Long noncoding RNA MIR31HG is a bona fide prognostic marker with colorectal cancer cell-intrinsic properties

Elevated miR‐31 expression is associated with poor outcome in colorectal cancer (CRC). Whether the prognostic information is independent of known molecular subgroups and gene expression‐based consensus molecular subtypes (CMS) is currently unknown. To investigate this, we analyzed nearly 2000 CRC biopsies and preclinical models. The expression of miR‐31‐5p and its host transcript, long noncoding RNA MIR31HG, was strongly correlated (Spearman's ρ > 0.80). MIR31HG outlier expression was observed in 158/1265 (12%) of pCRCs and was associated with depletion of CMS2‐canonical subgroup (odds ratio = 0.21 [0.11–0.35]) and shorter relapse‐free survival (RFS) in multivariable analysis (adjusted hazard ratio = 2.2 [1.6–3.0]). For stage II disease, 5‐year RFS for patients with MIR31HG outlier status was 49% compared to 77% for those with normal‐like expression. MIR31HG outlier status was associated with inferior outcome also within clinical high risk groups and within the poor prognostic CMS4‐mesenchymal gene expression subtype specifically. Preclinical models with MIR31HG outlier expression were characterized by reduced expression of MYC targets as well as elevated epithelial‐mesenchymal transition, TNF‐α/NFκB, TGF‐β, and IFN‐α/γ gene expression signatures, indicating cancer cell‐intrinsic properties resembling the CMS4 subgroup—associations which were recapitulated in patient biopsies. Moreover, the prognostic value of MIR31HG outlier status was independent of cytotoxic T lymphocyte and fibroblast infiltration. We here present evidence that MIR31HG expression provides clinical stratification beyond major gene expression phenotypes and tumor immune and stromal cell infiltration and propose a model where increased expression is an indicator of a cellular state conferring intrinsic invasive and/or immuno‐evasive capabilities

Alternative splicing expands the prognostic impact of KRAS in microsatellite stable primary colorectal cancer

KRAS mutation is a well‐known marker for poor response to targeted treatment and patient prognosis in microsatellite stable (MSS) colorectal cancer (CRC). However, variation in clinical outcomes among patients wild‐type for KRAS underlines that this is not a homogeneous population. Here, we evaluated the prognostic impact of KRAS alternative splicing in relation to mutation status in a single‐hospital series of primary MSS CRCs (N = 258). Using splicing‐sensitive microarrays and RNA sequencing, the relative expression of KRAS‐4A versus KRAS‐4B transcript variants was confirmed to be down‐regulated in CRC compared to normal colonic mucosa (N = 41; p ≤ 0.001). This was independent of mutation status, however, gene set enrichment analysis revealed that the effect of splicing on KRAS signaling was specific to the KRAS wild‐type subgroup, in which low relative KRAS‐4A expression was associated with a higher level of KRAS signaling (p = 0.005). In concordance, the prognostic value of KRAS splicing was also dependent on mutation status, and for patients with Stage I–III KRAS wild‐type MSS CRC, low relative KRAS‐4A expression was associated with inferior overall survival (HR: 2.36, 95% CI: 1.07–5.18, p = 0.033), a result not found in mutant cases (pinteraction = 0.026). The prognostic association in the wild‐type subgroup was independent of clinicopathological factors, including cancer stage in multivariable analysis (HR: 2.68, 95% CI: 1.18–6.09, p = 0.018). This suggests that KRAS has prognostic value beyond mutation status in MSS CRC, and highlights the importance of molecular heterogeneity in the clinically relevant KRAS wild‐type subgroup

Technical differences between sequencing and microarray platforms impact transcriptomic subtyping of colorectal cancer

