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Next generation sequencing of FFPE human colon cancer samples for validation of ColoType consensus molecular subtype (CMS) assay

ABSTRACT: Purpose: To validate the novel ColoType assay for computing consensus molecular subtypes of colon cancer tumor samples Method: Whole-genome RNA-sequencing of colon cancer samples from formalin-fixed paraffin-embedded (FFPE) provided data for consensus molecular subtyping samples using multiple independent methods, including the novel ColoType method. A custom Illumina AmpliSeq library was also used to implement ColoType on these samples by targeted RNA-sequencing. Results: Multiple independent methods for computing CMS subtypes for FFPE colon cancer samples have widespread agreement with ColoType assay.

ORGANISM(S): Homo sapiens

PROVIDER: GSE152430 | GEO | 2020/07/27

REPOSITORIES: GEO

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Project description:Objective: In order to personalize standard therapies based on molecular profiles, we previously classified colorectal cancers (CRCs) into five distinct subtypes (CRCAssigner) and later into four consensus molecular subtypes (CMS) with different prognoses and treatment responses. For clinical application, here we developed an inexpensive multiplex biomarker-based assay.Objective: In order to personalise standard therapies based on molecular profiles, we previously classified colorectal cancers (CRCs) into five distinct subtypes (CRCAssigner) and later into four consensus molecular subtypes (CMS) with different prognoses and treatment responses. For clinical application, here we developed a low-cost multiplex biomarker assay. Design: Three cohorts of untreated fresh frozen CRC samples (n = 57) predominantly from primary tumours and profiled by microarray/RNA-Seq were analysed. A reduced 38-gene panel (CRCAssigner-38) was selected from the published 786-gene CRCAssigner signature (CRCAssigner-786) using an in-house gene selection approach. A customised NanoString Technologies nCounter platform-based assay (NanoCRCAssigner) was developed for comparison with different classifiers (CMS subtypes), platforms (microarrays and RNA-Seq), and gene sets (CRCAssigner-38 and CRCAssigner-786). An additional cohort of fresh-frozen paraffin embedded (FFPE) samples (n = 24) was subtyped using NanoCRCAssigner as a proof-of-concept. Results: NanoCRCAssigner classified fresh frozen samples (n = 48; except those showing a mixture of subtypes) into all five CRCAssigner subtypes with overall high concordance across platforms (>87%) and with CMS subtypes (81%) irrespective of variable tumour cellularity. The association of subtypes with their known molecular (microsatellite-instable and stemness), mutational (KRAS/BRAF), and clinical characteristics (including overall survival) further demonstrated assay validity. To reduce costs, we switched from the standard protocol to a low-cost protocol with a high Pearson correlation co-efficient (0.9) between protocols. Technical replicates were highly correlated (0.98). FFPE samples were also successfully classified and showed high reproducibility (0.96).

Project description:The Colorectal Cancer Subtyping Consortium established four Consensus Molecular Subtypes (CMS) in colorectal cancer: CMS1 (microsatellite-instability [MSI], Immune), CMS2 (Canonical, epithelial), CMS3 (Metabolic), and CMS4 (Mesenchymal). However, in the clinical practice only patients with MSI tumors have experienced a change in the management of their disease. Proteomics has experimented significant technical advances with the implementation of data-independent acquisition (DIA) as the gold-standard for the analysis of large cohort of patients, providing a dynamic framework of the disease and direct information about the effectors of the biological processes. The aim of this study is the characterization of the proteome of colon cancer CMS by DIA–based proteomics and expand the utility of CMS in the clinical practice. CMS were assigned and differences in the protein profiles between CMS were defined and evaluated using a Systems Biology approach. One hundred fifty-eight paraffin samples from colon cancer patients with stage II-III disease treated with adjuvant chemotherapy were analyzed. After applying quality criteria, 1,940 identified and quantified proteins were used for the analyses. CMS1 had an overexpression of proteins related to immune system, specifically neutrophils, phagocytosis and antimicrobial response, and a glycolytic profile. CMS3 had an overexpression of proteins related to metabolic pathways, while CMS2 showed an intermediate protein profile between CMS1-3, but with a characteristic profile regarding translation, adhesion, and mitochondria and metabolism biological processes. Two proteomics subtypes within CMS2 with different protein characteristics and prognosis were identified. CMS4 was the most different group with an overexpression of proteins related to angiogenesis, extracellular matrix, focal adhesion, and complement activation. In conclusion, DIA LC-MS/MS proteomics characterization of CRC CMS established new features for each CMS, such as a higher mitochondrial activity in CMS2 or an overexpression of proteins related to phagocytosis in CMS1, but also confirmed previous findings established using transcriptomics such as an overexpression of proteins related to angiogenesis in CMS4. In this CMS4 subtype, several adhesion-related proteins suggested a high metastatic profile and a possible chemoresistance that may explain the worse prognosis of this subtype. CMS1 was characterized as immune and glycolitic-active subtype, being the combination of immunotherapy and glycolytic inhibitors a possible therapeutic strategy. Additionally, two different CMS2 proteomics-based subtypes with different prognosis and possible therapeutic targets were identified. These proccesses could be used as therapeutic targets in the future.

Project description:Recent genome-scale ChIP-chip studies of transcription factors have shown that a low percentage of experimentally determined binding sites contain the consensus motif for the immunoprecipitated factor. In most cases, differences between in vivo target sites that contain or lack a consensus motif have not been explored. We have previously shown that most sites to which E2F family members are bound in vivo do not contain E2F consensus motifs. The main purpose of this study was to develop an understanding of how E2F binding specificity is achieved in vivo. In particular, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using promoter and ENCODE arrays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are a) the site must be in a core promoter and b) the promoter region must be utilized as a promoter by the transcriptional machinery in that particular cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including a) indirect recruitment, b) looping to the core promoter mediated by an E2F bound to a distal consensus motif, and c) assisted binding of E2F to a site that weakly resembles an E2F consensus motif within the core promoter. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated promoter fragments. Our findings suggest that in vivo a) the presence of a consensus motif is not sufficient to recruit E2Fs, b) E2Fs can bind to isolated regions that lack a consensus motif, and c) binding can require regions other than the best match to the E2F PWM in the core promoter. Keywords: E2F, ChIP-chip, transcription factor binding, consensus motifs 37 ChIP-chip arrays (of these, 14 array sets are biological duplicates). 22 samples are included in this series, the rest can be found in supplementary info to the following papers: Xu 2007, Jin 2006, Komashko 2008

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Next generation sequencing of FFPE human colon cancer samples for validation of ColoType consensus molecular subtype (CMS) assay

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