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Abstract Background Microsatellite instability (MSI) due to mismatch repair deficiency (dMMR) is common in colorectal cancer (CRC). These cancers are associated with somatic coding events, but the noncoding pathophysiological impact of this genomic instability is yet poorly understood. Here, we perform an analysis of coding and noncoding MSI events at the different steps of colorectal tumorigenesis using whole exome sequencing and search for associated splicing events via RNA sequencing at the bulk-tumor and single-cell levels. Results Our results demonstrate that MSI leads to hundreds of noncoding DNA mutations, notably at polypyrimidine U2AF RNA-binding sites which are endowed with cis-activity in splicing, while higher frequency of exon skipping events are observed in the mRNAs of MSI compared to non-MSI CRC. At the DNA level, these noncoding MSI mutations occur very early prior to cell transformation in the dMMR colonic crypt, accounting for only a fraction of the exon skipping in MSI CRC. At the RNA level, the aberrant exon skipping signature is likely to impair colonic cell differentiation in MSI CRC affecting the expression of alternative exons encoding protein isoforms governing cell fate, while also targeting constitutive exons, making dMMR cells immunogenic in early stage before the onset of coding mutations. This signature is characterized by its similarity to the oncogenic U2AF1-S34F splicing mutation observed in several other non-MSI cancer. Conclusions Overall, these findings provide evidence that a very early RNA splicing signature partly driven by MSI impairs cell differentiation and promotes MSI CRC initiation, far before coding mutations which accumulate later during MSI tumorigenesis.

Background & Aims: Recent studies have shown that cancers arise as a result of the positive selection of driver somatic events in tumor DNA, with negative selection playing only a minor role, if any. However, these investigations were concerned with alterations at nonrepetitive sequences and did not take into account mutations in repetitive sequences that have very high pathophysiological relevance in the tumors showing microsatellite instability (MSI) resulting from mismatch repair deficiency investigated in the present study. Methods: We performed whole-exome sequencing of 47 MSI colorectal cancers (CRCs) and confirmed results in an independent cohort of 53 MSI CRCs. We used a probabilistic model of mutational events within microsatellites, while adapting pre-existing models to analyze nonrepetitive DNA sequences. Negatively selected coding alterations in MSI CRCs were investigated for their functional and clinical impact in CRC cell lines and in a third cohort of 164 MSI CRC patients. Results: Both positive and negative selection of somatic mutations in DNA repeats was observed, leading us to identify the expected true driver genes associated with the MSI-driven tumorigenic process. Several coding negatively selected MSI-related mutational events (n = 5) were shown to have deleterious effects on tumor cells. In the tumors in which deleterious MSI mutations were observed despite the negative selection, they were associated with worse survival in MSI CRC patients (hazard ratio, 3; 95% CI, 1.1–7.9; P = .03), suggesting their anticancer impact should be offset by other as yet unknown oncogenic processes that contribute to a poor prognosis. Conclusions: The present results identify the positive and negative driver somatic mutations acting in MSI-driven tumorigenesis, suggesting that genomic instability in MSI CRC plays a dual role in achieving tumor cell transformation. Exome sequencing data have been deposited in the European genome–phenome archive (accession: EGAS00001002477). Keywords: Colorectal Cancer, Microsatellite Instability, Tumorigenic Process, Driver Gene Mutations, Positive and Negative Selection

