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

Peer reviewe

Heterogeneous radiological response to neoadjuvant therapy is associated with poor prognosis after resection of colorectal liver metastases

Introduction Surgery combined with perioperative chemotherapy has become standard of care in patients with resectable colorectal liver metastases. However, poor outcome is expected for a significant subgroup. The clinical implications of inter-metastatic heterogeneity remain largely unknown. In a prospective, population-based series of patients undergoing resection of multiple colorectal liver metastases, the aim was to investigate the prevalence and prognostic impact of heterogeneous response to neoadjuvant chemotherapy. Materials and Methods Radiological response to treatment was evaluated in a lesion-specific manner in 2–5 metastases per patient. Change of lesion diameter was evaluated and response/progression was classified according to three different size thresholds; 3, 4 and 5 mm. A heterogeneous response was defined as progression and response of different metastases in the same patient. Results In total, 142 patients with 585 liver metastases were examined with the same radiological method (MRI or CT) before and after neoadjuvant treatment. Heterogeneous response to treatment was seen in 16 patients (11%) using the 3 mm size change threshold, and this group had a 5-year cancer-specific survival of 19% compared to 49% for patients with response in all lesions (p = 0.003). Cut-off values of 4–5 mm were less sensitive for detecting a heterogeneous response, but the survival difference was similar and significant. Conclusion A subgroup of patients with multiple colorectal liver metastases had heterogeneous radiological response to neoadjuvant chemotherapy and poor prognosis. The evaluation of response pattern is easy to perform, feasible in clinical practice and, if validated, a promising biomarker for treatment decisions

Genomic and prognostic heterogeneity among RAS/BRAFV600E/TP53 co-mutated resectable colorectal liver metastases

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, BRAFV600, 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/BRAFV600E 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/BRAFV600E/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/BRAFV600E/TP53

Metastatic heterogeneity of the consensus molecular subtypes of colorectal cancer

Abstract 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

High Concordance and Negative Prognostic Impact of RAS/BRAF/PIK3CA Mutations in Multiple Resected Colorectal Liver Metastases

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 and their prognostic impact. Patients and Methods We analyzed the mutation status among 372 liver metastases and 78 primary tumors from 106 patients by methods used in clinical routine testing, by Sanger sequencing, by next-generation sequencing (NGS), and/or by droplet digital polymerase chain reaction. The 3-year cancer-specific survival (CSS) was analyzed using the Kaplan-Meier method. Results Although Sanger sequencing indicated inter-metastatic mutation heterogeneity in 14 of 97 patients (14%), almost all cases were refuted by high-sensitive NGS. Also, heterogeneity among metastatic deposits was concluded only for PIK3CA in 2 patients. Similarly, primary-to-metastatic heterogeneity was indicated in 8 of 78 patients (10%) using Sanger sequencing but for only 2 patients after NGS, showing the emergence of 1 KRAS and 1 PIK3CA mutation in the metastatic lesions. KRAS mutations were present in 53 of 106 patients (50%) and were associated with poorer 3-year CSS after liver resection (37% vs. 61% for KRAS wild-type; P = .004). Poor prognostic associations were found also for the combination of KRAS/NRAS/BRAF mutations compared with triple wild-type (P = .002). Conclusion Intra-patient mutation heterogeneity was virtually undetected, both between the primary tumor and the liver metastases and among the metastatic deposits. KRAS mutations separately, and KRAS/NRAS/BRAF mutations combined, were associated with poor patient survival after partial liver resection

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

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