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Jasperson KW, Samowitz WS, Burt RW. Constitutional mismatch repair-deficiency syndrome presenting as colonic adenomatous polyposis: clues from the skin. Constitutional mismatch repair-deficiency (CMMR-D) syndrome is an autosomal recessive condition characterized by hematologic malignancies, brain tumors, Lynch syndrome-associated cancers and skin manifestations reminiscent of neurofibromatosis type 1 (NF1). In contrast to Lynch syndrome, CMMR-D syndrome is exceptionally rare, onset typically occurs in infancy or early childhood and, as described in this report, may also present with colonic polyposis suggestive of attenuated familial adenomatous polyposis (AFAP) or MUTYH associated polyposis (MAP). Here we describe two sisters with CMMR-D syndrome due to germline bi-allelic MSH6 mutations. Both sisters are without cancer, are older than typical for this condition, have NF1 associated features and a colonic phenotype suspicious for an attenuated polyposis syndrome. This report highlights the role of skin examinations in leading to an underlying genetic diagnosis in individuals with colonic adenomatous polyposis, but without mutations associated with AFAP or MAP. [ABSTRACT FROM AUTHOR]

Introduction: We examined expression of genes in the p53-signaling pathway. We determine if genes that have significantly different expression in carcinoma tissue compared to normal mucosa also have significantly differentially expressed miRNAs. We utilize a sample of 217 CRC cases., Methods: We focused on fold change (FC) > 1.50 or <0.67 for genes and miRNAs, that were statistically significant after adjustment for multiple comparisons. We evaluated the linear association between the differential expression of miRNA and mRNA. miRNA:mRNA seed-region matches also were determined., Results: Eleven dysregulated genes were associated with 37 dysregulated miRNAs; all were down-stream from the TP53 gene. MiR-150-5p (HR = 0.82) and miR-196b-5p (HR 0.73) significantly reduced the likelihood of dying from CRC when miRNA expression increased in rectal tumors., Conclusions: Our data suggest that activation of p53 from cellular stress, could target downstream genes that in turn could influence cell cycle arrest, apoptosis, and angiogenesis through mRNA:miRNA interactions., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Patients with familial adenomatous polyposis (FAP) frequently undergo colectomy to reduce the 70 to 90% lifetime risk of colorectal cancer. After risk-reducing colectomy, duodenal cancer and complications from duodenal surgeries are the main cause of morbidity. Our objective was to prospectively describe the duodenal and gastric polyp phenotype in a cohort of 150 FAP patients undergoing pre-screening for a chemoprevention trial and analyze variables that may affect recommendations for surveillance., Methods: Individuals with a diagnosis of FAP underwent prospective esophagogastroduodenoscopy using a uniform system of mapping of size and number of duodenal polyps for a 10 cm segment. Gastric polyps were recorded as the total number., Results: The distribution of the count and sum diameter of duodenal polyps were statistically different in two genotype groups, those with APC mutations associated with classic FAP had a greater count (median 17) and sum diameter of polyps (median 32 mm) than those with APC mutations associated with attenuated FAP (median count 4 and median sum diameter of 7 mm) (p < 0.0001). The number of gastric polyps did not differ based on genotype (p = 0.67) but advancing age correlated with severity of gastric polyposis (p = 0.019). Spigelman (modified) staging of II or greater was found in 88% of classic FAP patients and 48% attenuated FAP patients. Examples of severe and mild upper GI phenotype are observed in patients with identical APC mutations, showing that the APC mutation location is not absolutely predictive of an upper GI phenotype., Conclusions: Most FAP patients have duodenal and gastric polyps which become more prevalent and advanced with age. Standard upper endoscopic surveillance is recommended based on personal history independent of APC mutation location., Trial Registration: NCT 01187901 registered August 24, 2010, prospective to enrollment.

Background: The TGFβ-signaling pathway plays an important role in the pathogenesis of colorectal cancer (CRC). Loss of function of several genes within this pathway, such as bone morphogenetic proteins (BMPs) have been seen as key events in CRC progression., Methods: In this study we comprehensively evaluate differential gene expression (RNASeq) of 81 genes in the TGFβ-signaling pathway and evaluate how dysregulated genes are associated with miRNA expression (Agilent Human miRNA Microarray V19.0). We utilize paired carcinoma and normal tissue from 217 CRC cases. We evaluate the associations between differentially expressed genes and miRNAs and sex, age, disease stage, and survival months., Results: Thirteen genes were significantly downregulated and 14 were significantly upregulated after considering fold change (FC) of > 1.50 or < 0.67 and multiple comparison adjustment. Bone morphogenetic protein genes BMP5, BMP6, and BMP2 and growth differentiation factor GDF7 were downregulated. BMP4, BMP7, INHBA (Inhibin beta A), TGFBR1, TGFB2, TGIF1, TGIF2, and TFDP1 were upregulated. In general, genes with the greatest dysregulation, such as BMP5 (FC 0.17, BMP6 (FC 0.25), BMP2 (FC 0.32), CDKN2B (FC 0.32), MYC (FC 3.70), BMP7 (FC 4.17), and INHBA (FC 9.34) showed dysregulation in the majority of the population (84.3, 77.4, 81.1, 80.2, 82.0, 51.2, and 75.1% respectively). Four genes, TGFBR2, ID4, ID1, and PITX2, were un-associated or slightly upregulated in microsatellite-stable (MSS) tumors while downregulated in microsatellite-unstable (MSI) tumors. Eight dysregulated genes were associated with miRNA differential expression. E2F5 and THBS1 were associated with one or two miRNAs; RBL1, TGFBR1, TGIF2, and INHBA were associated with seven or more miRNAs with multiple seed-region matches. Evaluation of the joint effects of mRNA:miRNA identified interactions that were stronger in more advanced disease stages and varied by survival months., Conclusion: These data support an interaction between miRNAs and genes in the TGFβ-signaling pathway in association with CRC risk. These interactions are associated with unique clinical characteristics that may provide targets for further investigations.

Colorectal cancer (CRC) accounts for about 8% of all new cancer cases diagnosed in the US. We used whole exome sequence data from triplet samples (colon carcinoma, colon adenoma, and normal tissue) from 18 individuals to assess gene mutation rates. Of the 2 204 genes that were mutated, APC, TTN, TP53, KRAS, OBSCN, SOX9, PCDH17, SIGLEC10, MYH6, and BRD9 were consistent with genes being an early driver of carcinogenesis, in that they were mutated in multiple adenomas and multiple carcinomas. Fifty-two genes were mutated in ≥12.5% of microsatellite stable (MSS) carcinomas but not in any of the adenomas, in line with the profile of a late driver event involved in tumor progression. Thirty-eight genes were sequenced in a larger independent set of 148 carcinoma/normal tissue pairs to obtain more precise mutation frequencies. Eight of the genes, APC, TP53, ATM, CSMD3, LRP1B, RYR2, BIRC6, and MUC17, contained mutations in >20% of the carcinomas. Interestingly, mutations in four genes in addition to APC that are associated with dysregulation of Wnt signaling, were all classified as early driver events. Most of the genes that are commonly associated with colon cancer, including APC, TP53, and KRAS, were all classified as being early driver genes being mutated in both adenomas and carcinomas. Classifying genes as potential early and late driver events points to candidate genes that may help dissect pathways involved in both tumor initiation and progression., (© 2018 The Authors Genes, Chromosomes and Cancer Published by Wiley Periodicals, Inc.)

