High-Throughput Profiling of Colorectal Cancer Liver Metastases Reveals Intra- and Inter-Patient Heterogeneity in the EGFR and WNT Pathways Associated with Clinical Outcome

SIMPLE SUMMARY: Tumor heterogeneity can greatly influence therapy outcome and patient survival. In this study, we aimed at unraveling inter- and intra-patient heterogeneity of colorectal cancer liver metastases (CRLM). To this end, we comprehensively characterized CRLM using state-of-the-art high-throughput technologies combined with bioinformatics analyses. We found a high degree of inter- and intra-patient heterogeneity among the metastases, in particular in genes of the WNT and EGFR pathways. Through analyzing the master regulators and effectors associated with the regulation of these genes, we identified a specific gene signature that was highly expressed in a large cohort of colorectal cancer patients and associated with clinical outcome. ABSTRACT: Seventy percent of patients with colorectal cancer develop liver metastases (CRLM), which are a decisive factor in cancer progression. Therapy outcome is largely influenced by tumor heterogeneity, but the intra- and inter-patient heterogeneity of CRLM has been poorly studied. In particular, the contribution of the WNT and EGFR pathways, which are both frequently deregulated in colorectal cancer, has not yet been addressed in this context. To this end, we comprehensively characterized normal liver tissue and eight CRLM from two patients by standardized histopathological, molecular, and proteomic subtyping. Suitable fresh-frozen tissue samples were profiled by transcriptome sequencing (RNA-Seq) and proteomic profiling with reverse phase protein arrays (RPPA) combined with bioinformatic analyses to assess tumor heterogeneity and identify WNT- and EGFR-related master regulators and metastatic effectors. A standardized data analysis pipeline for integrating RNA-Seq with clinical, proteomic, and genetic data was established. Dimensionality reduction of the transcriptome data revealed a distinct signature for CRLM differing from normal liver tissue and indicated a high degree of tumor heterogeneity. WNT and EGFR signaling were highly active in CRLM and the genes of both pathways were heterogeneously expressed between the two patients as well as between the synchronous metastases of a single patient. An analysis of the master regulators and metastatic effectors implicated in the regulation of these genes revealed a set of four genes (SFN, IGF2BP1, STAT1, PIK3CG) that were differentially expressed in CRLM and were associated with clinical outcome in a large cohort of colorectal cancer patients as well as CRLM samples. In conclusion, high-throughput profiling enabled us to define a CRLM-specific signature and revealed the genes of the WNT and EGFR pathways associated with inter- and intra-patient heterogeneity, which were validated as prognostic biomarkers in CRC primary tumors as well as liver metastases.