The prognostic effect of tumor-associated macrophages in stage I-III colorectal cancer depends on T cell infiltration.

BACKGROUND: Tumor-associated macrophages (TAMs) are associated with unfavorable patient prognosis in many cancer types. However, TAMs are a heterogeneous cell population and subsets have been shown to activate tumor-infiltrating T cells and confer a good patient prognosis. Data on the prognostic value of TAMs in colorectal cancer are conflicting. We investigated the prognostic effect of TAMs in relation to tumor-infiltrating T cells in colorectal cancers. METHODS: The TAM markers CD68 and CD163 were analyzed by multiplex fluorescence immunohistochemistry and digital image analysis on tissue microarrays of 1720 primary colorectal cancers. TAM density in the tumor stroma was scored in relation to T cell density (stromal CD3+ and epithelial CD8+ cells) and analyzed in Cox proportional hazards models of 5-year relapse-free survival. Multivariable survival models included clinicopathological factors, MSI status and BRAFV600E mutation status. RESULTS: High TAM density was associated with a favorable 5-year relapse-free survival in a multivariable model of patients with stage I-III tumors (p = 0.004, hazard ratio 0.94, 95% confidence interval 0.90-0.98). However, the prognostic effect was dependent on tumoral T-cell density. High TAM density was associated with a good prognosis in patients who also had high T-cell levels in their tumors, while high TAM density was associated with poorer prognosis in patients with low T-cell levels (pinteraction = 0.0006). This prognostic heterogeneity was found for microsatellite stable tumors separately. CONCLUSIONS: This study supported a phenotypic heterogeneity of TAMs in colorectal cancer, and showed that combined tumor immunophenotyping of multiple immune cell types improved the prediction of patient prognosis.

Spatial analysis and CD25-expression identify regulatory T cells as predictors of a poor prognosis in colorectal cancer.

Regulatory T cells (Tregs) are a heterogeneous cell population that can either suppress or stimulate immune responses. Tumor-infiltrating Tregs are associated with an adverse outcome from most cancer types, but have generally been found to be associated with a good prognosis in colorectal cancer (CRC). We investigated the prognostic heterogeneity of Tregs in CRC by co-expression patterns and spatial analyses with diverse T cell markers, using multiplex fluorescence immunohistochemistry and digital image analysis in two consecutive series of primary CRCs (total n = 1720). Treg infiltration in tumors, scored as FOXP3+ or CD4+/CD25+/FOXP3+ (triple-positive) cells, was strongly correlated to the overall amount of CD3+ and CD8+ T cells, and consequently associated with a favorable 5-year relapse-free survival rate among patients with stage I-III CRC who underwent complete tumor resection. However, high relative expression of the activation marker CD25 in triple-positive Tregs was independently associated with an adverse outcome in a multivariable model incorporating clinicopathological and known molecular prognostic markers (hazard ratio = 1.35, p = 0.028). Furthermore, spatial marker analysis based on Voronoi diagrams and permutation testing of cellular neighborhoods revealed a statistically significant proximity between Tregs and CD8+-cells in 18% of patients, and this was independently associated with a poor survival (multivariable hazard ratio = 1.36, p = 0.017). These results show prognostic heterogeneity of different Treg populations in primary CRC, and highlight the importance of multi-marker and spatial analyses for accurate immunophenotyping of tumors in relation to patient outcome.

E-cadherin is a robust prognostic biomarker in colorectal cancer and low expression is associated with sensitivity to inhibitors of topoisomerase, aurora, and HSP90 in preclinical models.

Cell-cell and cell-matrix adhesion proteins that have been implicated in colorectal epithelial integrity and epithelial-to-mesenchymal transition could be robust prognostic and potential predictive biomarkers for standard and novel therapies. We analyzed in situ protein expression of E-cadherin (ECAD), integrin ß4 (ITGB4), zonula occludens 1 (ZO-1), and cytokeratins in a single-hospital series of Norwegian patients with colorectal cancer (CRC) stages I-IV (n = 922) using multiplex fluorescence-based immunohistochemistry (mfIHC) on tissue microarrays. Pharmacoproteomic associations were explored in 35 CRC cell lines annotated with drug sensitivity data on > 400 approved and investigational drugs. ECAD, ITGB4, and ZO-1 were positively associated with survival, while cytokeratins were negatively associated with survival. Only ECAD showed independent prognostic value in multivariable Cox models. Clinical and molecular associations for ECAD were technically validated on a different mfIHC platform, and the prognostic value was validated in another Norwegian series (n = 798). In preclinical models, low and high ECAD expression differentially associated with sensitivity to topoisomerase, aurora, and HSP90 inhibitors, and EGFR inhibitors. E-cadherin protein expression is a robust prognostic biomarker with potential clinical utility in CRC.