Apoptosis is genetically regulated and involves intrinsic and extrinsic pathways. We examined 133 genes within these pathways to identify whether they are expressed differently in colorectal carcinoma (CRC) and normal tissue (N = 217) and if they are associated with similar differential miRNA expression. Gene expression data (RNA-Seq) and miRNA expression data (Agilent Human miRNA Microarray V19.0) were generated. We focused on dysregulated genes with a fold change (FC) of > 1.50 or < 0.67, that were significant after adjustment for multiple comparisons. miRNA:mRNA seed-region matches were determined. Twenty-three genes were significantly downregulated (FC < 0.67) and 18 were significantly upregulated (FC > 1.50). Of these 41 genes, 11 were significantly associated with miRNA differential expression. BIRC5 had the greatest number of miRNA associations (14) and the most miRNAs with a seed-region match (10). Four of these matches, miR-145-5p, miR-150-5p, miR-195-5p, and miR-650, had a negative beta coefficient. CSF2RB was associated with ten total miRNAs (five with a seed-region match, and one miRNA, miR-92a-3p, with a negative beta coefficient). Of the three miRNAs associated with CTSS, miR-20b-5p, and miR-501-3p, had a seed-region match and a negative beta coefficient between miRNA:mRNA pairs. Several miRNAs that were associated with dysregulated gene expression, seed-region matches, and negative beta coefficients also were associated with CRC-specific survival. Our data suggest that miRNAs could influence several apoptosis-related genes. BIRC5, CTSS, and CSF2R all had seed-region matches with miRNAs that would favor apoptosis. Our study identifies several miRNA associated with apoptosis-related genes, that if validated, could be important therapeutic targets.

Background: Sessile serrated polyps (SSPs) have emerged as important precursors for a large number of sporadic colorectal cancers. They are difficult to detect during colonoscopy due to their flat shape and the excessive amounts of secreted mucin that cover the polyps. The underlying genetic and epigenetic basis for the emergence of SSPs is largely unknown with existing genetic studies confined to a limited number of oncogenes and tumor suppressors. A full characterization of the genetic and epigenetic landscape of SSPs would provide insight into their origin and potentially offer new biomarkers useful for detection of SSPs in stool samples., Methods: We used a combination of genome-wide mutation detection, exome sequencing and DNA methylation profiling (via methyl-array and whole-genome bisulfite sequencing) to analyze multiple samples of sessile serrated polyps and compared these to familial adenomatous polyps., Results: Our analysis revealed BRAF-V600E as the sole recurring somatic mutation in SSPs with no additional major genetic mutations detected. The occurrence of BRAF-V600E was coincident with a unique DNA methylation pattern revealing a set of DNA methylation markers showing significant (~3 to 30 fold) increase in their methylation levels, exclusively in SSP samples. These methylation patterns effectively distinguished sessile serrated polys from adenomatous polyps and did so more effectively than parallel gene expression profiles., Conclusions: This study provides an important example of a single oncogenic mutation leading to reproducible global DNA methylation changes. These methylated markers are specific to SSPs and could be of important clinical relevance for the early diagnosis of SSPs using non-invasive approaches such as fecal DNA testing.

Mitogen-activated protein kinase (MAPK) pathways regulate many cellular functions including cell proliferation and apoptosis. We examined associations of differential gene and microRNA (miRNA) expression between carcinoma and paired normal mucosa for 241 genes in the KEGG-identified MAPK-signaling pathway among 217 colorectal cancer (CRC) cases. Gene expression data (RNA-Seq) and miRNA expression data (Agilent Human miRNA Microarray V19.0; Agilent Technologies Inc., Santa Clara, CA, USA) were analyzed. We first identified genes most strongly associated with CRC using a fold change (FC) of >1.50 or <0.67) that were statistically significant after adjustment for multiple comparisons. We then determined miRNAs associated with dysregulated genes and through miRNA:mRNA (messenger RNA) seed region matches discerned genes with a greater likelihood of having a direct biological association. Ninety-nine genes had a meaningful FC for all CRC, microsatellite unstable-specific tumors, or microsatellite stable-specific tumors. Thirteen dysregulated genes were associated with miRNAs, totaling 68 miRNA:mRNA associations. Thirteen of the miRNA:mRNA associations had seed region matches where the differential expression between the miRNA and mRNA was inversely related suggesting a direct association as a result of their binding. Several direct associations, upstream of ERK1/ERK2, JNK, and p38, were found for PDGFRA with 7 miRNAs; RASGRP3 and PRKCB with miR-203a; and TGFBR1 with miR-6071 and miR-2117. Other associations between miRNAs and mRNAs are most likely indirect, resulting from feedback and feed forward loops. Our results suggest that miRNAs may alter MAPK signaling through direct binding with key genes in this pathway. We encourage others to validate results in targeted CRC experiments that can help solidify important therapeutic targets., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: The nuclear factor-kappa B (NF-κB) signalling pathway is a regulator of immune response and inflammation that has been implicated in the carcinogenic process. We examined differentially expressed genes in this pathway and miRNAs to determine associations with colorectal cancer (CRC)., Methods: We used data from 217 CRC cases to evaluate differences in NF-κB signalling pathway gene expression between paired carcinoma and normal mucosa and identify miRNAs that are associated with these genes. Gene expression data from RNA-Seq and miRNA expression data from Agilent Human miRNA Microarray V19.0 were analysed. We evaluated genes most strongly associated and differentially expressed (fold change (FC) of > 1.5 or < 0.67) that were statistically significant after adjustment for multiple comparisons., Results: Of the 92 genes evaluated, 22 were significantly downregulated and nine genes were significantly upregulated in all tumours. Two additional genes (CD14 and CSNK2A1) were dysregulated in MSS tumours and two genes (CARD11 and VCAM1) were downregulated and six genes were upregulated (LYN, TICAM2, ICAM1, IL1B, CCL4 and PTGS2) in MSI tumours. Sixteen of the 21 dysregulated genes were associated with 40 miRNAs. There were 76 miRNA:mRNA associations of which 38 had seed-region matches. Genes were associated with multiple miRNAs, with TNFSRF11A (RANK) being associated with 15 miRNAs. Likewise several miRNAs were associated with multiple genes (miR-150-5p with eight genes, miR-195-5p with four genes, miR-203a with five genes, miR-20b-5p with four genes, miR-650 with six genes and miR-92a-3p with five genes)., Conclusions: Focusing on the genes and their associated miRNAs within the entire signalling pathway provides a comprehensive understanding of this complex pathway as it relates to CRC and offers insight into potential therapeutic agents.

The PI3K/AKT-signaling pathway is one of the most frequently activated signal-transduction pathways in cancer. We examined how dysregulated gene expression is associated with miRNA expression in this pathway in colorectal cancer (CRC). We used data from 217 CRC cases to evaluate differential pathway gene expression between paired carcinoma and normal mucosa and identify miRNAs that are associated with these genes. Gene expression data from RNA-Seq and miRNA expression data from Agilent Human miRNA Microarray V19.0 were analyzed. We focused on genes most associated with CRC (fold change (FC) of >1.5 or <0.67) that were statistically significant after adjustment for multiple comparisons. Of the 304 genes evaluated, 76 had a FC of <0.67, and 57 had a FC of >1.50; 47 of these genes were associated with miRNA differential expression. There were 145 mRNA:miRNA seed-region matches of which 26 were inversely associated suggesting a greater likelihood of a direct association. Most miRNA:mRNA associations were with factors that stimulated the pathway. For instance, both IL6R and PDGFRA had inverse seed-region matches with seven miRNAs, suggesting that these miRNAs have a direct effect on these genes and may be key elements in activation of the pathway. Other miRNA:mRNA associations with similar impact on the pathway were miR-203a with ITGA4, miR-6071 with ITGAV, and miR-375 with THBS2, all genes involved in extracellular matrix function that activate PI3Ks. Gene expression in the PI3K/Akt-signaling pathway is dysregulated in CRC. MiRNAs were associated with many of these dysregulated genes either directly or in an indirect manner., (© 2017 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.)

This corrects the article DOI: 10.1038/modpathol.2017.38.

