Transcriptomic subtyping of malignant peripheral nerve sheath tumours highlights immune signatures, genomic profiles, patient survival and therapeutic targets.

BACKGROUND: Malignant peripheral nerve sheath tumour (MPNST) is an aggressive orphan disease commonly affecting adolescents or young adults. Current knowledge of molecular tumour biology has been insufficient for development of rational treatment strategies. We aimed to discover molecular subtypes of potential clinical relevance. METHODS: Fresh frozen samples of MPNSTs (n = 94) and benign neurofibromas (n = 28) from 115 patients in a European multicentre study were analysed by DNA copy number and/or transcriptomic profiling. Unsupervised transcriptomic subtyping was performed and the subtypes characterized for genomic aberrations, clinicopathological associations and patient survival. FINDINGS: MPNSTs were classified into two transcriptomic subtypes defined primarily by immune signatures and proliferative processes. "Immune active" MPNSTs (44%) had sustained immune signals relative to neurofibromas, were more frequently low-grade (P = 0.01) and had favourable prognostic associations in a multivariable model of disease-specific survival with clinicopathological factors (hazard ratio 0.25, P = 0.003). "Immune deficient" MPNSTs were more aggressive and characterized by proliferative signatures, high genomic complexity, aberrant TP53 and PRC2 loss, as well as high relative expression of several potential actionable targets (EGFR, ERBB2, EZH2, KIF11, PLK1, RRM2). Integrated gene-wise analyses suggested a DNA copy number-basis for proliferative transcriptomic signatures in particular, and the tumour copy number burden further stratified the transcriptomic subtypes according to patient prognosis (P < 0.01). INTERPRETATION: Approximately half of MPNSTs belong to an "immune deficient" transcriptomic subtype associated with an aggressive disease course, PRC2 loss and expression of several potential therapeutic targets, providing a rationale for molecularly-guided intervention trials. FUNDING: Research grants from non-profit organizations, as stated in the Acknowledgements.

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.

De novo transcriptomic subtyping of colorectal cancer liver metastases in the context of tumor heterogeneity.

BACKGROUND: Gene expression-based subtyping has the potential to form a new paradigm for stratified treatment of colorectal cancer. However, current frameworks are based on the transcriptomic profiles of primary tumors, and metastatic heterogeneity is a challenge. Here we aimed to develop a de novo metastasis-oriented framework. METHODS: In total, 829 transcriptomic profiles from patients with colorectal cancer were analyzed, including primary tumors, liver metastases, and non-malignant liver samples. High-resolution microarray gene expression profiling was performed of 283 liver metastases from 171 patients treated by hepatic resection, including multiregional and/or multi-metastatic samples from each of 47 patients. A single randomly selected liver metastasis sample from each patient was used for unsupervised subtype discovery by nonnegative matrix factorization, and a random forest prediction model was trained to classify multi-metastatic samples, as well as liver metastases from two independent series of 308 additional patients. RESULTS: Initial comparisons with non-malignant liver samples and primary colorectal tumors showed a highly variable degree of influence from the liver microenvironment in metastases, which contributed to inter-metastatic transcriptomic heterogeneity, but did not define subtype distinctions. The de novo liver metastasis subtype (LMS) framework recapitulated the main distinction between epithelial-like and mesenchymal-like tumors, with a strong immune and stromal component only in the latter. We also identified biologically distinct epithelial-like subtypes originating from different progenitor cell types. LMS1 metastases had several transcriptomic features of cancer aggressiveness, including secretory progenitor cell origin, oncogenic addictions, and microsatellite instability in a microsatellite stable background, as well as frequent RAS/TP53 co-mutations. The poor-prognostic association of LMS1 metastases was independent of mutation status, clinicopathological variables, and current subtyping frameworks (consensus molecular subtypes and colorectal cancer intrinsic subtypes). LMS1 was also the least heterogeneous subtype in comparisons of multiple metastases per patient, and tumor heterogeneity did not confound the prognostic value of LMS1. CONCLUSIONS: We report the first large study of multi-metastatic gene expression profiling of colorectal cancer. The new metastasis-oriented subtyping framework showed potential for clinically relevant transcriptomic classification in the context of metastatic heterogeneity, and an LMS1 mini-classifier was constructed to facilitate prognostic stratification and further clinical testing.

