Characterization of cholangiocarcinoma heterogeneity: integration of digital histologic whole slide imaging with spatial molecular profiling

Cholangiocarcinomas (CCA) are a heterogeneous group of malignancies arising from the biliary tree. The tumour heterogeneity at anatomical and histological levels hampered the research for effective early diagnosis strategies and treatment options, leading to poor outcomes for the patients. Our aim was to combine digital pathology with spatial molecular profiling for characterizing tumour phenotypes with relevant clinical value. CCA samples (n=140) were obtained from dif- ferent European centres joining the European Network for the Study of CCA. Extensive histological/ immunohistochemical characterization of tumours has been performed. Slides were digitalized by Aperio Scanscope CS2. Spatial profiling was performed by using Nanostring GeoMx DSP. CCAs were divided in intrahepatic (n=85), perihilar (n=41) and distal (n=14) based on their anatomical loca- tion. Intrahepatic (i) CCAs were histologically distinguished into small (37%) or large (47%) bile duct (BD) types. Large BD type iCCAs were characterized by a higher mucin content (p=0.021) and more frequent peri- neural invasion (p<0.001) compared to small BD type. In a subgroup of cases (n=10), spatial molecular analysis was performed by selecting the CD45-positive cell fraction in the tumour centre or at invasion front, revealing CD68 cells at invasion front as significantly associated to prognosis. In the entire iCCA cohort, a lower num- ber of CD68+ cells at the invasion front resulted associated with a poor prognosis in terms of overall survival [HR:3.43 (1.62-7.27), p=0.028]. These results represent a proof-of-concept that the combination of digital histology with spatial molecular profiling could represent an integrative approach for tumour characterization and for development of a personalized approach.