To identify gene expression biomarkers and pathways targeted by sulindac and erlotinib given in a chemoprevention trial with a significant decrease in duodenal polyp burden at 6 months ( P < 0.001) in familial adenomatous polyposis (FAP) patients, we biopsied normal and polyp duodenal tissues from patients on drug versus placebo and analyzed the RNA expression. RNA sequencing was performed on biopsies from the duodenum of FAP patients obtained at baseline and 6-month endpoint endoscopy. Ten FAP patients on placebo and 10 on sulindac and erlotinib were selected for analysis. Purity of biopsied polyp tissue was calculated from RNA expression data. RNAs differentially expressed between endpoint polyp and paired baseline normal were determined for each group and mapped to biological pathways. Key genes in candidate pathways were further validated by quantitative RT-PCR. RNA expression analyses of endpoint polyp compared with paired baseline normal for patients on placebo and drug show that pathways activated in polyp growth and proliferation are blocked by this drug combination. Directly comparing polyp gene expression between patients on drug and placebo also identified innate immune response genes (IL12 and IFNγ) preferentially expressed in patients on drug. Gene expression analyses from tissue obtained at endpoint of the trial demonstrated inhibition of the cancer pathways COX2/PGE2, EGFR, and WNT. These findings provide molecular evidence that the drug combination of sulindac and erlotinib reached the intended tissue and was on target for the predicted pathways. Furthermore, activation of innate immune pathways from patients on drug may have contributed to polyp regression. Cancer Prev Res; 11(1); 4-15. ©2017 AACR See related editorial by Shureiqi, p. 1 ., (©2017 American Association for Cancer Research.)

The Wnt-signaling pathway functions in regulating cell growth and thus is involved in the carcinogenic process of several cancers, including colorectal cancer. We tested the hypothesis that multiple genes in this signaling pathway are dysregulated and that miRNAs are associated with these dysregulated genes. We used data from 217 colorectal cancer (CRC) cases to evaluate differences in Wnt-signaling pathway gene expression between paired CRC and normal mucosa and identify miRNAs that are associated with these genes. Gene expression data from RNA-Seq and miRNA expression data from Agilent Human miRNA Microarray V19.0 were analyzed. We focused on genes most strongly associated with CRC (fold change (FC) of >1.5 or <0.67) and that were statistically significant after adjustment for multiple comparisons. Of the 138 Wnt-signaling pathway genes examined, 27 were significantly down-regulated (FC<0.67) and 32 genes were significantly up-regulated (FC>1.50) after adjusting for multiple comparisons. Thirteen of the 66 Wnt-signaling genes that were differentially expressed in CRC tumors were associated with differential expression of miRNAs. A total of 93 miRNA:mRNA associations were detected for these 13 genes. Of these 93 associations, 36 miRNA seed-region matches were observed, suggesting that miRNAs have both direct and indirect effects on Wnt-signaling pathway genes. In summary, our data supports the hypothesis that the Wnt-signaling pathway is dysregulated in CRC and suggest that miRNAs may importantly influence that dysregulation., Competing Interests: CONFLICTS OF INTEREST None.

Tumor suppressor genes (TSGs) and oncogenes (OG) are involved in carcinogenesis. MiRNAs also contribute to cellular pathways leading to cancer. We use data from 217 colorectal cancer (CRC) cases to evaluate differences in TSGs and OGs expression between paired CRC and normal mucosa and evaluate how TSGs and OGs are associated with miRNAs. Gene expression data from RNA-Seq and miRNA expression data from Agilent Human miRNA Microarray V19.0 were used. We focus on genes most strongly associated with CRC (fold change (FC) of ≥1.5 or ≤0.67) that were statistically significant after adjustment for multiple comparisons. Of the 74 TSGs evaluated, 22 were associated with carcinoma/normal mucosa differential expression. Ten TSGs were up-regulated (FAM123B, RB1, TP53, RUNX1, MSH2, BRCA1, BRCA2, SOX9, NPM1, and RNF43); six TSGs were down-regulated (PAX5, IZKF1, GATA3, PRDM1, TET2, and CYLD); four were associated with MSI tumors (MLH1, PTCH1, and CEBPA down-regulated and MSH6 up-regulated); and two were associated with MSS tumors (PHF6 and ASXL1 up-regulated). Thirteen of these TSGs were associated with 44 miRNAs. Twenty-seven of the 59 OGs evaluated were dysregulated: 14 down-regulated (KLF4, BCL2, SSETBP1, FGFR2, TSHR, MPL, KIT, PDGFRA, GNA11, GATA2, FGFR3, AR, CSF1R, and JAK3), seven up-regulated (DNMT1, EZH2, PTPN11, SKP2, CCND1, MET, and MYC); three down-regulated for MSI (FLT3, CARD11, and ALK); two up-regulated for MSI (IDH2 and HRAS); and one up-regulated with MSS tumors (CTNNB1). These findings suggest possible co-regulatory function between TSGs, OGs, and miRNAs, involving both direct and indirect associations that operate through feedback and feedforward loops., (© 2017 Authors Genes, Chromosomes and Cancer Published by Wiley Periodicals, Inc.)

Half of miRNAs expressed in colorectal tissue are expressed < 50% of the population. Many infrequently expressed miRNAs have low levels of expression. We hypothesize that less frequently expressed miRNAs, when expressed at higher levels, influence both disease stage and survival after diagnosis with colorectal cancer (CRC); low levels of expression may be background noise. We examine 304 infrequently expressed miRNAs in 1893 population-based cases of CRC with paired carcinoma and normal mucosa miRNA profiles. We evaluate miRNAs with disease stage and survival after adjusting for age, study center, sex, MSI status, and AJCC stage. These miRNAs were further evaluated with RNA-Seq data to identify miRNA::mRNA associations that may provide insight into the functionality of miRNAs. Eleven miRNAs were associated with advanced disease stage among colon cancer patients ( Q value = 0.10). Eight infrequently expressed miRNAs influenced survival if highly expressed in overall CRC. Of these, five increased likelihood of dying if they were highly expressed, i.e. miR-124-3p, miR-143-5p, miR-145-3p, miR31-5p, and miR-99b-5p, while three were associated with better survival if highly expressed, i.e. miR-362-5p, miR-374a-5p, and miR-590-5p. Thirteen miRNAs infrequently expressed in colon-specific carcinoma tissue were associated with CRC survival if highly expressed. Evaluation of miRNAs::mRNA associations showed that mRNA expression influenced by infrequently expressed miRNA contributed to networks and pathways shown to influence disease progression and prognosis. Our large study enabled us to examine the implications of infrequently expressed miRNAs after removal of background noise. These results require replication in other studies. Confirmation of our findings in other studies could lead to important markers for prognosis., Competing Interests: CONFLICTS OF INTEREST None of the authors have financial and non-financial competing interests to report.

Advances in defining the mutational landscape of colorectal cancer (CRC) over the past decades have revolutionized the molecular understanding and clinical testing algorithms for this disease. Mutation testing is standard of care for the work-up of CRCs. This review focuses on the current indications and strategies for molecular testing in CRC and discusses the potential changes in CRC testing approach associated with the emerging clinical application of genomic-based technologies.

We have previously shown that commonly expressed miRNAs influenced tumor molecular phenotype in colorectal cancer. We hypothesize that infrequently expressed miRNAs, when showing higher levels of expression, help to define tumor molecular phenotype. In this study, we examine 304 miRNAs expressed in at least 30 individuals, but in <50% of the population and with a mean level of expression above 1.0 relative florescent unit. We examine associations in 1893 individuals who have the tumor molecular phenotype data as well as miRNA expression levels for both carcinoma and normal colorectal tissue. We compare miRNAs uniquely associated with tumor molecular phenotype to the RNAseq data to identify genes associated with these miRNAs. This information is used to further identify unique pathways associated with tumor molecular phenotypes of TP53-mutated, KRAS-mutated, CpG island methylator phenotype and microsatellite instability tumors. Thirty-seven miRNAs were uniquely associated with TP53-mutated tumors; 30 of these miRNAs had higher level of expression in TP53-mutated tumors, while seven had lower levels of expression. Of the 34 miRNAs associated with CpG island methylator phenotype-high tumors, 16 were more likely to have a CpG island methylator phenotype-high tumor and 19 were less likely to be CpG island methylator phenotype-high. For microsatellite instability, 13 of the 22 infrequently expressed miRNAs were significantly less likely to be expressed in microsatellite unstable tumors. KRAS-mutated tumors were not associated with any miRNAs after adjustment for multiple comparisons. Of the dysregulated miRNAs, 17 were more likely to be TP53-mutated tumors while simultaneously being less likely to be CpG island methylator phenotype-high and/or microsatellite instability tumors. Genes regulated by these miRNAs were involved in numerous functions and pathways that influence cancer risk and progression. In summary, some infrequently expressed miRNAs, when expressed at higher levels, appear to have significant biological meaning in terms of tumor molecular phenotype and gene expression profiles.

