Esocheck (Lucid Diagnostics) is a noninvasive, simple bedside/office based non-endoscopic detection device for Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). In a prior pilot study (PMID:29343623), a two marker methylated DNA marker panel detected BE/EAC in DNA extracted from samples obtained with the generation 1 (Gen 1) device with over 90% accuracy. However, 18% of subjects were unable to swallow the Gen 1 device and the DNA yield was insufficient for detecting BE/EAC in an additional 9%.

We report a biomarker-based non-endoscopic method for detecting Barrett’s esophagus (BE) based on detecting methylated DNAs retrieved via a swallowable balloon-based esophageal sampling device. BE is the precursor of, and a major recognized risk factor for, developing esophageal adenocarcinoma. Endoscopy, the current standard for BE detection, is not cost-effective for population screening. We performed genome-wide screening to ascertain regions targeted for recurrent aberrant cytosine methylation in BE, identifying high-frequency methylation within the CCNA1 locus. We tested CCNA1 DNA methylation as a BE biomarker in cytology brushings of the distal esophagus from 173 individuals with or without BE. CCNA1 DNA methylation demonstrated an area under the curve of 0.95 for discriminating BE-related metaplasia and neoplasia cases versus normal individuals, performing identically to methylation of VIM DNA, an established BE biomarker. When combined, the resulting two biomarker panel was 95% sensitive and 91% specific. These results were replicated in an independent validation cohort of 149 individuals who were assayed using the same cutoff values for test positivity established in the training population. To progress toward non-endoscopic esophageal screening, we engineered a well-tolerated, swallowable, encapsulated balloon device able to selectively sample the distal esophagus within 5 min. In balloon samples from 86 individuals, tests of CCNA1 plus VIM DNA methylation detected BE metaplasia with 90.3% sensitivity and 91.7% specificity. Combining the balloon sampling device with molecular assays of CCNA1 plus VIM DNA methylation enables an efficient, well-tolerated, sensitive, and specific method of screening at-risk populations for BE.

The molecular basis of aberrant protein glycosylation, a pathological alteration widespread in colorectal cancers (CRC) and the mechanisms by which it contributes to tumor progression remain largely unknown. We performed targeted re-sequencing of 430 glycosylation-associated genes in a series of patient-derived CRC cell lines (N = 31) and matched primary tumor tissues, identifying 12 new significantly mutated glycosylation-associated genes in colon cancer. In particular, we observed an enrichment of mutations in genes ( B3GNT2 , B4GALT 2, ST6GALNAC2 ) involved in the biosynthesis of N - and Cores 1–3 O -linked glycans in the colon, accounting for ~16% of the CRCs tested. Analysis of independent large-scale tumor tissue datasets confirmed recurrent mutations within these genes in colon and other gastrointestinal cancers. Systematic biochemical and phenotypic characterization of the candidate wild-type and mutant glycosyltransferases demonstrated these mutations as either markedly altering protein localization, post-translational modification, encoded enzymatic activities and/or the migratory potential of colon carcinoma cells. These findings suggest that functionally deleterious mutations in glycosyltransferase genes in part underlie aberrant glycosylation and contribute to the pathogenesis of molecular subsets of colon and other gastrointestinal malignancies.

We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors.

We are pleased to read the letter by Ashktorab et al. (1). We agree with Ashktorab et al. on the need for additional whole-exome sequencing of colon cancers arising in African Americans and on the value of continuing to add more cases to the numbers of African American colon cancers that have been characterized. The additional 11 African American colon cancers reported by Ashktorab et al. will be a helpful addition in this regard (2). We agree with Ashktorab et al. that the absence of mutations in EPH receptor A6 (EPHA6) and Folliculin (FLCN) from the 11 microsatellite-stable (MSS) samples they studied is not surprising, given that less than one mutation in either of these genes would be expected. In the interest of clarity, we point out that neither APC nor Kirsten rat sarcoma viral oncogene homolog (KRAS) were included in the list of new genes our study identified as significantly mutated in African American colon cancers (3). However, both our study (ref. 3, table S14) and studies by others (4) all detected a higher rate of KRAS mutations in colon cancers from African Americans compared with Caucasians. We also agree with Ashktorab et al. that WD repeat domain 87 (WDR87) is significantly mutated in colon cancers arising in African Americans, although our study found that WDR87 is also a target for mutations in colon cancers arising among Caucasians, and thus it is not in the top 15 genes whose mutations we found are highly associated with colon cancers arising in African Americans (3). We join Ashktorab et al. in hoping that the initial findings reported in PNAS will spur larger efforts in studies of ethnicity-associated differences in the mutational landscapes of colon and other cancers, as well as studies aimed at elucidating the underlying biological mechanisms by which these differences arise.

