Consensus Molecular Subtypes Research Articles

Abstract 7587: Fascin1 might be a promising molecular target for colorectal cancer patients at advanced stage

Abstract Background: Colorectal cancers (CRC) show high morbidity and mortality. Especially, the consensus molecular subtypes 4 (CMS4)-CRC shows extremely poor prognosis and high chemo-resistance rate, in compared with the other types of CRC. CMS4-CRC is histologically characteristic for the extensive stromal infiltration with poorly differentiated cancer cells in the tumor microenvironment. Several studies have reported that sessile serrated adenomas, which is characterized by disorganized crypt architecture and irregular serrations with high malignant potential such as epithelial-mesenchymal transition and TGFβ signaling, might be closely associated with CMS4-CRC. Fascin-1 is an actin-binding protein supposed to be associated with sessile serrated adenomas. However, the expression and clinical significance of fascin-1 in CMS4 CRC remains to be unclear. Aim: The aim of this study was to clarify the clinic-pathological significance of fascin-1 in colorectal cancer, and to evaluate the effects of fascin-1 inhibitors on the invasion and migration ability of CMS4 cancer cell lines. Materials and Methods: We conducted an immunohistochemical study on 351 colorectal cancer patients, where we examined the expression levels of Fascin-1 and analyzed their correlation with the patients’ clinicopathologic features. Using two human CMS4-CRC cell lines, SW480 and HCT116, we investigated the effects of the fascin-1 inhibitors, NP-G2-044 and BDP-13176, on the proliferation and the migration activity of CMS4-CRC cells via MTT assay and a wound-healing assay. Results: Fascin-1 is expressed both on cancer cells and on stromal cells, but mainly expressed on cancer cells. Significance of fascin-1 expression on cancer cells was evaluated in this study. Fascin-1 expression is significantly associated with histologically poorly differentiation (p < 0.001), deeper T invasion (p = 0.003), and proximal colon cancer (p < 0.001). High fascin-1 expression is significantly associated with worse prognosis (p = 0.0085, log-rank) and higher recurrence rates (p = 0.018). Univariate analysis demonstrated that overall survival of patients was significantly associated with Fascin1 expression in cancer cells, age ≥65 years old, poor histological type, lymphatic invasion, lymph node metastasis, and venous invasion. Multivariate analysis revealed that fascin1 expression (p = 0.037), poor histological type (p = 0.022), venous invasion (p = 0.078), and lymph node metastasis (p = 0.009) were significantly correlated with the overall survival rate of CRC patients. Both fascin-1 inhibitors significantly reduced the invasion and migration of colorectal cancer cells at the concentration of 100 nM. Conclusions: Fascin-1 is one of key molecules for the development of CRC. Fascin-1 might be a promising molecular target for CMS4-CRC patients at advanced stage. Citation Format: Canfeng Fan, Qiang Wang, Rika Aoyama, Hinano Nishikubo, Kyouka Kawabata, Imanishi Daiki, Takashi Sakuma, Koji Maruo, Gen Tsujio, Yurie Yamamoto, Tatsunari Fukuoka, Masakazu Yashiro. Fascin1 might be a promising molecular target for colorectal cancer patients at advanced stage [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 7587.

Abstract 1150: Unveiling the spatial landscape of tumor and stroma heterogeneity in colorectal cancer with spatial transcriptomics

Abstract Heterogeneity in colorectal cancer (CRC) manifests in the diversity and interactions among tumor cells, support stroma, and immune components, critically impacting patient prognosis and therapeutic efficacy. In this study, spatial transcriptomics (ST, 10x Genomics Visium Spatial Gene Expression) was performed on 40 retrospectively collected CRC samples, along with detailed clinicopathological parameters and long-term clinical outcomes, with the goal of unraveling the molecular topography of the tumor and surrounding stromal compartments, and associating their spatial interactions to clinical outcomes. Five primary RNA groups were identified through unsupervised clustering on ST data, correlating with tissue classes detected in H&E-stained images via a pre-trained deep learning model. Markers and pathways specific to these tissues were identified. Furthermore, immune cell infiltration in these regions was deconvoluted using reference single-cell RNA sequencing (scRNA-seq) data. Sub-clustering within the tumor groups revealed five major tumor subtypes, each characterized by a distinct set of differentially expressed marker genes. Enrichment analysis across these subtypes highlighted distinct activation and suppression in pathways including KRAS, p53, MAPK, and Integrin. Tumors exhibited intra- and inter-tumoral heterogeneity. Location-specific tumor subtypes were observed, with those in right-sided CRC displaying increased immune cell infiltration, linked to enriched pathways such as Type II Interferon Signaling and Cytokine Signaling. Conversely, left-sided CRC subtypes had reduced immune cell presence, and were characterized by enhanced pathways related to vascular development, Hypoxia, and p53 Signaling. Inferred clonal lineages from copy number variation (CNV) using ST suggested a spatially structured phylogenetic organization corresponding to transcriptional clusters. Additionally, the ST data unveiled distinct spatial distribution patterns across various cancer-associated fibroblast (CAF) phenotypes, such as matrix, inflammatory, vessel-associated, tumor-promoting, and antigen-presenting CAFs. Transcriptional patterns uncovered through scRNA-seq, including epithelial-mesenchymal transition (EMT) signatures and consensus molecular subtypes (CMS), displayed unique spatial layouts within the tumor microenvironment. To our knowledge, this is the most comprehensive study to date on spatial transcriptomic heterogeneity within CRC patients, revealing distinct tumor, stroma and immune compositions and spatial structures. This spatially detailed map provides novel insights into CRC heterogeneity, critical for advancing personalized treatment. Citation Format: Jie Zhou, Todd B. Sheridan, Sergii Domanskyi, Stephanie L. Cowles, Sunghee Park, Ilham Putra, Olga Anczukow, Jeffrey H. Chuang, Jill C. Rubinstein. Unveiling the spatial landscape of tumor and stroma heterogeneity in colorectal cancer with spatial transcriptomics [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 1150.

