Colorectal Cancer Intrinsic Subtypes Research Articles

Analysis of primary and acquired resistance to cetuximab from multiomic data in the New EPOC trial.

3577 Background: The use of cetuximab (cetux) with chemotherapy (chemo) in patients with operable liver metastases from colorectal cancer (CRC) conferred a survival disadvantage in New EPOC. Whilst genetic alterations such as emergent sub-clonal mutations in RAS/ BRAF, ERBB2 and MET are recognized as mechanisms of resistance to EGFR inhibition (EGFRi), divergent mechanisms such as transcriptomic changes may also be responsible. Of the CRC intrinsic subtypes (CRIS), CRIS-C has elevated EGFR signalling and is thought to have greater sensitivity to EGFRi. Methods: Resected primary tumors (n = 205; pre neoadjuvant treatment) and/or liver metastases (n = 144; post neoadjuvant treatment) from 223 patients, wild type for KRAS (codons 12/13/61) at trial entry, randomized to chemo +/- cetux underwent targeted NGS and/or transcriptome profiling. Primary clinical endpoints were progression free (PFS) and overall survival (OS). Results: Repeat NGS identified additional RAS/BRAF mutations in primary tumors from 49 patients (25 KRAS, 15 NRAS, 9 BRAF V6000E). Of the 148 patients with confirmed RAS/BRAF wild type primary tumors , 46% were classified as CRIS-C with the remainder being CRIS-A (9.5%), CRIS-B (11%), CRIS-D (8.8%) or CRIS-E (8.1%). CRIS-C patients had a similar PFS with ChemoCetux vs Chemo (HR 0.84 95% CI 0.47-1.5 p = 0.55) whereas non-CRIS-C had a shorter PFS (HR 2.12 95%CI 1.24-3.63 p = 0.006; p interaction 0.021). OS results were similar (p interaction 0.067). In liver metastases, genetic changes were comparable (Chemo vs ChemoCetux): KRAS (4.1% v 2.1%), NRAS (4.1% v 0%), BRAF V600E (0% v 0%), ERBB2 gain/amplification (20% v 21%) and MET gain/amplification (25% v 18%). However, gain/amplification of MET resulted in shorter OS with ChemoCetux vs Chemo (HR 5.13 95%CI 1.38-19.15 p = 0.015) whereas those without MET gain/amplification had a similar OS (HR 1.32 95%CI 0.76-2.28 p = 0.32; p interaction 0.029). The same association was found after excluding liver metastases with RAS/BRAF mutations (n = 101, p interaction 0.030). Differential expression/mutation analyses did not identify other stratifiers after false discovery correction. Conclusions: The shorter survival with ChemoCetux was restricted to patients with a non CRIS-C subtype before treatment supporting the use of this transcriptomic classifier to stratify for EGFRi. After neoadjuvant treatment, gain/amplification of MET was associated with a shorter survival with ChemoCetux. By contrast MAPK pathway mutations were largely unchanged supporting earlier findings that alternative resistance mechanisms predominate when EGFRi is combined with chemotherapy. In summary these results highlight the heterogeneity and differential responses to EGFRi that exist within RAS/RAF wild type CRC and provide insights into the substantial survival detriment observed in the New EPOC trial. Clinical trial information: ISRCTN22944367 .

Impact of genome and epigenome on intratumor heterogeneity in colorectal cancer

Impact of genome and epigenome on intratumor heterogeneity in colorectal cancer

Correlation of Immunological and Histopathological Features with Gene Expression-Based Classifiers in Colon Cancer Patients.