e14607 Background: Carcinoembryonic antigen (CEA) is a commonly used tumor marker in colorectal cancer (CRC) but has poor sensitivity (59%) and specificity (84%). Vimentin exon-1 sequences are hypermethylated in 53-83% of colon cancer compared to normal colorectal mucosae. We explored whether mVim could serve as a biomarker in patients with CRC. Methods: We performed a retrospective review of 47 patients (pts) treated for CRC at Case Comprehensive Cancer Center, who had banked plasma available for mVim and serum CEA within 30 days of mVim. Disease status, clinical outcomes and pathology information were obtained. Blood mVim levels were determined using next-generation sequencing and mVim level of ≥ 1% was considered positive. Results: mVim was obtained from 24 pre-operative CRC pts with locoregional (LR) disease and 23 CRC pts with active metastatic (Met) disease. We also measured mVim in 34 pts without CRC as a comparison group. In LR pts, mVim had a sensitivity and specificity of 21% and 97%, respectively. Sensitivity and specificity of CEA ≥ 2.5 was 38% and 87%, respectively and CEA ≥ 5 was 4% and 95%, respectively. Additionally, in these LR cases, a positive pre-op mVim had a positive predictive value (PPV) of 83% compared to CEA ≥ 2.5, 41% and CEA ≥ 5, 17%. Among Met pts, mVim had a sensitivity and specificity of 57% and 97%, respectively. Sensitivity and specificity of CEA ≥ 2.5 was 83% and 87%, respectively and CEA ≥ 5 was 74% and 95%, respectively. The PPV of a positive mVim in Met pts was 93% compared to CEA ≥ 2.5, 59% and CEA ≥ 5, 77%. Moreover, mVim (HR 5.07; p = 0.053, 95% CI 0.98-26.24) appeared to be a possible prognostic marker for survival in the Met group when compared to CEA (HR 1.00; p = 0.229, 95% CI 0.99-1.00). Conclusions: These preliminary data indicate that mVim has high specificity for detection of cancer in patients with LR or Met CRC. mVim appears to be more specific than CEA in both LR and Met CRC. In Met CRC, mVim may also have prognostic potential. Prospective studies are ongoing to validate these findings, including serial assessment of mVim and correlation with disease status. Given the high specificity of mVim, this biomarker should be explored in the surveillance setting as an indicator of early recurrence.

Reliable detection of somatic copy-number alterations (sCNAs) in tumors using whole-exome sequencing (WES) remains challenging owing to technical (inherent noise) and sample-associated variability in WES data. We present a novel computational framework, ENVE, which models inherent noise in any WES dataset, enabling robust detection of sCNAs across WES platforms. ENVE achieved high concordance with orthogonal sCNA assessments across two colorectal cancer (CRC) WES datasets, and consistently outperformed a best-in-class algorithm, Control-FREEC. We subsequently used ENVE to characterize global sCNA landscapes in African American CRCs, identifying genomic aberrations potentially associated with CRC pathogenesis in this population. ENVE is downloadable at https://github.com/ENVE-Tools/ENVE .