Prognostic role of tumour-infiltrating lymphocytes and macrophages in relation to MSI, CDX2 and BRAF status: a population-based study of metastatic colorectal cancer patients.

BACKGROUND: Tumour-infiltrating CD3, CD8 lymphocytes and CD68 macrophages are associated with favourable prognosis in localised colorectal cancer, but the effect in metastatic colorectal cancer (mCRC) is not established. METHODS: A Scandinavian population-based cohort of non-resectable mCRC patients was studied. Tissue microarrays (n = 460) were stained with CD3, CD8 and CD68 using fluorescence-based multiplex immunohistochemistry. Associations with clinicopathological variables, overall survival (OS) and progression-free survival were estimated. RESULTS: Two-thirds of microsatellite instable (MSI) and one-fourth of microsatellite stable (MSS) tumours displayed the highest quartile density of CD8. For CD3 high vs low cases, median OS was 20 vs 16 months (HR: 0.76, 95% CI: 0.59, 0.76, p = 0.025) with 3-year OS of 27 vs 13%. For CD68 high vs low cases, median OS was 23 vs 15 months (HR: 0.69, 95% CI: 0.54, 0.88, p = 0.003) with 3-year OS of 28 vs 12%. MSI, BRAF mutation and CDX2 loss were negative prognostic markers independent of tumour immune infiltration. CONCLUSIONS: In mCRC, high lymphocyte infiltration was found in proportions of MSI and MSS tumours-potential subgroups of immunotherapy response. Tumour-infiltrating CD3 lymphocytes and CD68 macrophages were associated with median and long-term survival. MSI was a significant negative prognostic marker despite high immunogenicity.

The expressed mutational landscape of microsatellite stable colorectal cancers.

BACKGROUND: Colorectal cancer is the 2nd leading cause of cancer-related deaths with few patients benefiting from biomarker-guided therapy. Mutation expression is essential for accurate interpretation of mutations as biomarkers, but surprisingly, little has been done to analyze somatic cancer mutations on the expression level. We report a large-scale analysis of allele-specific mutation expression. METHODS: Whole-exome and total RNA sequencing was performed on 137 samples from 121 microsatellite stable colorectal cancers, including multiregional samples of primary and metastatic tumors from 4 patients. Data were integrated with allele-specific resolution. Results were validated in an independent set of 241 colon cancers. Therapeutic associations were explored by pharmacogenomic profiling of 15 cell lines or patient-derived organoids. RESULTS: The median proportion of expressed mutations per tumor was 34%. Cancer-critical mutations had the highest expression frequency (gene-wise mean of 58%), independent of frequent allelic imbalance. Systematic deviation from the general pattern of expression levels according to allelic frequencies was detected, including preferential expression of mutated alleles dependent on the mutation type and target gene. Translational relevance was suggested by correlations of KRAS/NRAS or TP53 mutation expression levels with downstream oncogenic signatures (p < 0.03), overall survival among patients with stage II and III cancer (KRAS/NRAS: hazard ratio 6.1, p = 0.0070), and targeted drug sensitivity. The latter was demonstrated for EGFR and MDM2 inhibition in pre-clinical models. CONCLUSIONS: Only a subset of mutations in microsatellite stable colorectal cancers were expressed, and the "expressed mutation dose" may provide an opportunity for more fine-tuned biomarker interpretations.

Prediction of relapse-free survival according to adjuvant chemotherapy and regulator of chromosome condensation 2 (RCC2) expression in colorectal cancer.

