Multiregional genomic analysis of intraepithelial or superficial spreading extrahepatic bile duct cancer elucidated by minute dissection and deep sequencing

Background: Extrahepatic bile duct cancer (EHBC) sometimes shows unique spreading patterns including broad intraepithelial spread. These irregular spreading patterns could have important clinical significance, especially under the setting of operation procedure decision. However, intra- and inter-tumoral heterogeneity of genomic abnormality have not been revealed on EHBC. Methods: We performed detailed mapping on 6 cases of surgically resected EHBC cases (perihilar; 4, distal; 2). After that, targeted DNA sequence using 67 gene in-house panel were performed. DNA was extracted minutely dissected specimens of non-neoplastic, intraepithelial, and invasive foci (9-25 areas/case, 107 areas in total). Results: Among the 6 EHBC cases, four cases showed intraepithelial spread. For the intraepithelial spreading cases, oncogenic mutation(s) of tumor suppressor genes, TP53 and RBM10, were detected broadly in the background of invasive carcinoma, including morphologically identified intraepithelial spread and even some reactive areas. Multifocal subclonal expansions within invasive and intraepithelial carcinoma formed morphologically and genomically asymmetric lesions. In contrast, two cases without intraepithelial spread showed genetic abnormalities restricted to the invasive foci, suggestive of their de novo invasion. For all 6 cases, neither KRAS nor GNAS mutations were detected, which was different from previously proposed BilIN-associated carcinogenic process. These changes could be confirmed in subsequently planned analysis.