Metastatic heterogeneity of the consensus molecular subtypes of colorectal cancer.

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.

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.

Patient-Derived Organoids from Multiple Colorectal Cancer Liver Metastases Reveal Moderate Intra-patient Pharmacotranscriptomic Heterogeneity.

PURPOSE: Molecular tumor heterogeneity may have important implications for the efficacy of targeted therapies in metastatic cancers. Inter-metastatic heterogeneity of sensitivity to anticancer agents has not been well explored in colorectal cancer. EXPERIMENTAL DESIGN: We established a platform for ex vivo pharmacogenomic profiling of patient-derived organoids (PDO) from resected colorectal cancer liver metastases. Drug sensitivity testing (n = 40 clinically relevant agents) and gene expression profiling were performed on 39 metastases from 22 patients. RESULTS: Three drug-response clusters were identified among the colorectal cancer metastases, based primarily on sensitivities to EGFR and/or MDM2 inhibition, and corresponding with RAS mutations and TP53 activity. Potentially effective therapies, including off-label use of drugs approved for other cancer types, could be nominated for eighteen patients (82%). Antimetabolites and targeted agents lacking a decisive genomic marker had stronger differential activity than most approved chemotherapies. We found limited intra-patient drug sensitivity heterogeneity between PDOs from multiple (2-5) liver metastases from each of ten patients. This was recapitulated at the gene expression level, with a highly proportional degree of transcriptomic and pharmacological variation. One PDO with a multi-drug resistance profile, including resistance to EGFR inhibition in a RAS-mutant background, showed sensitivity to MEK plus mTOR/AKT inhibition, corresponding with low-level PTEN expression. CONCLUSIONS: Intra-patient inter-metastatic pharmacological heterogeneity was not pronounced and ex vivo drug screening may identify novel treatment options for metastatic colorectal cancer. Variation in drug sensitivities was reflected at the transcriptomic level, suggesting potential to develop gene expression-based predictive signatures to guide experimental therapies.

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.

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

BACKGROUND: 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 METHODS: In 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). RESULTS: TP53 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>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). CONCLUSIONS: 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.

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.

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-ß, 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.

Inferior survival for patients with malignant peripheral nerve sheath tumors defined by aberrant TP53.

Malignant peripheral nerve sheath tumor is a rare and aggressive disease with poor treatment response, mainly affecting adolescents and young adults. Few molecular biomarkers are used in the management of this cancer type, and although TP53 is one of few recurrently mutated genes in malignant peripheral nerve sheath tumor, the mutation prevalence and the corresponding clinical value of the TP53 network remains unsettled. We present a multi-level molecular study focused on aberrations in the TP53 network in relation to patient outcome in a series of malignant peripheral nerve sheath tumors from 100 patients and 38 neurofibromas, including TP53 sequencing, high-resolution copy number analyses of TP53 and MDM2, and gene expression profiling. Point mutations in TP53 were accompanied by loss of heterozygosity, resulting in complete loss of protein function in 8.2% of the malignant peripheral nerve sheath tumors. Another 5.5% had MDM2 amplification. TP53 mutation and MDM2 amplification were mutually exclusive and patients with either type of aberration in their tumor had a worse prognosis, compared to those without (hazard ratio for 5-year disease-specific survival 3.5, 95% confidence interval 1.78-6.98). Both aberrations had similar consequences on the gene expression level, as analyzed by a TP53-associated gene signature, a property also shared with the copy number aberrations and/or loss of heterozygosity at the TP53 locus, suggesting a common "TP53-mutated phenotype" in as many as 60% of the tumors. This was a poor prognostic phenotype (hazard ratio = 4.1, confidence interval:1.7-9.8), thus revealing a TP53-non-aberrant patient subgroup with a favorable outcome. The frequency of the "TP53-mutated phenotype" warrants explorative studies of stratified treatment strategies in malignant peripheral nerve sheath tumor.

Colorectal Cancer Consensus Molecular Subtypes Translated to Preclinical Models Uncover Potentially Targetable Cancer Cell Dependencies.