Abstract 3338: Development of a CK2α inhibitor for treatment of wnt-driven colorectal cancer

Abstract A novel, potent and highly selective orally bioavailable small molecule inhibitor of Casein Kinase 2α (CK2α) has been developed and validated in pre-clinical models of wnt-driven colorectal carcinoma; and is poised to enter clinical development.CK2α is considered as a positive regulator of wnt signaling and tumorigenesis. The wnt pathway is known to be sensitive to and amplified by CK2α activity at multiple nodes and can be inhibited by loss of CK2α function. The human genetics of colorectal cancer (CRC) have been well characterized and approximately 80% tumors are identified as being wnt pathway mutation driven. CRC tumors can be further stratified into consensus molecular subtypes (CMS), with iCMS2 representing 37% of CRC patients, being characterized by ongoing wnt pathway upregulation. Development of CK2α inhibitors as a targeted therapy for CRC is therefore mechanistically well aligned to the wnt signaling drive and may serve as a promising therapeutic strategy for this cancer. A novel small molecule binding site on CK2α has been exploited to design and develop an inhibitor with Ki of 95 pM and exquisite selectivity over the kinome. This inhibitor has been comprehensively characterized using in vitro, ex vivo and in vivo pharmacology studies. Cellular mechanism of action studies in CRC cell lines have quantified the potency of target engagement, inhibition of known CK2α phospho-sites, inhibition of tumorigenic wnt pathway activity, and induction of apoptosis. This CK2α inhibitor was also profiled in a high content screen of CRC patient-derived organoids established from various driver mutation backgrounds. Use of these ex vivo models has validated the potent cytotoxic activity of CK2α inhibition in tumor samples with wnt-driven characteristics and highlights the potential therapeutic efficacy of CK2α inhibition in this setting. In vivo CRC xenograft studies have confirmed anti-tumor efficacy and developed the understanding of pharmacokinetic pharmacodynamic relationship between free plasma concentration of drug and inhibition of CK2α-specific biomarker in the tumor. The preclinical data presented establishes the strong rationale for clinical development of a novel CK2α inhibitor for the treatment of locally advanced or metastatic solid tumors, with an emphasis on wnt-driven CRC. Citation Format: Darren Cawkill. Development of a CK2α inhibitor for treatment of wnt-driven 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 3338.

Abstract 1498: TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 colorectal cancer

Abstract Background: Colorectal cancer (CRC) is characterized by a metastatic pattern that shows tropism towards the liver which is the major cause of CRC-related deaths. The current CRC classification, Consensus Molecular Subtypes (CMS), suggests an association between transforming growth factor (TGF)-β signaling, cancer-associated fibroblasts (CAFs), and the risk of liver metastasis. However, the key downstream effectors of TGFβ signaling in CAFs and their role in hepatic metastases remain poorly understood. Methods: The aim of this study was to investigate the TGF-β/CAF dependent cross talk with the hepatocytes in pre-metastatic niche formation. CAFs from 18 primary CRC tissues and 7 liver-metastasized CRC were stimulated with TGFβ1 and qPCR arrays/ELISAs were performed to identify differentially expressed genes. Results were cross-referenced with RNAseq expression data of CMS-classified CRC and in fresh samples from CRC patients. To investigate CAF-liver crosstalk hepatocytes were stimulated with the TGFβ primed CAF-derived cytokines and the effect on pro-inflammatory gene expression and neutrophil migration was determined. Subsequently, blockade of these pathways via chemical inhibition and CRISPR/Cas9 genetic ablation was performed to study the molecular mechanism involved in the induction of this hepatic pro-inflammatory program. Results: TGFβ signaling in primary CRC-derived CAFs leads to increased expression of different IL-6 cytokine family members, which was also reflected in human CRC samples. CAF-derived IL6 family members induce upregulation of myeloid chemoattractants, including SAA1, in hepatocytes and increased neutrophil-to-hepatocyte migration in vitro. Chemical blockade and genetic ablation of the IL-6 family cytokine signaling pathway showed the critical role of the gp130 co-receptor in the regulation of this inflammatory response in hepatocytes and the potential formation of a premetastatic niche. Conclusion: TGFβ signaling in CAFs actively contributes to the formation of a neutrophil-dependent, pre-metastatic hepatic niche, and this mechanism might play a role in CMS4 subtype CRC. Citation Format: Subinuer Abudukelimu, Tom J. Harryvan, Stef G. Janson, Imke Stouten, Els M. Verdegaal, Lukas JAC Hawinkels. TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 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 1498.