The purpose of this study was to evaluate the association between four distinct histopathological features: (1) tumor infiltrating lymphocytes, (2) mucinous differentiation, (3) tumor-stroma ratio, plus (4) tumor budding and two gene expression-based classifiers—(1) consensus molecular subtypes (CMS) plus (2) colorectal cancer intrinsic subtypes (CRIS). All four histopathological features were retrospectively scored on hematoxylin and eosin sections of the most invasive part of the primary tumor in 218 stage II and III colon cancer patients from two independent cohorts (AMC-AJCC-90 and AC-ICAM). RNA-based CMS and CRIS assignments were independently obtained for all patients. Contingency tables were constructed and a χ2 test was used to test for statistical significance. Odds ratios with 95% confidence intervals were calculated. The presence of tumor infiltrating lymphocytes and a mucinous phenotype (>50% mucinous surface area) were strongly correlated with CMS1 (p < 0.001 and p = 0.008) and CRIS-A (p = 0.006 and p < 0.001). The presence of mucus (≥ 10%) was associated with CMS3: mucus was present in 64.1% of all CMS3 tumors (p < 0.001). Although a clear association between tumor-stroma ratio and CMS4 was established in this study (p = 0.006), still 32 out of 61 (52.5%) CMS4 tumors were scored as stroma-low, indicating that CMS4 tumors cannot be identified solely based on stromal content. Higher budding counts were seen in CMS4 and CRIS-B tumors (p = 0.045 and p = 0.046). No other associations of the measured parameters were seen for any of the other CRIS subtypes. Our analysis revealed clear associations between histopathologic features and CMS or CRIS subtypes. However, identification of distinct molecular subtypes solely based on histopathology proved to be infeasible. Combining both molecular and morphologic features could potentially improve patient stratification.

De novo transcriptomic subtyping of colorectal cancer liver metastases in the context of tumor heterogeneity

BackgroundGene expression-based subtyping has the potential to form a new paradigm for stratified treatment of colorectal cancer. However, current frameworks are based on the transcriptomic profiles of primary tumors, and metastatic heterogeneity is a challenge. Here we aimed to develop a de novo metastasis-oriented framework.MethodsIn total, 829 transcriptomic profiles from patients with colorectal cancer were analyzed, including primary tumors, liver metastases, and non-malignant liver samples. High-resolution microarray gene expression profiling was performed of 283 liver metastases from 171 patients treated by hepatic resection, including multiregional and/or multi-metastatic samples from each of 47 patients. A single randomly selected liver metastasis sample from each patient was used for unsupervised subtype discovery by nonnegative matrix factorization, and a random forest prediction model was trained to classify multi-metastatic samples, as well as liver metastases from two independent series of 308 additional patients.ResultsInitial comparisons with non-malignant liver samples and primary colorectal tumors showed a highly variable degree of influence from the liver microenvironment in metastases, which contributed to inter-metastatic transcriptomic heterogeneity, but did not define subtype distinctions. The de novo liver metastasis subtype (LMS) framework recapitulated the main distinction between epithelial-like and mesenchymal-like tumors, with a strong immune and stromal component only in the latter. We also identified biologically distinct epithelial-like subtypes originating from different progenitor cell types. LMS1 metastases had several transcriptomic features of cancer aggressiveness, including secretory progenitor cell origin, oncogenic addictions, and microsatellite instability in a microsatellite stable background, as well as frequent RAS/TP53 co-mutations. The poor-prognostic association of LMS1 metastases was independent of mutation status, clinicopathological variables, and current subtyping frameworks (consensus molecular subtypes and colorectal cancer intrinsic subtypes). LMS1 was also the least heterogeneous subtype in comparisons of multiple metastases per patient, and tumor heterogeneity did not confound the prognostic value of LMS1.ConclusionsWe report the first large study of multi-metastatic gene expression profiling of colorectal cancer. The new metastasis-oriented subtyping framework showed potential for clinically relevant transcriptomic classification in the context of metastatic heterogeneity, and an LMS1 mini-classifier was constructed to facilitate prognostic stratification and further clinical testing.

Transcriptomic and proteomic intra-tumor heterogeneity of colorectal cancer varies depending on tumor location within the colorectum.