BACKGROUND: There is a need for improved selection of patients for adjuvant chemotherapy after resection of non-metastatic colorectal cancer (CRC). Regulator of chromosome condensation 2 (RCC2) is a potential prognostic biomarker. We report on the establishment of a robust protocol for RCC2 expression analysis and prognostic tumour biomarker evaluation in patients who did and did not receive adjuvant chemotherapy. MATERIALS AND METHODS: RCC2 was analysed in 2916 primary CRCs from the QUASAR2 randomised trial and two single-hospital Norwegian series. A new protocol using fluorescent antibody staining and digital image analysis was optimised. Biomarker value for 5-year relapse-free survival was analysed in relation to tumour stage, adjuvant chemotherapy and the molecular markers microsatellite instability, KRAS/BRAFV600E/TP53 mutations and CDX2 expression. RESULTS: Low RCC2 expression was scored in 41% of 2696 evaluable samples. Among patients with stage I-III CRC who had not received adjuvant chemotherapy, low RCC2 expression was an independent marker of inferior 5-year relapse-free survival in multivariable Cox models including clinicopathological factors and molecular markers (HR 1.45, 95% CI 1.09 to 1.94, p=0.012, N=521). RCC2 was not prognostic in patients who had received adjuvant chemotherapy, neither in QUASAR2 nor the pooled Norwegian series. The interaction between RCC2 and adjuvant chemotherapy for prediction of patient outcome was significant in stage III, and strongest among patients with microsatellite stable tumours (pinteraction=0.028). CONCLUSIONS: Low expression of RCC2 is a biomarker for poor prognosis in patients with stage I-III CRC and seems to be a predictive biomarker for effect of adjuvant chemotherapy.

Molecular correlates of sensitivity to PARP inhibition beyond homologous recombination deficiency in pre-clinical models of colorectal cancer point to wild-type TP53 activity.

BACKGROUND: PARP inhibitors are active in various tumour types beyond BRCA-mutant cancers, but their activity and molecular correlates in colorectal cancer (CRC) are not well studied. METHODS: Mutations and genome-wide mutational patterns associated with homologous recombination deficiency (HRD) were investigated in 255 primary CRCs with whole-exome sequencing and/or DNA copy number data. Efficacy of five PARP inhibitors and their molecular correlates were evaluated in 93 CRC cell lines partly annotated with mutational-, DNA copy number-, and/or gene expression profiles. Post-treatment gene expression profiling and specific protein expression analyses were performed in two pairs of PARP inhibitor sensitive and resistant cell lines. FINDINGS: A subset of microsatellite stable (MSS) CRCs had truncating mutations in homologous recombination-related genes, but these were not associated with genomic signatures of HRD. Eight CRC cell lines (9%) were sensitive to PARP inhibition, but sensitivity was not predicted by HRD-related genomic and transcriptomic signatures. In contrast, drug sensitivity in MSS cell lines was strongly associated with TP53 wild-type status (odds ratio 15.7, p = 0.023) and TP53-related expression signatures. Increased downstream TP53 activity was among the primary response mechanisms, and TP53 inhibition antagonized the effect of PARP inhibitors. Wild-type TP53-mediated suppression of RAD51 was identified as a possible mechanism of action for sensitivity to PARP inhibition. INTERPRETATION: PARP inhibitors are active in a subset of CRC cell lines and preserved TP53 function may increase the likelihood of response.

Patient-Derived Organoids from Multiple Colorectal Cancer Liver Metastases Reveal Moderate Intra-patient Pharmacotranscriptomic Heterogeneity.

PURPOSE: Molecular tumor heterogeneity may have important implications for the efficacy of targeted therapies in metastatic cancers. Inter-metastatic heterogeneity of sensitivity to anticancer agents has not been well explored in colorectal cancer. EXPERIMENTAL DESIGN: We established a platform for ex vivo pharmacogenomic profiling of patient-derived organoids (PDO) from resected colorectal cancer liver metastases. Drug sensitivity testing (n = 40 clinically relevant agents) and gene expression profiling were performed on 39 metastases from 22 patients. RESULTS: Three drug-response clusters were identified among the colorectal cancer metastases, based primarily on sensitivities to EGFR and/or MDM2 inhibition, and corresponding with RAS mutations and TP53 activity. Potentially effective therapies, including off-label use of drugs approved for other cancer types, could be nominated for eighteen patients (82%). Antimetabolites and targeted agents lacking a decisive genomic marker had stronger differential activity than most approved chemotherapies. We found limited intra-patient drug sensitivity heterogeneity between PDOs from multiple (2-5) liver metastases from each of ten patients. This was recapitulated at the gene expression level, with a highly proportional degree of transcriptomic and pharmacological variation. One PDO with a multi-drug resistance profile, including resistance to EGFR inhibition in a RAS-mutant background, showed sensitivity to MEK plus mTOR/AKT inhibition, corresponding with low-level PTEN expression. CONCLUSIONS: Intra-patient inter-metastatic pharmacological heterogeneity was not pronounced and ex vivo drug screening may identify novel treatment options for metastatic colorectal cancer. Variation in drug sensitivities was reflected at the transcriptomic level, suggesting potential to develop gene expression-based predictive signatures to guide experimental therapies.

