Investigating the Concordance in molecular subtypes of primary colorectal tumors and their matched synchronous liver metastasis

To date, no systematic analyses are available assessing concordance of molecular classifications between primary tumors (PT) and matched liver metastases (LM) of metastatic colorectal cancer (mCRC). We investigated concordance between PT and LM for four clinically relevant CRC gene signatures. Twenty-seven fresh and 55 formalin-fixed paraffin-embedded pairs of PT and synchronous LM of untreated mCRC patients were retrospectively collected and classified according to the MSI-like, BRAF-like, TGFB activated-like and the Consensus Molecular Subtypes (CMS) classification. We investigated classification concordance between PT and LM and association of TGFBa-like and CMS classification with overall survival. Fifty-one successfully profiled matched pairs were used for analyses. PT and matched LM were highly concordant in terms of BRAF-like and MSI-like signatures, (90.2% and 98% concordance, respectively). In contrast, 40% to 70% of PT that were classified as mesenchymal-like, based on the CMS and the TGFBa-like signature, respectively, lost this phenotype in their matched LM (60.8% and 76.5% concordance, respectively). This molecular switch was independent of the microenvironment composition. In addition, the significant change in subtypes was observed also by using methods developed to detect cancer cell-intrinsic subtypes. More importantly, the molecular switch did not influence the survival. PT classified as mesenchymal had worse survival as compared to nonmesenchymal PT (CMS4 vs CMS2, hazard ratio [HR] = 5.2, 95% CI = 1.5-18.5, P = .0048; TGFBa-like vs TGFBi-like, HR = 2.5, 95% CI = 1.1-5.6, P = .028). The same was not true for LM. Our study highlights that the origin of the tissue may have major consequences for precision medicine in mCRC

Neutrophil recruitment and barrier impairment in celiac disease:A genomic study

Background & Aims: Celiac disease is an enteropathy featuring villous atrophy, crypt hyperplasia, and lymphocytosis. Tissue remodeling is driven by an inflammatory reaction to gluten in genetically susceptible individuals. The adaptive pathway is considered the major immune response but recent evidence has indicated the involvement of innate immunity as well. To assess the contribution of either immune response we performed global gene expression profiling of the regenerating mucosa. Methods: microarray hybridizations were performed with biopsy samples from 13 untreated patients, 31 patients on a gluten-free diet in various stages of remission, and 21 controls. Additional data were generated using low-density array and conventional quantitative reverse-transcription polymerase chain reaction, and immunohistochemistry. Results: A total of 108 differentially expressed immune-related genes were identified (50 innate, 43 adaptive, 9 both innate/adaptive, and 6 immunoregulatory). Expression levels showed a gradual change as opposed to the discrete histological transitions. In addition to details provided on the adaptive and innate immune pathways used, we observed a chronic recruitment of activated neutrophils. Neutrophil involvement was unabated in otherwise completely normalized remission patients. Conclusions: We observed a contribution of both the innate and adaptive immune response in celiac disease pathogenesis. The discrepancy between the histological classification and the observed incremental change in immune-gene expression may have consequences for current diagnostic inclusion criteria. Enhanced neutrophil. infiltration in both active and remission patients points to a genetic impairment of the intestinal barrier that may contribute to the cause rather than the consequence of celiac disease

Oncogenic Role of miR-15a-3p in 13q Amplicon-Driven Colorectal Adenoma-to-Carcinoma Progression.

Progression from colorectal adenoma to carcinoma is strongly associated with an accumulation of genomic alterations, including gain of chromosome 13. This gain affects the whole q arm and is present in 40%-60% of all colorectal cancers (CRCs). Several genes located at this amplicon are known to be overexpressed in carcinomas due to copy number dosage. A subset of these genes, including the mir-17~92 cluster, are functionally involved in CRC development. The present study set out to explore whether apart from mir-17~92, other miRNAs located at the 13q amplicon show a copy number dependent dosage effect that may contribute to 13q-driven colorectal adenoma-to-carcinoma progression. Integration of publically available miRNA expression, target mRNA expression and DNA copy number data from 125 CRCs yielded three miRNAs, miR-15a, -17, and -20a, of which high expression levels were significantly correlated with a 13q gain and which influenced target mRNA expression. These results could be confirmed by qRT-PCR in a series of 100 colon adenomas and carcinomas.Functional analysis of both mature miRNAs encoded by mir-15a, i.e. miR-15a-5p and miR-15a-3p, showed that silencing of miR-15a-3p significantly inhibited viability of CRC cells. Integration of miR-15a expression levels with mRNA expression data of predicted target genes identified mitochondrial uncoupling protein 2 (UCP2) and COP9 signalosome subunit 2 (COPS2) as candidates with significantly decreased expression in CRCs with 13q gain. Upon silencing of miR-15a-3p, mRNA expression of both genes increased in CRC cells, supporting miR-15a-3p mediated regulation of UPC2 and COPS2 expression. In conclusion, significant overexpression of miR-15a-3p due to gain of 13q is functionally relevant in CRC, with UCP2 and COPS2 as candidate target genes. Taken together our findings suggest that miR-15a-3p may contribute to adenoma-to-carcinoma progression

Oncogenic Role of miR-15a-3p in 13q Amplicon-Driven Colorectal Adenoma-to-Carcinoma Progression.

