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Gene expression-based subtypes of colorectal cancer have clinical relevance, but the representativeness of primary tumors and the consensus molecular subtypes (CMS) for metastatic cancers is not well known. We investigated the metastatic heterogeneity of CMS. The best approach to subtype translation was delineated by comparisons of transcriptomic profiles from 317 primary tumors and 295 liver metastases, including multi-metastatic samples from 45 patients and 14 primary-metastasis sets. Associations were validated in an external data set (n = 618). Projection of metastases onto principal components of primary tumors showed that metastases were depleted of CMS1-immune/CMS3-metabolic signals, enriched for CMS4-mesenchymal/stromal signals, and heavily influenced by the microenvironment. The tailored CMS classifier (available in an updated version of the R package CMScaller) therefore implemented an approach to regress out the liver tissue background. The majority of classified metastases were either CMS2 or CMS4. Nonetheless, subtype switching and inter-metastatic CMS heterogeneity were frequent and increased with sampling intensity. Poor-prognostic value of CMS1/3 metastases was consistent in the context of intra-patient tumor heterogeneity.

About 80% of colorectal cancers (CRCs) have chromosomal instability, which is an integral part of aggressive malignancy development, but the importance of specific copy number aberrations (CNAs) in modulating gene expression, particularly within the framework of clinically relevant molecular subtypes, remains mostly elusive. We performed DNA copy number profiling of 257 stage I-IV primary CRCs and integrative gene expression analysis in 151 microsatellite stable (MSS) tumors, focusing on high-level amplifications and the effect of CNAs on the characteristics of the gene expression-based consensus molecular subtypes (CMS). The results were validated in 323 MSS tumors from TCGA. Novel recurrent high-level amplifications (≥15 additional copies) with a major impact on gene expression were found for TOX3 (16q) at 1.5% frequency, as well as for CCND2 (12p) and ANXA11 (10q) at 1% frequency, in addition to the well-known targets ERBB2 (17q) and MYC (8q). Focal amplifications with ≥15 or ≥5 additional copies of at least one of these regions were associated with a poor overall survival among patients with stage I-III MSS CRCs (multivariable hazard ratio ≥3.2, p ≤ 0.01). All high-level amplifications were focal and had a more consistent relationship with gene expression than lower amplitude and/or broad-range amplifications, suggesting specific targeting during carcinogenesis. Genome-wide, copy number driven gene expression was enriched for pathways characteristic of the CMS2-epithelial/canonical subtype, including DNA repair and cell cycle progression. Furthermore, 50% of upregulated genes in CMS2-epithelial/canonical MSS CRCs were driven by CNAs, an enrichment compared with the other CMS groups, and associated with the stronger correspondence between CNAs and gene expression in malignant epithelial cells than in the cells of the tumor microenvironment (fibroblasts, endothelial cells, leukocytes). In conclusion, we identify novel recurrent amplifications with impact on gene expression in CRC and provide the first evidence that CMS2 may have a stronger copy-number related genetic basis than subtypes more heavily influenced by gene expression signals from the tumor microenvironment. publishedVersion

Background Gene 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. Methods In 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. Results Initial 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. Conclusions We 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.

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.

Testicular germ cell tumours (TGCTs) appear as different histological subtypes or mixtures of these. They show similar, multiple DNA copy number changes, where gain of 12p is pathognomonic. However, few high-resolution analyses have been performed and focal DNA copy number changes with corresponding candidate target genes remain poorly described for individual subtypes. We present the first high-resolution DNA copy number aberration (CNA) analysis on the subtype embryonal carcinomas (ECs), including 13 primary ECs and 5 EC cell lines. We identified recurrent gains and losses and allele-specific CNAs. Within these regions, we nominate 30 genes that may be of interest to the EC subtype. By in silico analysis of data from 150 TGCTs from The Cancer Genome Atlas (TCGA), we further investigated CNAs, RNA expression, somatic mutations and fusion transcripts of these genes. Among primary ECs, ploidy ranged between 2.3 and 5.0, and the most common aberrations were DNA copy number gains at chromosome (arm) 7, 8, 12p, and 17, losses at 4, 10, 11, and 18, replicating known TGCT genome characteristics. Gain of whole or parts of 12p was found in all samples, including a highly amplified 100 kbp segment at 12p13.31, containing SLC2A3. Gain at 7p21, encompassing ETV1, was the second most frequent aberration. In conclusion, we present novel CNAs and the genes located within these regions, where the copy number gain of SLC2A3 and ETV1 are of interest, and which copy number levels also correlate with expression in TGCTs.

