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The purpose of this study was to provide an evidence base for colorectal cancer research activity that might influence policy, mainly at the national level. Improvements in healthcare delivery have lengthened life expectancy, but within a situation of increased cancer incidence. The disease burden of CRC has risen significantly, particularly in Africa, Asia and Latin America. Research is key to its control and reduction, but few studies have delineated the volume and funding of global research on CRC. We identified research papers in the Web of Science (WoS) from 2007 to 2021, and determined the contributions of the leading countries, the research domains studied, and their sources of funding. We identified 62 716 papers, representing 5.7% of all cancer papers. This percentage was somewhat disproportionate to the disease burden (7.7% in 2015), especially in Eastern Europe. International collaboration increased over the time period in almost all countries except in China. Genetics, surgery and prognosis were the leading research domains. However, research on palliative care and quality-of-life in CRC was lacking. In Western Europe, the main funding source was the charity sector, particularly in the UK, but in most other countries government played the leading role, especially in China and the USA. There was little support from industry. Several Asian countries provided minimal contestable funding, which may have reduced the impact of their CRC research. Certain countries must perform more CRC research overall, especially in domains such as screening, palliative care and quality-of-life. The private-non-profit sector should be an alternative source of support.

Background Colorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1–4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue. Methods Using transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n = 577) and mouse (n = 57 across n = 8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers. Results We developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours. Conclusions When applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package enables researchers to select suitable mouse models of human CRC subtype for their experimental testing.

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.

Purpose: Precise mechanism-based gene expression signatures (GES) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signaling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumor samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumor stroma percentage can directly influence GESs, undermining the intended mechanistic signaling. Experimental Design: Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single-cell RNA sequencing and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely used GESs to be influenced, or confounded, by stromal content in tumor tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets. Results: Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumor models not fully recapitulating the stromal and immune microenvironment. Conclusions: Efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.

Background. Two pre-operative radiotherapy (RT) regimens are in common use for patients (pts) with locally advanced rectal cancer (LARC): Conventionally fractionated, long course chemoradiation (LCCRT, 25x1.8Gy with concomitant chemotherapy) or hypofractionated short-course RT (5X5Gy) followed by systemic chemotherapy (SCRT-C). RT is thought to induce immune responses to cellular damage, but the understanding of their evolution and relation to fractionation is limited. We are conducting a serial biospecimen collection in such pts with baseline, 2, 6 and 12 week (wk) sampling. We report serial measurement of tumor and peripheral blood lymphocyte responses for each regimen. Method. Multiplex immunofluorescence quantified tumor infiltrating lymphocytes (CD8, FOXP3). Routine diagnostic flow cytometry quantified circulating lymphocytes. A multiplex ELISA quantified cytokines in blood plasma. Bulk RNA-sequencing (QuantSeq) quantified gene expression within the tumor. Result. We report results for 20pts who received RT for stage III/IV LARC (13 LCCRT, 7 SCRT-C). Biopsy results are available for 10pts, circulating lymphocytes for 20pts and peripheral blood cytokines for 16pts. In LCCRT patients (n=8), relative to baseline, tumor infiltrating cytotoxic (CD8+) T cells were uniformly decreased during (2wks, P Conclusion. LCCRT caused a drop in T cells during treatment, whilst SCRT-C appears to induce intra-tumoral T cell responses from wk2 and abrogates systemic reactions to a lesser extent. These results require evaluation in a larger cohort but have implications for understanding how RT induces microenvironmental changes and impacts pelvic bone marrow. We show in vivo that SCRT may be more immunostimulatory in LARC, with implications for trials combining RT with immunotherapy. Citation Format: Lily V. Hillson, Ross K. McMahon, Kathryn A. Pennel, Jean A. Quinn, Leia Jones, Raheleh Amirkhah, Aula Ammar, Phimmada Hatthakarnkul, Annabelle Ferguson, Simon W. Milling, Alec McDonald, Philip D. Dunne, Joanne Edwards, Sean M. O'Cathail, Campbell S. Roxburgh. Temporal changes in intratumoral and systemic lymphocytes in response to short and long course radiotherapy regimens in locally advanced rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3231.

Cancer cells’ ability to inhibit apoptosis is key to malignant transformation and limits response to therapy. Here, we performed multiplexed immunofluorescence analysis on tissue microarrays with 373 cores from 168 patients, segmentation of 2.4 million individual cells, and quantification of 18 cell lineage and apoptosis proteins. We identified an enrichment for BCL2 in immune, and BAK, SMAC, and XIAP in cancer cells. Ordinary differential equation-based modeling of apoptosis sensitivity at single-cell resolution was conducted and an atlas of inter- and intra-tumor heterogeneity in apoptosis susceptibility generated. Systems modeling at single-cell resolution identified an enhanced sensitivity of cancer cells to mitochondrial permeabilization and executioner caspase activation compared to immune and stromal cells, but showed significant inter- and intra-tumor heterogeneity.

Inhibitors of apoptosis proteins (IAPs) are intracellular proteins, with important roles in regulating cell death, inflammation, and immunity. Here, we examined the clinical and therapeutic relevance of IAPs in colorectal cancer. We found that elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in patients with microsatellite stable (MSS) stage III colorectal cancer treated with 5-fluorouracil (5FU)–based adjuvant chemotherapy, suggesting their involvement in promoting chemoresistance. A novel IAP antagonist tolinapant (ASTX660) potently and rapidly downregulated cIAP1 in colorectal cancer models, demonstrating its robust on-target efficacy. In cells co-cultured with TNFα to mimic an inflammatory tumor microenvironment, tolinapant induced caspase-8–dependent apoptosis in colorectal cancer cell line models; however, the extent of apoptosis was limited because of inhibition by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinapant-induced apoptosis was augmented by FOLFOX in human colorectal cancer and murine organoid models in vitro and in vivo, due (at least in part) to FOLFOX-induced downregulation of class I histone deacetylases (HDAC), leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, the effects of FOLFOX could be phenocopied using the clinically relevant class I HDAC inhibitor, entinostat, which also induced acetylation of Ku70 and FLIP downregulation. Further analyses revealed that caspase-8 knockout RIPK3-positive colorectal cancer models were sensitive to tolinapant-induced necroptosis, an effect that could be exploited in caspase-8–proficient models using the clinically relevant caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX in poor prognosis MSS colorectal cancer with elevated cIAP1/2 expression.

Modern methods of acquiring molecular data have improved rapidly in recent years, making it easier for researchers to collect large volumes of information. However, this has increased the challenge of recognizing interesting patterns within the data. Atlas Correlation Explorer (ACE) is a user-friendly workbench for seeking associations between attributes in The Cancer Genome Atlas (TCGA) database. It allows any combination of clinical and genomic data streams to be searched using an evolutionary algorithm approach. To showcase ACE, we assessed which RNA sequencing transcripts were associated with estrogen receptor (ESR1) in the TCGA breast cancer cohort. The analysis revealed already well-established associations with XBP1 and FOXA1, but also identified a strong association with CT62, a potential immunotherapeutic target with few previous associations with breast cancer. In conclusion, ACE can produce results for very large searches in a short time and will serve as an increasingly useful tool for biomarker discovery in the big data era. Significance: ACE uses an evolutionary algorithm approach to perform large searches for associations between any combinations of data in the TCGA database.

153 Background: Treatment response to neoadjuvant therapy in locally advanced rectal cancer (LARC) remains heterogenous. Clinicians are guided by pre-treatment clinical assessment alone in determining neoadjuvant strategy. More must be done to uncover biological mechanisms underpinning response and resistance. We have developed a biospecimen collection protocol in LARC performing serial sampling of tumors and peripheral blood samples prior to, during and after treatment to characterize the biological evolution of this heterogenous response. Here we present early proof of concept results with a focus on the intra-tumoral immune response relating to radiotherapy (RT). Methods: Patients receiving standard-of-care neoadjuvant RT were recruited to an ethically approved study between Dec 2018 - Aug 2021. The protocol consisted of a baseline biopsy and blood sample prior to RT followed by repeat sampling at 2, 6 and 12wks after Day 1 of RT. Standard immunohistochemistry (IHC) was performed for markers of immune activity. Target capture sequencing was performed using RNA baits extracted from serial biopsies to target a 276 genes panel. Paired tumor-normal sequencing was performed. Bulk 3’ RNA seq (Lexogen Illumina Quantseq) characterized immune and inflammatory gene expression. A multiplex bead array (Luminex xPONENT) of 24 cytokines and chemokines was performed using serial plasma samples. Results: 17pts were recruited, 3 with stage IV disease. 14pts received chemoradiation and 3 pts had short-course based regimens. Treatment responses were evaluable in 14pts: graded complete in 2pts; good/ near complete in 7pts and partial/ poor in 5pts. All tumors were MSS, and most frequently mutated genes were APC (75%), KRAS (38%), NRAS (25%) , NOTCH1 (25%) and PIK3CA (25%) (n = 8). Quantseq demonstrated that the immune/ inflammatory response, as measured by interferon-gamma response and IL-6/ JAK-STAT signaling, was significantly elevated up to 12wks after Day 1 RT, with a peak at around 6wks (n = 3). Correlative IHC showed an increase in innate immune cells in pts with a favorable response at 6wks (n = 8). Cytokine/ chemokine analysis suggested patients with a favorable response demonstrated strong inflammatory (MCP1 & IL-17a) responses 2 and 6wks post-RT and strong CTL (Granzyme B) and Th1 (GM-CSF & IP-10) responses 12wks post-RT (n = 10). Conclusions: We show acquisition of meaningful genomic and transcriptomic material from serial biopsies in rectal cancer is possible. Early data suggest that dynamic profiling of rectal tumors demonstrates transcriptomic evolution during treatment. Specifically, we show that the immune response to radiotherapy peaks at around 6wks after initiation of RT and persists to 12wks. This supports ongoing trials of immunomodulatory treatments in combination with, and following, RT in rectal cancer. Further work is required to define differences between responders and non-responders.

