Consensus Molecular Subtypes Classification Research Articles

TP53 mutation status and consensus molecular subtypes of colorectal cancer in patients from Rwanda

BackgroundMutations in the TP53 tumor suppressor gene are well-established drivers of colorectal cancer (CRC) development. However, data on the prevalence of TP53 variants and their association with consensus molecular subtype (CMS) classification in patients with CRC from Rwanda are currently lacking. This study addressed this knowledge gap by investigating TP53 mutation status concerning CMS classification in a CRC cohort from Rwanda.MethodsFormalin-fixed paraffin-embedded (FFPE) tissue blocks were obtained from 51 patients with CRC at the University Teaching Hospital of Kigali, Rwanda. Exons 4 to 11 and their flanking intron-exon boundaries in the TP53 gene were sequenced using Sanger sequencing to identify potential variants. The recently established immunohistochemistry-based classifier was employed to determine the CMS of each tumor.ResultsSequencing analysis of cancerous tissue DNA revealed TP53 pathogenic variants in 23 of 51 (45.1%) patients from Rwanda. These variants were predominantly missense types (18/23, 78.3%). The most frequent were c.455dup (p.P153Afs*28), c.524G > A (p.R175H), and c.733G > A (p.G245S), each identified in three tumors. Trinucleotide sequence context analysis of the 23 mutations (20 of which were single-base substitutions) revealed a predominance of the [C > N] pattern among single-base substitutions (SBSs) (18/20; 90.0%), with C[C > T]G being the most frequent mutation (5/18, 27.8%). Furthermore, pyrimidine bases (C and T) were preferentially found at the 5ʹ flanking position of the mutated cytosine (13/18; 72.2%). Analysis of CMS subtypes revealed the following distribution: CMS1 (microsatellite instability-immune) (6/51, 11.8%), CMS2 (canonical) (28/51, 54.9%), CMS3 (metabolic) (9/51, 17.6%), and CMS4 (mesenchymal) (8/51, 15.7%). Interestingly, the majority of TP53 variants were in the CMS2 subgroup (14/23; 60.1%).ConclusionOur findings indicate a high frequency of TP53 variants in CRC patients from Rwanda. Importantly, these variants are enriched in the CMS2 subtype. This study, representing the second investigation into molecular alterations in patients with CRC from Rwanda and the first to explore TP53 mutations and CMS classification, provides valuable insights into the molecular landscape of CRC in this understudied population.

Molecular profiling of risk factors for relapse in Japanese patients with stage II colorectal cancer: a retrospective cohort study.

The association between the molecular profiles and prognosis of Stage II colorectal cancer remains unclear. This study aimed to examine the risk factors for relapse of Stage II colorectal cancer using molecular profiling. We retrospectively enrolled patients with pStage II colorectal cancer who did not receive perioperative adjuvant therapy and whose surgically resected specimens were evaluated using gene expression and whole-exome analyses between January 2014 and December 2018. We evaluated the long-term outcomes and examined the risk factors for relapse-free survival. We evaluated 322 patients with pStage II colorectal cancer, including 126 (39.1%) with right colon cancer. Eighty-seven patients (27.0%) had pT4 tumor, 175 (54.3%) had positive venous invasion, 120 (37.3%) had positive lymphatic invasion, and 68 (21.1%) had perineural invasion. The presence of mutations in key genes for colorectal cancer development based on whole-exome analyses was as follows: APC, 245 (76.1%); TP53, 208 (64.6%); and KRAS, 134 (41.6%). According to the consensus molecular subtype classification based on gene expression, 76 patients (23.6%) had consensus molecular subtype 4 and a significantly lower relapse-free survival than the other patients (5-year relapse-free survival: 83.8% vs. 92.9%, p = 0.017). Perineural invasion (hazard ratio: 5.316, p < 0.001) and consensus molecular subtype 4 (hazard ratio: 2.399, p = 0.020) were identified as independent risk factors for relapse-free survival. Molecular profiling of Stage II colorectal cancer to assess the risk factors for relapse may contribute to the indication and drug selection for adjuvant chemotherapy.

Correction: A consensus molecular subtypes classification strategy for clinical colorectal cancer tissues

Correction: A consensus molecular subtypes classification strategy for clinical colorectal cancer tissues