Aberrant glycosylation is a hallmark of many human cancers including colon cancer and has been proposed to affect cell growth, differentiation, adhesion, transformation and metastasis. However, the molecular basis for aberrant glycosylation remains largely unknown. We previously reported the finding of somatic and germline inactivating mutations in the gene encoding for GALNT12 transferase, a key enzyme involved in the initiating step of mucin type O-glycosylation, in individuals with colon cancer (PNAS 106(31):12921-25). This study provided the first evidence for the presence of genetic defects in the glycosylation pathway that contribute to the pathogenesis of colon cancer, and is consistent with the hypothesis that multiple rare germline gene variants may account for many of the cases of colon neoplasia that arise in the population. Given that aberrant glycosylation is a recurrent alteration in colon cancers, it is highly likely that additional genes (other than GALNT12) within the glycosylation pathway may be targeted for somatic mutations and contribute to colon tumor development. Accordingly, we performed massively parallel targeted re-sequencing of 430 glycosylation pathway genes in 31 microsatellite stable colon cancer cell lines. We identified a total of 41 somatic mutations in 36 unique genes. Using the statistical method detailed in our prior study, we found 12 of the genes to be significantly mutated above the background rate (P≤0.01, FDR Citation Format: Srividya Venkitachalam, Leslie Revoredo, Lakshmeswari Ravi, James Lutterbaugh, Sanford Markowitz, Thomas Gerken, Kishore Guda. Novel recurrent glycosylation-associated gene mutations in colon cancer. [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 1113. doi:10.1158/1538-7445.AM2015-1113

BACKGROUND & AIMS: Genetic and epigenetic alterations contribute to the pathogenesis of colorectal cancer (CRC). There is considerable molecular heterogeneity among colorectal tumors, which appears to arise as polyps progress to cancer. This heterogeneity results in different pathways to tumorigenesis. Although epigenetic and genetic alterations have been detected in conventional tubular adenomas, little is known about how these affect progression to CRC. We compared methylomes of normal colon mucosa, tubular adenomas, and colorectal cancers to determine how epigenetic alterations might contribute to cancer formation. METHODS: We conducted genome-wide array-based studies and comprehensive data analyses of aberrantly methylated loci in 41 normal colon tissue, 42 colon adenomas, and 64 cancers using HumanMethylation450 arrays. RESULTS: We found genome-wide alterations in DNA methylation in the nontumor colon mucosa and cancers. Three classes of cancers and 2 classes of adenomas were identified based on their DNA methylation patterns. The adenomas separated into classes of high-frequency methylation and low-frequency methylation. Within the high-frequency methylation adenoma class a subset of adenomas had mutant KRAS. Additionally, the high-frequency methylation adenoma class had DNA methylation signatures similar to those of cancers with low or intermediate levels of methylation, and the low-frequency methylation adenoma class had methylation signatures similar to that of nontumor colon tissue. The CpG sites that were differentially methylated in these signatures are located in intragenic and intergenic regions. CONCLUSIONS: Genome-wide alterations in DNA methylation occur during early stages of progression of tubular adenomas to cancer. These findings reveal heterogeneity in the pathogenesis of colorectal cancer, even at the adenoma step of the process.

impact metastatic potential which account for the low survival rate for this particular cancer.Aim: To quantitatively assess complex cancer cell phenotypes after gene inhibition by RNAi interference (RNAi), and assess the effect of these distinct morphologies on cell viability and cell motility using semi-automated image-based high-throughput screening.Method: GOhTRT cells were seeded and treated with the siRNA Human Druggable Genome Library (Dharmacon) by reverse transfection.Cells immunostained for DNA, tubulin and actin were imaged with the InCell Analyzer 1000 and processed using the InCell Analyzer software, CellHTS2 and RNAither.Statistical z-score analysis was performed on the combined A-T metric (F-actin area-α-tubulin area).The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay.Results:127 high confidence hits (Z-factor>2) was refined to six genes (RRM2, ITGB8, GPS1, SPRY1, NOL1, MYO9B) on the basis of distinct morphologies, reproducible metrics, and functional pathway analysis.In siRRM2 cells nuclear displacement (ND) and A-T area was 1.598±0.076;p <0.0001 and 285.70±35.48;p<0.05, respectively.siGPS1 cells had an ND and A-T area of 0.845±0.036;p =0.0369 and 266.201±25.629;p =0.0326, respectively.Silencing of GPS1, MYO9B and SPRY1 increased the rate of migration in a scratch wound assay, with 86.98%±3.097%,75.78%±5.454%and 72.97%±5.463%(p =0.0022) respectively.No significant difference in cell viability existed for siGPS1 (0.9037 ± 0.06575; p = 0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), suggesting that wound closure is by virtue of migratory signalling and not proliferation.Cell viability was decreased considerably in siRRM2 cells (0.2492±0.02798; p <0.0001) and siMYO9B (0.4048±0.04663; p <0.0001) in comparison to siNT cells (1.046±0.07712).Discussion:In summary, this approach successfully identified genes regulating oesophageal cancer cell cytoskeletal remodelling and metastasis using in-vitro assays, some of which are already associated with metastasis in literature and database searches.Further mechanistic studies and gene pathway analysis of candidate genes will provide novel therapeutic targets which can be utilised to block the spread of cancer in oesophageal adenocarcinoma patients.