Stromal cells of mesenchymal origin reside below the epithelial compartment and provide structural support in the intestine. These intestinal stromal cells interact with both the epithelial cell compartments, as well as infiltrating hematopoietic immune cells. The importance of these cells in regulating immune homeostasis during inflammation is well recognized. However, little is known about their function and phenotype in the inflammatory tumor microenvironment. Using a syngeneic, immunogenic model of colorectal cancer, we showed that TNFa-initiated inflammatory signaling in CT26 colorectal cancer cells selectively induced PD-L1 expression in stromal cells. Using CD274 shRNA and antibodymediated approaches, we showed that stromal cell PD-L1 potentiated enhanced immunosuppression, characterized by inhibition of activated CD8(+) granzyme B-secreting T cells in vitro, and the inhibition of CD8(+) effector cells was associated with enhanced tumor progression. Stromal cell immunosuppressive and tumor-promoting effects could be reversed with administration of anti-PD-1 in vivo. We validated our findings of stromal cell CD274 expression in two cohorts of clinical samples and also observed PD-L1 induction on human stromal cells in response to exposure to the inflammatory secretome fromhuman colon cancer cells, irrespective of microsatellite instability. Collectively, our data showed that tumor-associated stromal cells support T-cell suppression by PD-L1 induction, which is dependent on colon cancer inflammatory signaling. Our findings reveal a key role of mesenchymal stromal cells PD-L1 in suppression of CD8(+) antitumor immune responses and potentiation of colorectal cancer progression. (C) 2018 AACR. The authors would like to acknowledge financial support from the Irish Cancer Society (CRF12RYA) and Science Foundation Ireland (15/SIRG/3456) and Galway University Foundation to Dr. A.E. Ryan, a postgraduate scholarship from the Irish Research Council (GOIPG/2013/998) to G O’Malley, and a grant from Science Foundation Ireland (12/IA/1624) to T. Ritter. The authors thank Professor Matthew Griffin, Professor Rhodri Ceredig, and Dr. Declan McKernan for helpful discussions; Ms. Georgina Shaw, Dr. Joana Cabral, and Ms. Athina Rigalou for technical assistance; and Dr. Shirley Hanley in the NUIG Flow Cytometry core facility for technical expertise. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. peer-reviewed 2019-11-01

We read with interest the description by Niu et al of punctate immunostaining for the mismatch repair (MMR) protein MLH1 in a small series of cases of endometrial cancer, in association with complete loss of PMS2 immunostaining. (1) The authors conclude that unawareness of this rare pattern of aberrant MLH1 staining can result in erroneous reporting of such cases as isolated loss of PMS2 staining, interpreted as representing likely Lynch syndrome due to germline mutation, whereas in fact somatic hypermethylation of the MLH1 promoter is the likely underlying molecular mechanism leading to defective mismatch repair in most such cases. We have observed this phenomenon similarly in a series of colon cancers, to the best of our knowledge not previously described in peer-reviewed literature, and herein describe our series, which support the conclusions of Niu et al.

BackgroundImmunosuppressive tumour microenvironments (TME) reduce the effectiveness of immune responses in cancer. Non-haematopoietic mesenchymal stromal cells, precursors to cancer-associated fibroblasts (CAFs), dictate tumour progression by enhancing immune cell suppression. Sialic acids, which exist as terminal sugars of glycans (known as sialoglycans), are highly expressed on cancer cells and hyper-sialylation of glycans is known to promote immune evasion in cancer. Sialoglycans are recognized by sialic acid-binding immunoglobulin-like lectins (Siglecs), a family of immunomodulatory receptors, which are analogous to the immune checkpoint inhibitor PD-1.1 The role of sialyation in stromal cell-mediated immunosuppression, however, is unknown. Using models of solid (colorectal cancer - CRC) and haematological (multiple myeloma - MM) stromal-rich tumours in both mouse and human, the aim of this study was to investigate if stromal cell sialylation contributes to enhanced immunosuppression in the TME.MethodsFlow cytometric analysis of sialic acid expression was performed initially on bone marrow-derived stromal cells isolated from healthy human donor bone marrow aspirates, from wild-type Balb/c mice or from 5T33 multiple myeloma mice. Stromal cells were also isolated and expanded from colorectal cancer patient tumour biopsies (CAFs) with matched controls isolated from tumour-adjacent non-cancerous tissue (normal-associated fibroblasts - NAFs) or from whole blood from primary multiple myeloma bone aspirates. Informed consent was obtained from all patients prior to sampling. Immunosuppression assays were performed using these stromal cells with or without exposure to the tumour cell secretome from the mouse and human CRC cell lines CT26 or HCT116 and HT29, respectively, co-cultured with either murine lymphocytes or healthy human donor-derived peripheral blood mononuclear cells (PBMCs).ResultsOur results showed that tumour conditioned stromal cells have increased levels of sialyltransferase gene expression, α2,3/α2,6-linked sialic acid and Siglec ligands. Co-culture assays revealed that CAFs induced significantly higher frequencies of Siglec 7 and Siglec 9-expressing CD8 T cells, as well as Tim-3 and PD-1-expressing CD8 T cells, compared to NAFs. Inhibition of sialyltransferase activity using the inhibitor 3FAXNeu5Ac reversed these CAF-induced effects. Interestingly, sialyltransferase inhibition had no observed effects on T cells co-cultured with NAFs.ConclusionsThese results demonstrate that targeting stromal cell sialylation can reverse immune cell suppression and reactivate exhausted T cells. These novel data support a rationale for the assessment of stromal cell sialylation and Siglec ligand expression in order to better stratify patients for immunotherapeutic combination treatments that aim to reactivate exhausted T cells in stromal-enriched tumour microenvironments.AcknowledgementsThe authors would like to thank the Blood Cancer Network of Ireland Biobank for providing bone marrow aspirates.ReferenceGray MA, Stanczak MA, Mantuano NR, Xiao H, Pijnenborg JFA, Malaker SA, Miller CL, Weidenbacher PA, Tanzo JT, Ahn G, Woods EC, Läubli H, Bertozzi CR. Targeted glycan degradation potentiates the anticancer immune response in vivo. Nat Chem Biol 2020;16:1376–1384.Ethics ApprovalColorectal tumor and adjacent normal mucosal tissue were obtained from patients undergoing colon tumor resection at University Hospital Galway under an ethically approved protocol (Clinical Research Ethics Committee, Ref: C.A. 2074). Samples were collected and isolated by the Blood Cancer Network of Ireland under an ethically approved protocol. Written informed explicit consent was obtained from all patients prior to sampling. Mice were housed and maintained following the conditions approved by the Animals Care Research Ethics Committee of the National University of Ireland, Galway (NUIG) and procedures were conducted under individual and project authorisation licenses from the Health Products Regulatory Authority (HPRA) of Ireland or from the Ethical Committee for Animal Experiments, Vrije Universiteit Brussel (license no. LA1230281, 16-281-6).

BACKGROUNDSignet ring cell colorectal cancer (SRCCa) has a bleak prognosis. Employing molecular pathology techniques we investigated the potential of precision medicine in this disease.METHODSUsing test (n=26) and validation (n=18) cohorts, analysis of mutations, DNA methylation and transcriptome was carried out. Microsatellite instability (MSI) status was established and immunohistochemistry (IHC) was used to test for adaptive immunity (CD3) and the immune checkpoint PDL1.RESULTS DNA methylation data split the cohorts into hypermethylated (n=18, 41%) and hypomethylated groups (n=26, 59%). The hypermethylated group predominant in the proximal colon was enriched for CpG island methylator phenotype (CIMP), BRAF V600E mutation and MSI (PC) across both groups. No statistically significant difference in outcome was observed between the two groups.CONCLUSIONS Our data show that SRCCa phenotype comprises two distinct genotypes. The MSI(+)/CIMP(+)/BRAF V600E(+)/CD3(+)/PDL1(+) hypermethylated genotype is an ideal candidate for immune checkpoint inhibitor therapy. In addition, one fourth of SRCCa cases can potentially be targeted by KIT inhibitors.

Colorectal cancer (CRC) results from a transformation of colonic epithelial cells into adenocarcinoma cells due to genetic and epigenetic instabilities, alongside remodelling of the surrounding stromal tumour microenvironment. Epithelial-specific epigenetic variations escorting this process include chromatin remodelling, histone modifications and aberrant DNA methylation, which influence gene expression, alternative splicing and function of non-coding RNA. In this review, we first highlight epigenetic modulators, modifiers and mediators in CRC, then we elaborate on causes and consequences of epigenetic alterations in CRC pathogenesis alongside an appraisal of the complex feedback mechanisms realized through alternative splicing and non-coding RNA regulation. An emphasis in our review is put on how this intricate network of epigenetic and post-transcriptional gene regulation evolves during the initiation, progression and metastasis formation in CRC.

Neoadjuvant chemoradiotherapy is commonly used to treat rectal cancer but patients have different levels of response and/or toxic effects. As part of the Stratification in COloRecTal cancer (S:CORT) programme, we collected 257 rectal biopsies from two cohorts: Grampian (single hospital) and Aristotle (clinical trial). All patients had been subsequently treated with identical regimen of neoadjuvant radiotherapy and capecitabine. We performed trancriptomic, mutation and copy number profiling and aimed to identify biomarkers associated with the robust pathological endpoint of complete response (CR). Key biological determinants were identified by linear regression of different pre-defined, hypothesis-driven biomarkers for radiotherapy response, adjusted by the known confounders T and N stage. A novel RNA signature was derived using a personalised bioinformatical pipeline using a wide range of machine learning approaches. Results were validated in a publicly available transcriptomic cohort of 107 patients treated with similar dose of radiotherapy and 5-fluorouracil infusion. Further comparision of the biological determinants and the novel RNA signature were performed in the same cohorts and also TCGA by linear regression. Previously published transcriptomic signatures were retrieved and assessed in the validation, unseen cohort. Grampian and Aristotle cohorts had similar statistical power and showed similar associations of CR with biological candidates, 10 of them being significant or borderline (p The immune, stromal and epithelial components of rectal tumours are important players for prediction of CR to radiotherapy in rectal cancer. A 33-gene transcriptomic biomarker can be used to effectively select patients that are highly likely to achieve CR allowing organ preservation while modulation of the relevant biological features in the other patients may be tested to improve their poor outcome with current treatment strategies. Citation Format: Enric Domingo, Sanjay Rathee, Andrew Blake, Leslie M. Samuel, Graeme I. Murray, David Sebag-Montefiore, Simon Gollins, Nicholas West, Rubina Begum, Marian Duggan, Laura White, Susan Richman, Philip Quirke, James Robineau, Keara Redmond, Aikaterini Chatzipli, Ultan McDermott, Ian Tomlinson, Philip Dunne, Francesca Buffa, Tim Maughan. Stratification of radiotherapy and fluoropyrimidine-based chemotherapy from multi-omic profiling in rectal cancer biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB129.