Objectives: Fundic gland polyps (FGPs) can rarely exhibit dysplasia of the surface epithelium. Based on retrospective data, FGPs with dysplasia (FGPDs) are thought to be a strong marker for familial adenomatous polyposis (FAP), although sporadic, non-syndromic FGPDs also occur. Owing to the significant syndromic association, diagnosis of an apparently sporadic FGPD may prompt clinical evaluation for FAP, especially its attenuated variant. We sought to evaluate the positive predictive value of incidental FGPDs for FAP. We also characterized the clinicopathologic features of incidental FGPDs to advance clinical management., Methods: Incidental FGPDs were identified from 2004 to 2015 in patients without FAP at biopsy. All clinical follow-up data were reviewed, and germline analysis for APC and MUTYH mutations was performed in consenting patients., Results: We identified 25 incidental FGPDs in patients not known to have FAP (11.6% of FGPDs, 1.0% of all FGPs). Four patients had a family history of gastric polyps or gastrointestinal cancers. Clinical management included completion polypectomy and gastric endoscopic surveillance (44%), endoscopic surveillance alone (32%), no follow-up (24%), colonoscopy referral (12%), and genetic counseling (4%). Colonoscopies on record revealed 0-7 cumulative adenomas. Follow-up averaged 4.4 years (range 0.3-10.6). No clinical evidence of FAP, gastric cancer, death, or surgery occurred. None of the 11 patients consenting to germline APC and MUTYH testing had genomic alterations., Conclusions: Incidental FGPDs in this series were all found to be sporadic (25/25) by endoscopic, clinical, and molecular findings, and thus FGPDs were not harbingers of FAP. As isolated findings, FGPDs do not appear to warrant follow-up genetic counseling or testing.

Background/aims: The serrated pathway accounts for 15-25% of sporadic colorectal cancer (CRC). In our study, we sought to accurately characterize sessile serrated polyps (SSP) in a population by electronically interrogating colonoscopy patients' endoscopy and pathology reports using a rules-based text search of pre-defined SSP-related terms. To this aim, we compared a sample of putative SSP and hyperplastic polyps (HP) using our algorithm to a determination of SSP or HP by pathologist and molecular examination to determine the feasibility of large-scale identification of SSP in electronic medical records., Methods: In 23,990 endoscopy reports from colonoscopies with pathology performed at a University of Utah Healthcare facility in 2000-2012, we identified serrated lesions and categorized each as putative SSP or HP using a text search algorithm. We obtained 93 tissue samples for histologic and molecular analysis., Results: Serrated polyps were categorized as putative SSP (N = 920) and putative HP (N = 7159) by text search algorithm. Histologic examination of 93 samples identified 37 SSP, 11 probable SSP, and 45 HP. Of 26 putative SSP, 25 were SSP/probable SSP (96%) by histology. Of 67 putative HP, 44 were HP (66%) by histology. Reducing size criterion from ≥1 to ≥5 mm in the search algorithm caused improved sensitivity (77.1%) without decline in specificity (97.8%)., Conclusions: A simple rules-based search to identify SSP provides "proof of principle" that SSP can be identified in a large electronic record set. Pilot data indicate defining large, right-sided polyps as ≥5 mm provides adequate sensitivity to detect SSP from electronic records while maintaining high specificity.

Endoscopic mucosal biopsy may misplace mucosal elements into the submucosa of colonic adenomas, mimicking invasive adenocarcinoma. Biopsy-related misplacement can be more challenging to recognize than typical misplaced epithelium (pseudoinvasion) in pedunculated polyps. We compared the features of 16 polyps with biopsy-related misplaced epithelium with those of 10 adenomas with pseudoinvasion and 10 adenomas with invasive adenocarcinoma and performed Ki67 and p53 immunostaining on all cases. Features of misplaced epithelium in polyps referred to the Bowel Cancer Screening Program Expert Board in the United Kingdom were also evaluated for the same morphologic features. Biopsy-related epithelial misplacement occurred in adenomas throughout the colon and often appeared infiltrative (69%), including epithelial cells singly dispersed within reactive fibroinflammatory stroma or granulation tissue (44%). Misplaced epithelium displayed only low-grade cytologic features and was associated with extruded mucin (75%), tattoo pigment (63%), and misplaced normal glands (38%); scant lamina propria and muscularis mucosae were often present (88% and 44%, respectively). Cases referred to the Bowel Cancer Screening Program Expert Board also contained infiltrative-appearing misplaced epithelium (91%) that was cytologically low grade (72%), contained nondysplastic glands (11%), and showed other signs of injury. In contrast, misplaced epithelium in pedunculated polyps always had a lobular contour with a rim of lamina propria, hemorrhage, and/or hemosiderin. Invasive carcinomas showed malignant cytology and desmoplasia; most (70%) lacked features of trauma. Ki67 and p53 staining was patchy and weak in the misplaced epithelium, whereas invasive carcinomas showed increased staining for one or both markers. Pathologists should be aware that endoscopically manipulated adenomas may contain misplaced epithelium that simulates malignancy.

MiRNAs are small, non-protein-coding RNA molecules that regulate gene expression either by post-transcriptionally suppressing mRNA translation or by mRNA degradation. We examine differentially expressed miRNAs in colorectal carcinomas, adenomas and normal colonic mucosa. Data come from population-based studies of colorectal cancer conducted in Utah and the Kaiser Permanente Medical Care Program. A total of 1893 carcinoma/normal-paired samples and 290 adenoma tissue samples were run on the Agilent Human miRNA Microarray V19.0 which contained 2006 miRNAs. We tested for significant differences in miRNA expression between paired carcinoma/adenoma/normal colonic tissue samples. Fewer than 600 miRNAs were expressed in >80% of people for colonic tissue; of these 86.5% were statistically differentially expressed between carcinoma and normal colonic mucosa using a false discovery rate of 0.05. Roughly half of these differentially expressed miRNAs showed a progression in levels of expression from normal to adenoma to carcinoma tissue. Other miRNAs appeared to be altered at the normal to adenoma stage, while others were only altered at the adenoma to carcinoma stage or only at the normal to carcinoma stage. Evaluation of the Agilent platform showed a high degree of repeatability (r = 0.98) and reasonable agreement with the NanoString platform. Our data suggest that miRNAs are highly dysregulated in colorectal tissue among individuals with colorectal cancer; the pattern of disruption varies by miRNA as tissue progresses from normal to adenoma to carcinoma., (© The Author 2016. Published by Oxford University Press.)

Most sporadic colonic adenocarcinomas are microsatellite stable (MSS) and arise from conventional adenomas by dysregulation of the APC/β-catenin/Wnt signaling pathway. Sporadic adenocarcinomas with a high degree of microsatellite instability (MSI) likely arise from sessile serrated polyps through the serrated neoplastic pathway. These polyps contain BRAF mutations and are prone to epigenetic methylation that ultimately silences MLH1, leading to MSI and heralding progression of dysplasia to invasive adenocarcinoma. Most investigators believe that these 2 models of cancer progression are mutually exclusive, although recent studies describe Wnt signaling activation in serrated polyps and propose that it plays a role in the development of sporadic colonic adenocarcinomas with MSI. We sought to test this hypothesis by evaluating β-catenin immunoexpression in 44 sporadic microsatellite unstable adenocarcinomas and 44 MSS colon cancers. We defined sporadic MSI-high carcinomas as those with loss of MLH1 and PMS2 immunostaining and BRAF V600E mutations that occurred in patients 50 years of age or older without a family history of colonic adenocarcinoma or Lynch syndrome. Forty-one (93%) of these carcinomas displayed membranous β-catenin staining only, compared with 28 (64%) site-matched MSS tumors with abnormal nuclear β-catenin staining.