Overexpression of TSPAN8 in consensus molecular subtype 3 colorectal cancer

BackgroundRecently, consensus molecular subtypes (CMSs) have been proposed as a robust transcriptome-based classification system for colorectal cancer (CRC). Tetraspanins (TSPANs) are transmembrane proteins. They have been associated with the development of numerous malignancies, including CRC, through their role as “master organizers” for multi-molecular membrane complexes. No previous study has investigated the correlation between TSPANs and CMS classification. Herein, we investigated the expression of TSPANs in patient-derived primary CRC tissues and their CMS classifications. MethodsRNA samples were derived from primary CRC tissues (n = 100 patients diagnosed with colorectal adenocarcinoma) and subjected to RNA sequencing for transcriptome-based CMS classification and TSPAN-relevant analyses. Immunohistochemistry (IHC) and immunofluorescence (IF) stains were conducted to observe the protein expression level. To evaluate the relative biological pathways, gene-set enrichment analysis was performed. ResultsOf the highly expressed TSPAN genes in CRC tissues (TSPAN8, TSPAN29, and TSPAN30), TSPAN8 was notably overexpressed in CMS3-classified primary tissues. The overexpression of TSPAN8 protein in CMS3 CRC was also observed by IHC and IF staining. As a result of gene-set enrichment analysis, TSPAN8 may potentially play a role in organizing signaling complexes for kinase-based metabolic deregulation in CMS3 CRC. ConclusionsThe present study reports the overexpression of TSPAN8 in CMS3 CRC. This study proposes TSPAN8 as a subtype-specific biomarker for CMS3 CRC. This finding provides a foundation for future CMS-based studies of CRC, a complex disease and the second leading cause of cancer mortality worldwide.

A consensus molecular subtypes classification strategy for clinical colorectal cancer tissues.

Consensus Molecular Subtype (CMS) classification of colorectal cancer (CRC) tissues is complicated by RNA degradation upon formalin-fixed paraffin-embedded (FFPE) preservation. Here, we present an FFPE-curated CMS classifier. The CMSFFPE classifier was developed using genes with a high transcript integrity in FFPE-derived RNA. We evaluated the classification accuracy in two FFPE-RNA datasets with matched fresh-frozen (FF) RNA data, and an FF-derived RNA set. An FFPE-RNA application cohort of metastatic CRC patients was established, partly treated with anti-EGFR therapy. Key characteristics per CMS were assessed. Cross-referenced with matched benchmark FF CMS calls, the CMSFFPE classifier strongly improved classification accuracy in two FFPE datasets compared with the original CMSClassifier (63.6% versus 40.9% and 83.3% versus 66.7%, respectively). We recovered CMS-specific recurrence-free survival patterns (CMS4 versus CMS2: hazard ratio 1.75, 95% CI 1.24-2.46). Key molecular and clinical associations of the CMSs were confirmed. In particular, we demonstrated the predictive value of CMS2 and CMS3 for anti-EGFR therapy response (CMS2&3: odds ratio 5.48, 95% CI 1.10-27.27). The CMSFFPE classifier is an optimized FFPE-curated research tool for CMS classification of clinical CRC samples.

Microenvironment, systemic inflammatory response and tumor markers considering consensus molecular subtypes of colorectal cancer.

Introduction: Colorectal carcinomas (CRC) are one of the most frequent malignancies worldwide. Based on gene expression profile analysis, CRCs can be classified into four distinct subtypes also known as the consensus molecular subtypes (CMS), which predict biological behaviour. Besides CMS, several other aspects of tumor microenvironment (TME) and systemic inflammatory response (SIR) influence the outcome of CRC patients. TME and inflammation have important role in the immune (CMS1) and mesenchymal (CMS4) subtypes, however, the relationship between these and systemic inflammation has not been assessed yet. Our objective was to evaluate the connection between CMS, TME and SIR, and to analyze the correlation between these markers and routinely used tumor markers, such as CEA (Carcinoembryonic Antigen) and CA19-9 (Carbohydrate Antigen 19-9). Methods: FFPE (Formalin Fixed Paraffin Embedded) samples of 185 CRC patients were collected. TME was described using tumor-stroma ratio (TSR), Klintrup-Makinen (KM) grade, and Glasgow Microenvironment Score (GMS). CMS classification was performed on tissue microarray using MLH1, PMS2, MSH2 and MSH6, and pan-cytokeratin, CDX2, FRMD6, HTR2B and ZEB1 immunohistochemical stains. Pre-operative tumor marker levels and inflammatory markers [C-reactive protein - CRP, albumin, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute platelet count (APC)] and patient history were retrieved using MedSolution database. Results: Amongst TME-markers, TSR correlated most consistently with adverse clinicopathological features (p < 0.001) and overall survival (p < 0.001). Elevated CRP and modified Glasgow Prognostic Score (mGPS) were associated with worse outcome and aggressive phenotype, similarly to tumor markers CEA and CA19-9. Stroma-Tumor Marker score (STM score), a new combined score of CA19-9 and TSR delivered the second best prognostication after mGPS. Furthermore, CMS4 showed association with TSR and several laboratory markers (albumin and platelet derived factors), but not with other SIR descriptors. CMS did not show any association with CEA and CA19-9 tumor markers. Conclusion: More routinely available TME, SIR and tumor markers alone and in combination deliver reliable prognostic data for choosing the patients with higher risk for propagation. CMS4 is linked with high TSR and poor prognosis, but in overall, CMS-classification showed only limited effect on SIR- and tumor-markers.