A recent phase II study of patients with metastatic colorectal carcinoma showed that mismatch repair gene status was predictive of clinical response to PD-1–targeting immune checkpoint blockade. Further examination revealed strong correlation between PD-L1 protein expression and microsatellite instability (MSI) in stage IV colorectal carcinoma, suggesting that the amount of PD-L1 protein expression could identify late-stage patients who might benefit from immunotherapy. To assess whether the clinical associations between PD-L1 gene expression and MSI identified in metastatic colorectal carcinoma are also present in stage II/III colorectal carcinoma, we used in silico analysis to elucidate the cell types expressing the PD-L1 gene. We found a statistically significant association of PD-L1 gene expression with MSI in early-stage colorectal carcinoma (P < 0.001) and show that, unlike in non–colorectal carcinoma tumors, PD-L1 is derived predominantly from the immune infiltrate. We demonstrate that PD-L1 gene expression has positive prognostic value in the adjuvant disease setting (PD-L1low vs. PD-L1high HR = 9.09; CI, 2.11–39.10). PD-L1 gene expression had predictive value, as patients with high PD-L1 expression appear to be harmed by standard-of-care treatment (HR = 4.95; CI, 1.10–22.35). Building on the promising results from the metastatic colorectal carcinoma PD-1–targeting trial, we provide compelling evidence that patients with PD-L1high/MSI/immunehigh stage II/III colorectal carcinoma should not receive standard chemotherapy. This conclusion supports the rationale to clinically evaluate this patient subgroup for PD-1 blockade treatment. Cancer Immunol Res; 4(7); 582–91. ©2016 AACR.

The Colorectal Cancer (CRC) Subtyping Consortium (CRCSC) recently published four consensus molecular subtypes (CMS’s) representing the underlying biology in CRC. The Microsatellite Instable (MSI) immune group, CMS1, has a favorable prognosis in early stage disease, but paradoxically has the worst prognosis following relapse, suggesting the presence of factors enabling neoplastic cells to circumvent this immune response. To identify the genes influencing subsequent poor prognosis in CMS1, we analyzed this subtype, centered on risk of relapse.In a cohort of early stage colon cancer (n=460), we examined, in silico, changes in gene expression within the CMS1 subtype and demonstrated for the first time the favorable prognostic value of chemokine-like factor (CKLF) gene expression in the adjuvant disease setting [HR=0.18, CI=0.04-0.89]. In addition, using transcription profiles originating from cell sorted CRC tumors, we delineated the source of CKLF transcription within the colorectal tumor microenvironment to the leukocyte component of these tumors. Further to this, we confirmed that CKLF gene expression is confined to distinct immune subsets in whole blood samples and primary cell lines, highlighting CKLF as a potential immune cell-derived factor promoting tumor immune-surveillance of nascent neoplastic cells, particularly in CMS1 tumors. Building on the recently reported CRCSC data, we provide compelling evidence that leukocyte-infiltrate derived CKLF expression is a candidate biomarker of favorable prognosis, specifically in MSI-immune stage II/III disease.

Background: Molecular subtyping of colon cancer (CC) has repeatedly identified a poor-prognostic group of patients, characterized by high levels of stroma (particularly fibroblast) in the tumor microenvironment. To date, the benefit of standard 5FU-based adjuvant chemotherapy in these high-fibroblast (HiFi) patients has been unclear. Given TGF-β signaling is associated with HiFi tumors, a number of recent clinical trials have focussed on targeting TGF-β in these patients. While these efforts are ongoing, we set out to identify novel therapeutically-relevant biological signaling within HiFi tumors. Methods: Untreated stage II (n=215) and stage II/III (n=258) tumors were assigned a fibroblast score, using single-sample gene set enrichment analysis, enabling stratification into HiFi and LoFi groups based on their histology and transcriptome. Supervised stratification, based on relapse-free survival, within the HiFi group allowed for in silico discovery, interrogation and independent validation of the HiFi-specific biology underpinning relapse. Upstream regulators of these processes were identified as potential therapeutic targets, and assessed in an in vitro co-culture model, to confirm mechanistic signaling, and an in vivo HiFi model, to confirm efficacy. Results: We confirmed the poor prognosis of the HiFi group (p = 0.008), followed by discovery and independent validation of the prognostic value of STAT1-related signaling in stratifying HiFi tumors based on disease relapse (HR 0.2 (0.1-0.5) and 0.09 (0.02-0.47)). This signaling was significantly associated with activation of antigen processing and presentation in specific immune lineages (p < 0.001). In line with the upstream regulator analysis, treatment with poly I:C (a TLR3 agonist) increased STAT1-related signaling and antigen processing in an in vitro macrophage-stromal co-culture system. Conclusions: We have found that increased levels of STAT1-related signaling, resulting in antigen processing and presentation in specific subclasses of immune cells, is associated with reduced risk of recurrence in the otherwise poor-prognostic HiFi subtype of CC. Using in silico and in vitro methods, we demonstrate that poly I:C is a potential therapeutic option for patients with stromal-rich tumors. Results from ongoing in vivo validation in a HiFi mouse model will provide preclinical evidence of the utility of poly I:C in this setting and support a phase II clinical trial. Citation Format: Amy M. McCorry, Niamh A. Leonard, Rene Jackstadt, Dustin J. Flanagan, Owen J. Sansom, Tim Maughan, Simon Leedham, Emma M. Kerr, Aideen E. Ryan, Mark Lawler, Philip D. Dunne, ACRCelerate and S:CORT consortia. STAT1-related antigen processing and presentation dictates prognosis in the fibroblast-rich subtype of stage II/III colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3867.

Colorectal cancer (CRC) has one of the highest Worldwide incidences and mortality rates. Genotoxic chemotherapy following surgery in stage III patients confers treatment benefit to less than 20% of the patients, with more than 50% of stage III patients going on to develop distant metastases. Currently, there are no predictive biomarkers that can identify which stage III patients will recur, which patients will benefit from chemotherapy and which should be redirected towards alternative therapeutic interventions. A major challenge in identifying such a universal biomarker is that CRC is a heterogeneous disease with multiple subtypes. In the current study, we assessed clinically-relevant immune cell populations in the tumour microenvironment (TME) of stage III tumours using a novel hyperplex in situ immunofluorescence imaging technology (Cell DIVETM, GE Healthcare, Issaquah, WA). Tissue microarrays (TMAs) with up to three 1mm diameter cores per patient were prepared from 139 stage III CRC patients treated with adjuvant FOLFOX chemotherapy. Single sections (5 µm) were iteratively stained with Cy3- and Cy5-conjugated antibodies for immune cell markers as well as markers of cell death and metabolism. The images underwent illumination correction, DAPI-based registration and autofluorescence removal. After image quality control corrections, single cell segmentation was performed using a combination of DAPI [nuclear], pan-cytokeratin [epithelial], NaKATPase [membrane] and S6 [cytoplasmic] segmentation markers and an average of ~3,000 stromal cells and ~ 4,000 epithelial were segmented per tumour core. A machine learning-based algorithm for immune cell classification and quantification was used to analyse the immune markers CD45, CD3, CD4, CD8, FOXP3 and PD1 to identify: cytotoxic T cells, T helper cells, regulatory T cells and potential relevance of immune checkpoint therapy. In the tumour tissues, the median proportion of CD3+ segmented cells was ~8%. Classified immune cells were counted within epithelial and stromal regions, with patients categorised as Low, Intermediate and High (based on 75th percentile, respectively) for each cell type. Preliminary survival analyses show that patients with ‘CD8 High' intratumoural cytotoxic T cells have better Disease-Free Survival compared to ‘CD8 Low' patients in this FOLFOX-treated cohort. By combining single-cell data with clinicopathological patient data, we aim to identify immune-, cell death- and metabolism-related signatures that can predict benefit from adjuvant FOLFOX chemotherapy for Stage III CRC patients. Citation Format: Xanthi Stachtea, Andreas Lindner, Manuela Salvucci, Sanghee Cho, Anup Sood, Elizabeth McDonough, Alberto Santamaria-Pang, John Graf, Philip Dunne, Mark Lawler, Jochen Prehn, Fiona Ginty, Daniel Longley. Hyperplexed immunofluorescence analysis (Cell DIVETM) of immune-related tumor heterogeneity in stage III colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2676.

Background: Transcriptional predictors are increasingly important for sub-typing cancer patients and understanding disease aetiology, predicting patient outcomes and response to treatment. Colorectal cancers (CRC) can be classified by transcriptionally inferred consensus (CMS) and intrinsic (CRIS) sub-types, annotating transcriptional profiles derived from colorectal cancer samples with these sub-types and other transcriptionally inferred predictors requires an experienced bioinformatician and is time-consuming. Methods: Publically available R packages CMSclassifier, CRISclassifier, MCP-counter and DoRothEA have been integrated into a web interface using the R/Shiny framework which allows users to upload gene expression data which can be additionally normalized with DESeq2. Visualization of this data can be interacted with through using plotly. Results: To address this bottleneck, we developed the classifieRC Shiny app, which enables rapid analysis, annotation of colorectal cancer transcriptional profiles with state-of-the-art transcriptional CRC sub-typing (CMS and CRIS) as well as estimation of cellular composition (MCP-Counter) and transcription factor activity predictions (DoRothEA). classifieRC can be accessed through a web-based interface, a locally deployable R-script or executable software, with capability of publishing datasets and their resulting analysis to Shiny.IO. Conclusions: classifieRc enables researchers to rapidly annotate colorectal transcriptomic datasets with molecular sub-types and of functional predictions without the need for a dedicated bioinformatician, expediting insights related patient cohort analyses and novel discoveries. classifieRc provides an easy to use flexible framework for functional annotation transcriptomic datasets and a platform for development of other disease specific apps. Citation Format: Gerard Quinn, Tamas Sessler, Wendy Allen, Sarah Maguire, Philip Dunne, Darragh McArt, Harper VanSteenhouse, Peter Gallagher, Andrea Lees, Dan Longley, Bruce Seligmann, Mark Wappett, Simon McDade. classifieRc: An interactive web interface for the molecular classification of colorectal cancer from RNA-sequencing data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3205.

