Effect of phosphoproteomic subtyping of gastric cancer on chemotherapy and guidance of targeted cancer therapy.

4050 Background: Gastric cancer is the fifth most common malignancy and the fourth leading cause of death from malignant neoplasms worldwide, and advanced unresectable or recurrent advanced gastric cancer (AGC) has a poor prognosis, so further therapeutic development is needed. However, only a few molecular targeted agents are effective for AGC, and precision medicine using genomic profiling has limited scalability due to the low rate of alterations matched to single pharmacotherapy and complex networks of signal transduction activated by multiple oncogenic proteins. Thus, it is imperative to establish a new concept of precision medicine adapt to the dynamic changes in cancer. Methods: We collected endoscopic biopsy specimens from 84 chemo-naïve gastric cancer patients. We also serially collected endoscopic biopsy specimens from 9 AGC patients receiving 2nd line therapy (pre-treatment: 9 patients, post-treatment 2 months later: 6 patients, and end of treatment: 2 patients). Each specimen was immediately snap frozen in liquid nitrogen within 20 s after collection. Ultra deep proteome and phosphoproteome analysis were performed by multiplex analysis using TMT reagent. Genomic analysis was performed by using a targeted high-multiplex PCR-based NGS panel. Results: Ultrasensitive mass spectrometry-based proteomics using 84 chemo-naïve gastric cancer specimens quantified an average of 21000 phosphorylation sites and clearly divided gastric cancer into subtype 1 (proliferative type, 35%), subtype 2 (EMT type, 15%), and subtype 3. (OxPhos type, 50%). The association between these subtypes with TCGA subtypes was low. Subsequent analysis using serially collected gastric cancer specimens from 9 patients unveiled the dynamic transitions within cancer tissues, along with the concomitant rewiring of the kinome network, ultimately resulting in the acquisition of the EMT subtype throughout the course of treatment. We investigated elaborate intracellular signaling related to EMT in gastric cancer and validate therapeutic approaches targeting AXL in vitro and in vivo. Conclusions: Our proof-of-concept of phosphoproteome analysis of fresh-frozen endoscopic biopsy specimens is capable of not only defining cancer subtyping but also capturing dynamic cancer signaling changes in response to therapy. In particular, serial biopsy analysis can track the cancer evolution which is expected to provide resources for the development of new treatments and biomarkers for AGC adaptively in the future.