Intra-tumor heterogeneity (ITH) of colorectal cancer (CRC) complicates molecular tumor classification, such as transcriptional subtyping. Differences in cellular states, biopsy cell composition, and tumor microenvironment may all lead to ITH. Here we analyze ITH at the transcriptomic and proteomic levels to ascertain whether subtype discordance between multiregional biopsies reflects relevant biological ITH or lack of classifier robustness. Further, we study the impact of tumor location on ITH. Multiregional biopsies from stage II and III CRC tumors were analyzed by RNA sequencing (41 biopsies, 14 tumors) and multiplex immune protein analysis (89 biopsies, 29 tumors). CRC subtyping was performed using consensus molecular subtypes (CMS), CRC intrinsic subtypes (CRIS), and TUMOR types. ITH-scores and network maps were defined to determine the origin of heterogeneity. A validation cohort was used with one biopsy per tumor (162 tumors). Overall, inter-tumor transcriptional variation exceeded ITH, and subtyping calls were frequently concordant between multiregional biopsies. Still, some tumors had high transcriptional ITH and were classified discordantly. Subtyping of proximal MSS tumors were discordant for 50% of the tumors, this ITH was related to differences in the microenvironment. Subtyping of distal MSS tumors were less discordant, here the ITH was more cancer-cell related. The subtype discordancy reflected actual molecular ITH within the tumors. The relevance of the subtypes was reflected at protein level where several inflammation markers were significantly increased in immune related transcriptional subtypes, which was verified in an independent cohort (Wilcoxon rank sum test; p<0.05). Unsupervised hierarchical clustering of the protein data identified large ITH at protein level; as the multiregional biopsies clustered together for only 9 out of 29 tumors. Our transcriptomic and proteomic analyses show that the tumor location along the colorectum influence the ITH of CRC, which again influence the concordance of subtyping.

RALB GTPase: a critical regulator of DR5 expression and TRAIL sensitivity in KRAS mutant colorectal cancer

RAS mutant (MT) metastatic colorectal cancer (mCRC) is resistant to MEK1/2 inhibition and remains a difficult-to-treat group. Therefore, there is an unmet need for novel treatment options for RASMT mCRC. RALA and RALB GTPases function downstream of RAS and have been found to be key regulators of several cell functions implicated in KRAS-driven tumorigenesis. However, their role as regulators of the apoptotic machinery remains to be elucidated. Here, we found that inhibition of RALB expression, but not RALA, resulted in Caspase-8-dependent cell death in KRASMT CRC cells, which was not further increased following MEK1/2 inhibition. Proteomic analysis and mechanistic studies revealed that RALB depletion induced a marked upregulation of the pro-apoptotic cell surface TRAIL Death Receptor 5 (DR5) (also known as TRAIL-R2), primarily through modulating DR5 protein lysosomal degradation. Moreover, DR5 knockdown or knockout attenuated siRALB-induced apoptosis, confirming the role of the extrinsic apoptotic pathway as a regulator of siRALB-induced cell death. Importantly, TRAIL treatment resulted in the association of RALB with the death-inducing signalling complex (DISC) and targeting RALB using pharmacologic inhibition or RNAi approaches triggered a potent increase in TRAIL-induced cell death in KRASMT CRC cells. Significantly, high RALB mRNA levels were found in the poor prognostic Colorectal Cancer Intrinsic Subtypes (CRIS)-B CRC subgroup. Collectively, this study provides to our knowledge the first evidence for a role for RALB in apoptotic priming and suggests that RALB inhibition may be a promising strategy to improve response to TRAIL treatment in poor prognostic RASMT CRIS-B CRC.

Technical differences between sequencing and microarray platforms impact transcriptomic subtyping of colorectal cancer.

Gene expression profiling has increasing relevance in the molecular screening of patients with colorectal cancer (CRC). We investigated potential platform-specific effects on transcriptomic subtyping according to established frameworks by comparisons of expression profiles from RNA sequencing and exon-resolution microarrays in 126 primary microsatellite stable CRCs. There was a strong platform correspondence in global gene expression levels, albeit with systematic technical bias likely attributed to few sequencing reads covering short (<2000 nucleotides) and/or lowly expressed genes (<1 FPKM), as well as over-saturation of highly expressed genes on microarrays. Classification concordances according to both the consensus molecular subtypes and CRC intrinsic subtypes (CRIS) were also strong, but with disproportionate subtype distributions between platforms caused by frequent disagreements in adherence to sample classification thresholds. Subtypes defined largely by genes expressed at low levels, including the CRIS-D subtype and the estimated level of tumor-infiltrating cytotoxic lymphocytes, had a weaker correspondence in classification metrics between platforms. In conclusion, even subtle differences between platforms suggest that clinical translation of transcriptomic CRC subtyping frameworks is dependent on assay standardization, and systematic technical biases reinforce the need for careful selection of classifier genes.