Colorectal cancer commonly metastasizes into multiple liver foci, but intermetastatic heterogeneity remains poorly described. Here, we demonstrate that mutations of RAS/BRAF/TP53 are homogeneous within patients, while DNA copy number aberrations can vary greatly. RAS/BRAF/TP53 co‐mutations conferred a dismal prognosis, and co‐mutations combined with a high burden and heterogeneity of copy number aberrations identified patients with the poorest outcome., Hepatic resection is potentially curative for patients with colorectal liver metastases, but the treatment benefit varies. KRAS/NRAS (RAS)/TP53 co‐mutations are associated with a poor prognosis after resection, but there is large variation in patient outcome within the mutation groups, and genetic testing is currently not used to evaluate benefit from surgery. We have investigated the potential for improved prognostic stratification by combined biomarker analysis with DNA copy number aberrations (CNAs), and taking tumor heterogeneity into account. We determined the mutation status of RAS, BRAF V600, and TP53 in 441 liver lesions from 171 patients treated by partial hepatectomy for metastatic colorectal cancer. CNAs were profiled in 232 tumors from 67 of the patients. Mutations and high‐level amplifications of cancer‐critical genes, the latter including ERBB2 and EGFR, were predominantly homogeneous within patients. RAS/BRAF V600E and TP53 co‐mutations were associated with a poor patient outcome (hazard ratio, HR, 3.9, 95% confidence interval, CI, 1.3–11.1, P = 0.012) in multivariable analyses with clinicopathological variables. The genome‐wide CNA burden and intrapatient intermetastatic CNA heterogeneity varied within the mutation groups, and the CNA burden had prognostic associations in univariable analysis. Combined prognostic analyses of RAS/BRAF V600E/TP53 mutations and CNAs, either as a high CNA burden or high intermetastatic CNA heterogeneity, identified patients with a particularly poor outcome (co‐mutation/high CNA burden: HR 2.7, 95% CI 1.2–5.9, P = 0.013; co‐mutation/high CNA heterogeneity: HR 2.5, 95% CI 1.1–5.6, P = 0.022). In conclusion, DNA copy number profiling identified genomic and prognostic heterogeneity among patients with resectable colorectal liver metastases with co‐mutated RAS/BRAF V600E/TP53.

Structured Abstract Background The prevalence and clinical implications of genetic heterogeneity in patients with multiple colorectal liver metastases remain largely unknown. In a prospective series of patients undergoing resection of colorectal liver metastases, the aim was to investigate the inter-metastatic and primary-to-metastatic heterogeneity of mutations in KRAS, NRAS, BRAF and PIK3CA, as well as their prognostic impact. Materials and Methods We analyzed mutation status among 371 liver metastases and 78 primary tumors from 106 patients by several methods used in clinical routine testing, Sanger sequencing, next-generation sequencing (NGS) and/or droplet digital PCR. Three-year cancer-specific survival (3y-CSS) was analyzed with the Kaplan-Meier method. Results Although Sanger sequencing indicated inter-metastatic mutation heterogeneity in 14/96 patients (15%), almost all cases were refuted by high-sensitive NGS, and heterogeneity among metastatic deposits were concluded only for PIK3CA in two patients. Similarly, primary-to-metastatic heterogeneity was indicated in 8/78 patients (10%) using Sanger sequencing, but concluded for only two patients after NGS, showing the emergence of one KRAS and one PIK3CA mutation in the metastatic lesions. In total, KRAS mutations were present in 53/106 patients (50%) and associated with a poorer 3y-CSS after liver resection (37% versus 61% for KRAS wild type; p=0.004). Poor prognostic associations were found also for the combination of KRAS/NRAS/BRAF mutations in comparison with triple wild type (p=0.002). Conclusion Intra-patient mutation heterogeneity was virtually non-detected, neither between the primary tumor and liver metastases, nor among metastatic deposits. KRAS mutations separately, and KRAS/NRAS/BRAF mutations combined, were associated with a poor patient survival after partial liver resection.

