, and overall accuracy of 93%.Conclusion—Pathological examination of CRC allows assignment of RER+ status; assignment is specific and relatively sensitive. Conversely RER− and RER+/− CRC are indistinguishable.

Background—Familial adenomatous polyposis usually results in colonic polyposis with hundreds to thousands of polyps, congenital hypertrophy of the retinal pigment epithelium (CHRPE), and variable extracolonic features. Recent reports indicate that patients with distal mutations between codons 1445 and 1578 do not express CHRPE and have a high incidence of desmoid tumours.Patients—The family studied has an unusual phenotype of sparse colonic polyposis but profuse upper gastrointestinal polyposis. Affected subjects do not have CHRPE.Methods—The protein truncation test followed by sequencing identified a 2 base pair deletion at codon 1520 in the APC gene. This results in a frameshift creating a stop codon 13 codons downstream.Results—This family demonstrates that sparse colonic polyposis but severe upper tract polyposis may be associated with mutations between codons 1445 and 1578.Conclusions—Study of duodenal and colonic polyps in further cases with mutations in this region is warranted. Such mutations may preferentially cause duodenal adenomas and desmoid tumours as somatic mutations in these tumours also occur in this region, unlike colorectal tumours where somatic mutations occur more proximally. This study emphasises the importance of screening the upper gastrointestinal tract even when the colonic disease is mild.

Genomic instability at simple repeated sequences has been observed in various types of human cancers and is considered an important mechanism in tumorigenesis. Alterations at microsatellite loci have been reported scattered throughout the genome. Recently, the transforming growth factor-beta receptor type II (TGF-beta RII) and the insulin-like growth factor II receptor (IGF-IIR) genes were shown to have inactivating mutations within coding microsatellite sequences. The demonstration of mutations in two growth regulatory genes supports the idea that other regulatory genes with repeat sequences may also be targets in tumours with defective mismatch repair. We examined genes involved in tumour suppression, cell adhesion and cell cycle regulation for mutations at small repeat sequences in replication error positive gastrointestinal cancers. Several polymorphisms were found which exhibited instability, but no other instability was present in the regions examined.

PURPOSE: Metachronous colorectal cancer still occurs in a small percentage of patients, despite colonoscopic surveillance. Cancers in hereditary nonpolyposis colorectal cancer for which there is a high risk of metachronous cancer show distinctive DNA changes termed replication errors (RER+). Ten to 20 percent of sporadic colorectal cancers are also RER+. The aim of this study was to identify factors predictive of metachronous colorectal cancer, despite colonoscopic surveillance. Clinicopathologic characteristics and RER status of cancers were examined. METHODS: Colorectal cancer patients, who entered into a surveillance program of being examined with colonoscopy within six months of surgery and then at intervals of three years thereafter, were reviewed. The 433 patients compliant with the protocol who had had more than one colonoscopy had been followed up for a mean of 3.8±2.2 years. DNA was extracted from archival paraffin-embedded cancer tissue for determination of RER status. RESULTS: Ten cases of metachronous cancer were identified, giving a rate of 0.61 percent per year. The site of the index cancer in patients who later developed metachronous cancer was predominantly proximal (P=0.0007), and these cancers were more likely to have mucinous histology (P

In many countries in the world, iron deficiency is clearly a much greater problem than iron overload. In Australia the situation is less clear. A large population study undertaken in the Liver Unit of the University of Queensland Department of Medicine and the Queensland Institute of Medical Research under the direction of Professors June Halliday and Lawrie Powell has shed some light on this question. The study originally started out with the hypothesis that the genetic disease haemochromatosis was common in the Australian population and, therefore, significant num- bers of Australians had too much iron.

Allelic loss is a common mechanism of inactivation of tumour-suppressor genes in colorectal carcinomas. A number of known or putative tumour-suppressor genes including NF1, BRCA1, NME1, NME2 and prohibitin are present on the long arm of chromosome 17, and this region has not been extensively analysed in colorectal tumours. In this study 72 colorectal carcinomas were examined for allelic loss at eight loci on chromosome 17. Allelic loss was frequent both at the p53 locus, which is known to be important in colorectal carcinoma, and also telomeric to p53 on 17p. Allelic loss continued to be present in more than 50% of cases in the pericentromeric region and on proximal 17q to the marker LEW101 (D17S40) at 17q22-23. The most telomeric markers on 17q showed lower rates of allelic loss. Analysis of cases with partial deletions which did not include the p53 locus showed a common region of overlap of the deletions centred on D17S40. This suggests the target of allelic loss on 17q is a tumour-suppressor gene in this region. Images Figure 1 Figure 2

