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In colorectal cancer, peritoneal metastases (PMs) associate with severe morbidity and dismal prognosis. Given the incidence of this disease and the lack of adequate treatments currently available, PMs pose a large unmet clinical need. Although PMs can be accompanied by more widespread metastatic disease, it often occurs as the only sign of dissemination. This implies that the route of metastatic spread to the peritoneum differs from that to distant organs. PMs are thought to result from cancer cells that spill into the abdominal cavity, and are able to attach to the peritoneal lining and form tumor deposits. This cascade places specific demands on the cancer cells. Here, we report that colorectal cancers that present with PMs almost universally classify as consensus molecular subtype 4 (CMS4). This previously recognized disease entity is characterized by mesenchymal features, poor prognosis, and resistance to therapies currently used against peritoneal metastases, which explains their limited efficacy. By leveraging disease models that capture CMS4-specific features, including the ability to form PMs in vivo, we identified elesclomol as a highly effective agent. Elesclomol kills cancer cells in a copper-dependent fashion by targeting the oxidative phosphorylation machinery, which we found to be a specific vulnerability of CMS4 cancers. Elesclomol-Cu2+ was effective following only minutes of exposure to CMS4 cell lines and organoids, supporting its use in intra-abdominal treatment procedures. It is therefore a promising candidate for the local treatment of peritoneal metastases of colorectal cancer. Citation Format: Sanne Bootsma, Mark P. Dings, Leandro Ferreira Moreno, Kristiaan J. Lenos, Louis Vermeulen, Maarten F. Bijlsma. Effective treatment of colorectal peritoneal metastases by exploiting a molecular subtype specific vulnerability [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 272.

Cancers develop in a stepwise fashion as mutations in oncogenes and tumor suppressor genes accumulate. The mutations provide the cells in which they occur typically an advantage over the surrounding wild type neighbors, resulting in clonal expansion. Understanding the nature of the competitive benefit of oncogenic cells might result in the identification of targets for prevention of cancer. In particular in individuals affected by hereditary cancer syndromes, in which the initiating mutations are well-described, this is a promising strategy. I will discuss our work on intestinal carcinogenesis and the impact on stem cell properties of mutations associated with familial cancer syndromes affecting the gut. In particular I will discuss how inactivation of the tumor suppressor gene APC, which is affected in individuals suffering from Familial Adenomatous Polyposis (FAP), generates supercompetitor cells that actively suppress their neighbors. As we will see, the uncovered mechanism directly suggests a potent chemopreventive strategy that is currently being tested in a proof-of-concept clinical trial in FAP individuals. Citation Format: Louis Vermeulen. Outcompeting cancer in the intestine [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr IA007.

Personalized cancer medicine is becoming increasingly important in colorectal cancer treatment. Especially for targeted therapies, large variations between individual treatment responses exist. Predicting therapy response is of utmost significance, as it prevents overtreatment and adverse effects in patients. For EGFR-targeted therapy, many mechanisms of resistance have been uncovered, for example, mutations in KRAS and BRAF, and upregulation of alternative receptors. Currently, routine testing for all known modifiers of response is unpractical, and as a result, decision-making for anti-EGFR therapy is still largely based on assessing the mutation status of an individual gene (KRAS). Recently, comprehensive classifications of colorectal cancer have been presented that integrate many of the (epi-)genetic and microenvironmental factors that contribute to colorectal cancer heterogeneity. These classification systems are not only of prognostic value but also predict therapy efficacy, including the response to anti-EGFR agents. Therefore, molecular subtype–based stratification to guide therapeutic decisions is a promising new strategy that might overcome the shortcomings of single gene testing in colorectal cancer as well as in other malignancies. Furthermore, the development of new agents in a disease subtype–specific fashion has the potential to transform drug-discovery studies and generate novel, more effective therapies. Cancer Res; 75(2); 245–9. ©2014 AACR.