Purpose: EphA2, a member of the Eph receptor tyrosine kinases family, is an important regulator of tumor initiation, neovascularization, and metastasis in a wide range of epithelial and mesenchymal cancers; however, its role in colorectal cancer recurrence and progression is unclear. Experimental Design: EphA2 expression was determined by immunohistochemistry in stage II/III colorectal tumors (N = 338), and findings correlated with clinical outcome. The correlation between EphA2 expression and stem cell markers CD44 and Lgr5 was examined. The role of EphA2 in migration/invasion was assessed using a panel of KRAS wild-type (WT) and mutant (MT) parental and invasive colorectal cancer cell line models. Results: Colorectal tumors displayed significantly higher expression levels of EphA2 compared with matched normal tissue, which positively correlated with high CD44 and Lgr5 expression levels. Moreover, high EphA2 mRNA and protein expression were found to be associated with poor overall survival in stage II/III colorectal cancer tissues, in both univariate and multivariate analyses. Preclinically, we found that EphA2 was highly expressed in KRASMT colorectal cancer cells and that EphA2 levels are regulated by the KRAS-driven MAPK and RalGDS-RalA pathways. Moreover, EphA2 levels were elevated in several invasive daughter cell lines, and downregulation of EphA2 using RNAi or recombinant EFNA1 suppressed migration and invasion of KRASMT colorectal cancer cells. Conclusions: These data show that EpHA2 is a poor prognostic marker in stage II/III colorectal cancer, which may be due to its ability to promote cell migration and invasion, providing support for the further investigation of EphA2 as a novel prognostic biomarker and therapeutic target. Clin Cancer Res; 22(1); 230–42. ©2015 AACR.

In this study, we developed an image analysis algorithm for quantification of two potential apoptotic biomarkers in non-small-cell lung cancer (NSCLC): FLIP and procaspase-8. Immunohistochemical expression of FLIP and procaspase-8 in 184 NSCLC tumors were assessed. Individual patient cores were segmented and classified as tumor and stroma using the Definiens Tissue Studio. Subsequently, chromogenic expression of each biomarker was measured separately in the nucleus and cytoplasm and reported as a quantitative histological score. The software package pROC was applied to define biomarker thresholds. Cox proportional hazards analysis was applied to generate hazard ratios (HRs) and associated 95% CI for survival. High cytoplasmic expression of tumoral (but not stromal) FLIP was associated with a 2.5-fold increased risk of death in lung adenocarcinoma patients, even when adjusted for known confounders (HR 2.47, 95% CI 1.14–5.35). Neither nuclear nor cytoplasmic tumoral procaspase-8 expression was associated with overall survival in lung adenocarcinoma patients; however, there was a significant trend (P for trend=0.03) for patients with adenocarcinomas with both high cytoplasmic FLIP and high cytoplasmic procaspase-8 to have a multiplicative increased risk of death. Notably, high stromal nuclear procaspase-8 expression was associated with a reduced risk of death in lung adenocarcinoma patients (adjusted HR 0.31, 95% CI 0.15–0.66). On further examination, the cells with high nuclear procaspase-8 were found to be of lymphoid origin, suggesting that the better prognosis of patients with tumors with high stromal nuclear procaspase-8 is related to immune infiltration, a known favorable prognostic factor. No significant associations were detected in analysis of lung squamous cell carcinoma patients. Our results suggest that cytoplasmic expression of FLIP in the tumor and nuclear expression of procaspase-8 in the stroma are prognostically relevant in non-small-cell adenocarcinomas but not in squamous cell carcinomas of the lung.

Histopathology-based staging of colorectal cancer (CRC) has utility in assessing the prognosis of patient subtypes, but as yet cannot accurately predict individual patient's treatment response. Transcriptomics approaches, using array based or next generation sequencing (NGS) platforms, of formalin fixed paraffin embedded tissue can be harnessed to develop multi-gene biomarkers for predicting both prognosis and treatment response, leading to stratification of treatment. While transcriptomics can shape future biomarker development, currently

Colorectal cancer (CRC) has one of the highest worldwide incidences and mortality rates. About 25% of stage II patients develop recurrence within 5 years. However, use of adjuvant chemotherapy yields minimal therapeutic benefit. Clinical decision-making in this population would be informed by prognostic and predictive biomarkers that help indicate more targeted interventions and/or intensified disease monitoring. Higher cMET expression has been shown to be a prognostic marker in CRC and has been evaluated as a drug target in several cancers, including CRC. cMET acts as the receptor for hepatocyte growth factor (HGF), and is associated with increased proliferation, migration and morphogenesis of epithelial cells via activation of multiple downstream pathways including PI3K/AKT, MAPK and the NF-kB pathways. Here, we investigated whether increased cellular expression of multiple markers in the cMET/adjacent pathways was correlated with clinical outcome. The study population consisted of 283 patients with stage II CRC, without neoadjuvant treatment. Using a multiplexed immunofluorescence method (MxIF) with cell-level quantification (Cell DIVETM, GE Healthcare), 41 biomarkers in the cMET, mTOR, MAPK and associated pathways, and lymphocyte markers were analyzed in an iterative sequence of staining, imaging and dye inactivation, followed by image registration, cell segmentation and biomarker intensity-quantitation. Poor quality images and cells were filtered based on poor segmentation and tissue quality following manual review. Images that did not perfectly register were also excluded. For epithelial cell analysis, all stromal cells were filtered, resulting in a total of 559,952 epithelial cells. K-means clustering was conducted on cMET and related pathways (cMET, pGSK3β, 4EBP1, p4EBP1, S6, pS6, pStat3, pp38MAPK, pNFkBp65, pNFkBp105, EGFR, pERK1/2, HER2, IGF1R, CA-IX, Glut1, SLC7A5 and Ki67). Cluster analysis identified 6 cell clusters with varying expression levels of cMET and other pathway markers. A cluster corresponding to low cMET network activation (cMETLow) was correlated with reduced risk of recurrence (cox pH model Likelihood ratio test p-value = 0.056). Furthermore, MMR proficient patients (who had a higher recurrence rate) had a significantly greater proportion of cells with elevated cMET and related pathway marker (cMETHigh) s(p= 0.007). They also had a significantly lower fraction of infiltrating helper T cells (CD3+CD4+, pvalue 0.076) and CD68+ cells in the epithelial region, compared to MMR deficient patients (p=0.005). Further research will include continuing in-depth analysis of MMR status, cMET pathway and lymphocyte response as well as the role of cellular heterogeneity. In summary, comprehensive cMET pathway analysis using a multiplexed single cell approach indicates for the first time an association between low cMET network activation and superior clinical outcomes. Citation Format: Elizabeth McDonough, Anup Sood, Fiona Ginty, Sanghee Cho, John Graf, Jochen Prehn, Philip Dunne, Andreas Lindner, Manuela Salvucci, Daniel Longley, Mark Lawler, Travis Hollmann, Jinru Shia. Lower cellular activation of cMET signaling network is associated with reduced recurrence risk in stage II colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A039. doi:10.1158/1535-7163.TARG-19-A039

Apoptosis is essential for chemotherapy responses. We previously developed the mathematical models APOPTO-CELL and DR_MOMP to estimate the magnitude of tumor cells resilience to undergo and execute apoptosis in response to chemotherapy, and demonstrated that these models are prognostic markers of clinical responses in stage 3 colorectal cancer patients [Salvucci M et al., Clin Cancer Res 2017; Lindner AU et al., Gut 2018]. However, the heterogeneity of colorectal cancer tissue represents a hurdle that we have yet to overcome on our way to entirely understand differences in response and clinical outcome in patients. Quantification of protein levels as input for systems studies routinely reflects a local cell population average rather than protein levels in individual cells. In this study we aim to gain a better understanding of apoptosis resilience in tumors with respect to tumor cell heterogeneity and cell composition and local aspects of the tumor microenvironment. Two to three core slices of formalin-fixed and paraffin-embedded primary colorectal cancer tissue were collected from 165 stage II-IV patients from a previously used cohort [Salvucci M et al. 2017; Lindner AU et al. 2018]. The levels of 25 protein markers for cell type and immune status, apoptosis, metabolism and cell proliferation, as well as cell segmentation markers, were measured on TMA slides using a multiplexed tissue immunofluorescence imaging platform (Cell DIVE [Gerdes et al., Proc Natl Acad Sci USA 2013]). Batch correction between slides was performed using linear regression (using markers for regulatory T cell in patient tissues as housekeeping cells. Formalin-fixed gene deficient cells were used in parallel for antibody validation. The layout allowed us to create protein profiles for on average 13,079 (± 2,282 SD) cells per core per patient, and to categorize these as epithelial-like cells, regulatory T cells, T helper cells, cytotoxic T cells, other leukocytes and other cells. The data will be used to identify cells contributing most to the signal measured with traditional methods (such as reverse protein phase arrays) that were used to predict patients clinical response to therapy in our previous studies. Further, we will assess spatial differences in distribution and frequency of different cells and their contribution to clinical outcome in colorectal cancer patients. Specifically, we are investigating metrics to characterize the spatial features and layout found in each core in terms of textures, degree of lacunarity, neighboring cells composition and patterns of protein expression (diffuse vs. patchy) and co-expression among cell types. Further, we will characterize the degree of intra- and inter-patient spatial heterogeneity and will examine the association between the identified spatial motifs with macroscopical characteristics of the tumor and outcome. Citation Format: Andreas Urlich Lindner, Manuela Salvucci, Xanthi Stachtea, Steven Carberry, Philip D. Dunne, Anup Sood, Elizabeth McDonough, Sanghee Cho, Pierre Laurent-Puig, Sandra Van Schaeybroeck, Manuel Salto-Tellez, John F. Graf, Markus Rehm, Mark Lawler, Daniel B. Longley, Fiona Ginty Fiona, Jochen H. Prehn. Exploratory multiplex tissue image analysis of the impact of heterogeneity in the microenvironment of primary colorectal cancer on apoptosis markers in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-088.