A panel of intestinal differentiation markers (CDX2, GPA33, and LI-cadherin) identifies gastric cancer patients with favourable prognosis.

BACKGROUND: Gastric cancer is the fifth most common cancer and the third cause of global cancer mortality. CDX2 is an intestinal differentiation marker with prognostic value in gastric cancer and transcriptionally regulates the expression of glycoprotein A33 (GPA33) and liver intestine cadherin (LI-cadherin). METHODS: This study evaluated the clinical significance of the combined expression of CDX2 and its targets GPA33 and LI-cadherin in gastric cancer by fluorescence-based multiplex immunohistochemistry together with digital image analysis and chromogenic immunohistochemistry in 329 gastric cancer samples arranged in tissue microarrays. Additionally, publicly available RNA-seq expression data from 354 gastric cancer samples from the TCGA database were used to validate the immunohistochemistry results. RESULTS: Expression of the three markers (CDX2, GPA33, and LI-cadherin) was strongly correlated, defining an intestinal differentiation panel. Low or negative protein expression of the intestinal differentiation panel identified patients with particularly poor overall survival, irrespective of the methodology used, and was validated in the independent series at the RNA-seq level. CONCLUSIONS: Expression of the intestinal differentiation panel (CDX2, GPA33, and LI-cadherin) defines a set of biomarkers with a strong biological rationale and favourable impact for prognostication of gastric cancer patients.

Digital image analysis of multiplex fluorescence IHC in colorectal cancer recognizes the prognostic value of CDX2 and its negative correlation with SOX2.

Flourescence-based multiplex immunohistochemistry (mIHC) combined with multispectral imaging and digital image analysis (DIA) is a quantitative high-resolution method for determination of protein expression in tissue. We applied this method for five biomarkers (CDX2, SOX2, SOX9, E-cadherin, and ß-catenin) using tissue microarrays of a Norwegian unselected series of primary colorectal cancer. The data were compared with previously obtained chromogenic IHC data of the same tissue cores, visually assessed by the Allred method. We found comparable results between the methods, although confirmed that DIA offered improved resolution to differentiate cases with high and low protein expression. However, we experienced inherent challenges with digital image analysis of membrane staining, which was better assessed visually. DIA and mIHC enabled quantitative analysis of biomarker coexpression on the same tissue section at the single-cell level, revealing a strong negative correlation between the differentiation markers CDX2 and SOX2. Both methods confirmed known prognostic associations for CDX2, but DIA improved data visualization and detection of clinicopathological and biological associations. In summary, mIHC combined with DIA is an efficient and reliable method to evaluate protein expression in tissue, here shown to recapitulate and improve detection of known clinicopathological and survival associations for the emerging biomarker CDX2, and is therefore a candidate approach to standardize CDX2 detection in pathology laboratories.

Correction: Tumour-infiltrating CD8+ lymphocytes and colorectal cancer recurrence by tumour and nodal stage.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tumour-infiltrating CD8+ lymphocytes and colorectal cancer recurrence by tumour and nodal stage.