Abstract 659: TR-107 and erastin produce synergistic inhibition of colorectal cancer cell viability in vitro

Abstract Introduction: Ferroptosis is an emerging potential target for the treatment of colorectal cancer (CRC) and has been shown to sensitize cancer cells to combinatory chemotherapy. Our studies show that the TR-107 compound, an agonist of ClpP - the proteolytic subunit of mitochondrial maintenance complex - can synergize with Erastin, an inducer of ferroptosis cell death. TR-107 potently disrupts mitochondrial proteostasis and attenuates oxidative phosphorylation, resulting in excessive oxidative stress. Here, we show that Erastin synergizes with TR-107 activity by downregulating the cofactor of GPX4, a major intracellular antioxidant defense resulting in CRC cell apoptosis. Methods: We first evaluated the efficacy of TR-107 against eight CRC cell lines belonging to four established consensus molecular subtypes. Cells were treated with control (DMSO) or TR-107 (10 nM, 50 nM, 1 µM) for 24, 48, and 72 hours. We determined IC50 values with Cell Titer-Glo, cell proliferation with the Beckman Coulter cell counter, cell cycle with flow cytometry, OXPHOS activity with Seahorse Real-Time ATP Rate assay, mitochondrial protein levels with western blot and proteomics, and differential gene expression using RNA sequencing and RT-PCR. Subsequently, we assessed the efficacy of TR-107 and Erastin co-treatment. Cells were treated with DMSO, TR-107 (10, 50 nM), Erastin (1, 10 µM), or combinations of the abovementioned concentrations. Cell viability was measured 72 hours post-treatment with Cell Titer-Glo. Synergy scores (HSA) were calculated with Synergyfinder on RStudio. Ferroptosis-related protein expression 24 hours after co-treatment was assessed with western blot and densitometry. Results: TR-107 IC50 values ranged from 2-70 nM, producing a dose- and time-dependent inhibitory effect in cell proliferation. Western blot showed that 50 nM TR-107 abolished the expression of critical mitochondrial proteins ClpX, TFAM, and TUFM. Seahorse data showed a significant reduction in OXPHOS activity. Multi-omics revealed downregulation of respiratory chain complex subunits, mtDNA transcription/translation factors, and upregulation of mitophagy and ferroptosis-related pathways in treated cells. Co-treatment with Erastin produced synergistic HSA scores in 63% (5/8) of assessed CRC cell lines. RKO and NCI-H508 were highly sensitive to Erastin treatment, suggesting that lower Erastin concentrations may synergize with TR-107 and thus reduce dose toxicity. Western blot analysis 24 hours post-treatment showed a loss of GPX4 protein levels in co-treated cells compared to 50 nM TR-107 treatment. This suggests a rapid downregulation of intracellular antioxidant defense that may induce ferroptosis sooner and more effectively than single-agent treatment. Conclusion: In vitro results suggest that co-treating with TR-107 and Erastin produces a significant, synergistic reduction in CRC cell viability. Citation Format: Michael Giarrizzo, Joseph F. LaComb, Hetvi R. Patel, Agnieszka B. Bialkowska, Rohan Reddy, Lee M. Graves, Edwin J. Iwanowicz. TR-107 and erastin produce synergistic inhibition of colorectal cancer cell viability in vitro [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 659.

Functional proteomics of colon cancer Consensus Molecular Subtypes.

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, only MSI tumour patients have seen a change in their disease management in clinical practice. This study aims to characterise the proteome of colon cancer CMS and broaden CMS's clinical utility. One-hundred fifty-eight paraffin samples from stage II-III colon cancer patients treated with adjuvant chemotherapy were analysed through DIA-based mass-spectrometry proteomics. CMS1 exhibited overexpression of immune-related proteins, specifically related to neutrophils, phagocytosis, antimicrobial response, and a glycolytic profile. These findings suggested potential therapeutic strategies involving immunotherapy and glycolytic inhibitors. CMS3 showed overexpression of metabolic proteins. CMS2 displayed a heterogeneous protein profile. Notably, two proteomics subtypes within CMS2, with different protein characteristics and prognoses, were identified. CMS4 emerged as the most distinct group, featuring overexpression of proteins related to angiogenesis, extracellular matrix, focal adhesion, and complement activation. CMS4 showed a high metastatic profile and suggested possible chemoresistance that may explain its worse prognosis. DIA proteomics revealed new features for each colon cancer CMS subtype. These findings provide valuable insights into potential therapeutic targets for colorectal cancer subtypes in the future.