Abstract 5059: Robust single sample consensus molecular subtype classification for primary and metastatic colorectal cancer

Abstract Introduction Consensus Molecular Subtypes (CMS) represent a well-established molecular stratification framework for colorectal cancer (CRC). Existing methods for CMS classification rely on a representative input cohort as a preprocessing step (CMScaller) or have difficulty generalizing to metastatic samples (CMSclassifier). We developed Tempus CMS to overcome these limitations. Our method normalizes gene expression data from input samples to a static reference cohort to enable single sample classification of both primary and metastatic tumors. We evaluated the performance of this classifier on a large, de-identified CRC cohort comprising 8,489 samples from primary and metastatic sites. Methods To normalize input data, our method shifts and scales each gene expression value based on the mean and standard deviation of the expression of that gene in the reference cohort (n=2,787 primary and metastatic CRC). CMS calls are then generated via nearest template prediction similar to CMScaller (Eide et al., 2017). The analysis cohort was selected from clinical biopsies within 30 days of primary or metastatic diagnosis excluding reference cohort samples. CMS calls were assessed by comparing subtype prevalence to reported rates and by testing for known enrichments of pathways and genomic markers. Results Subtype prevalences for lower gastrointestinal (GI) biopsies and resections (n=5,090) were comparable to reported rates for primary CRC tumors: CMS1 - 12%, CMS2 - 28%, CMS3 - 19%, and CMS4 - 33% with 7.6% no calls. Biopsies from metastatic tissue showed site-specific changes in prevalence: CMS2 increased to 43% in liver samples (n=1,715) and CMS4 was identified in 49% of samples from metastatic sites excluding the liver (n=1,684). In addition, we found that purity had an effect on prevalence: in lower GI tumors with ≥ 50% tumor purity rates of CMS2 and CMS4 were 34% and 26%, respectively, while rates in tumors with purity between 20% and 50% were 20% CMS2 and 41% CMS4. Across all tissue groups, pathway enrichment showed significant associations, including TGF-ꞵ signaling in CMS4 (adj P ≤ 4.4e-15) and cell differentiation in CMS3 (adj P ≤ 3.0e-27). Expected enrichments of CMS markers were also observed: BRAF mutations and microsatellite instability in CMS1 and KRAS mutations in CMS3. In liver tissue, higher KRAS mutation rates were noted in CMS1 (57%) and CMS3 (83%) compared to GI tissue (35% and 67%, respectively). Conclusions In this study, we developed an enhanced, single sample CMS-calling algorithm optimized for primary and metastatic sites. Application of this algorithm in a large cohort recapitulated known biology, which suggests that the tool can be used to support clinical studies requiring robust molecular stratification. Citation Format: Andrew J. Sedgewick, Tushar Chandra, Timothy Taxter, Justin Guinney. Robust single sample consensus molecular subtype classification for primary and metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5059.

Molecular Subtypes and Outcomes in a Multi-Institutional Review of Rectal Cancer Patients

Colorectal cancer (CRC) is a heterogeneous malignancy associated with a variety of genetic mutations. More recent data indicates that CRC can be broken down into unique biologically distinct consensus molecular subtypes (CMS) based on different pathological and genetic signatures. The purpose of this study is to evaluate the outcomes of rectal cancer patients in a multi-institutional network based on tumor mutational assessment and CMS group. Patient exome and transcriptome sequencing data and clinical outcomes were collected under the Total Cancer Care Protocol and Avatar® project within the Oncology Research Information Exchange Network (ORIEN). A total of 101 patients with demographic and outcomes information had data for microsatellite instability (MSI), tumor mutation burden (TMB), transcriptome, and whole exome sequencing (WES). Molecular subclasses (CMS1, CMS2, CMS3, CMS4, CMS-Mixed) were assigned based on transcriptional signatures by the R package "CMS Caller." Survival analysis was performed with the R packages "Survival" and "Survminer." A total of 101 rectal cancer patients, with a median age of 56.8, had a median follow up of 3.5 years (range 0.26-23.5). 78 patients were treated with curative intent for clinically localized disease and 35% of these patients developed metastatic disease. The remaining 23 patients had synchronous metastatic disease at presentation. There were 5 (5%) CMS1, 29 (29%) CMS2, 13 (13%) CMS 3, 49 (49%) CMS 4, and 5 (5%) CMS-Mixed patients in our cohort respectively. The cohort included 5 (5%) BRAF, 51 (50%) KRAS, and 63 (62%) TP53 mutated patients and 5 (5%) MSI high patients. Median survival was 18.8, 117.2, 125.7 and 119 months for CMS1, CMS2, CMS3, and CMS4 patients respectively, with insufficient events in CMS-mixed for calculation (p = 0.15). CMS1 patients had a significantly shorter survival compared to the other cohorts (p = 0.02), with 2 of 5 of these patients having received immunotherapy. 40% (2,0) CMS1, 52% (7,8) of CMS2, 15% (1,1) of CMS3, 59% (13,15) of CMS4, and 40% (1,1) of CMS-Mixed presented with or developed metastatic disease respectively. When divided into mutation groups, median survival was 43 versus 119, 119 versus 117, and 126 versus 119 months for BRAF, KRAS, and TP53 mutated and wild type patients respectively (p = 0.18, p = 0.48, p = 0.93). Evaluation of TMB and MSI status did not reveal significant differences in outcomes (p = 0.54, p = 0.7), with median survival of 126 months versus 117 in TMB high versus low patients and unreached versus 119 months in MSI versus MSS patients respectively. Of note, 3 of the 5 MSI patients were also CMS1, with the other two coming from CMS4 and CMS-mixed cohorts. CMS classification and tumor mutation status are associated with differential outcomes for rectal cancer patients, with some groups having a large likelihood of developing metastatic disease. Further work on optimizing and personalizing treatments for these high-risk populations is necessary.

