담도암에서 ROS1 발현과 그 억제제의 효과에 관한 연구

Abstract

Introduction: More knowledge about genetic and molecular features of biliary tract cancer is needed to develop effective therapeutic strategies. In the present study, the clinical and pathological significance of protein expression and gene rearrangement of ROS1, which is one of targets of crizotinib, in biliary tract cancer was assessed in tumor tissue obtained from patients. Also, the effect of crizotinib in biliary tract cancer cells was investigated in vitro.

Material and Methods: The effect of crizotinib was assessed in biliary tract cancer cell lines (SNU-245, SNU-308, SNU-478, SNU-1079, SNU-1196, TFK-1, HuCCT-1). Tetrazolium dye (MTT) assay, flow cytometry, Western immunoblot, and receptor tyrosine kinase array were used to assess the molecular mechanisms of growth inhibition by crizotinib. Tumor tissue specimens from 194 patients with curatively resected intrahepatic cholangiocarcinoma were collected and analyzed for ROS1 gene rearrangement using fluorescence in situ hybridization (FISH) and ROS1 protein expression using immunohistochemistry (IHC).

Results: SNU-478 and SNU-1196 showed sensitivity to crizotinib, with IC50 values of 0.90 μM and 0.50 μM, respectively. Sensitive cells showed increase in sub-G1 fraction and apoptotic cell death, whereas resistant cells did not show any difference after crizotinib treatment. Phosphor-Erk and its upstream signals (phosphor-C-raf and MEK) decreased in a dose-dependent manner in the sensitive cell lines (SNU-478 and SNU-1196). However, gene rearrangement of ROS1, gene amplification of c-MET, or alteration of ALK was not observed in the tested cell lines. Out of 194 tumor tissue from BTC patients, ROS1 IHC was positive (moderate or strong staining) in 72 tumors (37.1%). ROS1 protein expression was significantly correlated with well differentiated tumors, papillary or mucinous histology, oncocytic/hepatoid or intestinal type tumors, and periductal infiltrating or intraductal growing tumors (vs. mass-forming cholangiocarcinoma). ROS-expressing tumors were associated with better disease-free survival (30.1 months for ROS1 expression (+) tumors vs. 9.0 months for ROS1 (-) tumors, p=0.006). Median overall survival was 43.0 months and 21.7 months, respectively (p=0.071). Although break-apart FISH was successfully performed in 102 samples, a split pattern indicative of ROS1 gene rearrangement was not found in the examined samples.

Conclusion: Protein expression of ROS1 was observed in 37.1% of intrahepatic cholangiocarcinoma and associated with with well-differentiated histology and better disease-free survival, but ROS1 gene rearrangement was not found. Some biliary tract cancer cells showed sensitivity to crizotinib, and further studies to select optimal candidate for crizotinib are warranted.