The purpose of this study is to determine the genetic frequency of GNAS activating mutations in colorectal cancer and the corresponding pathology of GNAS mutant tumors. Oncogenic mutations in GNAS have been described in a number of neoplasms including those of the pituitary, kidney, pancreas, and, more recently, in colon cancer. To ascertain the frequency in colon cancer we employed a sensitive pyrosequencing platform for mutation detection of the R201C and R201H GNAS hotspots in tumor samples representing all clinical stages. We additionally assayed for KRAS and BRAF mutations as previous reports have shown that these often co-occur with activating GNAS mutations. Of the 428 colon tumors assayed, mutations in GNAS were present in 10 of the samples (2.3%), indicating this is a significant, albeit infrequent, mutation in colorectal tumors. Nine GNAS mutant tumors (90%) harbored concomitant activating mutations in either the KRAS or BRAF oncogene, which was significantly greater than the mutation frequency of these genes in the tumor population (56%, p<0.0305). All ten of the GNAS mutant tumors arose in the right (proximal) colon (p<0.007), and 7 of 8 reviewed cases exhibited a marked villous morphology. Taken together, these data indicate that GNAS mutant colon tumors commonly have synchronous mutations in KRAS or BRAF, are right-sided in location, and are associated with a villous morphology.

, 732 (2012); 336 Science et al. Da-Qiao Ding Homologous Chromosomes in Meiosis Meiosis-Specific Noncoding RNA Mediates Robust Pairing of This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): December 2, 2013 www.sciencemag.org (this information is current as of The following resources related to this article are available online at http://www.sciencemag.org/content/336/6082/732.full.html version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/content/suppl/2012/05/09/336.6082.732.DC1.html can be found at: Supporting Online Material http://www.sciencemag.org/content/336/6082/732.full.html#related found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/content/336/6082/732.full.html#ref-list-1 , 6 of which can be accessed free: cites 26 articles This article http://www.sciencemag.org/content/336/6082/732.full.html#related-urls 5 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/molec_biol Molecular Biology subject collections: This article appears in the following

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a metabolic antagonist of COX-2, catalyzing the degradation of inflammation mediator prostaglandin E2 (PGE2) and other prostanoids. Recent studies have established the 15-PGDH gene as a colon cancer suppressor.We evaluated 15-PDGH as a colon cancer susceptibility locus in a three-stage design. We first genotyped 102 single-nucleotide polymorphisms (SNPs) in the 15-PGDH gene, spanning ∼50 kb up and down-stream of the coding region, in 464 colon cancer cases and 393 population controls. We then genotyped the same SNPs, and also assayed the expression levels of 15-PGDH in colon tissues from 69 independent patients for whom colon tissue and paired germline DNA samples were available. In the final stage 3, we genotyped the 9 most promising SNPs from stages 1 and 2 in an independent sample of 525 cases and 816 controls (stage 3).In the first two stages, three SNPs (rs1365611, rs6844282 and rs2332897) were statistically significant (p<0.05) in combined analysis of association with risk of colon cancer and of association with 15-PGDH expression, after adjustment for multiple testing. For one additional SNP, rs2555639, the T allele showed increased cancer risk and decreased 15-PGDH expression, but just missed statistical significance (p-adjusted = 0.063). In stage 3, rs2555639 alone showed evidence of association with an odds ratio (TT compared to CC) of 1.50 (95% CI = 1.05-2.15, p = 0.026).Our data suggest that the rs2555639 T allele is associated with increased risk of colon cancer, and that carriers of this risk allele exhibit decreased expression of 15-PGDH in the colon.