Abstract PR013: Transcriptomic profiling of liquid biopsy in colorectal cancer

Abstract Objective: Consensus molecular subtype (CMS) is a predictive factor for treatment outcomes of chemotherapies for metastatic colorectal cancer (CRC). CMS classification is based on transcriptomic profiles of CRC specimens obtained by tumor biopsy. Tumor biopsy often provides limited information due to the heterogeneity within tumor and spatial heterogeneity between the primary tumor and distant metastases. In addition, repeated tissue biopsy to monitor the treatment response is not practical. Liquid biopsy is a minimally invasive method for the real-time monitoring of cancer-derived biomarkers. Among liquid biopsy biomarkers, extracellular vesicles (EVs) have a unique potential because they possess nucleic acids. We explored the use of CRC plasma EVs in predicting the molecular subtype of CRCs using RNA-seq. Methods: EVs were isolated from 10 different CMS-stratified colorectal cancer cell lines and a normal control. Whole transcriptome RNA-seq on cellular RNA (cRNA) and cell-derived evRNA was done to determine if EVs could be used to predict the CMS subtype of their cells of origin. We then sought to perform molecular subtyping of plasma EVs from patients with CRC. RNA-seq was performed on tumor tissues and matched plasma EVs from 46 patients with CRC and 59 healthy controls (age/gender matched). The bulk transcriptome from CRC plasma EVs was deconvoluted to predict the cancer percentage (CIBERSORTx) and to utilize cancer-specific transcriptome for CMS subtyping (DeMixT). Artificial neural network (ANN)-based CMS subtype classifier was used to classify molecular subtypes. Results: There was 100% concordance between CMS subtype of evRNA with that of the cRNA. We showed that, RNA mixtures containing as low as 1% cancer cell RNA could be accurately classified into the correct CMS class. Imputed proportions of cancer in plasma of CRC patients ranged from 0.89% to 2.08%. Receiver operating characteristic (ROC) curve showed area under the curve of 0.961 with specificity and sensitivity of 0.96 and 0.9, respectively. Plasma evRNA was classified into CMS classes and there was 67% (31/46) concordance between the predicted subtype of liquid biopsies and the tumor samples. In patients with tumor purity greater than 10%, the concordance was higher at 75% (27/36). Conclusions: EVs could be used to accurately predict CMS subtypes of their cells of origin. We created a pipeline using low-input RNA library preparation from plasma EV to estimate the cancer RNA portion present in the bulk transcriptome and predict the molecular subtypes of colorectal cancers. Molecular subtyping of evRNA may help to track CMS changes of the tumor in patients undergoing treatment. Citation Format: Vahid Bahrambeigi, Jaewon J. Lee, Kimal I. Rajapakshe, Bret M. Stephens, Jason T. Henry, Sarah Dhebat, Mark W. Hurd, Ryan Sun, Scott Kopetz, Anirban Maitra, Paola A. Guerrero. Transcriptomic profiling of liquid biopsy in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR013.

Abstract 5743: Comprehensive next generation sequencing profiling in combination with transcriptomic-based tumor molecular subtyping and harmonized TMB calculation using paired specimens from late-stage CRC patients