Su1465 The Prevalence of Sessile Serrated Adenomas in a Fecal Immunochemical Test Positive Colorectal Cancer Screening Cohort Nicholas J. Tutticci*, Barbara A. Leggett, Mark N. Appleyard, David G. Hewett Gastroenterology and Hepatology, Royal Brisbane and Womens Hospital, Brisbane, QLD, Australia; School of Medicine, University of Queensland, Brisbane, QLD, Australia Background: Sessile serrated adenomas (SSAs) are important, colonoscopicallysubtle precursor lesions in the serrated pathway of colorectal neoplasia. SSA prevalence rates have been described in cohorts undergoing colonoscopy for average risk screening and for clinical indications, but not in a high risk screening cohort (fecal immunochemical test positive, FIT ). We aimed to assess the prevalence of SSAs in participants from the Australian Bowel Cancer Screening Program, a national, population-based screening program offering fecal immunochemical testing to adults aged 50, 55 and 65, and colonoscopy for those returning a positive result. Methods: We enrolled 503 consecutive FIT screening program participants from a defined geographic catchment between 2008 and 2011. We assembled a comparison, non-screening control cohort of 1056 patients 50 years of age undergoing colonoscopy by matched colonoscopists for clinical indications from the same center. We used multiple logistic regression to analyze the predictors of SSAs in FIT screening participants, and compare the prevalence of SSAs between screening participants and controls, adjusting for age, sex, bowel preparation, colonoscopist and year of colonoscopy. Results: 164 SSAs were found in 64 (13%) of 503 FIT participants, representing 18% of all polyps; 114 (70%) of SSAs were proximal. Adenomas were found in 259 (52%) FIT screening program participants. Of these, 129 (26%) had advanced adenomas and 46 (5%) had synchronous SSAs. 10 cancers were found, none with synchronous SSAs. Among FIT screening participants, the presence of an SSA was significantly associated with the presence of synchronous adenomas (OR 2.67, p 0.002) and colonoscopist (OR 3.87, p 0.002) but not participant age, sex, year of procedure or bowel preparation. In the control group, adenomas were found in 284 (27%) patients, advanced adenomas in 80 (8%) and SSAs in 61 (6%). The crude prevalence of SSAs was 13% in FIT screening participants, compared with 6% in control patients. This difference was significant (adjusted OR 1.9, p 0.01) after controlling for age, sex, bowel preparation, colonoscopist and year, but not when controlling for the presence of an adenoma (adjusted OR 1.43, p 0.157). Conclusions: FIT screening program participants undergoing colonoscopy have a higher prevalence of SSAs than patients undergoing colonoscopy for clinical indications. However, this difference likely mirrors the higher prevalence of adenomas also seen in this screening cohort, rather than the detection of SSAs by FIT. Further, any differences need to be interpreted with caution given the variation in SSA prevalence between colonoscopists.

BRAF mutant colorectal cancers can be stratified according to the presence of microsatellite instability (MSI). MSI cancers with a BRAF mutation are associated with an excellent patient prognosis. By contrast, in microsatellite stable (MSS) cancers, a BRAF mutation confers a very poor patient prognosis. The causes of such a detrimental outcome are yet to be thoroughly determined. We have previously reported that BRAF mutant, MSS cancers (BRAFmut/MSS) have a significantly higher rate of p53 mutation and present at more advanced stages than MSI cancers. Frequencies of these features in the BRAFmut/MSS cancers are similar to those observed in BRAF wild type, MSS cancers (BRAFwt/MSS). Chromosomal instability (CIN) is a dominant feature of BRAFwt/MSS cancers, whilst MSI cancers are considered to be diploid. In MSS cancers, CIN has been associated with a worse prognosis. We therefore hypothesised that CIN would be common in BRAF mutant/MSS cancers. BRAFmut/MSS (n=60), and BRAFwt/MSS cancers (n=90) were investigated for CIN using Loss of Heterozygosity (LOH) analysis over 12 loci encompassing chromosomal regions 5q, 8p, 17p and 18q. CIN was assigned if at least 1 marker was positive for LOH, and a cancer was reported as showing no loss if at least 5 markers were informative and none demonstrated LOH. CIN was frequently found in BRAFmut/MSS cancers (41/57, 72%), and this rate was comparable to that observed in BRAFwt/MSS cancers (74/90, 82%). The greatest rate of CIN in BRAFmut/MSS cancers occurred at 8p (26/44, 59%), and the least at 5q (19/49, 39%). BRAFwt/MSS cancers showed most CIN at 17p (43/65, 66.2%), and the least at 5q (43/80, 53.8%). Within the BRAFmut/MSS cancer subgroup, CIN was highly correlated with the CpG Island Methylator Phenotype (17/23, 74%). CIN in BRAFmut/MSS cancers was significantly predominant in those at advanced (15/17, 88%), compared to early AJCC stages of presentation (6/13, 46%) (p=0.02). BRAFmut/MSS cancers with CIN at 18q and 8p significantly associated with worse survival compared to those with no loss (p=0.003, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 106. doi:1538-7445.AM2012-106