Lynch syndrome is the most common inherited colorectal cancer syndrome, accounting for 2-4% of all colorectal cancer cases. This review focuses on the tissue workup of Lynch syndrome, including methods to determine whether or not a tumor is mismatch repair deficient, and whether a mismatch repair-deficient tumor is sporadic or Lynch syndrome-associated. Strategies for determining which tumors to test and how best to implement a Lynch syndrome screening program are also discussed, as well as potential developments in the future.

Lynch syndrome is characterized by mutations in one of four mismatch repair genes, MLH1, MSH2, MSH6, or PMS2. Clinical mutation analysis of these genes includes sequencing of exonic regions and deletion/duplication analysis. However, detection of deletions and duplications in PMS2 has previously been confined to Exons 1-11 due to gene conversion between PMS2 and the pseudogene PMS2CL in the remaining 3' exons (Exons 12-15). We have recently described an MLPA-based method that permits detection of deletions of PMS2 Exons 12-15; however, the frequency of such deletions has not yet been determined. To address this question, we tested for 3' deletions in 58 samples that were reported to be negative for PMS2 mutations using previously available methods. All samples were from individuals whose tumors exhibited loss of PMS2 immunohistochemical staining without concomitant loss of MLH1 immunostaining. We identified seven samples in this cohort with deletions in the 3' region of PMS2, including three previously reported samples with deletions of Exons 13-15 (two samples) and Exons 14-15. Also detected were deletions of Exons 12-15, Exon 13, and Exon 14 (two samples). Breakpoint analysis of the intragenic deletions suggests they occurred through Alu-mediated recombination. Our results indicate that ∼12% of samples suspected of harboring a PMS2 mutation based on immunohistochemical staining, for which mutations have not yet been identified, would benefit from testing using the new methodology., (Copyright © 2012 Wiley Periodicals, Inc.)

The purpose of this review is to discuss important molecular changes that aid decision making in patient management and play a role in emerging treatment strategies for gastrointestinal malignancies. Although screening and surveillance practices have had an impact on the natural history of some tumor types, gastric carcinoma is a major cause of morbidity and mortality in high prevalence regions and colorectal carcinoma is still the fourth leading cause of cancer related death in the United States., (Copyright © 2012. Published by Elsevier Inc.)

Phosphatidylinositol 3'-kinase gene (PIK3CA) encodes a lipid kinase that regulates signaling pathways downstream of epidermal growth factor receptor and is mutated in 10% to 30% of colorectal cancers. Activating mutations in this gene up-regulates the AKT signaling pathway, making it a potentially useful therapeutic target. Mutations in PIK3CA are not exclusive of mutations in KRAS, BRAF, or NRAS. We designed a pyrosequencing assay to detect mutations in all three positions of codons 542 and 545 in exon 9 and codon 1047 in exon 20 of this gene. The exon 9 reverse PCR primer was designed to avoid amplifying a pseudogene in chromosome 22 that has >95% homology with exons 9 through 13 in PIK3CA. Two hundred colorectal cancers from FFPE tissue previously characterized for KRAS mutation status were evaluated for PIK3CA mutations. In KRAS-mutated samples, 20% had an additional mutation in PIK3CA. The mutation rate in KRAS wild-type samples was 7.5%. When using our assay, pseudogene was not observed in any of these samples. In addition, pseudogene- and gene-specific amplification was performed on DNA from 40 additional colorectal cancers. Sequencing of these PCR products yielded the expected gene or pseudogene sequence in all cases. Thus, we have developed a PIK3CA pyrosequencing assay capable of detecting mutations in all three positions in the three hot spot codons with no pseudogene interference., (Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: About half of Americans 50 to 75 years old do not follow recommended colorectal cancer (CRC) screening guidelines, leaving 40 million individuals unscreened. A simple blood test would increase screening compliance, promoting early detection and better patient outcomes. The objective of this study is to demonstrate the performance of an improved sensitivity blood-based Septin 9 (SEPT9) methylated DNA test for colorectal cancer. Study variables include clinical stage, tumor location and histologic grade., Methods: Plasma samples were collected from 50 untreated CRC patients at 3 institutions; 94 control samples were collected at 4 US institutions; samples were collected from 300 colonoscopy patients at 1 US clinic prior to endoscopy. SEPT9 methylated DNA concentration was tested in analytical specimens, plasma of known CRC cases, healthy control subjects, and plasma collected from colonoscopy patients., Results: The improved SEPT9 methylated DNA test was more sensitive than previously described methods; the test had an overall sensitivity for CRC of 90% (95% CI, 77.4% to 96.3%) and specificity of 88% (95% CI, 79.6% to 93.7%), detecting CRC in patients of all stages. For early stage cancer (I and II) the test was 87% (95% CI, 71.1% to 95.1%) sensitive. The test identified CRC from all regions, including proximal colon (for example, the cecum) and had a 12% false-positive rate. In a small prospective study, the SEPT9 test detected 12% of adenomas with a false-positive rate of 3%., Conclusions: A sensitive blood-based CRC screening test using the SEPT9 biomarker specifically detects a majority of CRCs of all stages and colorectal locations. The test could be offered to individuals of average risk for CRC who are unwilling or unable to undergo colonscopy.

Context: Rational anticancer therapy is beginning to expand the practice of surgical pathology beyond a primarily morphologic and immunophenotypic analysis into the molecular arena. Molecular testing of tumors can have both diagnostic and therapeutic value, which guides treatment decisions. This is true for colorectal cancer in which mutations in signaling mediators predict resistance to anti-epidermal growth factor receptor (anti-EGFR) therapy., Objective: To review the clinically relevant mutations that currently guide treatment decisions in metastatic colorectal cancer, summarize additional mutations that are expected to improve the prognostic sensitivity of molecular testing, and provide practical suggestions for submitting specimens for molecular analysis., Data Sources: Peer-reviewed literature reporting pertinent clinical trial data, mutation analysis, and molecular mechanisms of drug resistance, as well as comprehensive review articles germane to the topic and published testing recommendations from the College of American Pathologists., Conclusions: Molecular analysis of colorectal cancer is now mandated before initiation of anti-EGFR therapy and directly impacts treatment options and outcomes. Familiarity with the mutations that determine utility and efficacy of therapy, as well as the importance of careful sample selection, will facilitate appropriate testing and optimize patient care.

Context: About 15% of colorectal cancers are characterized by genomic microsatellite instability, and of these, about 1 in 5 (2%-4% overall) are due to Lynch syndrome, a dominantly inherited condition predisposing the patient to cancers of multiple organ systems, including the gastrointestinal tract. Identification of individuals with Lynch syndrome allows for increased surveillance of the affected individual and of potentially affected family members., Objective: To review the literature on microsatellite instability in colorectal cancer and current laboratory diagnostic testing strategies for the detection of Lynch syndrome., Data Sources: This review is based on peer-reviewed literature, published guidelines from professional organizations (Evaluation of Genomic Applications in Practice and Prevention Working Group, National Comprehensive Cancer Network), and information from clinical laboratories performing microsatellite instability testing., Conclusions: Universal screening for Lynch syndrome in all individuals affected with colorectal cancer has been recommended by the Evaluation of Genomic Applications in Practice and Prevention Working Group. Preliminary screening tests can identify individuals unlikely to be affected by Lynch syndrome, thereby reducing the need for full gene analysis. Immunohistochemistry and polymerase chain reaction-based tests for microsatellite instability have similar clinical sensitivity and specificity, and each method has advantages and limitations. BRAF and MLH1 methylation testing are useful reflex tests for those with a defect in MLH1 identified by immunohistochemistry. Emerging technologies, such as high-throughput sequencing, may substantially affect diagnostic algorithms in the future.

Lynch syndrome is characterized by mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. In PMS2, detection of mutations is confounded by numerous pseudogenes. Detection of 3' deletions is particularly complicated by the pseudogene PMS2CL, which has strong similarity to PMS2 exons 9 and 11-15, due to extensive gene conversion. A newly designed multiplex ligation-dependent probe amplification (MLPA) kit incorporates probes for variants found in both PMS2 and PMS2CL. This provides detection of deletions, but does not allow localization of deletions to the gene or pseudogene. To address this, we have developed a methodology incorporating reference samples with known copy numbers of variants, and paired MLPA results with sequencing of PMS2 and PMS2CL. We tested a subset of clinically indicated samples for which mutations were either unidentified or not fully characterized using existing methods. We identified eight unrelated patients with deletions encompassing exons 9-15, 11-15, 13-15, 14-15, and 15. By incorporating specific, characterized reference samples and sequencing the gene and pseudogene it is possible to identify deletions in this region of PMS2 and provide clinically relevant results. This methodology represents a significant advance in the diagnosis of patients with Lynch syndrome caused by PMS2 mutations., (© 2011 Wiley-Liss, Inc.)