BACKGROUND: Intratumoural T-cell infiltrate intensity cortes wrelaith clinical outcome in stage II/III colorectal cancer (CRC). We aimed to determine whether this association varies across this heterogeneous group. METHODS: We performed a pooled analysis of 1804 CRCs from the QUASAR2 and VICTOR trials. Intratumoural CD8+ and CD3+ densities were quantified by immunohistochemistry in tissue microarray (TMA) cores, and their association with clinical outcome analysed by Cox regression. We validated our results using publicly available gene expression data in a pooled analysis of 1375 CRCs from seven independent series. RESULTS: In QUASAR2, intratumoural CD8+ was a stronger predictor of CRC recurrence than CD3+ and showed similar discriminative ability to both markers in combination. Pooled multivariable analysis of both trials showed increasing CD8+ density was associated with reduced recurrence risk independent of confounders including DNA mismatch repair deficiency, POLE mutation and chromosomal instability (multivariable hazard ratio [HR] for each two-fold increase = 0.92, 95%CI = 0.87-0.97, P = 3.6 × 10-3). This association was not uniform across risk strata defined by tumour and nodal stage: absent in low-risk (pT3,N0) cases (HR = 1.03, 95%CI = 0.87-1.21, P = 0.75), modest in intermediate-risk (pT4,N0 or pT1-3,N1-2) cases (HR = 0.92, 95%CI = 0.86-1.0, P = 0.046) and strong in high-risk (pT4,N1-2) cases (HR = 0.87, 95%CI = 0.79-0.97, P = 9.4 × 10-3); PINTERACTION = 0.090. Analysis of tumour CD8A expression in the independent validation cohort revealed similar variation in prognostic value across risk strata (PINTERACTION = 0.048). CONCLUSIONS: The prognostic value of intratumoural CD8+ cell infiltration in stage II/III CRC varies across tumour and nodal risk strata.

Exploratory analyses of consensus molecular subtype-dependent associations of TP53 mutations with immunomodulation and prognosis in colorectal cancer.

BACKGROUND: Accumulating evidence suggests immunomodulatory and context-dependent effects of TP53 mutations in cancer. We performed an exploratory analysis of the transcriptional, immunobiological and prognostic associations of TP53 mutations within the gene expression-based consensus molecular subtypes (CMSs) of colorectal cancer (CRC). MATERIALS AND METHODS: In a single-hospital series of 401 stage I-IV primary CRCs, we sequenced the whole coding region of TP53 and analysed CMS-dependent transcriptional consequences of the mutations by gene expression profiling. Immunomodulatory associations were validated by multiplex, fluorescence-based immunohistochemistry of immune cell markers. Prognostic associations of TP53 mutations were analysed in an aggregated series of 635 patients classified according to CMS, including publicly available data from a French multicentre cohort (GSE39582). RESULTS: TP53 mutations were found in 60% of the CRCs. However, gene set enrichment analyses indicated that their transcriptional consequences varied among the CMSs and were most pronounced in CMS1-immune and CMS4-mesenchymal. Subtype specificity was primarily seen as an upregulation of gene sets reflecting cell cycle progression in CMS4 and a downregulation of T cell activity in CMS1. The subtype-dependent immunomodulatory associations were reinforced by significant depletion of several immune cell populations in mutated tumours compared with wild-type (wt) tumours exclusively in CMS1, including cytotoxic lymphocytes (adjusted p value in CMS1=0.002 and CMS2-4>0.9, Microenvironment Cell Populations (MCP)-counter algorithm). This was validated by immunohistochemistry-based quantification of tumour infiltrating CD8+ cells. Within CMS1, the immunomodulatory association of TP53 mutations was strongest among microsatellite stable (MSS) tumours, and this translated into a propensity for metastatic disease and poor prognostic value of the mutations specifically in the CMS1/MSS subtype (both series overall survival: TP53 mutation vs wt: HR 5.52, p=0.028). CONCLUSIONS: Integration of TP53 mutation status with the CMS framework in primary CRC suggested subtype-dependent immunobiological associations with prognostic and potentially immunotherapeutic implications, warranting independent validation.

Cancer stemness, intratumoral heterogeneity, and immune response across cancers.

Regulatory programs that control the function of stem cells are active in cancer and confer properties that promote progression and therapy resistance. However, the impact of a stem cell-like tumor phenotype ("stemness") on the immunological properties of cancer has not been systematically explored. Using gene-expression-based metrics, we evaluated the association of stemness with immune cell infiltration and genomic, transcriptomic, and clinical parameters across 21 solid cancers. We found pervasive negative associations between cancer stemness and anticancer immunity. This occurred despite high stemness cancers exhibiting increased mutation load, cancer-testis antigen expression, and intratumoral heterogeneity. Stemness was also strongly associated with cell-intrinsic suppression of endogenous retroviruses and type I IFN signaling, and increased expression of multiple therapeutically accessible immunosuppressive pathways. Thus, stemness is not only a fundamental process in cancer progression but may provide a mechanistic link between antigenicity, intratumoral heterogeneity, and immune suppression across cancers.

Combination therapies with HSP90 inhibitors against colorectal cancer.