Background: There has been enormous effort to develop a prognostic genomic classification of colorectal cancer (CRC). As a result, mismatch repair (MMR) status was established as a prognostic marker in addition to TNM-staging. Phenotypic subtypes were recently proposed that stratified patient survival based on assessing the immune infiltrate (KM grade), tumour growth (Ki67) and stromal infiltrate (TSP). However their clinical utility had not been compared to other proposed classification systems including consensus molecular subtypes (CMS) or current prognostic markers including MMR status. Methods: Three patient cohorts, a pilot cohort of 237 stage I-III CRC patients, a validation cohort of 879 stage I-III CRC patients, and the AMC-AJCCII-90 cohort with 81 stage II colon cancer patients were utilised to investigate associations between phenotypic subtypes, MMR status, CMS and patient survival. Results: In the pilot cohort, phenotypic subtype stratified cancer-specific survival (P Conclusions: Phenotypic subtype is a more effective prognostic classification than CMS and MMR status. Phenotypic subtype is clinically relevant to all patients irrespective of site and adjuvant treatment. Phenotypic subtypes should be incorporated alongside MMR status as a clinical aid for the prognosis of patients with CRC. Citation Format: Antonia Kathryn Roseweir, James H. Park, Sanne ten Hoorn, Arfon G. Powell, Campbell S. Roxburgh, Donald C. McMillan, Paul G. Horgan, Louis Vermeulen, Joanne Edwards. Phenotypic subtypes successfully stratify prognosis of patient with colorectal cancer: A step towards precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4615.

The recently developed concept of cancer stem cells (CSC) sheds new light on various aspects of tumor growth and progression. Here, we present a mathematical model of malignancies to investigate how a hierarchical organized cancer cell population affects the fundamental properties of solid malignancies. We establish that tumors modeled in a CSC context more faithfully resemble human malignancies and show invasive behavior, whereas tumors without a CSC hierarchy do not. These findings are corroborated by in vitro studies. In addition, we provide evidence that the CSC model is accompanied by highly altered evolutionary dynamics compared with the ones predicted to exist in a stochastic, nonhierarchical tumor model. Our main findings indicate that the CSC model allows for significantly higher tumor heterogeneity, which may affect therapy resistance. Moreover, we show that therapy which fails to target the CSC population is not only unsuccessful in curing the patient, but also promotes malignant features in the recurring tumor. These include rapid expansion, increased invasion, and enhanced heterogeneity. Cancer Res; 70(1); 46–56

Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Accurately identifying stage II CRC patients at high risk of recurrence and stage III patients at low risk of recurrence are key unmet clinical needs. We previously identified KCNQ1 as a tumour suppressor gene of which loss of expression was associated with poor survival in patients with CRC liver metastases. The present study aimed to examine the prognostic value of KCNQ1 in stage II and III colon cancer patients. Methods: KCNQ1 mRNA expression was assessed in 90 stage II colon cancer patients (AMC-AJCCII-90) using microarray gene expression data. KCNQ1 protein expression was evaluated by immuno-histochemistry on tissue microarrays of 386 stage II and III colon cancer patients. Results: Low KCNQ1 mRNA expression in microsatellite stable (MSS) stage II colon cancers was associated with poor disease free survival (DFS) (HR 3.35; 95% CI 1.16-9.66; p Conclusion: We conclude that KCNQ1 is a strong prognostic biomarker for prediction of disease recurrence (HR∼4) and may aid stratification of patients with stage II MSS colon cancer and stage III MSI CRC for adjuvant chemotherapy. Because KCNQ1 protein expression is determined by immuno-histochemistry, this biomarker can be implemented in standard clinical care using existing workflows. Citation Format: Sjoerd H. den Uil, Veerle M.H. Coupe, Janneke F. Linnekamp, Evert van den Broek, Jeroen A.C.M. Goos, Pien M. Delis-van Diemen, Eric J.T. Belt, Nicole C.T. van Grieken, Patricia M. Scott, Louis Vermeulen, Jan Paul Medema, Herman Bril, Hein B.A.C. Stockmann, Robert T. Cormier, Gerrit A. Meijer, Remond J. Fijneman. KCNQ1 expression is a strong prognostic biomarker for disease recurrence in stage II and III colon cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3125.