Gene expression profiling has increasing relevance in the molecular screening of patients with colorectal cancer (CRC). We investigated potential platform-specific effects on transcriptomic subtyping according to established frameworks by comparisons of expression profiles from RNA sequencing and exon-resolution microarrays in 126 primary microsatellite stable CRCs. There was a strong platform correspondence in global gene expression levels, albeit with systematic technical bias likely attributed to few sequencing reads covering short (<2000 nucleotides) and/or lowly expressed genes (<1 FPKM), as well as over-saturation of highly expressed genes on microarrays. Classification concordances according to both the consensus molecular subtypes and CRC intrinsic subtypes (CRIS) were also strong, but with disproportionate subtype distributions between platforms caused by frequent disagreements in adherence to sample classification thresholds. Subtypes defined largely by genes expressed at low levels, including the CRIS-D subtype and the estimated level of tumor-infiltrating cytotoxic lymphocytes, had a weaker correspondence in classification metrics between platforms. In conclusion, even subtle differences between platforms suggest that clinical translation of transcriptomic CRC subtyping frameworks is dependent on assay standardization, and systematic technical biases reinforce the need for careful selection of classifier genes

CMS-dependent prognostic impact of KRAS and BRAFV600E mutations in primary colorectal cancer

Background: The prognostic impact of KRAS and BRAFV600E mutations in primary colorectal cancer (CRC) varies with microsatellite instability (MSI) status. The gene expression–based consensus molecular subtypes (CMSs) of CRC define molecularly and clinically distinct subgroups, and represent a novel stratification framework in biomarker analysis. We investigated the prognostic value of these mutations within the CMS groups. Patients and methods: Totally 1197 primary tumors from a Norwegian series of CRC stage I–IV were analyzed for MSI and mutation status in hotspots in KRAS (codons 12, 13 and 61) and BRAF (codon 600). A subset was analyzed for gene expression and confident CMS classification was obtained for 317 samples. This cohort was expanded with clinical and molecular data, including CMS classification, from 514 patients in the publically available dataset GSE39582. Gene expression signatures associated with KRAS and BRAFV600E mutations were used to evaluate differential impact of mutations on gene expression among the CMS groups. Results: BRAFV600E and KRAS mutations were both associated with inferior 5-year overall survival (OS) exclusively in MSS tumors (BRAFV600E mutation versus KRAS/BRAF wild-type: Hazard ratio (HR) 2.85, P < 0.001; KRAS mutation versus KRAS/BRAF wild-type: HR 1.30, P = 0.013). BRAFV600E-mutated MSS tumors were strongly enriched and associated with metastatic disease in CMS1, leading to negative prognostic impact in this subtype (OS: BRAFV600E mutation versus wild-type: HR 7.73, P = 0.001). In contrast, the poor prognosis of KRAS mutations was limited to MSS tumors with CMS2/CMS3 epithelial-like gene expression profiles (OS: KRAS mutation versus wild-type: HR 1.51, P = 0.011). The subtype-specific prognostic associations were substantiated by differential effects of BRAFV600E and KRAS mutations on gene expression signatures according to the MSI status and CMS group. Conclusions: BRAFV600E mutations are enriched and associated with metastatic disease in CMS1 MSS tumors, leading to poor prognosis in this subtype. KRAS mutations are associated with adverse outcome in epithelial (CMS2/CMS3) MSS tumors

Molecular correlates of sensitivity to PARP inhibition beyond homologous recombination deficiency in pre-clinical models of colorectal cancer point to wild-type TP53 activity

Background PARP inhibitors are active in various tumour types beyond BRCA-mutant cancers, but their activity and molecular correlates in colorectal cancer (CRC) are not well studied. Methods Mutations and genome-wide mutational patterns associated with homologous recombination deficiency (HRD) were investigated in 255 primary CRCs with whole-exome sequencing and/or DNA copy number data. Efficacy of five PARP inhibitors and their molecular correlates were evaluated in 93 CRC cell lines partly annotated with mutational-, DNA copy number-, and/or gene expression profiles. Post-treatment gene expression profiling and specific protein expression analyses were performed in two pairs of PARP inhibitor sensitive and resistant cell lines. Findings A subset of microsatellite stable (MSS) CRCs had truncating mutations in homologous recombination-related genes, but these were not associated with genomic signatures of HRD. Eight CRC cell lines (9%) were sensitive to PARP inhibition, but sensitivity was not predicted by HRD-related genomic and transcriptomic signatures. In contrast, drug sensitivity in MSS cell lines was strongly associated with TP53 wild-type status (odds ratio 15.7, p = 0.023) and TP53-related expression signatures. Increased downstream TP53 activity was among the primary response mechanisms, and TP53 inhibition antagonized the effect of PARP inhibitors. Wild-type TP53-mediated suppression of RAD51 was identified as a possible mechanism of action for sensitivity to PARP inhibition. Interpretation PARP inhibitors are active in a subset of CRC cell lines and preserved TP53 function may increase the likelihood of response