A comprehensive multi-omics analysis identifies a robust scoring system for cancer-associated fibroblasts and intervention targets in colorectal cancer

BackgroundCancer-associated fibroblasts (CAF) play a critical role in promoting tumor growth, metastasis, and immune evasion. While numerous studies have investigated CAF, there remains a paucity of research on their clinical application in colorectal cancer (CRC).MethodsIn this study, we collected differentially expressed genes between CAF and normal fibroblasts (NF) from previous CRC studies, and utilized machine learning analysis to differentiate two distinct subtypes of CAF in CRC. To enable practical application, a CAF-related genes (CAFGs) scoring system was developed based on multivariate Cox regression. We then conducted functional enrichment analysis, Kaplan–Meier plot, consensus molecular subtypes (CMS) classification, and Tumor Immune Dysfunction and Exclusion (TIDE) algorithm to investigate the relationship between the CAFGs scoring system and various biological mechanisms, prognostic value, tumor microenvironment, and response to immune checkpoint blockade (ICB) therapy. Moreover, single-cell transcriptomics and proteomics analyses have been employed to validate the significance of scoring system-related molecules in the identity and function of CAF.ResultsWe unveiled significant distinctions in tumor immune status and prognosis not only between the CAF clusters, but also across high and low CAFGs groups. Specifically, patients in CAF cluster 2 or with high CAFGs scores exhibited higher CAF markers and were enriched for CAF-related biological pathways such as epithelial–mesenchymal transition (EMT) and angiogenesis. In addition, CAFGs score was identified as a risk index and correlated with poor overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and recurrence-free survival (RFS). High CAFGs scores were observed in patients with advanced stages, CMS4, as well as lymphatic invasion. Furthermore, elevated CAFG scores in patients signified a suppressive tumor microenvironment characterized by the upregulation of programmed death-ligand 1 (PD-L1), T-cell dysfunction, exclusion, and TIDE score. And high CAFGs scores can differentiate patients with lower response rates and poor prognosis under ICB therapy. Notably, single-cell transcriptomics and proteomics analyses identified several molecules related to CAF identity and function, such as FSTL1, IGFBP7, and FBN1.ConclusionWe constructed a robust CAFGs score system with clinical significance using multiple CRC cohorts. In addition, we identified several molecules related to CAF identity and function that could be potential intervention targets for CRC patients.

Seed and Soil: Consensus Molecular Subgroups (CMS) and Tumor Microenvironment Features Between Primary Lesions and Metastases of Different Organ Sites in Colorectal Cancer.

Consensus molecular subtypes (CMS) are mainly used for biological interpretability and clinical stratification of colorectal cancer (CRC) in primary tumors (PT) but few in metastases. The heterogeneity of CMS distribution in metastases and the concordance of CMS between PT and metastases still lack sufficient study. We used CMS to classify CRC metastases and combine it with histopathological analysis to explore differences between PT and distant metastases. We obtained gene expression profiles for 942 PT samples from TCGA database (n=376) and GEO database (n=566), as well as 442 metastasis samples from GEO database. Among these, 765 PT samples and 442 metastasis samples were confidently identified with CMS using the "CMS classifier" and enrolled for analysis. Clinicopathological manifestation and CMS classification of CRC metastases were assessed with data from GEO, TCGA, and cBioPortal. Overall, 105 PT-metastasis pairs were extracted from 10 GEO datasets to assess CMS concordance. Tumor microenvironment (TME) features between PT and metastases were analyzed by immune-stromal infiltration with ESTIMATE and xCell algorithms. Finally, TME features were validated with multiplex immunohistochemistry in 27 PT-metastasis pairs we retrospectively collected. Up to 64% of CRC metastases exhibited concordant CMS groups with matched PT, and the TME of metastases was similar to that of PT. For most common distant metastases, liver metastases were predominantly CMS2 and lung and peritoneal metastases were mainly CMS4, highlighting "seed" of tumor cells of different CMS groups had a preference for metastasis to "soil" of specific organs. Compared with PT, cancer-associated fibroblasts (CAF) reduced in liver metastases, CD4+T cells and M2-like macrophages increased in lung metastases, and M2-like macrophages and CAF increased in peritoneal metastases. Our findings underscore the importance of CMS-guided specific organ monitoring and treatment post-primary tumor surgery for patients. Differences in immune-stromal infiltration among different metastases provide targeted therapeutic opportunities for metastatic CRC.