Tumors are being increasingly perceived as abnormal organs that, in many respects, recapitulate the outgrowth and differentiation patterns of normal tissues. In line with this idea is the observation that only a small fraction of tumor cells is capable of initiating a new tumor. Because of the features that these cells share with somatic stem cells, they have been termed cancer stem cells (CSC). Normal stem cells reside in a “stem cell niche” that maintains them in a stem-like state. Recent data suggest that CSCs also rely on a similar niche, dubbed the “CSC niche,” which controls their self-renewal and differentiation. Moreover, CSCs can be generated by the microenvironment through induction of CSC features in more differentiated tumor cells. In addition to a role in CSC maintenance, the microenvironment is hypothesized to be involved in metastasis by induction of the epithelial-mesenchymal transition, leading to dissemination and invasion of tumor cells. The localization of secondary tumors also seems to be orchestrated by the microenvironment, which is suggested to form a premetastatic niche. Thus, the microenvironment seems to be of crucial importance for primary tumor growth as well as metastasis formation. Combined with its role in the protection of CSCs against genotoxic insults, these data strongly put forward the niche as an important target for novel therapies. Cancer Res; 71(3); 634–9. ©2011 AACR.

Colon cancer stem cells (CSC) can be identified with AC133, an antibody that detects an epitope on CD133. However, recent evidence suggests that expression of CD133 is not restricted to CSCs, but is also expressed on differentiated tumor cells. Intriguingly, we observed that detection of the AC133 epitope on the cell surface decreased upon differentiation of CSC in a manner that correlated with loss of clonogenicity. However, this event did not coincide with a change in CD133 promoter activity, mRNA, splice variant, protein expression, or even cell surface expression of CD133. In contrast, we noted that with CSC differentiation, a change occured in CD133 glycosylation. Thus, AC133 may detect a glycosylated epitope, or differential glycosylation may cause CD133 to be retained inside the cell. We found that AC133 could effectively detect CD133 glycosylation mutants or bacterially expressed unglycosylated CD133. Moreover, cell surface biotinylation experiments revealed that differentially glycosylated CD133 could be detected on the membrane of differentiated tumor cells. Taken together, our results argue that CD133 is a cell surface molecule that is expressed on both CSC and differentiated tumor cells, but is probably differentially folded as a result of differential glycosylation to mask specific epitopes. In summary, we conclude that AC133 can be used to detect cancer stem cells, but that results from the use of this antibody should be interpreted with caution. Cancer Res; 70(2); 719–29

BACKGROUND Recent molecular profiling of colorectal cancer has identified three distinct subtypes: two good-prognosis subtypes characterised by chromosomal instability (Subtype 1) and microsatellite instability (Subtype 2), and a poor prognosis subtype defined by epithelial-mesenchymal transition and stemness (Subtype 3). A key challenge lies in the subsequent implementation of this classification system in the clinic, where the requirement for sufficient bulk tumour and high cost hampers the widespread use of genomic profiling. In this proof-of-concept study, we have adapted our molecular signatures to an automated immunohistochemistry (IHC)-based classifier which can be used a rapid screening tool, and validated the feasibility of this approach in a multi-centre study. PATIENTS AND METHODS A total of 1080 patients from four different centres were used in this study. Tumour microarrays (TMAs) from each patient were stained using immunohistochemistry for a panel of five stains in conjunction with microsatellite instability status (MSS). An automated image analysis pipeline was developed to quantitate and normalize all staining. A classifier to distinguish colorectal cancer subtypes was trained on 74 patients from a single centre and applied to the three remaining cohorts. RESULTS We have developed an automated IHC-based classification system which demonstrated 84% correlation with our genomic profiling system. The prognostic value of colorectal cancer subtyping was validated in three independent cohorts, taking into account age, sex and stage (Hazards Ratios with 95% Confidence Interval: 1.8 (1.2 - 2.6), 1.4 (1.1 - 1.7) and 1.2 (1.0 - 1.6)). We evaluated the predictive value of the subtyping system in a retrospective analysis of a late-stage clinical trial and demonstrated the benefit of adjuvant cetuximab in KRAS/BRAF wild type Subtype 1 patients (Hazards Ratio 0.6 (0.4-0.9)) but not in Subtype 3 patients. CONCLUSION In this proof-of-concept study we have demonstrated the effectiveness of an automated IHC-TMA classifier as a surrogate for genomic profiling. Using this approach, we have validated the prognostic value of colorectal subtyping in a multi-centre study, and shown the predictive value of subtyping for adjuvant cetuximab therapy. This approach has demonstrated high clinical potential as a rapid screening tool, in particular in retrospective examination of patient cohorts where only formalin-fixed paraffin-embedded (FFPE) tissue is available. Citation Format: Anne Trinh, Filipe De Sousa E Melo, Xin Wang, Joan H de Jong, Evelyn Fessler, Marnix Jansen, Gerrit KJ Hooijer, Jan Paul Medema, Florian Markowetz, Louis Vermeulen. Automated immunohistochemistry-based identification of molecular subtypes in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-108. doi:10.1158/1538-7445.AM2015-LB-108