Abstract 5365: Prospective patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies

Abstract 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.

PO-523 Sensitivity of colorectal cancer (CRC) CRIS subtypes to SMAC mimetic birinapant with standard of care chemotherapeutics: implications for personalised medicine

IntroductionColorectal cancer (CRC) is a worldwide cause of death and requires better treatment options. The stratification of CRC cell-lines into tumour representative molecular subtypes may improve drug screening in-vitro to identify improved chemotherapeutics. The CRIS classifier identifies five ‘CRC intrinsic subtypes’ within the cancer cell transcriptome: A, B, C, D and E. This classifier improves cell line subtype mapping by considering only tumour cell transcripts. The inhibitor of apoptosis (IAP) proteins are promising targets in personalised medicine as CRC tumours dysregulate IAPs to avoid apoptosis. This study explored the efficacy of Birinapant - a potent SMAC mimetic and antagonist of IAPs, along with synergistic agents to promote optimal cell death in vitro using a model of CRC intrinsic subtypes.Material and methodsThe CRIS classifier analysis was performed on 150 CRC cell transcriptomes to select 10 cell lines: CRIS A (DLD-1, LS174T), CRIS B (LoVo, HT29), CRIS C (Gp5D, LIM1215), CRIS D (HCT116, RKO) and CRIS E (HCT116 p53-/-, LS513). The cells were treated with 1 µM Birinapant alone, and/or the cytotoxic pair Oxaliplatin and 5-Fluorouracil (2 µM, 10 µM), and/or TNF-alpha. Post-treatment cellular sensitivity was measured at 24, 48 and 72 hours by assessing Annexin V/Propidium Iodide positivity using high content microscopy.Results and discussionsThe cell death data from the entire panel showed that Birinapant cannot induce apoptosis when administered alone. However, Birinapant elicits synergistic effects in combination with Oxaliplatin/5-Flourouracil, or the pro-inflammatory cytokine TNF-alpha. The greatest degree of sensitivity was observed in the HCT116 and RKO CRIS D cell lines and least in the DLD1 and HT29 CRIS A subtypes. Of note, the p53-deficient HCT116 CRIS E model was less sensitive to the combined effects of Birinapant and chemotherapy, suggesting an important role for p53 in mediating this synergy.ConclusionFuture work will expand the analysis to more CRIS D cell lines and investigate the molecular mechanisms behind the p53-dependence of the chemotherapy/IAP antagonist interactions. These results have clinical relevance as in parallel studies, we have shown that patients with CRIS D subtype tumours have a suboptimal response to standard-of-care chemotherapy.1Reference1. WL Allen et al., JCO Precision Oncology, 2018 (in press)

Transcriptional subtyping and CD8 immunohistochemistry identifies poor prognosis stage II/III colorectal cancer patients who benefit from adjuvant chemotherapy.

PurposeTranscriptomic profiling of colorectal cancer (CRC) has led to the identification of four consensus molecular subtypes (CMS1 to 4) that have prognostic value in stage II and III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors; however, the clinical value of these classification systems in the prediction of response to standard-of-care adjuvant chemotherapy remains unknown.Patients and MethodsUsing samples from four European sites, we assembled a novel cohort of patients with stage II and III CRC (n = 156 samples) and performed transcriptomic profiling and targeted sequencing and generated a tissue microarray to enable integrated multiomics analyses. We also accessed data from two published cohorts of patients with stage II and III CRC: GSE39582 and GSE14333 (n = 479 and n = 185 samples, respectively).ResultsThe epithelial-rich CMS2 subtype of CRC benefitted significantly from treatment with adjuvant chemotherapy in both stage II and III disease (P = .02 and P < .001, respectively), whereas the CMS3 subtype significantly benefitted in stage III only (P = .001). After CRIS substratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from treatment with adjuvant chemotherapy in stage II and III disease (P = .0081 and P < .001, respectively), whereas the CRIS-D subtype significantly benefitted in stage III only (P = .0034). We also observed that CRIS-C patients with low levels of CD8+ tumor-infiltrating lymphocytes were most at risk for relapse in both stage II and III disease (log-rank P = .0031; hazard ratio, 12.18 [95% CI, 1.51 to 98.58]).ConclusionPatient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying patients with poor prognostic stage II and III disease who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for patients with stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.