Comprehensive RNA-sequencing analysis of colorectal cancer in a Korean cohort

Considering the recent increase in the number of colorectal cancer (CRC) cases in South Korea, we aimed to clarify the molecular characteristics of CRC unique to the Korean population. To gain insights into the complexities of CRC and promote the exchange of critical data, RNA-sequencing (RNA-Seq) analysis was performed to reveal the molecular mechanisms that drive the development and progression of CRC; this analysis is critical for developing effective treatment strategies. We performed RNA-Seq analysis of CRC and adjacent normal tissue samples from 214 Korean participants (comprising a total of 381 including 169 normal and 212 tumor samples) to investigate differential gene expression between the groups. We identified 19,575 genes expressed in CRC and normal tissues, with 3,830 differentially expressed genes (DEGs) between the groups. Functional annotation analysis revealed that the upregulated DEGs were significantly enriched in pathways related to the cell cycle, DNA replication, and IL-17, whereas the downregulated DEGs were enriched in metabolic pathways. We also analyzed the relationship between clinical information and subtypes using the Consensus Molecular Subtype (CMS) classification. Furthermore, we compared groups clustered within our dataset to CMS groups and performed additional analysis of the methylation data between DEGs and CMS groups to provide comprehensive biological insights from various perspectives. Our study provides valuable insights into the molecular mechanisms underlying CRC in Korean patients and serves as a platform for identifying potential target genes for this disease. The raw data and processed results have been deposited in a public repository for further analysis and exploration. Data availability statementThe RNA-Seq data have been deposited in the Korean Nucleotide Archive under the accession number KAP230608 (https://www.kobic.re.kr/kona/search_bioproject?bioproject_id=KAP230608#none). Clinical data, preprocessed gene quantification (RSEM), normalized gene expression data (log2CPM), and downstream analysis results have been deposited in Zenodo (https://doi.org/10.5281/zenodo.8333650). Further information and requests for experimental resources should be directed to and will be fulfilled by the corresponding authors, Kil-yong Lee (cyboryee@hanmail.net), Jin Ok Yang (joy@kribb.re.kr), and Young-Joon Kim (yjkim@yonsei.ac.kr).

Biological background of colorectal polyps and carcinomas with heterotopic ossification: A national study and literature review

The biological mechanisms and potential clinical impact of heterotopic ossification (HO) in colorectal neoplasms are not fully understood. This study investigates the clinicopathological characteristics of colorectal neoplasms associated with HO and examines the potential role of the bone morphogenetic protein (BMP) pathway in development of HO. An artificial intelligence (AI) based classification of colorectal cancers (CRC) exhibiting HO and their association with consensus molecular subtypes (CMS) is performed.The study included 77 cases via the Dutch nationwide Pathology databank. Immunohistochemistry for BMP2, SMAD4, and Osterix was performed. An AI algorithm assessed the tumour-stroma ratio to approximate the CMS. A literature search yielded 96 case reports, which were analysed and compared with our cases for clinicopathological parameters.HO was more frequently observed in our cohort in traditional serrated adenomas (25%), tubulovillous adenomas (25%) and juvenile polyps (25%), while in the literature it was most often seen in juvenile polyps (38.2%) and inflammatory polyps (29.4%). In both cohorts, carcinomas were mostly conventional (>60%) followed by mucinous and serrated adenocarcinomas. Higher expression of BMP2, SMAD4, and Osterix was observed in tumour and/or stromal cells directly surrounding bone, indicating activation of the BMP pathway. The tumour-stroma analysis appointed >50% of the cases to the mesenchymal subtype (CMS4) (59%).HO has a predilection for serrated and juvenile/inflammatory polyps, mucinous and serrated adenocarcinomas. BMP signalling is activated and seems to play a role in formation of HO in colorectal neoplasms. In line with TGFβ/BMP pathway activation associated with CMS4 CRC, HO seems associated with CMS4.

Pathway level subtyping identifies a slow-cycling biological phenotype associated with poor clinical outcomes in colorectal cancer

Molecular stratification using gene-level transcriptional data has identified subtypes with distinctive genotypic and phenotypic traits, as exemplified by the consensus molecular subtypes (CMS) in colorectal cancer (CRC). Here, rather than gene-level data, we make use of gene ontology and biological activation state information for initial molecular class discovery. In doing so, we defined three pathway-derived subtypes (PDS) in CRC: PDS1 tumors, which are canonical/LGR5+ stem-rich, highly proliferative and display good prognosis; PDS2 tumors, which are regenerative/ANXA1+ stem-rich, with elevated stromal and immune tumor microenvironmental lineages; and PDS3 tumors, which represent a previously overlooked slow-cycling subset of tumors within CMS2 with reduced stem populations and increased differentiated lineages, particularly enterocytes and enteroendocrine cells, yet display the worst prognosis in locally advanced disease. These PDS3 phenotypic traits are evident across numerous bulk and single-cell datasets, and demark a series of subtle biological states that are currently under-represented in pre-clinical models and are not identified using existing subtyping classifiers.

Stratification of Colorectal Patients Based on Survival Analysis Shows the Value of Consensus Molecular Subtypes and Reveals the CBLL1 Gene as a Biomarker of CMS2 Tumours.