Objective: To characterize the current state of evidence and apply simulation modeling to support decision making about provision and coverage of a Lynch syndrome (LS) screening program among colorectal cancer (CRC) patients in our integrated healthcare delivery system., Study Design: Application of multiple methods for synthesizing evidence guided by needs of our clinical and administrative decision makers., Methods: Narrative and focused reviews, computerized simulation models of multiple screening options, queries of our electronic data warehouse, and extensive communication with decision makers., Results: Review of published evidence at the time of the study period revealed that screening unselected CRC patients for LS would likely cost less than $25,000 per life-year saved (compared with no screening) and that screening with immunohistochemistry is substantially more efficient than other options. Our simulation models suggest that not only does including BRAF mutation testing substantially improve efficiency but that adding methylation testing improves it further. We characterized a variety of other metrics that contributed not only to local decisions but to the broader evidence base on this topic., Conclusion: The current state of evidence at the time of the study period suggests an LS screening program can be both effective in reducing mortality from CRC and cost-effective. However, direct evidence remains limited and multiple factors could threaten success of such a program. We have identified opportunities for optimizing the efficiency of available screening protocols. While there was enough evidence for our system to proceed with an LS screening program, we recognize the threats to program success and will prospectively collect outcome data supporting empirical examination of the program.

C-reactive protein (CRP), a biomarker of inflammation, has been shown to be influenced by genetic variation in the CRP gene. In this study, we test the hypothesis that genetic variation in CRP influences both the risk of developing colon and rectal cancer and survival. Two population-based studies of colon cancer (n = 1,574 cases, 1,970 controls) and rectal (n = 791 cases, 999 controls) were conducted. We evaluated four CRP tagSNPs: rs1205 (G > A, 3' UTR); rs1417938 (T > A, intron); rs1800947 (G > C, L184L); and rs3093075 (C > A, 3' flanking). The CRP rs1205 AA genotype was associated with an increased risk of colon cancer (OR 1.3, 95%CI 1.1-1.7), whereas the rs3093075 A allele was associated with a reduced risk of rectal cancer (OR 0.7, 95%CI 0.5-0.9). The strongest association for the rs1205 polymorphism and colon cancer was observed among those with KRAS2 mutations (OR 1.5, 95%CI 1.1-2.0). The CRP rs1205 AA genotype also was associated with an increased risk of CIMP+ rectal tumors (OR 2.5, 95%CI 1.2-5.3); conversely, the rs1417938 A allele was associated with a reduced risk of CIMP+ rectal tumors (OR 0.5, 95%CI 0.3-0.9). We observed interactions between CRP rs1800947 and BMI and family history of CRC in modifying risk of both colon and rectal cancer. These data suggest that genetic variation in the CRP gene influences risk of both colon and rectal cancer development., (Copyright © 2010 UICC.)

Mutational analysis of KRAS codons 12 and 13 is standard for patients with metastatic colorectal cancer since mutations in these codons predict lack of response to anti-EGFR therapies. However, even among patients whose tumors are wildtype for KRAS codons 12 and 13, only a subset respond to therapy. Since additional activating mutations downstream of EGFR may also play a role in treatment resistance, we sought to establish the frequency of these mutations. We evaluated 2121 colorectal tumors for mutations in codons 12 and 13 of the KRAS gene. A subset of these samples, comprised of 513 samples wildtype for KRAS codons 12 and 13, were tested for mutations in codons 61 and 146 of KRAS, codon 600 of BRAF, and codons 12, 13, and 61 of NRAS. Mutation status was determined by targeted pyrosequencing. Mutations in KRAS codon 12 or 13 were identified in 900/2121 (42.4%) samples. Of the 513 wildtype samples tested for additional mutations, 78 samples were mutant for BRAF, 19 for KRAS codon 61, 17 for KRAS codon 146, and 26 for NRAS. In total, 140/513 (27.3%) tumors wildtype for KRAS codons 12 and 13 harbored a mutation in another of the RAS pathway genes. While further study is needed to determine the full therapeutic implications of mutations in these codons, mutational testing of these codons may be useful for identifying a significant proportion of patients who may also be resistant to anti-EGFR therapies., (2011 Wiley-Liss, Inc.)

The concept of a CpG island methylator phenotype, or CIMP, quickly became the focus of several colorectal cancer studies describing its clinical and pathological features after its introduction in 1999 by Toyota and colleagues. Further characterization of CIMP in tumors lead to widespread acceptance of the concept, as expressed by Shen and Issa in their 2005 editorial, "CIMP, at last." Since that time, extensive research efforts have brought great insights into the epidemiology and prognosis of CIMP+ tumors and other epigenetic mechanisms underlying tumorigenesis. With the advances in technology and subsequent cataloging of the human methylome in cancer and normal tissue, new directions in research to understand CIMP and its role in complex biological systems yield hope for future epigenetically based diagnostics and treatments.

In an investigation of rectal tumors characterized by CpG island methylator phenotype (CIMP), KRAS2 mutation, and TP53 mutation, we examined associations with dietary and supplemental folate, riboflavin, vitamins B(6) and B(12), and methionine, nutrients involved in folate-mediated 1-carbon metabolism. We also examined folate intake and common MTHFR polymorphisms in relation to CIMP. Data from a population-based study of 951 cases (750 with tumor markers) and 1,205 controls were evaluated using multiple logistic regression models and generalized estimating equations. Reduced risk of methylated tumors was suggested in women with the upper tertile of folate intake (≥0.42 mg/day) vs. the lower tertile: OR = 0.6, 95%CI = 0.3-1.2. In men, a significant 3-fold increased risk of CIMP+ tumor was observed for the upper tertile of folate (≥0.75 mg/day) vs. the lower tertile (<0.44 mg/day): OR = 3.2, 95%CI = 1.5-6.7. These men consumed a greater proportion of folic acid fortified foods relative to natural, primarily plant-based sources (52% vs. 48%) than women with CIMP+ tumors (22% vs. 78%). MTHFR 1298A>C influenced folate in male CIMP+ risk (P interaction < 0.01). Our findings suggest folate supplementation effects may differ between genders, perhaps due to variation in MTHFR and/or endogenous/exogenous hormones, and may be important in the initiation and progression of methylated rectal tumors in men.

We examined candidate polymorphisms in genes involved in the folate-mediated, one-carbon metabolism pathway, DNMT1 1311V, MTHFD1 R134K and R653Q, MTHFR R594Q, MTR D919G, MTRR H595Y and I22M, SHMT1 L474F, SLC19A1 H27R, and TDG G199S, and associations with rectal tumor characteristics. We hypothesized that these candidate genes would influence CpG Island Methylator Phenotype and potentially KRAS2 or TP53 tumors. Data from a population-based study of 747 rectal cases (593 with tumor markers) and 956 controls were evaluated using generalized estimating equations. We observed an increased risk of TP53 tumor mutations in homozygous carriers of the MTHFD1 134K allele (0R=2.0, 95%CI 1.2-3.1, P- trend=0.02). In the presence of low folate intake, the R134K variant was associated with increased risk of CIMP+ tumors (OR=2.8, 95%CI 1.04-7.7). The MTRR I22M variant genotype was associated with a modest increased risk of TP53 mutations (OR=1.7, 95%CI 1.2-2.5, P-trend=0.001). Our findings offer limited support that polymorphisms in one-carbon metabolism genes influence rectal tumor phenotype, and that folate may interact with MTHFD1 to alter CIMP+ risk.