Abstract Background: Colorectal Cancer (CRC) can be classified into transcriptomics subtypes such as, stromal or immunogenic. A previously demonstrated consensus molecular subtype (CMS) classification method was utilized on CRC samples. We explored the potential clinical utility of combining a transcriptome derived subtypes approach with WES by demonstrating the association among subtypes and tumor characteristics such as whole exome based microsatellite instability (MSI), tumor mutational burden (TMB) and karyotype. Methods: Tumor tissue and paired blood samples were collected from 19 late-stage, treatment-naïve colorectal cancer (CRC) patients. gDNA and RNA were extracted and analyzed by the Personalis® ImmunoID NeXT Platform®. RNA-seq results were normalized with the R DESeq2 package, with CRC CMS classification performed by the R package CMScaller. Somatic variants, copy number variants and MSI were evaluated using paired tumor/normal (T/N) samples. Copy number (CN) was characterized both genome-wide as a ploidy estimate and focally as the number of amplified regions. TMB was computed in alignment with the Friends of Cancer Research phase I guidelines. Results: The most commonly mutated genes included TP53 (19/19), APC (16/19), and KRAS (9/19). CMScaller identified molecular subtypes (CMS1 immune/MSI n=1; CMS2 canonical/epithelial n=6; CMS3 metabolic/epithelial n=2; CMS4 mesenchyme/stromal n=8; unspecified n=2) in 17/19 samples. CMS2 and CMS4, the two most common molecular subtypes in this cohort, differed in terms of exome-wide MSI percent (p=0.03, Student’s t) and purity (p&amp;lt;0.007, Student’s t). TMB, in silico ploidy estimates, and focal copy number changes were assessed for associations with tumor purity, with a significant association only between purity and ploidy (p=0.014; Bonferroni q=0.42; Student’s t) at the highest and lowest purity quartiles. Exome-wide percent unstable MSI was significantly associated with tumor purity (p=0.0053, lowest vs. highest quartile purity), with high purity tumors possessing more MSI. No association between TMB and Union for International Cancer Control stage was identified. Conclusion: It has been demonstrated that the ImmunoID NeXT Platform’s transcriptomics capabilities enable CMS classification in most samples (17/19, 89%), an overall performance superior or comparable to previously reported results. An association between tumor purity and WES MSI using a broader exome-wide measurement was identified with transcriptomic based molecular subtyping. It has been corroborated that future comprehensive molecular classifiers can expand on transcriptomics based classification by leveraging DNA-based measurements to further delineate subtypes and eventually lead to biomarker driven precision oncology focused patient selection. Citation Format: Danyi Wang, Lee McDaniel, Sean Boyle, Juergen Scheuenpflug, Zheng Feng. Comprehensive next generation sequencing profiling in combination with transcriptomic-based tumor molecular subtyping and harmonized TMB calculation using paired specimens from late-stage CRC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5743.

Postoperative circulating tumor DNA combined with consensus molecular subtypes can better predict outcomes in stage III colon cancers: A prospective cohort study

BackgroundPrecise methods for risk stratification to guide adjuvant chemotherapy for stage III colon cancers are needed. Here, we combined circulating tumor DNA (ctDNA) with consensus molecular subtype (CMS) to improve risk stratification in stage III colon cancers. MethodsWe conducted a prospective, observational cohort study of 165 patients with stage III colon cancers. Somatic variants in tumor tissues and plasmas collected pre- and post-chemo were detected via a targeted sequencing panel of 197 cancer-related genes. CMSs classification was characterized using a targeted RNA sequencing panel of 788 genes. ResultsWe analyzed 151 pre-chemo and 124 post-chemo plasmas, while 130 patients were CMSs classified. ctDNA was detectable in 15.9% pre-chemo and 8.9% post-chemo samples. Significantly worse recurrence-free survival (RFS) was seen if ctDNA was detectable in pre-chemo samples (hazard ratio [HR], 3.585; P < 0.001) or in post-chemo samples (HR, 3.337; P = 0.005). Pre-chemo ctDNA (HR, 5.538; P < 0.001) and post-chemo ctDNA status (HR, 3.272; P = 0.037) remained independently associated with RFS in multivariate analysis. According to the redefined recurrence risk stratification, mid-risk patients (ctDNA-negative with CMS4/T4 or N2 tumors) were 5.3 times (HR, 5.269; P = 0.025) more likely to relapse than low-risk patients (ctDNA-negative with CMS1-3/T3N1 tumors), while high-risk patients (ctDNA-positive) were 14.6 times (HR, 14.590; P < 0.001) more likely to relapse. ConclusionsPostoperative ctDNA indicating residual disease, combined with CMSs classification and clinical risk reflecting the intrinsic characteristics of tumors, can redefine risk stratification of stage III colon cancers and better predict relapse.

The autocrine loop of ALK receptor and ALKAL2 ligand is an actionable target in consensus molecular subtype 1 colon cancer

BackgroundIn the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs).MethodsIn this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested cell lines, patient-derived organoids and mice with potent ALK inhibitors, already approved for clinical use.ResultsALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D tumor spheroids. Consistently, ALK appeares constitutively phosphorylated in CMS1, and it signals mainly through the AKT axis. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Consequently, disruption of ALK axis mediates the pro-apoptotic action of CMS1 cell lines, both in 2D and 3D and enhanced cell-cell adhesion and e-cadherin organization. In agreement with all these findings, the ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, as a proof of concept, the efficacy of ALK inhibition was demonstrated in both patient-derived organoids and in tumor xenografts in vivo.ConclusionsCollectively, these findings suggest that ALK targeting may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.

Consensus molecular subtypes of colorectal cancer in clinical practice: A translational approach.