The consensus molecular subtypes (CMSs) classification of colorectal cancer (CRC) is a system for patient stratification that can be potentially applied to therapeutic decisions. Hakai (CBLL1) is an E3 ubiquitin-ligase that induces the ubiquitination and degradation of E-cadherin, inducing epithelial-to-mesenchymal transition (EMT), tumour progression and metastasis. Using bioinformatic methods, we have analysed CBLL1 expression on a large integrated cohort of primary tumour samples from CRC patients. The cohort included survival data and was divided into consensus molecular subtypes. Colon cancer tumourspheres were used to analyse the expression of stem cancer cells markers via RT-PCR and Western blotting. We show that CBLL1 gene expression is specifically associated with canonical subtype CMS2. WNT target genes LGR5 and c-MYC show a similar association with CMS2 as CBLL1. These mRNA levels are highly upregulated in cancer tumourspheres, while CBLL1 silencing shows a clear reduction in tumoursphere size and in stem cell biomarkers. Importantly, CMS2 patients with high CBLL1 expression displayed worse overall survival (OS), which is similar to that associated with CMS4 tumours. Our findings reveal CBLL1 as a specific biomarker for CMS2 and the potential of using CMS2 with high CBLL1 expression to stratify patients with poor OS.

Multiomic molecular characterization of the response to combination immunotherapy in MSS/pMMR metastatic colorectal cancer

BackgroundImmune checkpoint inhibitor (ICI) combinations represent an emerging treatment strategies in cancer. However, their efficacy in microsatellite stable (MSS) or mismatch repair-proficient (pMMR) colorectal cancer (CRC) is variable. Here, a...

Association of class II and III BRAF mutations with EGFR blockade therapy response and representation of molecularly distinct subgroups of BRAF mutations in MMR proficient CRC.

168 Background: BRAF mutations (mts) represent a highly heterogeneous group of molecular alterations seen in colorectal cancer (CRC). Class I BRAF mts (V600) render aggressive biology to CRC and poor response to anti EGFR inhibitors. Currently there are limited data on clinical and molecular features of class II and III BRAF mts. In this study, we investigated the clinical and molecular characteristics of BRAF mutation classes and their impact on clinical outcomes in a large cohort of patients (pts) with mismatch proficient (pMMR) CRC. Methods: A total of 18575 pMMR CRC specimens were profiled by next-generation sequencing (592-gene, NextSeq; WES, WTS NovaSeq) (Caris Life Sciences, Phoenix, AZ). BRAF mutations were detected by NGS and classified using published literature (Sahin et al. JCO OP 2021). Interferon gamma signature and MAPK pathway activity score (MPAS) were calculated using RNA expression data (TPM). Real-world overall survival information was obtained from insurance claims and calculated from tissue collection to last contact, while post-treatment survival from first of treatment to last contact. KM estimates were calculated for molecularly defined cohorts. Significance was determined as p values of <0.05. Results: A total of 930,105, and 262 pts with class I, II, and III BRAF mts were identified. Pts with class III BRAF mts were significantly more common among younger pts (age<45) compared to class I and class II (8.8% vs. 4.8% vs. 1.0 % respectively; P<0.05). Class I BRAF mts were significantly enriched with consensus molecular subtype 1 (CMS1) (Class I, II and III: 44% vs. 17% vs. 18%) while class II and III BRAF mts were more often CMS2 subtype (canonical) compared to class I (2%, 30% and 35%, p<0.05). Class I BRAF and KRAS/ NRAS mts were mutually exclusive, while KRAS mts incidences were 15% and 22% for class II and class III, and NRAS mts incidence were 8% and 12%, respectively. MPAS score was significantly lower for class III (-0.32, arbitrary unit) compared to Class I (p<0.05), but similar MPAS scores were seen in class I and II (1.3 versus 1.38). No difference in interferon gamma signature was noted between classes. Pts with class II and III mts had significantly better overall survival compared to patients with class I mts (HR=0.69 CI: 0.597-0.804 p<0.0001) and slightly worse overall survival compared to wild-type BRAF pts. (HR; 0.85 CI: 0.74-0.96 P=0.011). Among pts treated with anti-EGFR, pts with class II and III BRAF mts had significantly better post-anti-EGFR survival compared to class I BRAF mts (HR 0.498 CI 0.32-0.766 P=0.001 and similar survival compared to those with BRAF wild-type (P=0.21). Conclusions: Class II and III BRAF mts are associated with improved outcomes with EGFR blockade and represent a distinct biological subgroup of pMMR CRC. Class III BRAF mts have lower MAPK activation, consistent with the pattern of kinase-dead mutations.

LMTK3 gene expression and the molecular landscape of colorectal cancer (CRC).