BackgroundColon cancer (CC) pathological staging fails to accurately predict recurrence, and to date, no gene expression signature has proven reliable for prognosis stratification in clinical practice, perhaps because CC is a heterogeneous disease. The aim of this study was to establish a comprehensive molecular classification of CC based on mRNA expression profile analyses.Methods and findingsFresh-frozen primary tumor samples from a large multicenter cohort of 750 patients with stage I to IV CC who underwent surgery between 1987 and 2007 in seven centers were characterized for common DNA alterations, including BRAF, KRAS, and TP53 mutations, CpG island methylator phenotype, mismatch repair status, and chromosomal instability status, and were screened with whole genome and transcriptome arrays. 566 samples fulfilled RNA quality requirements. Unsupervised consensus hierarchical clustering applied to gene expression data from a discovery subset of 443 CC samples identified six molecular subtypes. These subtypes were associated with distinct clinicopathological characteristics, molecular alterations, specific enrichments of supervised gene expression signatures (stem cell phenotype-like, normal-like, serrated CC phenotype-like), and deregulated signaling pathways. Based on their main biological characteristics, we distinguished a deficient mismatch repair subtype, a KRAS mutant subtype, a cancer stem cell subtype, and three chromosomal instability subtypes, including one associated with down-regulated immune pathways, one with up-regulation of the Wnt pathway, and one displaying a normal-like gene expression profile. The classification was validated in the remaining 123 samples plus an independent set of 1,058 CC samples, including eight public datasets. Furthermore, prognosis was analyzed in the subset of stage II-III CC samples. The subtypes C4 and C6, but not the subtypes C1, C2, C3, and C5, were independently associated with shorter relapse-free survival, even after adjusting for age, sex, stage, and the emerging prognostic classifier Oncotype DX Colon Cancer Assay recurrence score (hazard ratio 1.5, 95% CI 1.1-2.1, p = 0.0097). However, a limitation of this study is that information on tumor grade and number of nodes examined was not available.ConclusionsWe describe the first, to our knowledge, robust transcriptome-based classification of CC that improves the current disease stratification based on clinicopathological variables and common DNA markers. The biological relevance of these subtypes is illustrated by significant differences in prognosis. This analysis provides possibilities for improving prognostic models and therapeutic strategies. In conclusion, we report a new classification of CC into six molecular subtypes that arise through distinct biological pathways.

Mismatch repair-deficient colorectal cancers (CRC) display widespread instability at DNA microsatellite sequences (MSI). Although MSI has been reported to commonly occur at coding repeats, leading to alterations in the function of a number of genes encoding cancer-related proteins, nothing is known about the putative impact of this process on non-coding microRNAs. In miRbase V15, we identified very few human microRNA genes with mono- or di-nucleotide repeats (n = 27). A mutational analysis of these sequences in a large series of MSI CRC cell lines and primary tumors underscored instability in 15 of the 24 microRNA genes successfully studied at variable frequencies ranging from 2.5% to 100%. Following a maximum likelihood statistical method, microRNA genes were separated into two groups that differed significantly in their mutation frequencies and in their tendency to represent mutations that may or may not be under selective pressures during MSI tumoral progression. The first group included 21 genes that displayed no or few mutations in CRC. The second group contained three genes, i.e., hsa-mir-1273c, hsa-mir-1303 and hsa-mir-567, with frequent (≥ 80%) and sometimes bi-allelic mutations in MSI tumors. For the only one expressed in colonic tissues, hsa-mir-1303, no direct link was found between the presence or not of mono- or bi-allelic alterations and the levels of mature miR expression in MSI cell lines, as determined by sequencing and quantitative PCR respectively. Overall, our results provide evidence that DNA repeats contained in human miRNA genes are relatively rare and preserved from mutations due to MSI in MMR-deficient cancer cells. Functional studies are now required to conclude whether mutated miRNAs, and especially the miR-1303, might have a role in MSI tumorigenesis.

BACKGROUND: Insulin-like growth factors (IGF-I and -II) are pleiotropic regulators of somatic growth and development in vertebrate species. Endocrine and paracrine effects of both hormones are mediated by a common IGF type 1 receptor (IGF-1R). Lethal respiratory failure in neonatal IGF-1R knockout mice suggested a particular role for this receptor in pulmonary development, and we therefore investigated the consequences of IGF-1R inactivation in lung tissue. METHODS AND FINDINGS: We first generated compound heterozygous mutant mice harboring a hypomorphic (Igf1r(neo)) and a null (Igf1r(-)) allele. These IGF-1R(neo/-) mice express only 22% of normal IGF-1R levels and are viable. In adult IGF-1R(neo/-) mice, we assessed lung morphology and respiratory physiology and found normal histomorphometric characteristics and normal breathing response to hypercapnia. We then generated homozygous IGF-1R knockout mutants (IGF-1R(-/-)) and analyzed their lung development during late gestation using histomorphometric and immunohistochemical methods. IGF-1R(-/-) embryos displayed severe lung hypoplasia and markedly underdeveloped diaphragms, leading to lethal neonatal respiratory distress. Importantly, IGF-1R(-/-) lungs from late gestation embryos were four times smaller than control lungs and showed markedly thickened intersaccular mesenchyme, indicating strongly delayed lung maturation. Cell proliferation and apoptosis were significantly increased in IGF-1R(-/-) lung tissue as compared with IGF-1R(+/+) controls. Immunohistochemistry using pro-SP-C, NKX2-1, CD31 and vWF as markers revealed a delay in cell differentiation and arrest in the canalicular stage of prenatal respiratory organ development in IGF-1R(-/-) mutant mice. CONCLUSIONS/SIGNIFICANCE: We found that low levels of IGF-1R were sufficient to ensure normal lung development in mice. In contrast, complete absence of IGF-1R significantly delayed end-gestational lung maturation. Results indicate that IGF-1R plays essential roles in cell proliferation and timing of cell differentiation during fetal lung development.