Oncogene stability and homeostasis mediated by the HSP90 chaperone is a crucial protection trait of cancer cells. Therefore, HSP90 represents an attractive therapeutic target for many cancers, including colorectal cancer. Although monotherapy has limited clinical efficacy, preclinical and early-phase clinical studies indicate improved antitumor activity when HSP90 inhibitors are combined with chemotherapies or targeted agents. This may be further improved with a biomarker-guided approach based on oncogenic HSP90 clients, or stratification based on the consensus molecular subtypes of colorectal cancer, suggesting a synergistic activity with 5-fluorouracil in preclinical models of the chemorefractory mesenchymal subtype. Furthermore, HSP90 inhibition may activate mechanisms to turn non-immunogenic tumors hot and improve their recognition by the immune system, suggesting synergy with immune checkpoint blockade.

Prognostic, predictive, and pharmacogenomic assessments of CDX2 refine stratification of colorectal cancer.

We aimed to refine the value of CDX2 as an independent prognostic and predictive biomarker in colorectal cancer (CRC) according to disease stage and chemotherapy sensitivity in preclinical models. CDX2 expression was evaluated in 1045 stage I-IV primary CRCs by gene expression (n = 403) or immunohistochemistry (n = 642) and in relation to 5-year relapse-free survival (RFS), overall survival (OS), and chemotherapy. Pharmacogenomic associations between CDX2 expression and 69 chemotherapeutics were assessed by drug screening of 35 CRC cell lines. CDX2 expression was lost in 11.6% of cases and showed independent poor prognostic value in multivariable models. For individual stages, CDX2 was prognostic only in stage IV, independent of chemotherapy. Among stage I-III patients not treated in an adjuvant setting, CDX2 loss was associated with a particularly poor survival in the BRAF-mutated subgroup, but prognostic value was independent of microsatellite instability status and the consensus molecular subtypes. In stage III, the 5-year RFS rate was higher among patients with loss of CDX2 who received adjuvant chemotherapy than among patients who did not. The CDX2-negative cell lines were significantly more sensitive to chemotherapeutics than CDX2-positive cells, and the multidrug resistance genes MDR1 and CFTR were significantly downregulated both in CDX2-negative cells and in patient tumors. Loss of CDX2 in CRC is an adverse prognostic biomarker only in stage IV disease and appears to be associated with benefit from adjuvant chemotherapy in stage III. Early-stage patients not qualifying for chemotherapy might be reconsidered for such treatment if their tumor has loss of CDX2 and mutated BRAF.

Multispectral imaging for quantitative and compartment-specific immune infiltrates reveals distinct immune profiles that classify lung cancer patients.

Semiquantitative assessment of immune markers by immunohistochemistry (IHC) has significant limitations for describing the diversity of the immune response in cancer. Therefore, we evaluated a fluorescence-based multiplexed immunohistochemical method in combination with a multispectral imaging system to quantify immune infiltrates in situ in the environment of non-small-cell lung cancer (NSCLC). A tissue microarray including 57 NSCLC cases was stained with antibodies against CD8, CD20, CD4, FOXP3, CD45RO, and pan-cytokeratin, and immune cells were quantified in epithelial and stromal compartments. The results were compared with those of conventional IHC, and related to corresponding RNA-sequencing (RNAseq) expression values. We found a strong correlation between the visual and digital quantification of lymphocytes for CD45RO (correlation coefficient: r = 0.52), FOXP3 (r = 0.87), CD4 (r = 0.79), CD20 (r = 0.81) and CD8 (r = 0.90) cells. The correlation with RNAseq data for digital quantification (0.35-0.65) was comparable to or better than that for visual quantification (0.38-0.58). Combination of the signals of the five immune markers enabled further subpopulations of lymphocytes to be identified and localized. The specific pattern of immune cell infiltration based either on the spatial distribution (distance between regulatory CD8+ T and cancer cells) or the relationships of lymphocyte subclasses with each other (e.g. cytotoxic/regulatory cell ratio) were associated with patient prognosis. In conclusion, the fluorescence multiplexed immunohistochemical method, based on only one tissue section, provided reliable quantification and localization of immune cells in cancer tissue. The application of this technique to clinical biopsies can provide a basic characterization of immune infiltrates to guide clinical decisions in the era of immunotherapy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Colorectal Cancer Consensus Molecular Subtypes Translated to Preclinical Models Uncover Potentially Targetable Cancer Cell Dependencies.