Background: The tumor microenvironment is a key feature to understand cancer biology and may be used clinically. Quantification of tissue composition is usually based either on visual pathological review (VPR) or deconvolution of whole genome molecular data. Although the former is a direct measurement it has modest reproducibility while the latter is an indirect measurement of unclear accuracy, expensive and not always available. Here we test digital pathology coupled with machine learning as a new tool to assess tissue composition. Methods: As part of the Stratification in COloRecTal cancer (S:CORT) programme, a set of over 500 colorectal cancer (CRC) archival paraffin blocks from resections and biopsies were sequentially sectioned for Hematoxylin and Eosin staining (H&E), RNA extraction, a second H&E and DNA extraction. RNA expression microarrays, targeted DNA sequencing and DNA methylation arrays were applied. Tissue composition from the H&Es was obtained by VPR of expert pathologists and by a deep neural net (DNN) algorithm after supervised training on >1,500 tissue areas from S:CORT, TCGA, TEM and CORGI CRC cohorts. Tumor purity estimates were obtained from RNA and methylation arrays. Results: DNN estimates including area and cell counts were obtained for tumor, desmoplastic stroma, inflamed stroma, mucin/hypocellular stroma, muscle, necrosis and white space. An average of 6.8x105 total cells (range: 1.2x104-2.8x106) and 1.2x105 (range: 7.2x104-1.8x106) were classified for resections and biopsies respectively. Analyses performed twice on the same H&Es obtained matching results (r=1.0). Comparison of the paired first and second H&E showed very high correlations (r~0.9) and total cell counts correlated with DNA and RNA extraction yields (r~0.6). Tumor purity estimates by VPR mildly correlated with DNN (r~0.5) but they were underestimated and very variable. As a result, copy number adjusted by VPR purity tended to be overestimated compared to adjustment with DNN estimates. The improved performance of DNN is reflected in an accurate capture of non-linear association between area and cell counts in invasive cancer. In contrast, tumor purity estimates derived from RNA or DNA methylation arrays showed better correlations compared with DNN (r~0.6) but both overestimated purity in cases with low cell counts by up to a three-fold difference. Conclusions: Tissue composition analysis with DNN allows analytical robustness, automatization and standardization and provides very high reproducibility at single cell resolution. DNN-based estimation of tumor purity is more accurate than VPR or extrapolation from molecular data derived from genome-wide omic platforms which tend to under and overestimate tumor purity respectively. DNN could be used to better plan and asses downstream molecular analyses and to investigate tissue-based metrics as potential clinical biomarkers in clinical trials. Citation Format: Enric Domingo, Aikaterini Chatzipli, Susan Richman, Andrew Blake, Claire Hardy, Celina Whalley, Keara Redmon, Ian Tomlinson, Philip Dunne, Steven Walker, Andrew Beggs, Ultan McDermott, Graeme I. Murray, Leslie M. Samuel, Matthew Seymour, Philip Quirke, Tim Maughan, Viktor H. Koelzer. Assessment of tissue composition with digital pathology in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4446.

Purpose: Despite the use of 5-fluorouracil (5-FU)–based adjuvant treatments, a large proportion of patients with high-risk stage II/III colorectal cancer will relapse. Thus, novel therapeutic strategies are needed for early-stage colorectal cancer. Residual micrometastatic disease from the primary tumor is a major cause of patient relapse. Experimental Design: To model colorectal cancer tumor cell invasion/metastasis, we have generated invasive (KRASMT/KRASWT/+chr3/p53-null) colorectal cancer cell subpopulations. Receptor tyrosine kinase (RTK) screens were used to identify novel proteins that underpin the migratory/invasive phenotype. Migration/invasion was assessed using the XCELLigence system. Tumors from patients with early-stage colorectal cancer (N = 336) were examined for AXL expression. Results: Invasive colorectal cancer cell subpopulations showed a transition from an epithelial-to-mesenchymal like phenotype with significant increases in migration, invasion, colony-forming ability, and an attenuation of EGF receptor (EGFR)/HER2 autocrine signaling. RTK arrays showed significant increases in AXL levels in all invasive sublines. Importantly, 5-FU treatment resulted in significantly increased migration and invasion, and targeting AXL using pharmacologic inhibition or RNA interference (RNAi) approaches suppressed basal and 5-FU–induced migration and invasion. Significantly, high AXL mRNA and protein expression were found to be associated with poor overall survival in early-stage colorectal cancer tissues. Conclusions: We have identified AXL as a poor prognostic marker and important mediator of cell migration/invasiveness in colorectal cancer. These findings provide support for the further investigation of AXL as a novel prognostic biomarker and therapeutic target in colorectal cancer, in particular in the adjuvant disease in which EGFR/VEGF–targeted therapies have failed. Clin Cancer Res; 20(1); 164–75. ©2013 AACR.

BACKGROUND: Combined MET and BRAF inhibition showed clinical benefit in a patient with rectal cancer carrying BRAF(V600E) and MET amplification. However after 4 months, acquired resistance emerged and the patient deceased shortly after disease progression. The mechanism of resistance to this drug combination is unknown.METHODS: We analysed plasma circulating tumour DNA obtained at progression by exome sequencing and digital PCR. MET gene and mRNA in situ hybridisation analyses in two bioptic specimens obtained at progression were used to confirm the plasma data.RESULTS: We identified in plasma MET gene hyper-amplification as a potential mechanism underlying therapy resistance. Increased MET gene copy and transcript levels were detected in liver and lymph node metastatic biopsies. Finally, transduction of MET in BRAF mutant colorectal cancer cells conferred refractoriness to BRAF and MET inhibition.CONCLUSIONS: We identified in a rectal cancer patient MET gene hyper-amplification as mechanism of resistance to dual BRAF and MET inhibition.

Glioblastoma (GBM) is the most prevalent and aggressive of malignant primary brain tumours. Despite multi-modal therapy, including surgical resection, radiotherapy and chemotherapy, GBM continues to be uniformly lethal, with a median survival of 12–18 months from the time of diagnosis. The resistance to therapy and ultimate recurrence is thought to originate from a sub-population of cells residing in the initial tumour, identified as glioma-initiating or glioma-stem cells (GSC). These GSC have been shown to be intrinsically more invasive, with enhanced repair and drug-efflux pathways mediating resistance to chemo-radiation therapies. The purpose of this study was to use and validate a novel connectivity mapping procedure to identify candidate compounds targeting therapy-resistant and recurrent GBM. This procedure was developed by O’Reilly and colleagues (“QUADrATiC: A Scalable Approach to Repurposing FDA-approved therapeutics”, under review in Bioinformatics), and in this study, was performed on publicly available NCBI GEO microarray datasets. Based on the cancer stem cell theory whereby the resistant GSCs repopulate and are enriched in recurrent tumours, primary and recurrent GBM gene expression profiles were compared and the connections between the identified gene lists and drug responses were mapped. To validate that this connectivity mapping procedure is reliable at identifying effective treatments to target therapeutic resistance and recurrence, we tested one of the top ten compounds identified, rosiglitazone (rosi), a ligand of the peroxisome proliferator activated receptor gamma (PPARγ). The efficacy of rosi alone and in combination with the current standard of care therapies, temozolomide and radiation, at killing GSC was assessed in vitro using the clonogenic survival assay. Our data indicated that rosi significantly reduced survival of both non-stem glioma cells and GSC, alone and in combination with radiation and temozolomide. The p53 pathway was identified as a pathway by which rosi may induce permanent cell cycle arrest and cell death. Further work is ongoing to fully elucidate the downstream mechanisms of cell death. This work is proof-of-principle that the current connectivity mapping procedure can be instrumental in identifying more targeted therapeutic interventions for GBM resistance and prevent recurrence.

// Conor A. Bradley 1, * , Philip D. Dunne 1, * , Victoria Bingham 1 , Stephen McQuaid 1, 2 , Hajrah Khawaja 1 , Stephanie Craig 1 , Jackie James 1, 2 , Wendy L. Moore 1 , Darragh G. McArt 1 , Mark Lawler 1 , Sonali Dasgupta 1 , Patrick G. Johnston 1 , Sandra Van Schaeybroeck 1 1 Drug Resistance Group, Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen’s University Belfast, Belfast, UK 2 Tissue Pathology, Belfast Health and Social Care Trust, Belfast City Hospital, Belfast, UK * Joint first authors Correspondence to: Sandra Van Schaeybroeck, email: s.vanschaeybroeck@qub.ac.uk Keywords: c-MET, colorectal cancer (CRC), tumor budding, invasion, metastasis Received: July 06, 2016 Accepted: October 19, 2016 Published: October 26, 2016 ABSTRACT c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors ( p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis.

Purpose: A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer with potential diagnostic utility, culminating in publication of a colorectal cancer Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled. Experimental Design: We performed multiregion tissue RNA extraction/transcriptomic analysis using colorectal-specific arrays on invasive front, central tumor, and lymph node regions selected from tissue samples from 25 colorectal cancer patients. Results: We identified a consensus 30-gene list, which represents the intratumoral heterogeneity within a cohort of primary colorectal cancer tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential HR = 2.914 (confidence interval 0.9286–9.162) in stage II/III colorectal cancer patients, but in addition, we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread epithelial–mesenchymal transition. Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analyzed. Conclusions: Gene expression profiles derived from the nonmalignant stromal region can influence assignment of colorectal cancer transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision making in colorectal cancer. Clin Cancer Res; 22(16); 4095–104. ©2016 AACR. See related commentary by Morris and Kopetz, p. 3989

The topoisomerase I inhibitor irinotecan is used to treat advanced colorectal cancer and has been shown to have p53-independent anticancer activity. The aim of this study was to identify the p53-independent signaling mechanisms activated by irinotecan. Transcriptional profiling of isogenic HCT116 p53 wild-type and p53 null cells was carried out following treatment with the active metabolite of irinotecan, SN38. Unsupervised analysis methods showed that p53 status had a highly significant impact on gene expression changes in response to SN38. Pathway analysis indicated that pathways involved in cell motility [adherens junction, focal adhesion, mitogen-activated protein kinase (MAPK), and regulation of the actin cytoskeleton] were significantly activated in p53 null cells, but not p53 wild-type cells, following SN38 treatment. In functional assays, SN38 treatment increased the migratory potential of p53 null and p53-mutant colorectal cancer cell lines, but not p53 wild-type lines. Moreover, p53 null SN38-resistant cells were found to migrate at a faster rate than parental drug-sensitive p53 null cells, whereas p53 wild-type SN38-resistant cells failed to migrate. Notably, cotreatment with inhibitors of the MAPK pathway inhibited the increased migration observed following SN38 treatment in p53 null and p53-mutant cells. Thus, in the absence of wild-type p53, SN38 promotes migration of colorectal cancer cells, and inhibiting MAPK blocks this potentially prometastatic adaptive response to this anticancer drug. Mol Cancer Ther; 11(8); 1724–34. ©2012 AACR.