The role of Helicobacter pylori and atrophic gastritis in the pathogenesis of gastric lesions in familial adenomatous polyposis (FAP) has not been clarified.Thirty one patients with FAP.The presence of fundic gland polyposis (FGP) and gastric adenoma (GA) was determined by upper endoscopy with biopsies. The degree of gastric mucosal atrophy and H pylori status were determined by serological and histological findings. Germline mutation in the adenomatous polyposis coli (APC) gene was determined by polymerase chain reaction based single strand conformation polymorphism and direct sequencing.Gastric lesions were detected in 23 patients (74%). FGP and GA were found in 52% and 39%, respectively. APC gene mutation was identified in 22 of 30 patients. Patients with FGP were less frequently infected with H pylori than those without FGP (13% v 67%). The former patients had a lower degree of atrophy than the latter. Patients with GA tended to be more frequently infected with H pylori and they had higher degrees of atrophy than those without GA. When subjects were subdivided by gastric lesions (FGP alone, FGP+GA, GA alone, and negative groups), the GA alone group had the lowest pepsinogen I/II ratio and the highest seropositivity for H pylori. GA was found more frequently in patients positive for the APC mutation whereas no such a trend was observed in FGP.In FAP, H pylori associated atrophic gastritis contributes negatively to FGP. It seems to contribute positively to GA, especially in patients with truncating APC gene mutation.

Background: In familial juvenile polyposis, multiple juvenile polyps occur throughout the colon. The genetic defect has not been characterized. The risk of colon cancer is increased, although the magnitude of the increased risk is controversial. The hypothesis of this study was that the genetic defect is within a tumor suppressor gene, possibly one already known to be inactivated in colorectal neoplasia. Methods: Linkage analysis using the short tandem repeat polymorphism D5S346 was performed to determine if juvenile polyposis was linked to either APC (adenomatous polyposis coli) or MCC (mutated in colorectal carcinoma) genes within a single large family. Results: A family in which eight subjects have been affected by juvenile polyposis over three generations is described. Six affected subjects had colectomies in childhood, but the two who have so far survived beyond 35 years of age have developed adenocarcinoma of the jejunum. Within this family, linkage analysis excluded linkage of the juvenile polyposis trait to either APC or MCC. Conclusions: In a family with juvenile polyposis with a clear predisposition to malignancy, including carcinoma of the jejunum, APC and MCC were not the defective genes causing the condition.

Iron overload, such as occurs in the genetic disease haemochromatosis, leads to synthesis of ferritin containing an increased proportion of L subunits. Inflammation also leads to clinically important increases in ferritin synthesis but the predominant subunit involved is unclear. Elevation of serum ferritin concentration during the acute phase response confounds its use as an indicator of body iron stores and identification of the major subunit involved may allow distinction of the ferritin associated with inflammation from that synthesized during iron overload. The present study examined H and L ferritin subunit mRNA levels in rats with: (i) longstanding iron overload, both parenteral and oral, in which changes should be maximal and stable; and (ii) inflammation of 24 and 48 h duration. A two-fold increase in L mRNA level was found in both groups of iron loaded animals while H mRNA level was unchanged. This finding would account for the observed preponderance of L subunits in ferritin during iron overload. During the course of inflammation there was a progressive decrease in L mRNA level in the liver but not the spleen. H mRNA relative to total RNA level was unchanged in both liver and spleen. It is concluded that the differential regulation of the two ferritin subunits in response to different stimuli and in different tissues occurs at the level of alteration in mRNA concentration.