Prospective patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies.

Colorectal cancer (CRC) biopsies underpin accurate diagnosis, but are also relevant for patient stratification in molecularly‐guided clinical trials. The consensus molecular subtypes (CMSs) and colorectal cancer intrinsic subtypes (CRISs) transcriptional signatures have potential clinical utility for improving prognostic/predictive patient assignment. However, their ability to provide robust classification, particularly in pretreatment 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) pretreatment biopsies from the phase II rectal cancer trial COPERNCIUS (NCT01263171; n = 44). Compared to previous results obtained using CRC resection material, we 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 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. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies may inform prospective clinical trials

Background: Biopsies taken from primary colorectal cancers (CRC) serve as an initial diagnostic tool, but are increasingly being considered for use in trials involving patient stratification. The recently published consensus molecular subtypes (CMS) and colorectal cancer intrinsic subtypes (CRIS), which identify subtypes based on overall tumour microenvironment (TME) and neoplastic-specific gene expression profiles respectively, have evident prognostic value and have been suggested as stratification tools in the clinic. However, both CMS and CRIS molecular subtyping techniques have been validated only in CRC resection specimens, therefore a validation of their potential utility in pre-treatment biopsies should be undertaken.

Abstract 1742: Cancer cell-selective transcriptome analysis reveals new colorectal cancer molecular subtypes with improved biological resolution and superior predictive and prognostic performance

Abstract Molecular classification of colorectal cancer (CRC) based on gene expression profiling of tumor samples is known to be heavily affected by transcripts of stromal origin. As a consequence, current CRC transcriptional subtypes reflect an admixture of cancer cell-intrinsic traits and tumor microenvironment features. Whether selective analysis of the cancer cell transcriptome could improve CRC subtyping, remains an open issue. In patient-derived xenografts (PDXs), human transcripts only originate from cancer cells, because stromal transcripts are of mouse origin. We therefore assessed cancer-cell intrinsic transcriptional features of CRC by generating human-specific expression profiles of 515 PDXs from 244 CRC patients, and performing unsupervised class discovery. We identified five “CRC intrinsic subtypes” (CRIS A-E) only partially overlapping with the current ones, and robustly enriched for distinct molecular, functional and phenotypic traits: (i) CRIS-A: mucinous, glycolytic, CIMP, and enriched for microsatellite instability or mutations in KRAS; (ii) CRIS-B: marked TGF-β pathway activity, epithelial-mesenchymal transition and poor prognosis; (iii) CRIS-C: MSS, elevated EGFR signaling and sensitivity to EGFR-targeted treatments; (iv) CRIS-D: MSS, WNT activation, and IGF2 overexpression and amplification; (v) CRIS-E: MSS, Paneth cell-like phenotype and higher frequency of TP53 mutation. CRIS subtypes successfully categorized independent sets of primary and metastatic CRCs and cell lines, for a total of over 3000 samples profiled by microarrays and RNAseq. The new subtypes displayed unprecedented predictive and prognostic performances, indipendent from known markers including stromal signatures, whose integration with CRIS further enhanced prognostic significance. Citation Format: Claudio Isella, Sara E. Bellomo, Francesco Brundu, Francesco Galimi, Elisa Ficarra, Luigi Marchionni, Livio Trusolino, Andrea Bertotti, Enzo Medico. Cancer cell-selective transcriptome analysis reveals new colorectal cancer molecular subtypes with improved biological resolution and superior predictive and prognostic performance [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 1742. doi:10.1158/1538-7445.AM2017-1742

Selective analysis of cancer-cell intrinsic transcriptional traits defines novel clinically relevant subtypes of colorectal cancer