Background Droplet digital PCR (ddPCR) allows absolute quantification of nucleic acids and has potential for improved non-invasive detection of DNA methylation. For increased precision of the methylation analysis, we aimed to develop a robust internal control for use in methylation-specific ddPCR. Methods Two control design approaches were tested: (a) targeting a genomic region shared across members of a gene family and (b) combining multiple assays targeting different pericentromeric loci on different chromosomes. Through analyses of 34 colorectal cancer cell lines, the performance of the control assay candidates was optimized and evaluated, both individually and in various combinations, using the QX200™ droplet digital PCR platform (Bio-Rad). The best-performing control was tested in combination with assays targeting methylated CDO1, SEPT9, and VIM. Results A 4Plex panel consisting of EPHA3, KBTBD4, PLEKHF1, and SYT10 was identified as the best-performing control. The use of the 4Plex for normalization reduced the variability in methylation values, corrected for differences in template amount, and diminished the effect of chromosomal aberrations. Positive Droplet Calling (PoDCall), an R-based algorithm for standardized threshold determination, was developed, ensuring consistency of the ddPCR results. Conclusion Implementation of a robust internal control, i.e., the 4Plex, and an algorithm for automated threshold determination, PoDCall, in methylation-specific ddPCR increase the precision of DNA methylation analysis.

Chromosomal instability is a well-defined hallmark of tumor aggressiveness and metastatic progression in colorectal cancer. The magnitude of genetic heterogeneity among distinct liver metastases from the same patient at the copy number level, as well as its relationship with chemotherapy exposure and patient outcome, remains unknown. We performed high-resolution DNA copy number analyses of 134 liver metastatic deposits from 45 colorectal cancer patients to assess: (i) intra-patient inter-metastatic genetic heterogeneity using a heterogeneity score based on pair-wise genetic distances among tumor deposits; and (ii) genomic complexity, defined as the proportion of the genome harboring aberrant DNA copy numbers. Results were analyzed in relation to the patients’ clinical course; previous chemotherapy exposure and outcome after surgical resection of liver metastases. We observed substantial variation in the level of intra-patient inter-metastatic heterogeneity. Heterogeneity was not associated with the number of metastatic lesions or their genomic complexity. In metachronous disease, heterogeneity was higher in patients previously exposed to chemotherapy. Importantly, intra-patient inter-metastatic heterogeneity was a strong prognostic determinant, stronger than known clinicopathological prognostic parameters. Patients with a low level of heterogeneity (below the median level) had a three-year progression-free and overall survival rate of 23% and 66% respectively, versus 5% and 18% for patients with a high level (hazard ratio0.4, 95% confidence interval 0.2–0.8, P = 0.01; and hazard ratio0.3,95% confidence interval 0.1–0.7, P = 0.007). A low patient-wise level of genomic complexity (below 25%) was also a favorable prognostic factor; however, the prognostic association of intra-patient heterogeneity was independent of genomic complexity in multivariable analyses. In conclusion, intra-patient inter-metastatic genetic heterogeneity is a pronounced feature of metastatic colorectal cancer, and the strong prognostic association reinforces its clinical relevance and places it as a key feature to be explored in future patient cohorts., Author Summary Liver metastasis from colorectal cancer confers a poor patient prognosis and is often manifested as multiple independent lesions. Clonal evolution is an important biological process in metastatic development, but the magnitude and clinical relevance of genetic heterogeneity among metastatic deposits in individual patients remains unknown. We describe for the first time a large variation among patients in the level of inter-metastatic heterogeneity at the DNA copy number level in liver metastases from colorectal cancer. We found this heterogeneity to be a biological feature independent both of the number of metastases per patient and of the extent of copy number aberrations (genomic complexity)of the metastases. Previous chemotherapy exposure was associated with a higher patient-wise level of heterogeneity in subsequent metastases. Importantly, we discovered inter-metastatic heterogeneity to be a key determinant of patient survival and a stronger prognostic factor than known clinicopathological parameters of metastatic colorectal cancer. This study reinforces the clinical relevance of tumor heterogeneity and we identify inter-metastatic heterogeneity as an important feature to explore in future cohorts of patients with metastatic colorectal cancer.

Introduction Colorectal cancer (CRC) is a molecularly diverse disease with few targeted treatment options. Focal and high-amplitude DNA copy number aberrations are potential tumour drivers, and their identification may contribute to improved therapeutic outcomes in small patient subgroups, as recently illustrated by targeting the over-expression of HER2 protein resulting from high-level ERBB2 amplification in KRAS wild-type metastatic CRC. However, few recurrent amplifications have been detected in CRC. Material and methods We analysed focal high-level amplifications in 203 microsatellite stable (MSS) primary colorectal tumours using genome-wide high-resolution Affymetrix SNP6.0 arrays. The ASCAT algorithm was used to derive discrete allele specific copy number estimates. Results and discussions The overall copy number profiles confirmed the frequent gains and losses previously described in MSS CRC studies. Extreme focal amplifications (defined as >15 copies and ERBB2 , which was amplified to 97, 27 and 22 additional copies in tumours from three individual patients (amplification frequency 1.5%). The transcription factor TOX3 (16q) was also recurrently amplified in 1.5% of the patients, while MYC (8q), CCND2 (12 p) and a region on 10q22.3-q23.1, where ANXA11 was nominated as a likely target by GISTIC analysis, were recurrent in 1% of the patients. Regions with extreme and focal amplifications were also investigated for lower-amplitude aberrations (5–15 additional DNA copies), revealing a 3% amplification frequency of TOX3 in our cohort. Conclusion We have identified several recurrent amplifications in cancer-critical genes in CRC MSS tumours, including the transcription factor TOX3 , suggesting novel drug targets for preclinical studies.