Background: Colorectal cancer is the fourth most common type of cancer and the second most common cause of cancer death. Fewer than 5% of colon cancers arise in the presence of a clear hereditary cancer condition; however, current estimates suggest that an additional 15-25% of colorectal cancers arise on the basis of unknown inherited factors., Aim: To identify additional genetic factors responsible for colon cancer., Methods: A large kindred with excess colorectal cancer was identified through the Utah Population Database and evaluated clinically and genetically for inherited susceptibility., Results: A major genetic locus segregating with colonic polyps and cancer in this kindred was identified on chromosome 13q with a non-parametric linkage score of 24 (LOD score of 2.99 and p=0.001). The genetic region spans 21 Mbp and contains 27 RefSeq genes. Sequencing of all candidate genes in this region failed to identify a clearly deleterious mutation; however, polymorphisms segregating with the phenotype were identified. Chromosome 13q is commonly gained and overexpressed in colon cancers and correlates with metastasis, suggesting the presence of an important cancer progression gene. Evaluation of tumours from the kindred revealed a gain of 13q as well., Conclusions: This identified region may contain a novel gene responsible for colon cancer progression in a significant proportion of sporadic cancers. Identification of the precise gene and causative genetic change in the kindred will be an important next step to understanding cancer progression and metastasis.

Germline mutation detection in PMS2, one of four mismatch repair genes associated with Lynch syndrome, is greatly complicated by the presence of numerous pseudogenes. We used a modification of a long-range PCR method to evaluate PMS2 in 145 clinical samples. This modification avoids potential interference from the pseudogene PMS2CL by utilizing a long-range product spanning exons 11-15, with the forward primer anchored in exon 10, an exon not shared by PMS2CL. Large deletions were identified by MLPA. Pathogenic PMS2 mutations were identified in 22 of 59 patients whose tumors showed isolated loss of PMS2 by immunohistochemistry (IHC), the IHC profile most commonly associated with a germline PMS2 mutation. Three additional patients with pathogenic mutations were identified from 53 samples without IHC data. Thirty-seven percent of the identified mutations were large deletions encompassing one or more exons. In 27 patients whose tumors showed absence of either another protein or combination of proteins, no pathogenic mutations were identified. We conclude that modified long-range PCR can be used to preferentially amplify the PMS2 gene and avoid pseudogene interference, thus providing a clinically useful germline analysis of PMS2. Our data also support the use of IHC screening to direct germline testing of PMS2., ((c) 2010 Wiley-Liss, Inc.)

Microsatellite unstable cancers account for up to 15% of sporadic colon cancers and are predominantly located in the proximal colon. These cancers commonly show MLH1 promoter methylation and the CpG island methylator phenotype (CIMP). A potential precursor of sporadic unstable cancers, the proximal hyperplastic polyp, is also reported to have CIMP and MLH1 methylation. However, this latter finding is not supported by MLH1 protein expression studies. To help resolve this apparent discrepancy, we determined MLH1 promoter methylation and CIMP by quantitative real-time PCR for 29 proximal hyperplastic polyps, 23 distal hyperplastic polyps, and 11 sporadic microsatellite unstable colon cancers. BRAF V600E mutation status was also determined. Positive methylation was defined as the percentage of methylated reference (PMR) >10. Only 1 of 29 proximal hyperplastic polyps showed positive MLH1 methylation (PMR of 13.0). Neither this polyp nor seven other proximal polyps with PMR values between 0 and 10 showed loss of MLH1 protein expression by immunohistochemistry. In contrast, all 11 microsatellite unstable cancers showed high degrees of MLH1 methylation, with PMR values >30. Fourteen of twenty-nine (48%) of the proximal hyperplastic polyps and 1 of 23 (4%) of the distal hyperplastic polyps showed CIMP (P<0.001). Of the unstable cancers, 10 of 11 showed CIMP. The PMR values in the CIMP-positive proximal hyperplastic polyps were significantly lower than those of the unstable cancers for 4 of the 5 CIMP markers (P<0.05). BRAF V600E mutations were seen in 83% of proximal and 74% of distal hyperplastic polyps. Quantitative analysis of MLH1 methylation does not support earlier reports of MLH1 methylation in proximal hyperplastic polyps. However, these lesions do harbor promoter methylation at other CIMP loci, although at a lower level than that seen in unstable cancers. If these polyps are the precursor for sporadic microsatellite unstable cancers, then MLH1 methylation and higher degrees of promoter methylation in general occur at a later stage of carcinogenesis.

DNA repair enzymes function in major pathways to reverse DNA damage, including base excision repair (BER). Missense polymorphisms in BER repair genes may contribute to differences in DNA repair capacity, specific mutations, and susceptibility to cancer in the presence of exposure to carcinogens such as cigarette smoking. In a study of 1,604 incident colon cancer cases and 1,969 matched population-based controls genotyped for BER variants OGG1 (S326C) and XRCC1 (R194W, R280H, and R399Q), we found no associations with colon cancer overall. However, a 2-fold increased risk of BRAF V600E tumor mutation was observed in current and former cigarette smokers homozygous for the OGG1 polymorphism (odds ratio, 2.2; 95% confidence interval, 1.02-4.9, recessive model); similar associations were not observed for microsatellite instability, CpG island methylator phenotype, KRAS2 mutations, or TP53 mutations. The XRCC1 R194W polymorphism was associated with a modest increased risk of TP53 tumor mutations in those who regularly smoked cigarettes (odds ratio, 1.4; 95% confidence interval, 1.02-1.9). These findings point to the importance of studying tumor mutations when examining DNA repair polymorphisms and cigarette smoke exposure to identify potentially relevant associations with colorectal cancer.

The MSH6 G39E germline polymorphism is not associated with an increased risk of either microsatellite stable or unstable sporadic colorectal cancer. Other than microsatellite instability, however, most genetic and epigenetic changes of tumors associated with this common variant have not been studied. The objective of our investigation was to evaluate associations between the MSH6 G39E (116G>A) polymorphism and CpG island methylator phenotype (CIMP) and BRAF V600E mutations in tumors from a sample of 1048 individuals with colon cancer and 1964 controls from Utah, Northern California, and Minnesota. The G39E polymorphism (rs1042821) was determined by the five prime nuclease assay. CIMP was determined by methylation-specific polymerase chain reaction (PCR) of CpG islands in MLH1, methylated in tumors (MINT)1, MINT2, MINT31, and CDKN2A. The BRAF V600E mutation was determined by sequencing exon 15. In microsatellite stable tumors, homozygous carriers of the G39E polymorphism had an increased risk of CIMP+ colon cancer (odds ratio (OR) 2.2, 95% confidence interval (CI) 1.1, 4.2) and BRAF V600E mutation (OR 3.1, 95% CI 1.01, 9.7) in a case-control comparison. This finding was not observed in unstable tumors; however, power may have been low to detect an association. Age at diagnosis, family history, and alcohol use did not interact with MSH6 G39E and CIMP. The MSH6 G39E germline polymorphism may be associated with CIMP+ colon cancer.

Objective: High levels of microsatellite instability (MSI-H) have been associated in many studies with improved prognosis in colon cancer. Very few studies have evaluated the effect of MSI-H on rectal cancer survival. We assessed MSI-H and other genetic and epigenetic changes on survival of 990 individuals diagnosed with first primary rectal cancer., Methods: MSI was assessed primarily by instability in the mononucleotide repeat BAT-26. The BRAF V600E mutation was assessed by TaqMan assay. The CpG island methylator phenotype (CIMP) was determined by methylation-specific PCR of CpG islands in MLH1, methylated in tumors (MINT)1, (MINT)2, (MINT)31 and CDKN2A. KRAS2 codons 12 and 13 mutations, and TP53 mutations in exons 5-8 were determined by sequencing., Results: Multivariate analysis revealed that MSI-H (HRR 2.47, 95% CI 1.13-5.40) and KRAS2 mutations (HRR 1.37, 95% CI 1.04-1.81) were associated with a significantly higher risk of dying of rectal cancer. Only one of 22 MSI-H tumors showed a BRAF V600E mutation. Of 15 MSI-H rectal cancers evaluated for methylation, two exhibited MLH1 methylation and four exhibited CIMP., Conclusion: The genetic and epigenetic characteristics of MSI-H rectal cancers suggest that they are enriched for Lynch-associated tumors; adverse prognosis associated with MSI-H in these tumors may reflect the relatively high frequency of Lynch-associated cancers and/or the effect of radiation or chemotherapy on Lynch-associated rectal cancers or MSI tumors in general.