Exploratory analyses of consensus molecular subtype-dependent associations of TP53 mutations with immunomodulation and prognosis in colorectal cancer

Accumulating evidence suggests immunomodulatory and context-dependent effects of TP53 mutations in cancer. We performed an exploratory analysis of the transcriptional, immunobiological and prognostic associations of TP53 mutations within the gene expression-based consensus molecular subtypes (CMSs) of colorectal cancer (CRC).Materials and methodsIn a single-hospital series of 401 stage I–IV primary CRCs, we sequenced the whole coding region of TP53 and analysed CMS-dependent transcriptional consequences of the mutations by gene expression profiling. Immunomodulatory associations were validated by multiplex, fluorescence-based immunohistochemistry of immune cell markers. Prognostic associations of TP53 mutations were analysed in an aggregated series of 635 patients classified according to CMS, including publicly available data from a French multicentre cohort (GSE39582). mutations were found in 60% of the CRCs. However, gene set enrichment analyses indicated that their transcriptional consequences varied among the CMSs and were most pronounced in CMS1-immune and CMS4-mesenchymal. Subtype specificity was primarily seen as an upregulation of gene sets reflecting cell cycle progression in CMS4 and a downregulation of T cell activity in CMS1. The subtype-dependent immunomodulatory associations were reinforced by significant depletion of several immune cell populations in mutated tumours compared with wild-type (wt) tumours exclusively in CMS1, including cytotoxic lymphocytes (adjusted p value in CMS1=0.002 and CMS2−4&gt;0.9, Microenvironment Cell Populations (MCP)-counter algorithm). This was validated by immunohistochemistry-based quantification of tumour infiltrating CD8+ cells. Within CMS1, the immunomodulatory association of TP53 mutations was strongest among microsatellite stable (MSS) tumours, and this translated into a propensity for metastatic disease and poor prognostic value of the mutations specifically in the CMS1/MSS subtype (both series overall survival: TP53 mutation vs wt: HR 5.52, p=0.028).Integration of TP53 mutation status with the CMS framework in primary CRC suggested subtype-dependent immunobiological associations with prognostic and potentially immunotherapeutic implications, warranting independent validation

Transcriptional and functional consequences of TP53 splice mutations in colorectal cancer

TP53 mutations are common in colorectal cancer (CRC). Most TP53 sequencing studies have been restricted to coding regions, but recent studies have revealed that splice mutations can generate transcript variants with distinct tumorigenic and prognostic properties. Here, we performed unrestricted sequencing of all coding sequences and splice regions of TP53 in a single-hospital series of 401 primary CRCs. TP53 splice mutations were detected in 4% of the cases (N = 16), considerably more frequent than reported in major databases, and they were mutually exclusive to exon mutations. RNA sequencing revealed high-level expression of aberrant transcript variants in the majority of splice mutated tumors (75%). Most variants were predicted to produce truncated TP53 proteins, including one sample expressing the potentially oncogenic and druggable p53ψ isoform. Despite heterogeneous transcript structures, downstream transcriptional profiling revealed that TP53 splice mutations had similar effects on TP53 target gene expression and pathway activity as exonic mutations. Intriguingly, TP53 splice mutations were associated with worse 5-year relapse-free survival in stage II disease, compared to both TP53 wild-type and exon mutations (P = 0.007). These data highlight the importance of including splice regions when examining the biological and clinical consequences of TP53 mutations in CRC

Metastatic heterogeneity of the consensus molecular subtypes of colorectal cancer

