Proteogenomics of Colorectal Cancer Liver Metastases: Complementing Precision Oncology with Phenotypic Data

: Hotspot testing for activating KRAS mutations is used in precision oncology to select colorectal cancer (CRC) patients who are eligible for anti-EGFR treatment. However, even for KRAS^wildtype tumors anti-EGFR response rates are <30%, while mutated-KRAS does not entirely rule out response, indicating the need for improved patient stratification. We performed proteogenomic phenotyping of KRAS^wildtype and KRAS^G12V CRC liver metastases (mCRC). Among >9000 proteins we detected considerable expression changes including numerous proteins involved in progression and resistance in CRC. We identified peptides representing a number of predicted somatic mutations, including KRAS^G12V. For eight of these, we developed a multiplexed parallel reaction monitoring (PRM) mass spectrometry assay to precisely quantify the mutated and canonical protein variants. This allowed phenotyping of eight mCRC tumors and six paired healthy tissues, by determining mutation rates on the protein level. Total KRAS expression varied between tumors (0.47-1.01 fmol/µg total protein) and healthy tissues (0.13-0.64 fmol/µg). In KRAS^G12V-mCRC, G12V-mutation levels were 42-100%, while one patient had only 10% KRAS^G12V but 90% KRAS^wildtype. This might represent a missed therapeutic opportunity: based on hotspot sequencing, the patient was excluded from anti-EGFR treatment and instead received chemotherapy, while PRM-based tumor-phenotyping indicates the patient might have benefitted from anti-EGFR therapy.