Available treatments for glioma have limited effectiveness rendering this a disease of poor clinical outcome. Gene expression profiling can uncover the biological mechanisms underlying disease, information that is crucial for drug development or repurposing. RNA sequencing (RNA-seq) is routinely used to assess gene expression but costs remain high. Whilst sample multiplexing reduces RNA-seq costs, the lowered cDNA sequencing depth of multiplexed samples may hinder accurate differential gene expression detection. The impact of sequencing depth alteration on RNA-seq-based downstream analyses such as gene expression connectivity mapping is not known. Connectivity mapping is a method used to identify potential therapeutic compounds for drug repurposing. In this study, published RNA-seq from brain tumour (glioma) patients were analysed and assembled into two gene signature contrasts for astrocytoma disease progression (WHO Grade II-III; III-IV). Gene signatures were subsampled to simulate sequencing depth alterations and analysed in connectivity mapping to investigate target compound robustness. Data loss to gene signatures led to the loss, gain and consistent identification of significant connections to target compounds. The most accurate gene signature contrast with consistent patient gene expression profiles was more resilient to data loss and identified robust target compounds. Target compounds lost included candidate compounds of potential clinical utility in glioma (e.g. Suramin, Dasatinib). Lost connections may have been linked to low abundance genes in the gene signature that closely characterised the disease phenotype. Consistently identified connections may have related to highly expressed abundant genes that were ever-present in gene signatures, despite data reductions. Potential noise surrounding findings included false positive connections that were gained as a result of gene signature modification with data loss. Findings highlight the necessity for gene signature accuracy for connectivity mapping, which should improve the clinical utility of future target compounds identified for drug repurposing.

BACKGROUND: Gene expression profiling can uncover biological mechanisms underlying disease and is important in drug development. RNA-seq is routinely used to assess gene expression but costs remain high. Sample multiplexing reduces RNA-seq costs, however, multiplexed samples have lower cDNA sequencing depth, which can hinder accurate differential gene expression detection. The impact of sequencing depth alteration on RNA-seq-based downstream analyses such as gene expression connectivity mapping is not known, where connectivity mapping can be used to identify potential therapeutic compounds for repurposing. MATERIAL AND METHODS: In this study, published RNA-seq profiles from brain tumour (glioma) patients were assembled into two disease progression gene signature contrasts for astrocytoma. Available treatments for glioma have limited effectiveness rendering this a disease of poor clinical outcome. Gene signatures were subsampled to simulate sequencing alterations and analysed in connectivity mapping to investigate target compound robustness. RESULTS: Data loss to gene signatures led to the loss, gain and consistent identification of significant connections. The most accurate gene signature contrast with consistent patient gene expression profiles was more resilient to data loss and identified robust target compounds. Target compounds lost included candidate compounds of potential clinical utility in glioma (e.g. Suramin, Dasatinib). Lost connections may have been linked to low abundance genes in the gene signature that closely characterised the disease phenotype. Consistently identified connections may have been related to highly expressed abundant genes that were ever-present in gene signatures, despite data reductions. Potential noise surrounding findings included false positive connections that were gained as a result of gene signature modification with data loss. CONCLUSION: Findings highlight the necessity for gene signature accuracy for connectivity mapping, which should improve the clinical utility of future target compound discoveries.

Colorectal cancer (CRC) biopsies underpin accurate diagnosis, but are also relevant for patient stratification in molecularly-guided clinical trials. The consensus molecular subtypes (CMS) and colorectal cancer intrinsic subtypes (CRIS) transcriptional signatures have potential clinical utility for improving prognostic/predictive patient assignment. However, their ability to provide robust classification, in pre-treatment biopsies from multiple regions or at different time points remains untested. In this study, we undertook a comprehensive assessment of the robustness of CRC transcriptional signatures, including CRIS and CMS, using a range of tumour sampling methodologies currently employed in clinical and translational research. These include analyses using (i) laser-capture microdissected CRC tissue, (ii) eight publically available rectal cancer biopsy data sets (n=543), (iii) serial biopsies (from AXEBeam trial, NCT00828672; n=10), (iv) multi-regional biopsies from colon tumours (n=29 biopsies, n=7 tumours) and (v) pre-treatment biopsies from the phase II rectal cancer trial COPERNCIUS (NCT01263171; n=44). Compared to previous results obtained using CRC resection material, we now demonstrate that CMS classification in biopsy tissue is significantly less capable of reliably classifying patient subtype (43% unknown in biopsy versus 13% unknown in resections, p=0.0001). In contrast, there was no significant difference in classification rate between biopsies and resections when using the CRIS classifier. Additionally, we demonstrated that CRIS provides significantly better spatially- and temporally- robust classification of molecular subtypes in CRC primary tumour tissue compared to CMS (p= 0.003 and p=0.02, respectively). These findings may have potential to inform ongoing biopsy-based patient stratification in CRC, enabling robust and stable assignment of patients into clinically-informative arms of prospective multi-arm, multi-stage clinical trials. Citation Format: Matthew Alderdice, Susan D. Richman, Simon Gollins, Peter Stewart, Chris Hurt, Richard Adams, Amy M. McCorry, Aideen Roddy, Dale Vimalachandran, Claudio Isella, Enzo Medico, Tim Maughan, Darrgh G. McArt, Mark Lawler, Philip D. Dunne. Prospective patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies [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 5365.

Technology advancements have enhanced our abilities to gain greater insight to the tumor environment. However, such emergent methods have brought new considerations in data storage, access and analysis. Modern large data projects and clinical trial materials could be explored to a greater degree if appropriate infrastructure could be built to support efforts. Such a scaffold would be an integrated and dynamic framework where novel hypotheses could be investigated in modern big data collections. Placing discovery back in the hands of the researcher through a reactive and supportive framework will enhance our understanding of cancer aetiology. The Cancer Integromics Research Application Framework (CIRAFm) was created to emulate current platforms that have a rigid analytical interface. We designed a robust architecture that supports a reactive user-friendly interface blended to several cross-platform coding technologies. It has been developed to accommodate an individualised framework where we can create an ‘app-store' of key software to fit research questions. In efforts to encapsulate and accelerate differing data types it sits astride of two NoSQL based database management systems, minimising data redundancy. Initial requirements have begun to create algorithms to investigate alignment-free applications on next generation sequencing (NGS) data enhancing analysis of spatial and temporal heterogeneity in cancer. Key drivers can be explored by assisting software to build correlative marker associations using Darwinian approaches such as genetic algorithms. Information requirements from external platforms are assisted through a novel domain specific language (DSL) to enable a singular interface. The modularised architecture of the platform is enabled through an Angular framework supported by interactive and dynamic data visualisation software, D3.js. Data analytics can be explored through the ‘apps' created, investigating new markers in large data and enhancing our understanding of tumor heterogeneity. Alignment-free phylogenetics of NGS data harnesses our capabilities to display fully sequence evolution in patient data. This highlights possible sub-types, markers of interest and maps to therapeutic compounds via the DSL to our externally developed drug discovery software, QUADrATiC. Future scientific endeavours further defining new tumor subtypes are paramount in efforts to help uncover treatment strategies. Unburdened by legacy pipelines and data, flexible and robust models of architecture provide an effective and efficient framework for research on our ever increasing search for precision medicine. Citation Format: Darragh G. McArt, Seedevi Senevirathne, Aideen Roddy, Jessica Black, Alan Gilmore, Suneil Jain, Philip Dunne, David Waugh. Integrative analytics: A framework for 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 290.

Modern cancer research on prognostic and predictive biomarkers demands the integration of established and emerging high-throughput technologies. However, these data are meaningless unless carefully integrated with patient clinical outcome and epidemiological information. Integrated datasets hold the key to discovering new biomarkers and therapeutic targets in cancer. We have developed a novel approach and set of methods for integrating and interrogating phenomic, genomic and clinical data sets to facilitate cancer biomarker discovery and patient stratification. Applied to a known paradigm, the biological and clinical relevance of TP53, PICan was able to recapitulate the known biomarker status and prognostic significance at a DNA, RNA and protein levels.

The application of transcriptional gene signatures to stratify tumors into prognostic and predictive subtypes has evolved rapidly since the original landmark studies demonstrated the clinical utility of this approach in breast cancer. While the number of published signatures continues to increase, the likelihood of individual signatures achieving clinical utility has remained very low, with some estimates putting this figure below 1%. Transcriptional intratumoral heterogeneity (ITH), resulting from variation in stromal components at different regions of a tumor, has been shown to undermine molecular stratification of colorectal cancer (CRC) patients into prognostic and predictive subgroups. The degree of gene expression changes associated with variation in tumor microenvironment (TME) content may even mask the relatively more subtle changes associated with genetic variability and heterogeneity within the tumor epithelium. This variation in TME content, and subsequent ITH, at different regions of the tumor has long been recognised; however, in the era of precision medicine, the implications of such microenvironmental alterations in confounding patient classification are of increased importance. To examine this issue, a panel of clinically relevant prognostic and predictive transcriptional gene signatures were selected in order to assess the potential for discordant classification of patient samples based on the tumoral region profiled. Using these clinically relevant CRC gene expression signatures, we assessed the susceptibility of each signature to the confounding effects of ITH using gene expression microarray data obtained from multiple tumor regions of a cohort of 24 patients, including central tumor, the tumor invasive front and lymph node metastasis. Consensus annotated gene lists for each of the gene signatures were used, followed by in silico analysis within our multiregional clinical dataset. Sample clustering alongside correlative assessment revealed variation in the ability of each signature to cluster samples according to patient-of-origin rather than region-of-origin within the multiregion dataset. Signatures focused on cancer-cell intrinsic gene expression were found to produce more clinically useful, patient-centred classifiers, as exemplified by the recently developed CRC intrinsic signature (CRIS), which robustly clustered samples by patient rather than sample region. These findings highlight the potential of cancer-cell intrinsic signatures to reliably stratify CRC patients by minimising the confounding effects of ITH. In conclusion, our data support the clinical evaluation of signatures based on intrinsic gene expression, such as CRIS, which are unaffected by the confounding variable of transcriptional ITH and therefore have the potential to be used clinically to inform precision medicine decisions, ultimately leading to improved patient outcomes. Citation Format: Philip D. Dunne, Paul O'Reilly, Aideen Roddy, Matthew Alderdice, Susan Richman, Tim Maughan, Simon McDade, Patrick Johnston, Daniel Longley, Elaine Kay, Darragh McArt, Mark Lawler. Cancer cell intrinsic gene expression signatures minimize the confounding effects of intratumoral heterogeneity in colorectal cancer patient classification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-042. doi:10.1158/1538-7445.AM2017-LB-042