Stromal content heavily impacts the transcriptional classification of colorectal cancer (CRC), with clinical and biological implications. Lineage-dependent stromal transcriptional components could therefore dominate over more subtle expression traits inherent to cancer cells. Since in patient-derived xenografts (PDXs) stromal cells of the human tumour are substituted by murine counterparts, here we deploy human-specific expression profiling of CRC PDXs to assess cancer-cell intrinsic transcriptional features. Through this approach, we identify five CRC intrinsic subtypes (CRIS) endowed with distinctive molecular, functional and phenotypic peculiarities: (i) CRIS-A: mucinous, glycolytic, enriched for microsatellite instability or KRAS mutations; (ii) CRIS-B: TGF-β pathway activity, epithelial–mesenchymal transition, poor prognosis; (iii) CRIS-C: elevated EGFR signalling, sensitivity to EGFR inhibitors; (iv) CRIS-D: WNT activation, IGF2 gene overexpression and amplification; and (v) CRIS-E: Paneth cell-like phenotype, TP53 mutations. CRIS subtypes successfully categorize independent sets of primary and metastatic CRCs, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances.

Abstract IA09: Expression profiling of colorectal cancer PDX models for disease stratification and tumor-stroma interaction analysis

Abstract Tumor-stroma interactions have long been implicated in neoplastic aggressiveness and resistance to treatment. In PDX models, after engraftment, the stromal components are contributed by the mouse host. As a consequence, transcripts expressed by cancer cells can be distinguished from stromal transcripts by their species of origin. We therefore exploited species-specific expression profiling of colorectal cancer (CRC) PDXs to evaluate the involvement of stromal components in recently defined CRC transcriptional subtypes. We found that stroma-derived transcripts profoundly affect the overall tumor transcriptome, and that a previously defined poor prognosis subtype is actually endowed with abundant stroma. To stratify CRC based on cancer cell-intrinsic transcriptional features, we performed class discovery on a human-specific gene expression dataset of 515 CRC PDXs, and identified five CRC intrinsic subtypes (CRIS). CRIS classification successfully categorized independent sets of primary and metastatic CRC tumors, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances. In a complementary approach, we focused on stroma-specific transcripts to define expression signatures reflecting the abundance in human CRC samples of fibroblasts, leukocytes and endothelial cells. Stromal scores were found to correlate with poor CRC prognosis and resistance of rectal cancer to preoperative radiotherapy, highlighting the microenvironment as a variable and dynamic component of CRC, with diagnostic and therapeutic potential. Citation Format: Enzo Medico. Expression profiling of colorectal cancer PDX models for disease stratification and tumor-stroma interaction analysis. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA09.

Abstract 107: Unsupervised analysis of cancer-cell intrinsic transcriptional traits defines a new classification system for colorectal cancer with improved predictive and prognostic value

Abstract Recent work built on published transcriptional subtypes of colorectal cancer (CRC) has shown that tumor stromal content heavily impacts CRC classification, with clinical and biological implications. Lineage-dependent transcriptional components of stromal origin could therefore dominate over more subtle, yet relevant, gene expression traits inherent to cancer cells. Based on the notion that in patient-derived xenografts (PDXs) stromal cells of the human tumor are substituted by their murine counterparts, we assessed cancer-cell intrinsic transcriptional features by human-specific expression profiling on 515 PDXs from 244 CRC patients. We identified five CRC intrinsic subtypes (CRIS) endowed with molecular, functional and phenotypic peculiarities, many of which were not previously evidenced: CRIS-A was a MIN-like, KRAS-mutated, and secretory subtype with sustained glycolytic metabolism and inflammatory traits; CRIS-B grouped a subset of poorly differentiated tumors with active TGFβ signaling and EMT features; CRIS-C clustered KRAS wild-type CIN tumors exhibiting elevated EGFR pathway activity and MYC copy number gains; CRIS-D tumors were characterized by a typical stem phenotype with high WNT signaling, associated with strong enrichment in IGF2 amplification/overexpression and FGFR autocrine stimulation; CRIS-E again displayed WNT-related features, but associated with a Paneth-like phenotype and a TP53-mutated genotype. CRIS subtypes successfully categorized independent sets of primary and metastatic CRC tumors, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances. Integration of stroma-centered transcriptional profiles with cancer-cell intrinsic classification by CRIS further enhanced prognostic significance. Citation Format: Andrea Bertotti, Claudio Isella, Sara E. Bellomo, Francesco Brundu, Francesco Galimi, Elisa Ficarra, Livio Trusolino, Enzo Medico. Unsupervised analysis of cancer-cell intrinsic transcriptional traits defines a new classification system for colorectal cancer with improved predictive and prognostic value. [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 107.