172 Background: Lemur tail kinase 3 (LMTK3) plays a critical role in multiple cellular pathways such as Wnt signaling, KIT modulation, and the estrogen receptor pathway. We previously reported that LMTK3 gene polymorphisms are associated with clinical outcome in patients with CRC, and that LMTK3 and estrogen-mediated signaling play a crucial role in CRC tumorigenesis in vitro. Here, we aimed to characterize the molecular features associated with LMTK3 gene expression in CRC. Methods: 20,219 CRC were tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes or WES) and RNA (WTS). Top quartile transcripts per million (TPM) for LMTK3 expression (Q4) were considered high while bottom quartile (Q1) low. Consensus molecular subtypes (CMS) were assessed using RNAseq. Cell infiltration (CI) in the tumor microenvironment (TME) was estimated by QuantiSEQ. X2 and Fisher-Exact tests were used, and significance was determined as p-value adjusted for multiple comparisons ( q < 0.05). Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy (interferon gamma [IFNγ] score; T-cell inflamed signature [TIS] score). Results: LMTK3 expression was highest in left-sided (0.98 median TPM) followed by right-sided (0.96) and lowest in rectal tumors (0.92). No difference in patient sex distribution was observed between Q1 and Q4 cohorts. Overall, LMTK3 Q1 was associated with TMB-high (≥ 10 Mut/Mb, 11.4 vs 8.7%) and dMMR/MSI-H (8.1 vs 4.6%) ( q < 0.0001); however, these associations were no longer significant in pMMR/MSS tumors. In the pMMR/MSS cohort, LMTK3 expression was highest in CMS4 (1.09 median TPM) followed by CMS2 (0.98), CMS1 (0.88), and lowest in CMS3 (0.70, all intergroup q < 0.05). LMTK3 high showed differential rates of mutations and copy number alterations (CNA) in several genes, including higher mutation rates of APC, TP53, and CDX2 CNA, while lower mutation rates of KMT2A/D, ASXL1, ATM, SMAD4, RNF43 and AMER1 in the overall cohort ( q < 0.05). In the pMMR/MSS cohort, the associations with APC, TP53, SMAD4 and ATM mutations and CDX2 CNA still held true ( q < 0.05). High LMTK3 was associated with higher immune CI in the TME, including B cells, neutrophils, NK cells, CD4+ T cells, monocytes and dendritic cells, while macrophages and Tregs were lower regardless of tumor MSI status (fold change: 0.83-2.4, all q < 0.0001). The IFNγ score was significantly lower in LMTK3 Q4 while the TIS score was significantly higher in LMTK3 Q4 ( q < 0.0001). Conclusions: Our data show a strong association between LMTK3 gene expression and distinct molecular features and TME immune cell infiltration in CRC. These findings suggest that LMTK3 may be an important molecular factor that plays a role in determining the composition of the TME, thus targeting LMTK3 could represent a novel strategy in selected CRC subgroups.

Clinical characteristics and outcomes of patients with metastatic colitis-associated cancer treated with EGFR inhibitors.

37 Background: Colitis-associated cancer (CAC) is a devastating complication of inflammatory bowel disease. CAC tumor biology differs from that of sporadic colorectal cancer (CRC). Patients (pts) with (w/) metastatic CAC (mCAC) treated w/ 1st line (1L) chemotherapy have worse outcomes than pts w/ sporadic metastatic CRC (mCRC). EGFR inhibitor (EGFRi) w/ chemotherapy is standard of care for mCRC that is left (L) sided, RAS wild type (WT), without BRAF V600E mutation (mut) or ERBB2 amplification (amp). There are no published data on efficacy of EGFRi in CAC. We report outcomes of mCAC pts treated w/ EGFRi. Methods: Cases were identified by querying a prospectively maintained database of CAC pts seen at Memorial Sloan Kettering Cancer Center from 1/2000-present. Among mCAC pts who received EGFRi (n = 16), outcomes of interest: best response, progression-free survival (PFS), and overall survival (OS). PFS was calculated from date of 1st treatment w/ EGFRi until progression or death. OS was calculated from date of 1st treatment w/ EGFRi until death or last follow-up. Survival analyses were performed using Kaplan-Meier method. Data were obtained through review of electronic medical records. Results: 84 pts w/ mCAC were identified (52 L sided, 24 right [R] sided, 8 small bowel). Genomics were consistent w/ our prior reports: 79% TP53 mut, 19% APC mut, 14% MYC amp/gain, 32% KRAS mut, 4% BRAF mut, 6% ERBB2 amp. Limiting to pts w/ tumors that were L sided, RAS WT, without BRAF V600E mut or ERBB2 amp, we identified 34 (40%) mCAC pts. 16 (47%) of these pts received EGFRi as 2L or 3L therapy. 3 pts (19%) received EGFRi alone, 13 pts (81%) received EGFRi w/ FOLFIRI or irinotecan. 8 pts (50%) had tumors that were high grade and/or w/ signet ring features. Metastatic sites at initiation of EGFRi: lymph node (69%), liver (50%), lung (50%), peritoneum (38%), bone (38%), soft tissue (38%). 2 pts (12.5%, 95% CI: 1.5-38.3%) had a response, 4 pts (25%) had stable disease (SD), and 10 pts (62.5%) had progressive disease (PD) as best response. Median PFS and OS: 3.0 months (95% CI: 1.8-8.7) and 11.5 months (95% CI: 8.8-17.4), respectively. 2 additional pts w/ R sided CAC received EGFRi w/ SD and PD as best responses. Conclusions: Pts w/ mCAC, who by established criteria are eligible for treatment w/ EGFRi, have rapid progression and low response rates to EGFRi. This suggests that unlike sporadic CRC, CAC may not be driven primarily by EGFR. Many had high grade histology and unusual sites of metastases (bone, soft tissue). Experiments are under way to elucidate mechanisms underlying resistance to EGFRi and will be reported. These include immunohistochemistry for EGFR and its ligands, transcriptomic analyses to classify consensus molecular subtypes, and assessment of EGF dependence and EGFRi sensitivity in CAC organoids.