Abstract Gene expression-based subtypes of colorectal cancer have clinical relevance, but the representativeness of primary tumors and the consensus molecular subtypes (CMS) for metastatic cancers is not well known. We investigated the metastatic heterogeneity of CMS. The best approach to subtype translation was delineated by comparisons of transcriptomic profiles from 317 primary tumors and 295 liver metastases, including multi-metastatic samples from 45 patients and 14 primary-metastasis sets. Associations were validated in an external data set ( n  = 618). Projection of metastases onto principal components of primary tumors showed that metastases were depleted of CMS1-immune/CMS3-metabolic signals, enriched for CMS4-mesenchymal/stromal signals, and heavily influenced by the microenvironment. The tailored CMS classifier (available in an updated version of the R package CMScaller) therefore implemented an approach to regress out the liver tissue background. The majority of classified metastases were either CMS2 or CMS4. Nonetheless, subtype switching and inter-metastatic CMS heterogeneity were frequent and increased with sampling intensity. Poor-prognostic value of CMS1/3 metastases was consistent in the context of intra-patient tumor heterogeneity

The expressed mutational landscape of microsatellite stable colorectal cancers

Background Colorectal cancer is the 2nd leading cause of cancer-related deaths with few patients benefiting from biomarker-guided therapy. Mutation expression is essential for accurate interpretation of mutations as biomarkers, but surprisingly, little has been done to analyze somatic cancer mutations on the expression level. We report a large-scale analysis of allele-specific mutation expression. Methods Whole-exome and total RNA sequencing was performed on 137 samples from 121 microsatellite stable colorectal cancers, including multiregional samples of primary and metastatic tumors from 4 patients. Data were integrated with allele-specific resolution. Results were validated in an independent set of 241 colon cancers. Therapeutic associations were explored by pharmacogenomic profiling of 15 cell lines or patient-derived organoids. Results The median proportion of expressed mutations per tumor was 34%. Cancer-critical mutations had the highest expression frequency (gene-wise mean of 58%), independent of frequent allelic imbalance. Systematic deviation from the general pattern of expression levels according to allelic frequencies was detected, including preferential expression of mutated alleles dependent on the mutation type and target gene. Translational relevance was suggested by correlations of KRAS/NRAS or TP53 mutation expression levels with downstream oncogenic signatures (p < 0.03), overall survival among patients with stage II and III cancer (KRAS/NRAS: hazard ratio 6.1, p = 0.0070), and targeted drug sensitivity. The latter was demonstrated for EGFR and MDM2 inhibition in pre-clinical models. Conclusions Only a subset of mutations in microsatellite stable colorectal cancers were expressed, and the “expressed mutation dose” may provide an opportunity for more fine-tuned biomarker interpretations

Epigenetic disruption of miR-130a promotes prostate cancer by targeting SEC23B and DEPDC1

MicroRNAs (miRNAs) are small, non-coding RNAs that mediate post-transcriptional gene silencing, fine tuning gene expression. In an initial screen, miRNAs were found to be globally down-regulated in prostate cancer (PCa) cell lines and primary tumors. Exposure of PCa cell lines to a demethylating agent, 5-Aza-CdR resulted in an increase in the expression levels of miRNAs in general. Using stringent filtering criteria miR-130a was identified as the most promising candidate and selected for validation analyses in our patient series. Down-regulation of miR-130a was associated with promoter hypermethylation. MiR-130a methylation levels discriminated PCa from non-malignant tissues (AUC=0.956), and urine samples revealed high specificity for non-invasive detection of patients with PCa (AUC=0.89). Additionally, repressive histone marks were also found in the promoter of miR-130a. Over-expression of miR-130a in PCa cells reduced cell viability and invasion capability, and increased apoptosis. Putative targets of miR-130a were assessed by microarray expression profiling and DEPD1C and SEC23B were selected for validation. Silencing of both genes resembled the effect of over-expressing miR-130a in PCa cells. Our data indicate that miR-130a is an epigenetically regulated miRNA involved in regulation of key molecular and phenotypic features of prostate carcinogenesis, acting as a tumor suppressor miRNA.info:eu-repo/semantics/publishedVersio