Purpose: Response to standard oncologic treatment is limited in colorectal cancer. The gene expression-based consensus molecular subtypes (CMS) provide a new paradigm for stratified treatment and drug repurposing; however, drug discovery is currently limited by the lack of translation of CMS to preclinical models.Experimental Design: We analyzed CMS in primary colorectal cancers, cell lines, and patient-derived xenografts (PDX). For classification of preclinical models, we developed an optimized classifier enriched for cancer cell-intrinsic gene expression signals, and performed high-throughput in vitro drug screening (n = 459 drugs) to analyze subtype-specific drug sensitivities.Results: The distinct molecular and clinicopathologic characteristics of each CMS group were validated in a single-hospital series of 409 primary colorectal cancers. The new, cancer cell-adapted classifier was found to perform well in primary tumors, and applied to a panel of 148 cell lines and 32 PDXs, these colorectal cancer models were shown to recapitulate the biology of the CMS groups. Drug screening of 33 cell lines demonstrated subtype-dependent response profiles, confirming strong response to EGFR and HER2 inhibitors in the CMS2 epithelial/canonical group, and revealing strong sensitivity to HSP90 inhibitors in cells with the CMS1 microsatellite instability/immune and CMS4 mesenchymal phenotypes. This association was validated in vitro in additional CMS-predicted cell lines. Combination treatment with 5-fluorouracil and luminespib showed potential to alleviate chemoresistance in a CMS4 PDX model, an effect not seen in a chemosensitive CMS2 PDX model.Conclusions: We provide translation of CMS classification to preclinical models and uncover a potential for targeted treatment repurposing in the chemoresistant CMS4 group. Clin Cancer Res; 24(4); 794-806. ©2017 AACR.

Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies.

BACKGROUND: Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Comprehensive insights into their molecular characteristics may improve model selection for biomedical studies. METHODS: We have performed DNA, RNA and protein profiling of 34 cell lines, including (i) targeted deep sequencing (n = 612 genes) to detect single nucleotide variants and insertions/deletions; (ii) high resolution DNA copy number profiling; (iii) gene expression profiling at exon resolution; (iv) small RNA expression profiling by deep sequencing; and (v) protein expression analysis (n = 297 proteins) by reverse phase protein microarrays. RESULTS: The cell lines were stratified according to the key molecular subtypes of CRC and data were integrated at two or more levels by computational analyses. We confirm that the frequencies and patterns of DNA aberrations are associated with genomic instability phenotypes and that the cell lines recapitulate the genomic profiles of primary carcinomas. Intrinsic expression subgroups are distinct from genomic subtypes, but consistent at the gene-, microRNA- and protein-level and dominated by two distinct clusters; colon-like cell lines characterized by expression of gastro-intestinal differentiation markers and undifferentiated cell lines showing upregulation of epithelial-mesenchymal transition and TGFß signatures. This sample split was concordant with the gene expression-based consensus molecular subtypes of primary tumors. Approximately » of the genes had consistent regulation at the DNA copy number and gene expression level, while expression of gene-protein pairs in general was strongly correlated. Consistent high-level DNA copy number amplification and outlier gene- and protein- expression was found for several oncogenes in individual cell lines, including MYC and ERBB2. CONCLUSIONS: This study expands the view of CRC cell lines as accurate molecular models of primary carcinomas, and we present integrated multi-level molecular data of 34 widely used cell lines in easily accessible formats, providing a resource for preclinical studies in CRC.

Drug sensitivity and resistance testing identifies PLK1 inhibitors and gemcitabine as potent drugs for malignant peripheral nerve sheath tumors.