NTRK3 is a member of the neurotrophin receptor family and regulates cell survival. It appears to be a dependence receptor, and thus has the potential to act as an oncogene or as a tumor suppressor gene. NTRK3 is a receptor for NT-3 and when bound to NT-3 it induces cell survival, but when NT-3 free, it induces apoptosis. We identified aberrantly methylated NTRK3 in colorectal cancers through a genome-wide screen for hypermethylated genes. This discovery led us to assess whether NTRK3 could be a tumor suppressor gene in the colon. NTRK3 is methylated in 60% of colon adenomas and 67% of colon adenocarcinomas. NTRK3 methylation suppresses NTRK3 expression. Reconstitution of NTRK3 induces apoptosis in colorectal cancers, if NT-3 is absent. Furthermore, the loss of NTRK3 expression associates with neoplastic transformation in vitro and in vivo. We also found that a naturally occurring mutant NTRK3 found in human colorectal cancer inhibits the tumor suppressor activity of NTRK3. In summary, our findings suggest NTRK3 is a conditional tumor suppressor gene that is commonly inactivated in colorectal cancer by both epigenetic and genetic mechanisms whose function in the pathogenesis of colorectal cancer depends on the expression status of its ligand, NT-3.

Cancer is characterized by gene expression aberrations. Studies have largely focused on coding sequences and promoters, even though distal regulatory elements play a central role in controlling transcription patterns. We used the histone mark H3K4me1 to analyze gain and loss of enhancer activity genome-wide in primary colon cancer lines relative to normal colon crypts. We identified thousands of variant enhancer loci (VELs) that comprise a signature that is robustly predictive of the in vivo colon cancer transcriptome. Furthermore, VELs are enriched in haplotype blocks containing colon cancer genetic risk variants, implicating these genomic regions in colon cancer pathogenesis. We propose that reproducible changes in the epigenome at enhancer elements drive a specific transcriptional program to promote colon carcinogenesis.

BACKGROUND:In addition to mutations, epigenetic silencing of genes has been recognized as a fundamental mechanism that promotes human carcinogenesis. To date, characterization of epigenetic gene silencing has largely focused on genes in which silencing is mediated by hypermethylation of promoter-associated CpG islands, associated with loss of the H3K4me3 chromatin mark. Far less is known about promoters lacking CpG-islands or genes that are repressed by alternative mechanisms.METHODS:We performed integrative ChIP-chip, DNase-seq, and global gene expression analyses in colon cancer cells and normal colon mucosa to characterize chromatin features of both CpG-rich and CpG-poor promoters of genes that undergo silencing in colon cancer.RESULTS:Epigenetically repressed genes in colon cancer separate into two classes based on retention or loss of H3K4me3 at transcription start sites. Quantitatively, of transcriptionally repressed genes that lose H3K4me3 in colon cancer (K4-dependent genes), a large fraction actually lacks CpG islands. Nonetheless, similar to CpG-island containing genes, cytosines located near the start sites of K4-dependent genes become DNA hypermethylated, and repressed K4-dependent genes can be reactivated with 5-azacytidine. Moreover, we also show that when the H3K4me3 mark is retained, silencing of CpG island-associated genes can proceed through an alternative mechanism in which repressive chromatin marks are recruited.CONCLUSIONS:H3K4me3 equally protects from DNA methylation at both CpG-island and non-CpG island start sites in colon cancer. Moreover, the results suggest that CpG-rich genes repressed by loss of H3K4me3 and DNA methylation represent special instances of a more general epigenetic mechanism of gene silencing, one in which gene silencing is mediated by loss of H3K4me3 and methylation of non-CpG island promoter-associated cytosines.