The identification of several genetic mutations in colorectal cancer (CRC) has allowed a better comprehension of the prognosis and response to different antineoplastic treatments. Recently, through a systematic process, consensus molecular subtypes (CMS) have been described to characterize genetic and molecular mutations in CRC patients. Through CMS, CRC patients can be categorized into four molecular subtypes of CRC by wide transcriptional genome analysis. CMS1 has microsatellite instability and mutations in CIMP and BRAF pathways. CMS2, distinguished by mutations in specific pathways linked to cellular metabolism, also has a better prognosis. CMS3 has a KRAS mutation as a hallmark. CMS4 presents mutations in fibrogenesis pathways and mesenchymal-epithelial transition, associated with a worse prognosis. CMS classification can be a meaningful step in providing possible answers to important issues in CRC, such as the use of adjuvant chemotherapy in stage II, personalized first-line chemotherapy for metastasic CRC, and possible new target treatments that address specific pathways in each molecular subtype. Understanding CMS is a crucial step in personalized medicine, although prospective clinical trials selecting patients by CMS are required to pass proof-of-concept before becoming a routine clinical tool in oncology routine care.

Intratumor morphologic and transcriptomic heterogeneity in V600EBRAF-mutated metastatic colorectal adenocarcinomas

BackgroundIntratumor heterogeneity (ITH) is described as the presence of various clones within one tumor, each with their own unique features in terms of morphology, inflammation, genetics or transcriptomics. Heterogeneity provides the fuel for drug resistance; therefore, an accurate assessment of tumor heterogeneity is essential for the development of effective therapies. The purpose of this study was to dissect morphologic and molecular ITH in colorectal adenocarcinoma.Materials and methodsA series of 120 V600EBRAF-mutated (V600EBRAFmt) consecutive metastatic colorectal adenocarcinomas was assessed for morphologic heterogeneity. The two heterogeneous components of each specimen underwent a histopathological, immunohistochemical and molecular characterization to evaluate: histologic variant, grading, tumor-infiltrating lymphocytes (TILs), mismatch repair proteins' expression, KRAS/BRAF/NRAS mutations, microsatellite instability (MSI) status and consensus molecular subtype (CMS).ResultsThirty-one out of 120 (25.8%) V600EBRAFmt primary colorectal adenocarcinomas presented a heterogeneous morphology. Among these, eight cases had adequate material for molecular profiling. Five out of the eight (62.5%) cases resulted instable at MSI testing. The majority (62.5%) of the samples showed a CMS4 phenotype based on gene expression profiling. Heterogeneity in CMS classification was observed in four out of eight cases. One out of eight cases presented significant heterogeneity in the number of TILs between the two components of the tumor.ConclusionsAlthough the distribution of the immune infiltrate appears relatively conserved among heterogeneous areas of the same tumor, changes in gene expression profile and CMS occur in 50% of V600EBRAFmt adenocarcinoma cases in our small series and might contribute to variability in response to anticancer therapy and clinical outcomes. Assessment of morphological and molecular ITH is needed to improve colorectal cancer classification and to tailor anticancer treatments and should be included in the pathology report.

A panel of DNA methylation markers for the classification of consensus molecular subtypes 2 and 3 in patients with colorectal cancer.

3545 Background: Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). Currently available assays can identify CMS1 and CMS4 cases well, while a dedicated test to distinguish CMS2 and 3 is lacking. This study aimed to identify a panel of methylation markers to distinguish between CMS2 and 3 in patients with CRC. Methods: Fresh-frozen tumor tissue of 239 patients with stage I-III CRC was included. CMS classification was performed on RNA-seq data using the single-sample-prediction parameter from the “CMSclassifier” package. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group-regularised logistic ridge-regression with post-hoc group-weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3 based on 15, 10 or 5 markers. Data from TCGAwas used for validation. Results: Overall methylation profiles differed between CMS2 and CMS3. Group-regularisation of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only 5 markers, sensitivity, specificity, and accuracy were &gt; 90%. Validation showed comparable performances. Conclusions: Our highly sensitive and specific methylation marker panel can be used to distinguish CMS2 and 3. This enables development of a qPCR DNA methylation assay in patients with CRC to provide a specific and non-invasive classification tool.

RB1 and TP53 co-mutations correlate strongly with genomic biomarkers of response to immunity checkpoint inhibitors in urothelial bladder cancer