A partial epithelial-mesenchymal transition signature for highly aggressive colorectal cancer cells that survive under nutrient restriction.

Partial epithelial-mesenchymal transition (p-EMT) has recently been identified as a hybrid state consisting of cells with both epithelial and mesenchymal characteristics and is associated with the migration, metastasis, and chemoresistance of cancer cells. Here, we describe the induction of p-EMT in starved colorectal cancer (CRC) cells and identify a p-EMT gene signature that can predict prognosis. Functional characterisation of starvation-induced p-EMT in HCT116, DLD1, and HT29 cells showed changes in proliferation, morphology, and drug sensitivity, supported by in vivo studies using the chorioallantoic membrane model. An EMT-specific quantitative polymerase chain reaction (qPCR) array was used to screen for deregulated genes, leading to the establishment of an in silico gene signature that was correlated with poor disease-free survival in CRC patients along with the CRC consensus molecular subtype CMS4. Among the significantly deregulated p-EMT genes, a triple-gene signature consisting of SERPINE1, SOX10, and epidermal growth factor receptor (EGFR) was identified. Starvation-induced p-EMT was characterised by increased migratory potential and chemoresistance, as well as E-cadherin processing and internalisation. Both gene signature and E-cadherin alterations could be reversed by the proteasomal inhibitor MG132. Spatially resolving EGFR expression with high-resolution immunofluorescence imaging identified a proliferation stop in starved CRC cells caused by EGFR internalisation. In conclusion, we have gained insight into a previously undiscovered EMT mechanism that may become relevant when tumour cells are under nutrient stress, as seen in early stages of metastasis. Targeting this process of tumour cell dissemination might help to prevent EMT and overcome drug resistance. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Profiling the heterogeneity of colorectal cancer consensus molecular subtypes using spatial transcriptomics

The consensus molecular subtypes (CMS) of colorectal cancer (CRC) is the most widely-used gene expression-based classification and has contributed to a better understanding of disease heterogeneity and prognosis. Nevertheless, CMS intratumoral heterogeneity restricts its clinical application, stressing the necessity of further characterizing the composition and architecture of CRC. Here, we used Spatial Transcriptomics (ST) in combination with single-cell RNA sequencing (scRNA-seq) to decipher the spatially resolved cellular and molecular composition of CRC. In addition to mapping the intratumoral heterogeneity of CMS and their microenvironment, we identified cell communication events in the tumor-stroma interface of CMS2 carcinomas. This includes tumor growth-inhibiting as well as -activating signals, such as the potential regulation of the ETV4 transcriptional activity by DCN or the PLAU-PLAUR ligand-receptor interaction. Our study illustrates the potential of ST to resolve CRC molecular heterogeneity and thereby help advance personalized therapy.

CCR5 and CCL5 gene expression in colorectal cancer: comprehensive profiling and clinical value

BackgroundThe C-C motif chemokine receptor 5 (CCR5)/C-C motif chemokine ligand 5 (CCL5) axis plays a major role in colorectal cancer (CRC). We aimed to characterize the molecular features associated with...

Tumor-related IGF2BP1-derived molecular subtypes to predict prognosis and immune microenvironment in head and neck squamous cell carcinoma.

Recent studies have underscored the biological significance of RNA modifications in tumorigenicity and progression. However, the potential roles of RNA modifications in immune regulation and the formation of the tumor microenvironment (TME) in head and neck squamous carcinoma (HNSC) remain unclear. We collected 199 untreated HNSC samples and clinicopathological data from Fujian Provincial Cancer Hospital. MeRIP-seq and RNA-seq were performed to generate methylation and gene expression profiles, respectively. Consensus molecular subtyping was employed to identify prognosis-related genes and RNA modification patterns in HNSC. Experiments confirmed the potential oncogenic behavior influenced by key genes. Molecular subtypes were identified through consensus clustering and validated using external cohort validation sets. Among the RNA modification-related genes, IGF2BP1 emerged as the most prognostic. HNSC patients were categorized into high and low IGF2BP1 expression groups. High-expressing patients exhibited poorer survival and reduced chemosensitivity, coupled with increased tumor mutational burden, low PD-L1 expression, and limited immune cell infiltration, indicative of aggressive disease. Analysis revealed two distinct RNA modification patterns associated with IGF2BP1 expression: biosynthetically intense type (BIT) and oncogenically active type (OAT), each characterized by distinct clinical features, outcomes, and biological pathways. In an independent immunotherapy cohort, BIT patients displayed enhanced immune responses and sustained clinical benefits. This study highlights the crucial link between RNA modification and TME diversity. Evaluating RNA modification in tumors improves our understanding of TME features and supports the development of effective immunotherapy strategies.