Patients with malignant peripheral nerve sheath tumor (MPNST), a rare soft tissue cancer associated with loss of the tumor suppressor neurofibromin (NF1), have poor prognosis and typically respond poorly to adjuvant therapy. We evaluated the effect of 299 clinical and investigational compounds on seven MPNST cell lines, two primary cultures of human Schwann cells, and five normal bone marrow aspirates, to identify potent drugs for MPNST treatment with few side effects. Top hits included Polo-like kinase 1 (PLK1) inhibitors (volasertib and BI2536) and the fluoronucleoside gemcitabine, which were validated in orthogonal assays measuring viability, cytotoxicity, and apoptosis. DNA copy number, gene expression, and protein expression were determined for the cell lines to assess pharmacogenomic relationships. MPNST cells were more sensitive to BI2536 and gemcitabine compared to a reference set of 94 cancer cell lines. PLK1, RRM1, and RRM2 mRNA levels were increased in MPNST compared to benign neurofibroma tissue, and the protein level of PLK1 was increased in the MPNST cell lines compared to normal Schwann cells, indicating an increased dependence on these drug targets in malignant cells. Furthermore, we observed an association between increased mRNA expression of PLK1, RRM1, and RRM2 in patient samples and worse disease outcome, suggesting a selective benefit from inhibition of these genes in the most aggressive tumors.

Multilevel genomics of colorectal cancers with microsatellite instability-clinical impact of JAK1 mutations and consensus molecular subtype 1.

BACKGROUND: Approximately 15% of primary colorectal cancers have DNA mismatch repair deficiency, causing a complex genome with thousands of small mutations-the microsatellite instability (MSI) phenotype. We investigated molecular heterogeneity and tumor immunogenicity in relation to clinical endpoints within this distinct subtype of colorectal cancers. METHODS: A total of 333 primary MSI+ colorectal tumors from multiple cohorts were analyzed by multilevel genomics and computational modeling-including mutation profiling, clonality modeling, and neoantigen prediction in a subset of the tumors, as well as gene expression profiling for consensus molecular subtypes (CMS) and immune cell infiltration. RESULTS: Novel, frequent frameshift mutations in four cancer-critical genes were identified by deep exome sequencing, including in CRTC1, BCL9, JAK1, and PTCH1. JAK1 loss-of-function mutations were validated with an overall frequency of 20% in Norwegian and British patients, and mutated tumors had up-regulation of transcriptional signatures associated with resistance to anti-PD-1 treatment. Clonality analyses revealed a high level of intra-tumor heterogeneity; however, this was not associated with disease progression. Among the MSI+ tumors, the total mutation load correlated with the number of predicted neoantigens (P = 4 × 10-5), but not with immune cell infiltration-this was dependent on the CMS class; MSI+ tumors in CMS1 were highly immunogenic compared to MSI+ tumors in CMS2-4. Both JAK1 mutations and CMS1 were favorable prognostic factors (hazard ratios 0.2 [0.05-0.9] and 0.4 [0.2-0.9], respectively, P = 0.03 and 0.02). CONCLUSIONS: Multilevel genomic analyses of MSI+ colorectal cancer revealed molecular heterogeneity with clinical relevance, including tumor immunogenicity and a favorable patient outcome associated with JAK1 mutations and the transcriptomic subgroup CMS1, emphasizing the potential for prognostic stratification of this clinically important subtype. See related research highlight by Samstein and Chan 10.1186/s13073-017-0438-9.

NEDD4 is overexpressed in colorectal cancer and promotes colonic cell growth independently of the PI3K/PTEN/AKT pathway.

Ubiquitination controls multiple cellular processes relevant to cancer pathogenesis. Using Gene Set Enrichment Analysis of an mRNA transcriptome dataset, we have identified genes encoding components of the ubiquitin system that are differentially expressed in colorectal cancers as compared to normal colonic mucosa. Among the significantly overexpressed genes was NEDD4 (neural precursor cell-expressed developmentally down-regulated 4), the prototype member of the HECT (homologous to E6AP C-terminus) E3 ubiquitin ligase family. Previous studies have shown that NEDD4 may act as an oncoprotein by inducing ubiquitination and degradation of the tumor suppressor protein PTEN (phosphatase and tensin homolog). To investigate its functional importance in colorectal cancer, HCT-15 and LoVo colon cancer cells were depleted of NEDD4 by small interfering RNA. The depletion resulted in reduced growth and altered cell morphology in both cell lines. However, NEDD4 depletion did not affect the PTEN protein level or PI3K/AKT signaling pathway activation. Moreover, ectopic expression of NEDD4 did not influence the PTEN subcellular localization or protein level. Collectively, these data demonstrate that NEDD4 is overexpressed in colorectal cancers, and suggest that NEDD4 promotes growth of colon cancer cells independently of PTEN and PI3K/AKT signaling.