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3epsilon-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P

Alterations in the Wnt/APC (adenomatous polyposis coli) signalling pathway, resulting in β-catenin/T cell factor (Tcf)-dependent transcriptional gene activation, are frequently detected in familial and sporadic colon cancers. The neuropeptide neurotensin (NT) is widely distributed in the gastrointestinal tract. Its proliferative and survival effects are mediated by a G-protein coupled receptor, the NT1 receptor. NT1 receptor is not expressed in normal colon epithelial cells, but is over expressed in a number of cancer cells and tissues suggesting a link to the outgrowth of human colon cancer. Our results demonstrate that the upregulation of NT1 receptor occurring in colon cancer is the result of Wnt/APC signalling pathway activation. We first established the functionality of the Tcf response element within the NT1 receptor promoter. Consequently, we observed the activation of NT1 receptor gene by agents causing β-catenin cytosolic accumulation, as well as a strong decline of endogenous receptor when wt-APC was restored. At the cellular level, the re-establishment of wt-APC phenotype resulted in the impaired functionality of NT1 receptor, like the breakdown in NT-induced intracellular calcium mobilization and the loss of NT pro-invasive effect. We corroborated the Wnt/APC signalling pathway on the NT1 receptor promoter activation with human colon carcinogenesis, and showed that NT1 receptor gene activation was perfectly correlated with nuclear or cytoplasmic β-catenin localization while NT1 receptor was absent when β-catenin was localized at the cell–cell junction in early adenomas of patients with familial adenomatous polyposis, hereditary non-polyposis colorectal cancer and loss of heterozygosity tumours. In this report we establish a novel link in vitro between the Tcf/β-catenin pathway and NT1 receptor promoter activation. [ABSTRACT FROM AUTHOR]

Cyclooxygenase 2 (COX-2), also called prostaglandin endoperoxide synthase 2, is involved in colorectal tumor development. This review deals with particular questions raised in this field such as the mechanisms of COX-2 related tumor promotion, the role of the different types of cells (epithelial and interstitial) expressing COX-2, the factors that trigger COX-2 induction, and the clinical potential of selective COX-2 inhibitors to treat or prevent colorectal tumors. Several mechanisms of COX-2 related tumor promotion have been identified. Some are dependent on prostaglandin E 2 production (such as induction of cell proliferation, angiogenenis or local immunosuppression, inhibition of apoptosis, increase in cell motility) and others are not (such as carcinogen activation or malondialdehyde production). COX-2 expression has been demonstrated in epithelial cells of colorectal cancers and adenomas and also in interstitial cells. These cells correspond to macrophages and/or fibroblasts and endothelial cells. COX-2 expression in these interstitial cells participates in tumor development. Factors or events that trigger COX-2 expression include oncogene activation, antioncogene inactivation, cytokines, growth factors, some fatty acids, bile salts, and mucins. Finally, selective COX-2 inhibitors may be effective in preventing or treating colorectal adenomas or carcinomas. However, their real efficiency and the cost/benefit balance are currently evaluated, and no definite conclusion can be made at the moment. [ABSTRACT FROM AUTHOR]

BackgroundHuman chorionic gonadotrophin beta (hCGß) is expressed in several non-trophoblastic tumours, and this is usually associated with aggressive behaviour. Little is known about hCGß expression in Barrett’s adenocarcinoma.Materials and methodsWe determined the hCGß profile in a large series of surgically resected Barrett’s adenocarcinoma (a) at mRNA level using real-time quantitative reverse-transcription polymerase chain reaction analysis and (b) at protein level using immunohistochemistry with a polyclonal antibody and with a monoclonal antibody specific for free hCGß. We then sought links between the hCGß protein expression pattern and clinical and pathological parameters, including patient outcome as well as vascular endothelial growth factor (VEGF) expression.ResultshCGß protein expression was observed in 43 of 76 (57%) Barrett’s adenocarcinomas. We showed a strong correlation between hCGß protein abundance and CGB mRNA level. We observed a statistical link between hCGß protein expression and infiltrative tumour type ( P=0.023), perineural neoplastic invasion ( P=0.007) and VEGF protein expression ( P=0.016). hCGß expression tended to be associated with a poor outcome (16% versus 36% survival 8 years after resection).ConclusionExpression of hCGß correlates with specific infiltrative characteristics and is associated with higher VEGF expression. Both molecules may play a co-ordinated role in the development of Barrett’s adenocarcinomas. [ABSTRACT FROM AUTHOR]