The outcome of pancreatic ductal adenocarcinoma (PDAC) remains dismal. Factors that contribute to the lethality of this disease include its intrinsic resistance to therapeutics, an aggressive growth pattern, and a large degree of both inter- as well as intratumor heterogeneity. All these features have been attributed (at least in part) to the presence and function of a population of pluripotent cells called cancer stem cells. Despite the alleged importance of these cells for clinical outcome, the methods that have been used to study these cells in PDAC are troubled by some serious caveats. Defining important quantitative parameters for these cells in an unbiased, marker-agnostic, way is called for and we aim to achieve this by unbiased tracing methods in patient derived xenografts. A panel of PDX-derived lines generated in our institute was labeled using the LeGO (Lentiviral Gene Ontology; Weber et al. 2012 Nat. Prot) system to enable tracking of single cell clones by color. The system utilizes three vectors coding for red, green, and blue fluorophores much like a television uses these colors to generate an almost infinite range of colors in the visible range. Nuances in color and intensity are generated by the many possible combinations of fluorophores, the different integration numbers of the genes coding for them, and the variations in expression levels determined by for instance the integration sites. The end result is a population of cells in which each cell is endowed with a unique color. This will allow detection of the offspring of such a cell by identifying clones within a structural unit in for instance a tumor, or in a culture dish, of similar color. The ability to give rise to such a structure is then, depending on the experimental context and outcome, considered a proxy for stem cell function. Several primary lines have proven amenable to LeGO-marking. These lines showed stable color patterns during culturing in vitro as analyzed by continuous FACS measurements, and tumors derived from marked cells did not show a difference to umarked tumors as assessed by gross histology and growth rate. We are currently testing the ability of these cells to faithfully occupy and mark structural units in PDAC, much like intestinal stem cells would in a lineage tracing experiment in the gut, and derive numbers for stem cell number as well as replacement rate. Experiments are currently being performed to assess the system's potential to measure parameters associated with stemness, and possibly also differentiation status of the original tumor, as the cells grow in vitro. (Radio)chemotherapy regimens are included to formally address the purported resistance of cancer stem cells to these treatments without having to rely on an a priori chosen marker. Furthermore, more detailed comparison should reveal use of LeGO-derived data to measure the intra- and intertumor heterogeneity in PDAC tumor biology in our panel of PDXs. Citation Format: Veronique Veenstra, Helene Damhofer, Tom van Leusden, Jan Kessler, Jan Paul Medema, Hanneke van Laarhoven, Louis Vermeulen, Maarten Bijlsma. Heterogeneity of pancreatic ductal adenocarcinoma visualized. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2015-5177

Colon cancer is the fourth leading cause of cancer-related death worldwide. Extensive studies of the Wnt signal cascade have determined its role in colon cancer development and progress. Several targets of the Wnt-cascade like APCDD1, ASCL2, AXIN2 or DKK1 are thought to reduce Wnt activity by negative feedback. As a result, expression of these genes prevents pathway activation and silencing of these targets can cause proliferation of the tumor. Recently, we have shown that silencing of Wnt target genes by DNA-methylation predicts poor prognosis in colon cancer patients. Moreover, demethylation of those genes results in reexpression and is associated with reduced tumor growth in mice. Hence, treatment with demethylating agents can be beneficial for a subgroup of colon cancer patients with high methylation of Wnt target genes. The aim of this study is to clarify whether Wnt target gene methylation is an important driver in development of colon cancer and can be an effective target in colon cancer. To determine the role of Wnt target genes in colon cancer more extensively, we study the effect of selective expression of these genes via an inducible vector system in established colorectal cell lines and in patient derived cultures that display CpG island methylation on these genes. In that way, we can monitor the effect of induced overexpression on tumor growth, tumor invasion and sensitivity towards chemotherapeutic agents. As a control, baseline expression in the same cell population, but in an uninduced state will be used. Furthermore, we are also studying the effect of the demethylating agent decitabine on Wnt target gene methylation in a clinical trial in stage II/III colon cancer patients. Patients are pre-operatively treated with decitabine. Tumor material prior to decitabine treatment obtained during endoscopy is compared to resection material after treatment. Differences in methylation and expression of Wnt target genes are determined by respectively pyrosequencing and q-PCR. We hypothesize that reexpression of Wnt target genes via an inducible vector results in lower Wnt pathway activity through negative feedback. As a result, tumor growth and invasion will be reduced and might be more sensitive towards chemotherapeutics. To translate this into the clinic, demethylation of Wnt target genes by decitabine will hopefully give a similar effect in patients. Citation Format: Janneke Linnekamp, Raju Kandimalla, Louis Vermeulen, Hanneke van Laarhoven, Jan Paul Medema. Role of methylation of Wnt target genes in tumorigenesis and effect of re-expression with demethylating agent decitabine in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5273. doi:10.1158/1538-7445.AM2015-5273