Purpose: Response to standard oncologic treatment is limited in colorectal cancer. The gene expression-based consensus molecular subtypes (CMS) provide a new paradigm for stratified treatment and drug repurposing; however, drug discovery is currently limited by the lack of translation of CMS to preclinical models.Experimental Design: We analyzed CMS in primary colorectal cancers, cell lines, and patient-derived xenografts (PDX). For classification of preclinical models, we developed an optimized classifier enriched for cancer cell-intrinsic gene expression signals, and performed high-throughput in vitro drug screening (n = 459 drugs) to analyze subtype-specific drug sensitivities.Results: The distinct molecular and clinicopathologic characteristics of each CMS group were validated in a single-hospital series of 409 primary colorectal cancers. The new, cancer cell-adapted classifier was found to perform well in primary tumors, and applied to a panel of 148 cell lines and 32 PDXs, these colorectal cancer models were shown to recapitulate the biology of the CMS groups. Drug screening of 33 cell lines demonstrated subtype-dependent response profiles, confirming strong response to EGFR and HER2 inhibitors in the CMS2 epithelial/canonical group, and revealing strong sensitivity to HSP90 inhibitors in cells with the CMS1 microsatellite instability/immune and CMS4 mesenchymal phenotypes. This association was validated in vitro in additional CMS-predicted cell lines. Combination treatment with 5-fluorouracil and luminespib showed potential to alleviate chemoresistance in a CMS4 PDX model, an effect not seen in a chemosensitive CMS2 PDX model.Conclusions: We provide translation of CMS classification to preclinical models and uncover a potential for targeted treatment repurposing in the chemoresistant CMS4 group. Clin Cancer Res; 24(4); 794-806. ©2017 AACR.

CMScaller: an R package for consensus molecular subtyping of colorectal cancer pre-clinical models.

Colorectal cancers (CRCs) can be divided into four gene expression-based biologically distinct consensus molecular subtypes (CMS). This classification provides a potential framework for stratified treatment, but to identify novel CMS-drug associations, translation of the subtypes to pre-clinical models is essential. The currently available classifier is dependent on gene expression signals from the immune and stromal compartments of tumors and fails to identify the poor-prognostic CMS4-mesenchymal group in immortalized cell lines, patient-derived organoids and xenografts. To address this, we present a novel CMS classifier based on a filtered set of cancer cell-intrinsic, subtype-enriched gene expression markers. This new classifier, referred to as CMScaller, recapitulated the subtypes in both in vitro and in vivo models (551 in total). Importantly, by analyzing public drug response data from patient-derived xenografts and cell lines, we show that the subtypes are predictive of response to standard CRC drugs. CMScaller is available as an R package.

Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies.

BACKGROUND: Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Comprehensive insights into their molecular characteristics may improve model selection for biomedical studies. METHODS: We have performed DNA, RNA and protein profiling of 34 cell lines, including (i) targeted deep sequencing (n = 612 genes) to detect single nucleotide variants and insertions/deletions; (ii) high resolution DNA copy number profiling; (iii) gene expression profiling at exon resolution; (iv) small RNA expression profiling by deep sequencing; and (v) protein expression analysis (n = 297 proteins) by reverse phase protein microarrays. RESULTS: The cell lines were stratified according to the key molecular subtypes of CRC and data were integrated at two or more levels by computational analyses. We confirm that the frequencies and patterns of DNA aberrations are associated with genomic instability phenotypes and that the cell lines recapitulate the genomic profiles of primary carcinomas. Intrinsic expression subgroups are distinct from genomic subtypes, but consistent at the gene-, microRNA- and protein-level and dominated by two distinct clusters; colon-like cell lines characterized by expression of gastro-intestinal differentiation markers and undifferentiated cell lines showing upregulation of epithelial-mesenchymal transition and TGFß signatures. This sample split was concordant with the gene expression-based consensus molecular subtypes of primary tumors. Approximately » of the genes had consistent regulation at the DNA copy number and gene expression level, while expression of gene-protein pairs in general was strongly correlated. Consistent high-level DNA copy number amplification and outlier gene- and protein- expression was found for several oncogenes in individual cell lines, including MYC and ERBB2. CONCLUSIONS: This study expands the view of CRC cell lines as accurate molecular models of primary carcinomas, and we present integrated multi-level molecular data of 34 widely used cell lines in easily accessible formats, providing a resource for preclinical studies in CRC.