BackgroundMuscle invasive urothelial bladder carcinoma (MIBC) present RB1 and TP53 somatic alterations in a variable percentage of tumors throughout all molecular subtypes. MIBCs with neuroendocrine features have a high response rate to immunity checkpoint inhibitors (ICIs). Whether the presence of somatic co-alterations in these 2 genes in MIBCs is relevant to their responsiveness to ICIs is not known.MethodsThe potential correlation of different genomic biomarkers of response to ICIs like tumor mutational burden (TMB), single nucleotide variants (SNV) predicted neoantigens, DNA damage response (DDR) genes, DNA somatic signatures and TILs infiltrate was explored in patients with somatic co-alterations in RB1 and TP53 (RB1&TP53) as compared with patients with no alterations in any (double wild type, DWT) or with alterations in just one of the 2 genes. The Cancer Genome Atlas (TCGA) pancancer BLCA dataset of cystectomy specimens (n = 407) with mutation, copy number alterations and transcriptomic (RNA sequencing) data as well as the IMVigor 210 study (n = 348) of metastatic urothelial bladder cancers treated with atezolizumab (PD-L1 inhibitor) with clinical response data containing transcriptomic (RNA sequencing), along with a subset (n = 274) with mutation and copy number data were used for this purpose. A novel tumor microenvironment metascore (TMM) was developed based in a LASSO regularized Cox model with predictive and prognostic ability.ResultsSamples with co-altered RB1&TP53: a) were enriched in immunity effectors (CD8 cytotoxic lymphocytes, NK cells) and display higher scores of a T cell inflamed signature; b) have a higher TMB, higher number of SNV predicted neoantigens and higher TILs fractions; c) have a higher number of DDR mutated and deep deleted DDR genes; d) have DNA somatic signatures 2 and 13 related to APOBEC mutagenesis. Using the IMVigor 210 dataset, RB1&TP53 samples had the highest response rate to atezolizumab and a strong correlation with TMB and TMM. The consensus molecular subtype classification in the IMVigor 210 dataset showed a significant correlation with both the response to treatment (p = 0.001, Chisquare) and the presence of RB1 and TP53 genomic alterations (p < 0.001, Chisquare).ConclusionsRB1&TP53 co-alterations are strongly associated with genomic biomarkers of response to ICIs in MIBCs.

Colony-stimulating factor 3 signaling in colon and rectal cancers: Immune response and CMS classification in TCGA data.

Colorectal cancer is the 2nd leading cause of cancer-related deaths in the world. The mechanisms underlying CRC development, progression, and resistance to treatment are complex and not fully understood. The immune response in the tumor microenvironment has been shown to play a significant role in many cancers, including colorectal cancer. Colony-stimulating factor 3 (CSF3) has been associated with changes to the immune environment in colorectal cancer animal models. We hypothesized that CSF3 signaling would correlate with pro-tumor tumor microenvironment changes associated with immune infiltrate and response. We utilized publicly available datasets to guide future mechanistic studies of the role CSF3 and its receptor (CSF3R) play in colorectal cancer development and progression. Here, we use bioinformatics data and mRNA from patients with colon (n = 242) or rectal (n = 92) cancers, obtained from The Cancer Genome Atlas Firehose Legacy dataset. We examined correlations of CSF3 and CSF3R expression with patient demographics, tumor stage and consensus molecular subtype classification. Gene expression correlations, cell type enrichment, Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data scores and Gene Ontology were used to analyze expression of receptor and ligand, tumor microenvironment infiltration of immune cells, and alterations in biological pathways. We found that CSF3 and CSF3R expression is highest in consensus molecular subtype 1 and consensus molecular subtype 4. Ligand and receptor expression are also correlated with changes in T cell and macrophage signatures. CSF3R significantly correlates with a large number of genes that are associated with poor colorectal cancer prognosis.

Cofilin-1, LIMK1 and SSH1 are differentially expressed in locally advanced colorectal cancer and according to consensus molecular subtypes

BackgroundColorectal cancer (CRC) is among the deadliest cancers, wherein early dissemination of tumor cells, and consequently, metastasis formation, are the main causes of mortality and poor prognosis. Cofilin-1 (CFL-1) and its modulators, LIMK1/SSH1, play key roles in mediating the invasiveness by driving actin cytoskeleton reorganization in various cancer types. However, their clinical significance and prognostic value in CRC has not been fully explored. Here, we evaluated the clinical contribution of these actin regulators according to TNM and consensus molecular subtypes (CMSs) classification.MethodsCFL-1, LIMK1 and SSH1 mRNA/protein levels were assessed by real-time PCR and immunohistochemical analyses using normal adjacent and tumor tissues obtained from a clinical cohort of CRC patients. The expression levels of these proteins were associated with clinicopathological features by using the chi square test. In addition, using RNA-Seq data of CRC patients from The Cancer Genome Atlas (TCGA) database, we determine how these actin regulators are expressed and distributed according to TNM and CMSs classification. Based on gene expression profiling, Kaplan–Meier survival analysis was used to evaluated overall survival.ResultsBioinformatic analysis revealed that LIMK1 expression was upregulated in all tumor stages. Patients with high levels of LIMK1 demonstrated significantly lower overall survival rates and exhibited greater lymph node metastatic potential in a clinical cohort. In contrast, CFL-1 and SSH1 have expression downregulated in all tumor stages. However, immunohistochemical analyses showed that patients with high protein levels of CFL-1 and SSH1 exhibited greater lymph node metastatic potential and greater depth of local invasion. In addition, using the CMSs classification to evaluate different biological phenotypes of CRC, we observed that LIMK1 and SSH1 genes are upregulated in immune (CMS1) and mesenchymal (CMS4) subtypes. However, patients with high levels of LIMK1 also demonstrated significantly lower overall survival rates in canonical (CMS2), and metabolic (CMS3) subtypes.ConclusionsWe demonstrated that CFL-1 and its modulators, LIMK1/SSH1, are differentially expressed and associated with lymph node metastasis in CRC. Finally, this expression profile may be useful to predict patients with aggressive signatures, particularly, the immune and mesenchymal subtypes of CRC.

