Differential roles of EPS8 in carcinogenesis:Loss of protein expression in a subset of colorectal carcinoma and adenoma

AIM:To analyze the epidermal growth factor receptor pathway substrate 8(EPS8) expression status and role in colorectal carcinogenesis given that EPS8 has a conserved actin barbed-end capping function that is required for proper maturation in intestinal cells.METHODS:We studied 8 colon cancer cell lines and 58 colorectal tumors(19 adenomas and 39 carcinomas).We performed expression microarray analysis of colon cancer cell lines followed by loss of heterozygosity(LOH)analysis and immunohistochemistry for EPS8 expression in colon tumors.Subsequently,we performed mutation analysis by direct sequencing and methylation analysis by bisulfite sequencing and methylation-specific polymerase chain reaction assays.RESULTS:Expression microarray analysis of colon cancer cell lines showed overexpression of EPS8 transcript in all lines but RKO.Genome wide loss of heterozygosity(LOH) analysis of colon tumors,showed considerable LOH at the EPS8 gene locus.Immunohistochemically,EPS8 was constitutively expressed in normal colonic mucosa with a dot-like supranuclear localization with accentuation at the luminal surface supporting its proposed role in epithelial maturation.Nineteen colon tumors(4 adenoma,15 carcinoma) out of 51(37%) showed strikingly tumor specific EPS8 protein loss.Of the remaining tumors,5/51(2 adenoma,and 3 carcinoma,10%) showed marked overexpression,while 27/51 tumors(53%) showed retained expression.Mutation analysis revealed a missense mutation(c.794C>T,p.R265C) in exon 8 in RKO.The EPS8 promoter was also methylated in RKO,but there was no significant methylation in other cell lines or carcinoma specimens.CONCLUSION:The loss of EPS8 expression in colorectal adenomas and carcinomas suggests that down regulation of this gene contributes to the development of a subset of colorectal cancers,a finding which could have applications in diagnosis and treatment.

Gene mutations in stool from gastric and colorectal neoplasia patients by next-generation sequencing

AIM To study cancer hotspot mutations by next-generation sequencing(NGS) in stool DNA from patients with different gastrointestinal tract(GIT) neoplasms. METHODS Stool samples were collected from 87 Finnish patients diagnosed with various gastric and colorectal neoplasms, including benign tumors, and from 14 healthy controls. DNA was isolated from stools by usingthe PSP~? Spin Stool DNA Plus Kit. For each sample, 20 ng of DNA was used to construct sequencing libraries using the Ion AmpliS eq Cancer Hotspot Panel v2 or Ion AmpliS eq Colon and Lung Cancer panel v2. Sequencing was performed on Ion PGM. Torrent Suite Software v.5.2.2 was used for variant calling and data analysis.RESULTS NGS was successful in assaying 72 GIT samples and 13 healthy controls, with success rates of the assay being78% for stomach neoplasia and 87% for colorectal tumors. In stool specimens from patients with gastric neoplasia, five hotspot mutations were found in APC,CDKN2 A and EGFR genes, in addition to seven novel mutations. From colorectal patients, 20 mutations were detected in AKT1, APC, ERBB2, FBXW7, KIT, KRAS,NRAS, SMARCB1, SMO, STK11 and TP53. Healthy controls did not exhibit any hotspot mutations, except for two novel ones. APC and TP53 were the most frequently mutated genes in colorectal neoplasms, with five mutations, followed by KRAS with two mutations.APC was the most commonly mutated gene in stools of patients with premalignant/benign GIT lesions.CONCLUSION Our results show that in addition to colorectal neoplasms,mutations can also be assayed from stool specimens of patients with gastric neoplasms.