AimsTo establish the prevalence of inflammatory secretory phospholipase A2 (sPLA2) and cytoplasmic phospholipase A2 (cPLA2) expression in a surgical series of Barrett?s adenocarcinoma and associated preneoplastic lesions and to correlate this expression with clinicopathological data and prognosis.MethodssPLA2 and cPLA2 were analysed by means of immunohistochemistry in surgical specimens of 67 and 73 cases of Barrett?s adenocarcinomas, respectively. Barrett?s mucosa was analysed in 31 cases.ResultsExpression of sPLA2 was detected in 48% of Barrett?s mucosa negative for intraepithelial neoplasia and 63% of Barrett?s adenocarcinoma. Semi-quantitative analysis revealed a significant increase in sPLA2 expression between Barrett?s mucosa negative for intraepithelial neoplasia and adenocarcinoma. cPLA2 expression was detected in 18% of Barrett?s adenocarcinoma. An inverse correlation was found between cPLA2 expression and depth of tumour infiltration, neoplastic vascular invasion and neoplastic perineural invasion. Survival analysis showed no significant prognostic value for sPLA2 and cPLA2.ConclusionsPLA2 is frequently expressed in Barrett?s oesophagus. The increasing expression of sPLA2 that we observed from Barrett?s mucosa to adenocarcinoma suggests that sPLA2 could be involved in Barrett?s carcinogenesis. In contrast, cPLA2 expression is less frequently observed in Barrett?s oesophagus and is inversely associated with aggressive pathological features of the tumours. [ABSTRACT FROM AUTHOR]

Defective DNA mismatch repair results from genetic or epigenetic alterations that most frequently inactivate the genes hMLH1 and hMSH2. This is thought to promote tumourigenesis by accumulation of mutations in oncogenes and tumour suppressor genes. This pathway, first reported in colon cancer, has been recently demonstrated in a subgroup of sporadic pancreatic adenocarcinomas. Intraductal papillary-mucinous neoplasms of the pancreas are a special type of pancreatic tumours, characterised by a spectrum of morphological changes from mild to moderate and to non-invasive, and they may associate with adenocarcinoma. An immunohistochemical study of hmlh1 and hmsh2 protein expression was performed on 26 intraductal papillary-mucinous neoplasms. All tumours showed nuclear expression of hmlh1 and hmsh2 proteins. There were two distinctive patterns of protein expression on the basis of the location of cells expressing these markers: the ?normal? pattern, observed mainly in adenoma and rarely in intraductal papillary-mucinous neoplasms with moderate dysplasia and the ?dysplastic? pattern, frequently encountered in moderate dysplasia neoplasms, non-invasive and invasive carcinomas. These findings suggest that defective DNA mismatch repair, due to inactivation of hMLH1 and hMSH2, does not play a significant role in the pathogenesis of intraductal papillary-mucinous neoplasms of the pancreas. Two patterns of protein expression were observed and were correlated with the progression of dysplasia in intraductal papillary mucinous neoplasms. [ABSTRACT FROM AUTHOR]