Background: The c-MET proto-oncogene is frequently overexpressed (50-60%), amplified (1-3%), and mutated (1-3%) in colorectal cancer (CRC). Hepatocyte growth factor (HGF)-dependent and independent activation of c-MET has been associated with increased survival and resistance to targeted therapies. This study aimed to investigate the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Methods: In order to model CRC tumour cell invasion, we have generated invasive CRC subpopulations using Boyden Invasion chambers. To model the CRC microenvironment, we have used a range of co-culture techniques with CRC cells and colon fibroblasts. Migration/invasion was determined using xCELLigence System (Roche). c-MET expression in parental and invasive cell lines was measured using Western blotting and qRT-PCR. c-MET expression in CRC FFPE tissues was measured using IHC and RNAScope®. Results: We identified marked upregulated expression of c-MET at both the protein and transcript levels in our invasive CRC cell line models. Importantly, both parental and invasive subpopulations were found to be inherently dependent on c-MET for migration, as RNAi against c-MET abrogated migration/invasion in both parental and invasive models. We also demonstrated that stimulation of CRC cells with rh-HGF resulted in increased CRC cell migration/invasion. In addition, co-culture of CRC cells with colonic myofibroblasts, resulted in marked increases in migratory and invasive capacity, and this was dependent on HGF/c-MET signaling. Interestingly, stimulation with myofibroblast conditioned medium or HGF promotes rapid degradation of c-MET at the protein level, followed by recycling, while MET transcript remains unaltered, illustrating a dynamic expression of c-MET protein in response to activation. We further showed that MET is transcriptionally upregulated in tumour budding foci at the invasive front of a cohort of stage III CRC tumors. Intriguingly, c-MET protein levels do not correlate with the transcript, most likely due to a similar protein degradation process observed in our aforementioned in vitro models. Conclusions: We show for the first time a key role for transcriptional upregulation of MET as a molecular driver of tumour invasion, both in vitro and in stage III CRC tumours. Citation Format: Conor Bradley, Philip Dunne, Stephen McQuaid, Victoria Bingham, Mark Lawler, Manuel Salto-Tellez, Patrick Johnston, Sandra Van Schaeybroeck. Upregulation of the MET transcript is consistently associated with invasion and tumor budding in colorectal 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 924.

Glioblastoma (GBM) is the most prevalent and aggressive of malignant primary brain tumours. Despite multi-modal therapy, including surgical resection, radiotherapy and chemotherapy, GBM continues to be uniformly lethal, with a median survival of 12-18 months from the time of diagnosis. The resistance to therapy and ultimate recurrence is thought to originate from a sub-population of cells residing in the initial tumour, identified as glioma-initiating or glioma-stem cells (GSC). These GSC have been shown to be intrinsically more invasive, with enhanced repair and drug-efflux pathways mediating resistance to chemo-radiation therapies. The purpose of this study was to use and validate a novel connectivity mapping procedure to identify candidate compounds targeting therapy-resistant and recurrent GBM. This procedure was developed by O’Reilly and colleagues (“QUADrATiC: A Scalable Approach to Repurposing FDA-approved therapeutics”, under review in Bioinformatics), and in this study, was performed on publicly available NCBI GEO microarray datasets. Based on the cancer stem cell theory whereby the resistant GSCs repopulate and are enriched in recurrent tumours, primary and recurrent GBM gene expression profiles were compared and the connections between the identified gene lists and drug responses were mapped. To validate that this connectivity mapping procedure is reliable at identifying effective treatments to target therapeutic resistance and recurrence, we tested one of the top ten compounds identified, rosiglitazone (rosi), a ligand of the peroxisome proliferator activated receptor gamma (PPARγ). The efficacy of rosi alone and in combination with the current standard of care therapies, temozolomide and radiation, at killing GSC was assessed in vitro using the clonogenic survival assay. Our data indicated that rosi significantly reduced survival of both non-stem glioma cells and GSC, alone and in combination with radiation and temozolomide. The p53 pathway was identified as a pathway by which rosi may induce permanent cell cycle arrest and cell death. Further work is ongoing to fully elucidate the downstream mechanisms of cell death. This work is proof-of-principle that the current connectivity mapping procedure can be instrumental in identifying more targeted therapeutic interventions for GBM resistance and prevent recurrence. Citation Format: Shahnaz T. Al Rashid, Paul O’Reilly, Philip D. Dunne, Matthew Alderdice, Thomas J. Flannery, Kevin M. Prise, Shu-Dong Zhang, Darragh G. McArt. Therapeutic compound discovery targeting a recurrent glioblastoma (GBM) phenotype using LINCS compounds via QUADrATiC analyses. [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 72.

SummaryThere are currently no approved targeted therapies for advanced KRAS mutant (KRASMT) colorectal cancer (CRC). Using a unique systems biology approach, we identified JAK1/2-dependent activation of STAT3 as the key mediator of resistance to MEK inhibitors in KRASMT CRC in vitro and in vivo. Further analyses identified acute increases in c-MET activity following treatment with MEK inhibitors in KRASMT CRC models, which was demonstrated to promote JAK1/2-STAT3-mediated resistance. Furthermore, activation of c-MET following MEK inhibition was found to be due to inhibition of the ERK-dependent metalloprotease ADAM17, which normally inhibits c-MET signaling by promoting shedding of its endogenous antagonist, soluble “decoy” MET. Most importantly, pharmacological blockade of this resistance pathway with either c-MET or JAK1/2 inhibitors synergistically increased MEK-inhibitor-induced apoptosis and growth inhibition in vitro and in vivo in KRASMT models, providing clear rationales for the clinical assessment of these combinations in KRASMT CRC patients.

The c-MET proto-oncogene is frequently overexpressed (50-60%), amplified (5-10%), and mutated (5%) in colorectal cancer (CRC). Hepatocyte growth factor (HGF)-induced c-MET activation has been linked with migration, survival, invasion, and resistance to targeted therapy, and has been suggested as a possible prognostic biomarker for CRC. We are currently investigating the role of c-MET and microenvironment derived HGF in the acquisition of an invasive and migratory phenotype, and resistance to targeted therapy in CRC. In order to model CRC tumour cell invasion and metastasis, we have generated invasive subpopulations from CRC cells using Boyden Invasion chambers. Invasive cell lines were characterised for protein expression/activity by Western blotting, and analysed for migratory and invasive potential using the xCELLigence System (Roche). To model the CRC microenvironment, we have utilised a range of co-culture techniques with CRC cell lines and colon fibroblasts. c-MET expression in FFPE tissues was measured using IHC in a tissue microarray (TMA) derived from early stage CRC patients. HCT116 and LoVo invasive subpopulations showed an EMT-like, mesenchymal, migratory/invasive phenotype. In addition, increased expression and activation levels of c-MET were found in these sublines, which was determined to be ligand independent. Inhibition of c-MET using RNAi abrogated both basal and HGF-induced migration and invasion in CRC cell lines. Co-culture of CRC cells with HGF-expressing colon-derived fibroblasts leads to activation of c-MET, inducing CRC migration, invasion, and resistance to MEK inhibition, and this phenotype could be diminished using a HGF neutralising antibody. Significant increased expression levels of c-MET were also found in a CRC TMA compared to matched normal tissues, and also at the invasive edge of some CRC tumours, where it may be driving invasive biology. The identification of key pathways driving metastasis has a huge potential to change treatment strategies in CRC. We intend to further investigate the role of c-MET in regulation of migration, invasion, and resistance to therapy in CRC. We hypothesise that patients with high tumoural c-MET at the invasive front may benefit in particular from anti- c-MET therapies. Citation Format: Conor A. Bradley, Philip Dunne, Darragh McArt, Ken Arthur, Stephen McQuaid, Manuel Salto-Tellez, Patrick Johnston, Sandra Van Schaeybroeck. The role of c-MET/HGF signaling as a critical mediator of an invasive and resistant phenotype 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 4018. doi:10.1158/1538-7445.AM2015-4018

3573 Background: Despite the use of 5-FU-based adjuvant therapies, a large proportion of stage III (locally advanced) colorectal cancer (CRC) patients will relapse and die of metastatic disease. Re...

Background Inhibitor of Apoptosis Proteins (IAPs) are anti-apoptotic proteins which suppress the activation of caspases. IAP proteins have been shown to be overexpressed in several cancers, including colorectal cancer and correlate with poor prognosis. SMAC mimetics are a class of apoptosis inducing drugs that target IAPs by mimicking the N-terminal IAP-binding motif of SMAC. Colorectal cancer has a pro-inflammatory microenvironment with high levels of TNFα. The aims of this study were to assess the efficacy of SMAC mimetic treatment in colorectal cancer. Methods Colorectal cell lines were treated with SMAC mimetics and cell viability was determined using MTT assays. Apoptosis was assessed by Western blotting for caspase activation and PARP cleavage and Flow Cytometry using Annexin V/PI staining. Protein-protein interactions were assessed using co-immunoprecipitation experiments. The pro-inflammatory microenvironment was modelled by addition of exogenous TNFα and also media transfer from and direct co-cultures with macrophages (THP-1, monocytic cell line). Results Colorectal cell lines were resistant to SMAC mimetic treatment alone unless co-cultured with TNFα. Pretreatment of colorectal cancer cells with 5-FU and oxaliplatin sensitised the cells to SMAC mimetic treatment. THP-1 cells were found to secrete soluble TNFα and media transfer experiments and also co-culture with colorectal cell lines, sensitised the cancer cells to SMAC mimetic mediated cell death. Cell death under these conditions was found to be dependent on soluble TNFα through the utilisation of neutralising antibodies. Conclusion Our results to date suggest that the pro-inflammatory microenvironment of colorectal cancer could be exploited to target overexpressed IAP proteins leading to cell death. Citation Format: Nyree T. Crawford, Philip D. Dunne, Patrick G. Johnston, David J. Waugh, Daniel B. Longley. Rational targeting of inhibitor of apoptosis proteins (IAPs) for effective therapy of colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2290. doi:10.1158/1538-7445.AM2014-2290