Immunohistochemistry-Based Consensus Molecular Subtypes as a Prognostic and Predictive Biomarker for Adjuvant Chemotherapy in Patients with Stage II Colorectal Cancer.

BackgroundFor stage II colorectal cancer (CRC), the efficacy of adjuvant chemotherapy remains controversial. Consensus molecular subtype (CMS) has been validated to be a prognostic tool for CRCs. In this study, CMS status was investigated as a prognostic biomarker for the efficacy of adjuvant chemotherapy for stage II colorectal cancer.Materials and MethodsThe tissue microarray was retrospectively constructed of 165 nonconsecutive, primary, and sporadic stage II CRCs. CMS status was determined by immunohistochemistry staining of CDX2, HTR2B, FRMD6, and ZEB1, combining with microsatellite instability testing. The prognostic for adjuvant chemotherapy efficacy of CMS status was calculated by Kaplan‐Meier curves and Cox regression analysis. Subgroup analyses were conducted according to tumor location.ResultsKaplan‐Meier curves indicated that CMS was associated with overall survival (OS) and disease‐free survival for stage II CRCs. Cox regression analysis showed that CMS was an independent risk factor for OS. Among high‐risk clinicopathological factors, patients with CMS2/3 (hazard ratio [HR]: 0.445, 95% confidence interval [CI]: 0.227–0.875), left‐sided tumors (HR: 0.488, 95% CI: 0.247–0.968), or fewer than 12 lymph nodes examined (HR: 0.307, 95% CI: 0.097–0.974) had survival benefit from adjuvant chemotherapy. Subgroup analysis showed that adjuvant chemotherapy only improved OS for patients with left‐sided tumors of CMS2/3 subtype. Regardless of CMS, right‐sided tumors had no benefit from adjuvant chemotherapy.ConclusionCMS is a better prognostic factor for adjuvant chemotherapy for stage II CRCs. Together with tumor location, CMS classification will aid in personalized treatment for stage II CRCs.Implications for PracticeFor stage II colorectal cancer (CRC), the efficacy of adjuvant chemotherapy remains controversial, in that its minimal benefit (no more than 5% on average) is considered not worth the toxic effects of the drugs. There are still no effective prognostic and predictive biomarkers. This study showed that consensus molecular subtype (CMS) status is a predictive marker for adjuvant chemotherapy efficacy. Patients with left‐sided tumors of CMS2/3 subtype have survival benefit by receiving adjuvant chemotherapy, which will aid in personalized treatment for stage II CRCs. Moreover, this test of CMS based on immunohistochemistry is cheap, not time consuming, and easily conducted in the laboratories of most hospitals.

Improving clinical management of colon cancer through CONNECTION, a nation-wide colon cancer registry and stratification effort (CONNECTION II trial): rationale and protocol of a single arm intervention study

BackgroundIt is estimated that around 15–30% of patients with early stage colon cancer benefit from adjuvant chemotherapy. We are currently not capable of upfront selection of patients who benefit from chemotherapy, which indicates the need for additional predictive markers for response to chemotherapy.It has been shown that the consensus molecular subtypes (CMSs), defined by RNA-profiling, have prognostic and/or predictive value. Due to postoperative timing of chemotherapy in current guidelines, tumor response to chemotherapy per CMS is not known, which makes the differentiation between the prognostic and predictive value impossible. Therefore, we propose to assess the tumor response per CMS in the neoadjuvant chemotherapy setting. This will provide us with clear data on the predictive value for chemotherapy response of the CMSs.MethodsIn this prospective, single arm, multicenter intervention study, 262 patients with resectable microsatellite stable cT3–4NxM0 colon cancer will be treated with two courses of neoadjuvant and two courses of adjuvant capecitabine and oxaliplatin. The primary endpoint is the pathological tumor response to neoadjuvant chemotherapy per CMS. Secondary endpoints are radiological tumor response, the prognostic value of these responses for recurrence free survival and overall survival and the differences in CMS classification of the same tumor before and after neoadjuvant chemotherapy. The study is scheduled to be performed in 8–10 Dutch hospitals. The first patient was included in February 2020.DiscussionPatient selection for adjuvant chemotherapy in early stage colon cancer is far from optimal. The CMS classification is a promising new biomarker, but a solid chemotherapy response assessment per subtype is lacking. In this study we will investigate whether CMS classification can be of added value in clinical decision making by analyzing the predictive value for chemotherapy response. This study can provide the results necessary to proceed to future studies in which (neo) adjuvant chemotherapy may be withhold in patients with a specific CMS subtype, who show no benefit from chemotherapy and for whom possible new treatments can be investigated.Trial registrationThis study has been registered in the Netherlands Trial Register (NL8177) at 11–26-2019, https://www.trialregister.nl/trial/8177. The study has been approved by the medical ethics committee Utrecht (MEC18/712).