Background: Identifying determinants for histologically proven high-grade anal intraepithelial lesions (hHSIL) in HIV-positive MSM would allow better targeted screening. Methods: APACHES is a prospective study of anal HPV and related-lesions in 513 HIV-positive MSM aged ≥35 in six clinics across France. At baseline, participants underwent high resolution anoscopy (HRA) with biopsy of suspicious lesions, preceded by anal swabs for liquid-based cytology, p16/Ki67 immunostaining, and HPV DNA. hHSIL diagnosis was established by histopathological review panel consensus, and determinants assessed by logistic regression. Results: Baseline hHSIL prevalence was 10.4% and did not differ significantly by age, sexual behaviour or any HIV/immunodeficiency markers. hHSIL prevalence was significantly elevated in participants that smoked (ORadj=2.6, 95%CI 1.3–5.5) or who, in concurrent anal swabs, had ASCUS/LSIL (3.6, 95% CI 1.4–9.3) or ASC-H/HSIL (22.2, 95% CI 6.8–72.6) cytologic abnormalities, p16/Ki67 dual positivity (3.4, 95%CI 1.5–7.5), or non16-HR HPV (13.0, 95%CI 1.7–102), but most notably, HPV16 (46.3, 6.1–355) infection. Previous diagnosis of low- (2.3, 95%CI 1.0–5.4) or high- (3.8, 95%CI 1.5–9.9) grade anal lesion also conveyed higher hHSIL risk. After controlling for patient-specific determinants, there remained significant clinic-specific effects, especially in higher risk groups (HPV16-positive participants: 31.3% hHSIL in clinics A-D versus 5.1% in clinics E-F, p

Chromosomal instability (CIN) plays a crucial role in tumor development and occurs mainly as the consequence of either missegregation of normal chromosomes (MSG) or structural rearrangement (SR). However, little is known about the respective chromosomal targets of MSG and SR and the way these processes combined within tumors to generate CIN. To address these questions, we karyotyped a consecutive series of 96 near-diploid colorectal cancers (CRCs) and distinguished chromosomal changes generated by either MSG or SR in tumor cells. Eighty-three tumors (86%) presented with chromosomal abnormalities that contained both MSGs and SRs to varying degrees whereas all 13 others (14%) showed normal karyotype. Using a maximum likelihood statistical method, chromosomes affected by MSG or SR and likely to represent changes that are selected for during tumor progression were found to be different and mostly mutually exclusive. MSGs and SRs were not randomly associated within tumors, delineating two major pathways of chromosome alterations that consisted of either chromosome gains by MSG or chromosomal losses by both MSG and SR. CRCs showing microsatellite instability (MSI) presented with either normal karyotype or chromosome gains whereas MSS (microsatellite stable) CRCs exhibited a combination of the two pathways. Taken together, these data provide new insights into the respective involvement of MSG and SR in near-diploid colorectal cancers, showing how these processes target distinct portions of the genome and result in specific patterns of chromosomal changes according to MSI status.

Purpose: The MOSAIC (Multicenter International Study of Oxaliplatin/Fluorouracil/Leucovorin in the Adjuvant Treatment of Colon Cancer) study has demonstrated 3-year disease-free survival (DFS) and 6-year overall survival (OS) benefit of adjuvant oxaliplatin in stage II to III resected colon cancer. This update presents 10-year OS and OS and DFS by mismatch repair (MMR) status and BRAF mutation., Methods: Survival actualization after 10-year follow-up was performed in 2,246 patients with resected stage II to III colon cancer. We assessed MMR status and BRAF mutation in 1,008 formalin-fixed paraffin-embedded specimens., Results: After a median follow-up of 9.5 years, 10-year OS rates in the bolus/infusional fluorouracil plus leucovorin (LV5FU2) and LV5FU2 plus oxaliplatin (FOLFOX4) arms were 67.1% versus 71.7% (hazard ratio [HR], 0.85; P = .043) in the whole population, 79.5% versus 78.4% for stage II (HR, 1.00; P = .980), and 59.0% versus 67.1% for stage III (HR, 0.80; P = .016) disease. Ninety-five patients (9.4%) had MMR-deficient (dMMR) tumors, and 94 (10.4%) had BRAF mutation. BRAF mutation was not prognostic for OS (P = .965), but dMMR was an independent prognostic factor (HR, 2.02; 95% CI, 1.15 to 3.55; P = .014). HRs for DFS and OS benefit in the FOLFOX4 arm were 0.48 (95% CI, 0.20 to 1.12) and 0.41 (95% CI, 0.16 to 1.07), respectively, in patients with stage II to III dMMR and 0.50 (95% CI, 0.25 to 1.00) and 0.66 (95% CI, 0.31 to 1.42), respectively, in those with BRAF mutation., Conclusion: The OS benefit of oxaliplatin-based adjuvant chemotherapy, increasing over time and with the disease severity, was confirmed at 10 years in patients with stage II to III colon cancer. These updated results support the use of FOLFOX in patients with stage III disease, including those with dMMR or BRAF mutation., (© 2015 by American Society of Clinical Oncology.)