Background Colorectal cancer (CRC) is the third most common cancer worldwide. Among patients with early stage CRC, there are subsets of patients who will not benefit from adjuvant 5-FU or 5-FU/oxaliplatin treatment, highlighting the need for improved therapy options for early stage disease. In addition mutations in the KRAS proto-oncogene are found in 40%-45% of CRC patients and this mutation predicts resistance to EGFR mAb therapies. More recently it has been shown that treatments, such as bevacizumab or cetuximab, which prolong survival in the advanced disease setting, cannot be translated into successful adjuvant treatment strategies. The aim of this study was to identify therapy options which will be effective in the adjuvant setting and also whose knock-down is synthetically lethal with KRAS mutation in CRC models. Method A novel systems biology approach combined profiling of primary KRASMT/WT CRC tissues and isogenic paired KRASMT/WT CRC xenografts models. Metacore pathway analysis identified individual genes from novel KRAS-dependent pathways for incorporation into a primary RNAi screen. This screen was crossed with receptor tyrosine kinase analysis of our previously published pre-clinical adjuvant invasive cell line models. Our findings were validated by in vitro screening and expression analysis in CRC FFPE sections was measured using IHC. Results Our comprehensive approach identified 30 signalling pathways and 160 genes associated with KRASMT CRC cancer. Our siRNA screen confirmed a number of novel KRAS synthetic lethal target genes with a critical role in EMT and cell adhesion/migration. Down-regulation of members of the Eph/Ephrin family, in particular EpHA2, was found to be lethal in KRASMT but not KRASWT cells. This lethality was further synergistically enhanced in the presence of chemotherapy treatment. Overexpression of EphA2 was also identified in each of our invasive isogenic cell line models, regardless of genetic background, and EpHA2 silencing inhibited migration and invasion in a panel of CRC cells. EpHA2 expression has previously been reported as an essential component in cancer cells's ability to form tumors in vivo and in-line with this our EpHA2 overexpressing invasive subpopulations displayed an increased ability to form colonies. Importantly, high EpHA2 mRNA and protein expression were found to be associated with poor overall survival in both early stage CRC tissues and in advanced disease. Conclusion Using our novel comprehensive systems biology approach we have identified novel KRAS synthetic lethal targets and pathways. Combining these findings with targets which are effective against our pre-clinical adjuvant models we have identified EpHA2 as a key driver of invasion and migration and a synthetically lethal target in KRASMT CRC. In addition, we show that EpHA2 is a poor prognostic marker and an important novel target for KRASMT CRC tumors in both the adjuvant and advanced disease setting. Citation Format: Philip D. Dunne, Darragh G. McArt, Jaine K. Blayney, Sonali Dasgupta, Manuel Salto-Tellez, Patrick G. Johnston, Sandra Van Schaeybroeck. EpHA2 is an essential driver of invasion and a novel target in KRAS mutant colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2079. doi:10.1158/1538-7445.AM2014-2079

3581 Background: EpHA2 is a 130 kD transmembrane glycoprotein belonging to ephrin receptor subfamily and involved in angiogenesis/tumour neovascularisation. High EpHA2 mRNA level has recently been ...

The discovery of underlying mechanisms of drug resistance, and the development of novel agents to target these pathways, is a priority for patients with advanced colorectal cancer (CRC). We previously undertook a systems biology approach to design a functional genomic screen and identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of drug resistance. The aim of this study was to examine the role of FGFR4 in drug resistance using RNAi and the small-molecule inhibitor BGJ398 (Novartis). We found that FGFR4 is highly expressed at the RNA and protein levels in colon cancer tumour tissue compared with normal colonic mucosa and other tumours. Silencing of FGFR4 reduced cell viability in a panel of colon cancer cell lines and increased caspase-dependent apoptosis. A synergistic interaction was also observed between FGFR4 silencing and 5-fluorouracil (5-FU) and oxaliplatin chemotherapy in colon cancer cell lines. Mechanistically, FGFR4 silencing decreased activity of the pro-survival STAT3 transcription factor and expression of the anti-apoptotic protein c-FLIP. Furthermore, silencing of STAT3 resulted in downregulation of c-FLIP protein expression, suggesting that FGFR4 may regulate c-FLIP expression via STAT3. A similar phenotype and downstream pathway changes were observed following FGFR4 silencing in cell lines resistant to 5-FU, oxaliplatin and SN38 and upon exposure of parental cells to the FGFR small-molecule inhibitor BGJ398. Our results indicate that FGFR4 is a targetable regulator of chemo-resistance in CRC, and hence inhibiting FGFR4 in combination with 5-FU and oxaliplatin is a potential therapeutic strategy for this disease.

Background: Oncogenic mutations in BRAF occur in 8% of patients with advanced colorectal cancer (CRC) and have been shown to correlate with poor prognosis. In contrast to BRAF mutant (MT) melanoma, where the BRAF inhibitor PLX4032 has shown significant increases in response rates and overall survival compared to standard Dacarbazine treatment, only minor responses to PLX4032 treatment have been reported in BRAFMT CRC. Clear understanding of the vulnerabilities of BRAFMT CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumors has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. The aim of this study was to identify novel resistance mechanisms to MAPK inhibition in BRAFMT CRC. Methods: Paired BRAFMT/WT RKO and VACO432 CRC cell line models and non-isogenic BRAFMT LIM2405, WiDR and COLO205 CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Interaction between MEK1/2 and JAK1/2 inhibition was assessed using the MTT cell viability assays and flow cytometry. Apoptosis was measured using Western blotting for PARP, cleaved caspase 3/8 and caspase 8, 3/7 activity assays. Results: Treatment with MEK1/2 inhibitors AZD6244, GSK1120212, UO126 and PD98059 resulted in acute increases in STAT3 activity in the BRAFMT RKO and VACO432 cells but not in their BRAFWT clones and this was associated with increases in JAK2 activity. Inhibition of JAK/STAT3 activation using gene specific siRNA or small molecule inhibitors TG101348 or AZD1480, abrogated this survival response and resulted in significant increases in cell death when combined with MEK1/2 inhibitors AZD6244 or GSK1120212 in BRAFMT CRC cells. In addition, combination of MEK1/2 and JAK/STAT3 inhibition resulted in strong synergy with CI values between 0.3 and 0.7 in BRAFMT CRC cells. Conclusions: We have identified JAK/STAT3 activation as an important escape mechanism for BRAFMT CRC following MEK1/2 inhibition. These data provide a strong rationale for further investigation of combination of MEK1/2 and JAK/STAT3 inhibition in BRAFMT in vivo models. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B64. Citation Format: Robbie Carson, Basak Celtikci, Philip Dunne, Patrick Johnston, Sandra Van Schaeybroeck. The role of JAK1/2-STAT3 as acute resistance mechanism to MEK inhibition in BRAF mutant colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B64.

Mutations in the Kras proto-oncogene are found in 40%-45% of colorectal cancer (CRC) patients and confer resistance to EGFR monoclonal antibody (mAb) therapies. The identification of druggable targets uniquely required by KrasMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. We carried out microarray profiling of isogenic Kras wild type (WT) and mutant (MT) in vivo CRC models, KrasMT/WT primary colon tumours, pre-treatment metastatic CRC liver biopsies and drug resistant and sensitive CRC cell lines. We have used systems biology approach incorporating pathway analyses of the microarray data followed by siRNA screening to identify novel targets/pathways which are critical for the survival of KrasMT tumours, both basally and following chemotherapy treatment. We found that JAK1/2-STAT3 signaling was essential for the survival of KrasMT CRC cells. Moreover, suppression of JAK1/2-STAT3 activity using RNAi or small molecule inhibitors resulted in more potent increase in apoptosis in KrasMT CRC cells. In addition, treatment with chemotherapy and MEK1/2 inhibition resulted in significant increases in JAK2 and STAT3 activity and this was associated with strong increase in apoptosis when JAK1/2-STAT3 RNAi or small molecule inhibitors were combined with chemotherapy and MEK1/2 inhibition, especially in KrasMT cells. Moreover, we found that these effects were not drug- or cell line-specific. Collectively, using a systems biology approach, we have identified an essential role of JAK1/2-STAT3 signalling in survival of KrasMT CRC models. These findings support the further clinical investigation of JAK/STAT3 inhibitors in conjunction with chemotherapy or MEK inhibition in KrasMT CRC tumours. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 908. doi:1538-7445.AM2012-908

Background Colorectal cancer (CRC) is the third most common cancer in the UK with around 80% of patients undergoing surgery followed by adjuvant chemotherapy treatment. Among patients with clinicopathologically defined poor stage II and stage III CRC, there are subsets of patients who will not benefit from adjuvant 5-FU or 5-FU/oxaliplatin treatment. In fact, there is preclinical evidence to suggest that some of these patients may actually do worse when treated with adjuvant chemotherapy. The aim of this study was to develop in vitro adjuvant colon cancer models with the ultimate goal to identify novel treatment strategies for early stage CRC. Method Progressively invasive populations from parental HCT116 were generated using invasion chambers. Analysis of migration and invasion was carried out using the XCELLigence system, protein activity/expression using receptor tyrosine kinase array (RTK) and Western blotting, colony forming ability was assessed by survival assays and drug sensitivity using MTT assay and Flow cytometry. Results Six sublines of HCT116 cells were initially generated (I1-6) which displayed an increasing invasive/migratory phenotype compared to the parental cell line. At the molecular level, we found increases in basal activity of a number of RTK in the invasive sublines, such as Axl (a phenomenon which is characterized with a more aggressive nature in cancers) and also in other kinases such as ERK1/2, Akt and STAT3. Decreased activation of the EGFR and HER2 receptors was also noted in the invasive cell lines. In addition, exposure to sub-lethal 5-FU(IC20) doses resulted in significant increases in migration in these cell line models. Silencing of Axl resulted in significant decreases in migration of the invasive subpopulations even in the presence of 5-FU. Colony forming assays highlighted an increased ability to form colonies in the invasive sub-populations which also displayed drug resistance to both 5-FU and Oxaliplatin. The generation of invasive cell lines was also carried out in a number of other cell lines which confirmed that an increase in Axl expression could increase the migratory phenotype regardless of the genetic background of the cell and also that the results obtained are not an artefact of the cell line we have used. Conclusion Using our in-house developed in vitro preclinical cell line model we have begun to characterise the changes which tumour cells appear to go through during the transition to an invasive phenotype. We have highlighted Axl as a potential target for inhibition in early stage colorectal cancer as increases in total Axl protein levels correlate well with increases in invasive capacity in our models and silencing of the protein expression by siRNA results in ablation of the invasive potential in our model and also in a panel of CRC cell lines. Combination of an Axl small molecule inhibitor could potentially be a novel treatment strategy for early stage CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4324. doi:1538-7445.AM2012-4324