Association of colorectal cancer intrinsic subtypes with prognosis, chemotherapy response, deficient mismatch repair, and epithelial to mesenchymal transition (EMT).

3530 Background: Unbiased genome-wide analyses of gene expression patterns have been successfully used for molecular classification of breast cancer into subtypes that have clear relevance for prognosis and treatment. A similar classification is still missing for colorectal cancer (CRC). Methods: Using full genome expression data of 188 stage I-IV CRC patients, an unsupervised clustering revealed three major subtypes (A-, B-, C-type). A molecular subtype classification was developed and validated in 543 stage II and III patients. The subtypes were analyzed for correlation to clinical information, mutations in the kinome, known molecular markers status and chemotherapy response. In addition, subtypes were determined on 173 samples from The Cancer Genome Atlas (TCGA) colon dataset with Agilent genome expression data. Results: C-type patients have the worst outcome, a mesenchymal phenotype, and show no benefit from adjuvant chemotherapy treatment. Patients having A- or B-type tumors have a better clinical outcome, a more proliferative and epithelial phenotype and benefit from adjuvant chemotherapy. A- and C-type groups are enriched for tumors having oncogenic BRAF mutations and a deficient DNA mismatch repair system. B-type tumors showed a low overall kinome mutation frequency (1.6%), while both A- and C-type patients harbor a higher mutation frequency (respectively 4.2 and 6.2%), in agreement with their mismatch repair deficiency. Finally, CRC subtyping was confirmed in the colon TCGA dataset with 26 samples classified as A-type, 110 as B-type and 37 as C-type. In agreement with the different aggressiveness of the subtypes, A-type tumors were less prevalent in stage IV while C-type were less prevalent in stage I CRC. Conclusions: The heterogeneity of the intrinsic subtypes is largely based on three biological hallmarks of the tumor: an epithelial-to-mesenchymal transition, deficiency in mismatch repair genes that result in a high mutation frequency associated with MSI, and cellular proliferation. These subtypes are clinically relevant, as they differ in their underlying biology and might require different treatment strategies.

Association of colorectal cancer intrinsic subtypes with prognosis, chemotherapy response, deficient mismatch repair, and epithelial to mesenchymal transition (EMT).

333 Background: Unbiased genome-wide analyses of gene expression patterns have been successfully used for the molecular classification of breast cancer into subtypes that have clear relevance for prognosis and development of treatment plans. For colorectal cancer (CRC), however, a molecular classification is still missing. Methods: Using gene expression data of 188 stage I-IV colorectal cancer (CRC) patients, a molecular subtype classification was developed. The classifier was validated in 543 stage II and III patients and the subtypes were analyzed for correlation to clinical information, mutations in the kinome, known molecular marker status and chemotherapy response. Results: CRC is a heterogeneous disease that consists of at least three major intrinsic subtypes (A-, B-, C-type). The heterogeneity of the intrinsic subtypes is largely based on three biological hallmarks of the tumor: an epithelial-to-mesenchymal transition, deficiency in mismatch repair genes that result in a high mutation frequency associated with MSI, and cellular proliferation. C-type patients have the worst outcome, a mesenchymal gene expression phenotype, and show no benefit from adjuvant chemotherapy treatment. Patients having A-type or B-type tumors have a better clinical outcome, a more proliferative and epithelial phenotype, and benefit from adjuvant chemotherapy. B-type tumors showed a low overall kinome mutation frequency (1.6%), while both A-type and C-type patients harbor a higher mutation frequency (respectively 4.2 and 6.2%), in agreement with their mismatch repair deficiency. Conclusions: We have developed a diagnostic single sample predictor that allows the classification of CRC tumors of different intrinsic molecular subtypes. These subtypes are potentially clinically relevant, as they differ in their underlying biology and clinical outcome and consequently require different treatment strategies. [Table: see text]