Background: Gene expression-based subtyping is widely accepted as a relevant source of disease stratification. Despite the widespread use, its translational and clinical utility is hampered by discrepant results, likely related to differences in data processing and algorithms applied to diverse patient cohorts, sample preparation methods, and gene expression platforms. In the absence of a clear methodological gold standard to perform such analyses, a more general framework that integrates and compares multiple strategies is needed to define common disease patterns in a principled, unbiased manner. Methods: We formed a consortium of 6 independent experts groups - each with a previously published CRC classifier, ranging from 3 to 6 subtypes - to understand similarities and differences of their subtyping systems. Sage Bionetworks functioned as neutral party to aggregate public and proprietary data (Synapse platform) and perform meta-analysis. Each group applied its CRC subtyping signature to the collection of data sets with gene expression (n = 4,151, predominantly stage II and III). Using the resulting subtype labels, we developed a network-based model and applied a Markov cluster algorithm to detect robust network substructures that would indicate recurring subtype patterns and therefore a consensus subtyping system. Correlative analyses using clinico-pathological, genomic and epigenomic features was performed to robustly characterize the identified subtypes. Results: This analytical framework revealed significant interconnectivity between the six independent classification systems, leading to the identification of four biologically distinct consensus molecular subtypes (CMS) enriched for key pathway traits: CMS1 (MSI Immune), hypermutated, microsatellite unstable, with strong immune activation; CMS2 (Canonical), epithelial, chromosomally unstable, with marked WNT and MYC signaling activation; CMS3 (Metabolic), epithelial, with evident metabolic dysregulation; and CMS4 (Mesenchymal), prominent TGFβ activation, angiogenesis, stromal invasion. Patients diagnosed with MSI Immune tumors had worse survival after relapse and those with mesenchymal tumors had increased risk of metastasis and worse overall survival. Discussion: We describe a novel methodological paradigm for deriving benchmarks of disease subtyping. Our work represents the first example of a community of experts identifying and advocating for a single reproducible model for cancer subtyping, effectively unifying previous classifiers. In the CRC domain, the uniformity afforded by this new classification system and its application to a large data set revealed important subtype-specific biological associations that were previously unnoticed or marginally significant, supporting a new taxonomy of the disease. Citation Format: Justin Guinney, Rodrigo Dienstmann, Xin Wang, Aurelien de Reynies, Andreas Schlicker, Charlotte Soneson, Laetitia Marisa, Paul Roepman, Gift Nyamundanda, Paolo Angelino, Brian Bot, Jeffrey S. Morris, Iris Simon, Sarah Gerster, Evelyn Fessler, Felipe de Sousa e Melo, Edoardo Missiaglia, Hena Ramay, David Barras, Krisztian Homicsko, Dipen Maru, Ganiraju Manyam, Bradley Broom, Valerie Boige, Ted Laderas, Ramon Salazar, Joe W. Gray, Josep Tabernero, Rene Bernards, Stephen Friend, Pierre Laurent-Puig, Jan P. Medema, Anguraj Sadanandam, Lodewyk Wessels, Mauro Delorenzi, Scott Kopetz, Louis Vermeulen, Sabine Tejpar. Consensus molecular subtyping through a community of experts advances unsupervised gene expression-based disease classification and facilitates clinical translation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 603. doi:10.1158/1538-7445.AM2015-603