Cigarette smoking has been identified as a risk factor for rectal cancer. Our investigation evaluates associations between active and passive smoking and TP53, KRAS2, and BRAF V600E mutations, microsatellite instability (MSI), and CpG Island Methylator Phenotype (CIMP) in rectal tumors. We examine how genetic variants of GSTM1 and NAT2 alter these associations in a population-based, case-control study of 750 incident rectal cancer cases and 1,201 controls. Detailed tobacco exposure data were collected in an extensive questionnaire. DNA from blood was examined for GSTM1 and NAT2 variants. Tumor DNA was assessed to determine TP53 (exons 5-8), KRAS2 (codons 12-13) and BRAF mutations, MSI (BAT26 and TGFbetaRII analysis), and CIMP (methylation of CpG islands in CDKN2A, MLH1, MINT1, MINT2 and MINT31). Cigarette smoking (>20 pack-years, relative to nonsmokers) was associated with increased risk of TP53 mutations (OR = 1.4, 95% CI 1.02-2.0), BRAF mutations (OR = 4.2, 95% CI 1.3-14.2) and MSI (OR = 5.7, 95% CI 1.1-29.8) in rectal tumors. Long-term environmental tobacco smoke (ETS) exposure of >10 hr/wk was associated with increased risk of KRAS2 mutation (OR = 1.5, 95% CI 1.04-2.2). All smoking indicators were suggestive of increased risk in CIMP+ rectal cancer. GSTM1 and NAT2 were generally not associated with rectal tumor alterations; however, we observed an interaction of ETS and NAT2 in TP53-mutated tumors (p < 0.01). Our investigation shows active smoking is associated with increased risk of TP53, BRAF and MSI+ in rectal tumors and is suggestive of increased risk of CIMP+ tumors. ETS may increase risk of KRAS2 mutations; association with TP53 mutations and ETS may be influenced by NAT2.

Background: Germline mutations in DNA mismatch repair (MMR) genes cause Lynch syndrome colon cancers. Less understood is the risk of colon cancer associated with common polymorphisms in MMR genes and the potential interacting role of lifestyle factors known to damage DNA., Methods: A study was conducted to examine whether MLH1 (-93G>A and Ile219Val) and MSH6 (Gly39Glu) polymorphisms were associated with risk of colon cancer in data from 1609 colon cancer cases and 1972 controls. Genotype data were further stratified by microsatellite instability status, smoking, alcohol, Western diet, alcohol and obesity, to investigate potential heterogeneity., Results: The MSH6 39Glu allele was associated with increased risk of colon cancer among men (Gly/Glu or Glu/Glu vs Gly/Gly, OR 1.27; 95% CI 1.04 to 1.54). Neither MLH1 polymorphism was associated with colon cancer risk overall. When stratified by microsatellite stability status, however, the MLH1 -93A allele was associated with a more than doubling in microsatellite instability (MSI)-positive colon cancer risk (AA vs GG, OR 2.47; 95% CI 1.48 to 4.11); no associations were observed between the MMR polymorphisms examined and MSI-negative colon cancer. Statistically significant interactions were observed between: MLH1 -93G>A and smoking (MSI-negative colon cancer only, p value interaction: 0.005); and MLH1 Ile219Val and Western diet (p value interaction: 0.03)., Conclusions: The MSH6 Gly39Glu and MLH1 -93G>A polymorphisms were associated with risk of overall colon and MSI-positive colon cancers, respectively. Risk for colon cancer, stratified by MMR genotype, was further modified by smoking and Western diet.

Variation in genes associated with serum levels of proteins may be useful for examining specific disease pathways. Using data from a large study of colon cancer, we examine genetic variants in insulin, inflammation, estrogen, metabolizing enzymes, and energy homeostasis genes to explore associations with microsatellite instability (MSI), CpG Island methylator phenotype (CIMP), mutations of p53 in exons 5 through 8, and mutations in codons 12 and 13 of Ki-ras. Insulin-related genes were associated with CIMP-positive and MSI tumors, with the strongest associations among aspirin users. The Fok1 vitamin D receptor (VDR) polymorphism was associated with CIMP-positive/Ki-ras-mutated tumors; the Poly A and CDX2 VDR polymorphisms were associated only with Ki-ras-mutated tumors. NAT2 was associated with CIMP-positive/Ki-ras-mutated tumors but not with MSI tumors. The TCF7L2 rs7903146 polymorphism was associated with p53 mutated tumors. Most associations varied by recent aspirin/NSAID use: IL6 rs1800796 and rs1800795 polymorphisms were associated inversely with tumor mutations in the presence of aspirin/NSAIDs; POMC significantly reduced risk of Ki-ras-mutated tumors when aspirin/NSAIDs were not used; the TCF7L2 rs7903146 was associated with reduced risk of Ki-ras-mutated tumors in the presence of aspirin and increased risk in the absence of aspirin. These data, although exploratory, identify specific tumor subsets that may be associated with specific exposures/polymorphism combinations. The important modifying effects of aspirin/NSAIDs on associations with genetic polymorphisms reinforce the underlying role of inflammation in the etiology of colon cancer.

Our understanding of somatic alterations in colon cancer has evolved from a concept of a series of events taking place in a single sequence to a recognition of multiple pathways. An oncogenetic tree is a model intended to describe the pathways and sequence of somatic alterations in carcinogenesis without assuming that tumors will fall in mutually exclusive categories. We applied this model to data on colon tumor somatic alterations. An oncogenetic tree model was built using data on mutations of TP53, KRAS2, APC, and BRAF genes, methylation at CpG sites of MLH1 and TP16 genes, methylation in tumor (MINT) markers, and microsatellite instability (MSI) for 971 colon tumors from a population-based series. Oncogenetic tree analysis resulted in a reproducible tree with three branches. The model represents methylation of MINT markers as initiating a branch and predisposing to MSI, methylation of MHL1 and TP16, and BRAF mutation. APC mutation is the first alteration in an independent branch and is followed by TP53 mutation. KRAS2 mutation was placed a third independent branch, implying that it neither depends on, nor predisposes to, the other alterations. Individual tumors were observed to have alteration patterns representing every combination of one, two, or all three branches. The oncogenetic tree model assumptions are appropriate for the observed heterogeneity of colon tumors, and the model produces a useful visual schematic of the sequence of events in pathways of colon carcinogenesis.

The MLH1 -93 G>A promoter polymorphism has been reported to be associated with an increased risk of microsatellite unstable colorectal cancer. Other than microsatellite instability, however, the genetic and most epigenetic changes of tumors associated with this polymorphism have not been studied. We evaluated associations between the -93 G>A polymorphism and CpG island methylator phenotype (CIMP), BRAF V600E mutations, and MLH1 methylation in tumors from a sample of 1,211 individuals with colon cancer and 1,968 controls from Utah, Northern California, and Minnesota. The -93 G>A polymorphism was determined by the five prime nuclease assay. CIMP was determined previously by methylation-specific PCR of CpG islands in MLH1, methylated in tumors (MINT)1, MINT2, MINT31, and CDKN2A (p16). The BRAF V600E mutation was determined by sequencing exon 15. The MLH1 -93 G>A promoter polymorphism was associated with CIMP (odds ratio (OR) 3.44, 95% confidence interval (CI) 1.85, 6.42), MLH1 methylation (OR 4.16, 95%CI 2.20, 7.86), BRAF mutations (OR 4.26, 95%CI 1.83, 9.91), and older age at diagnosis (OR 3.65, 95%CI 2.08, 6.39) in microsatellite unstable tumors. These associations were not observed in stable tumors. Increased age at diagnosis and tumor characteristics of microsatellite unstable tumors associated with MLH1 -93 G>A suggests the polymorphism is acting at a relatively late stage of colorectal carcinogenesis to drive CIMP+ tumors down the microsatellite instability pathway.