Colorectal cancer is a heterogeneous disease and improved molecular stratification of patients is warranted for precise treatment. Several gene expression classification systems are reported in the literature, albeit many have shown low reproducibility in external validation. A robust random forest classification system based on gene expression profiles was recently proposed, suggesting the existence of four biologically different subtypes with associations to prognosis (CMS1, CMS2, CMS3 and CMS4) (Guinney,J., et al., Nat Med, 2015). By applying the CMS classifier to high-quality gene expression profiles from a consecutive series of >400 stage I-IV primary colorectal cancers we are able to reproduce results from Guinney et al in an independent cohort. Analyses of mutational hotspots in BRAF / KRAS and the complete coding sequence of TP53 confirms enrichment of BRAF mutations in CMS1 (p Citation Format: Kaja G. Berg, Ina A. Eilertsen, Sharmini Alagaratnam, Stine A. Danielsen, Arild Nesbakken, Anita Sveen, Ragnhild A. Lothe. Gene expression based subtypes of 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 103.

555 Background: Almost 50% of patients with colorectal cancer (CRC) develop liver metastases, often as multiple simultaneous metastatic deposits. Up to 25% of the patients are eligible for partial liver resection, but 70% will relapse after surgery and there are currently no good criteria to select patients who will benefit from the treatment. Genetic heterogeneity among metastatic deposits has great potential impact on disease progression, but has not been well described. Methods: Totally 134 liver metastatic deposits were collected from 45 patients undergoing partial liver resection for metastatic CRC according to a prospective study protocol. All deposits were analyzed for high-resolution DNA copy number variation using the Affymetrix SNP Array 6.0. A novel bioinformatic approach was used to measure intra-individual genetic heterogeneity among the metastatic deposits. Results: The patients showed a large variation in the level of intra-individual metastatic heterogeneity, and heterogeneity was independent of the number of liver metastases analyzed per patient. Heterogeneity was a strong and independent prognostic factor in multivariate analysis with known clinicopathological prognostic factors. Patients with a high level of heterogeneity (above the median) had a three-year overall survival rate of 18%, compared with 66% for patients with a low level (hazard ratio, HR = 3.7, P = 0.007). The corresponding survival rates for progression-free survival were 6% and 24% (HR = 2.6, P = 0.01). Conclusions: A high level of intra-individual genetic heterogeneity among liver metastatic deposits is associated with poor survival after partial liver resection in patients with metastatic CRC.

Major efforts have been invested in the identification of cancer biomarkers in plasma, but the extraordinary dynamic range in protein composition, and the dilution of disease specific proteins make discovery in plasma challenging. Focus is shifting towards using proximal fluids for biomarker discovery, but methods to verify the isolated sample’s origin are missing. We therefore aimed to develop a technique to search for potential candidate proteins in the proximal proteome, i.e. in the tumor interstitial fluid, since the biomarkers are likely to be excreted or derive from the tumor microenvironment. Since tumor interstitial fluid is not readily accessible, we applied a centrifugation method developed in experimental animals and asked whether interstitial fluid from human tissue could be isolated, using ovarian carcinoma as a model. Exposure of extirpated tissue to 106 g enabled tumor fluid isolation. The fluid was verified as interstitial by an isolated fluid:plasma ratio not significantly different from 1.0 for both creatinine and Na+, two substances predominantly present in interstitial fluid. The isolated fluid had a colloid osmotic pressure 79% of that in plasma, suggesting that there was some sieving of proteins at the capillary wall. Using a proteomic approach we detected 769 proteins in the isolated interstitial fluid, sixfold higher than in patient plasma. We conclude that the isolated fluid represents undiluted interstitial fluid and thus a subproteome with high concentration of locally secreted proteins that may be detected in plasma for diagnostic, therapeutic and prognostic monitoring by targeted methods. publishedVersion