Progression from colorectal adenoma to carcinoma is strongly associated with an accumulation of genomic alterations, including gain of chromosome 13. This gain affects the whole q arm and is present in 40%-60% of all colorectal cancers (CRCs). Several genes located at this amplicon are known to be overexpressed in carcinomas due to copy number dosage. A subset of these genes, including the mir-17~92 cluster, are functionally involved in CRC development. The present study set out to explore whether apart from mir-17~92, other miRNAs located at the 13q amplicon show a copy number dependent dosage effect that may contribute to 13q-driven colorectal adenoma-to-carcinoma progression. Integration of publically available miRNA expression, target mRNA expression and DNA copy number data from 125 CRCs yielded three miRNAs, miR-15a, -17, and -20a, of which high expression levels were significantly correlated with a 13q gain and which influenced target mRNA expression. These results could be confirmed by qRT-PCR in a series of 100 colon adenomas and carcinomas.Functional analysis of both mature miRNAs encoded by mir-15a, i.e. miR-15a-5p and miR-15a-3p, showed that silencing of miR-15a-3p significantly inhibited viability of CRC cells. Integration of miR-15a expression levels with mRNA expression data of predicted target genes identified mitochondrial uncoupling protein 2 (UCP2) and COP9 signalosome subunit 2 (COPS2) as candidates with significantly decreased expression in CRCs with 13q gain. Upon silencing of miR-15a-3p, mRNA expression of both genes increased in CRC cells, supporting miR-15a-3p mediated regulation of UPC2 and COPS2 expression. In conclusion, significant overexpression of miR-15a-3p due to gain of 13q is functionally relevant in CRC, with UCP2 and COPS2 as candidate target genes. Taken together our findings suggest that miR-15a-3p may contribute to adenoma-to-carcinoma progression

Schematic overview of in silico analysis pipeline used in this study.

<p>First level (association 1) shows a graphical representation of integration analysis of miRNA expression with copy number (CN) data. CN values for miRNAs on 13q were determined by combining values within a 2 Mb window surrounding the start of the miRNA. These values defined the covariate set used to determine association with miRNA expression. Second level (association 2) gives a graphical representation of the association of miRNA expression with predicted target mRNA expression. For each miRNA on 13q target mRNAs were determined by combining three or more target prediction tools. Expression levels of these target mRNAs defined the covariate set used to determine association with miRNA expression.</p

Association of miRNA expression with 13q copy number status and target mRNA expression.

<p>MiRNAs are ordered by significance of FDR of miRNA expression versus copy number. Chr, Chromosome; bp, base pair; exp, expression; n.d., no data; Significance considered at FDR<0.05.</p><p>Association of miRNA expression with 13q copy number status and target mRNA expression.</p

Analysis of mRNA expression levels of target genes in colorectal cancer cell line SW480.

<p>Relative expression of <i>UCP2</i> and <i>COPS2</i> in SW480 cells silenced for <i>miR-15a-5p</i>, <i>-3p</i> and <i>miR-17</i> compared to a non-targeting control (* p<0.05).</p

Functional analysis of the effect of <i>miR-15a-5p</i>, <i>-3p</i> and <i>miR-17</i> silencing in CRC cell line SW480.

<p>A) Representative example of expression levels of miRNAs upon silencing using antagomirs compared to a non-targeting control (**p<0.01; *p<0.05). B) Cell viability determined by MTT assay upon miRNA silencing in SW480 compared to non-targeting control (* p<0.05).</p

Analysis of miRNA expression in colorectal adenomas and carcinomas.

<p>A) Box plots comparing relative expression determined by qRT-PCR in adenomas (n = 48) versus carcinomas (n = 52) of four top candidate miRNAs, <i>miR-15a-3p</i>, <i>miR-15a-5p</i>, <i>miR-17</i> and <i>miR-20a</i>. B) Box plots comparing miRNA expression between tumours (adenomas and carcinomas) with (n = 24) or without (n = 73) 13q gain. P-values, determined by Mann-Whitney U-test, ≤ 0.05 are considered significant (* p ≤ 0.05; ** p < 0.01).</p