Genomic profiling of gallbladder adenosquamous carcinoma

Gallbladder carcinoma (GBC) is a rare and aggressive malignancy with a 5-year overall survival of less than 20%. Aggressive biological behavior and delayed diagnosis are the major causes of the poor outcome of the disease. The great majority of GBCs are biliary (or pancreatobiliary) type adenocarcinomas. Less than 5% of GBCs have a mixed phenotype, with at least 25% of the tumor having a squamous differentiation. These tumors have an even poorer prognosis than that of ordinary GBC and are classified as gallbladder adenosquamous carcinomas (GBASC). Little is known about the molecular characteristics of GBASCs, and the underlying factors contributing to their aggressive behavior remain poorly understood. Additionally, prognostic and predictive biomarkers for these tumors are not well established. Most research on GBASCs consists of case reports, and there is a lack of comprehensive molecular characterization for this cancer type. In this thesis we collected 25 cases of GBASC diagnosed between 2007 and 2022 from six Italian centers. Immunohistochemical characterization of predictive biomarkers like PD-L1, Mismatch repair proteins (MMR), HER2 and Claudin 18 (CLDN18) was performed along with genetic profiling using a Next Generation Sequencing (NGS) panel able to identify single nucleotide variations (SNV), insertions or deletions (indel) and copy number variations (CNV) in 67 driver genes. The immunohistochemical profile of the lesions revealed potentially druggable alterations: 24/25 cases (96%) had a PD-L1 CPS greater than 1 and 7/25 cases (28%) were positive for CLDN18. No cases harbored HER2 overexpression or exhibited a MMRd/MSI-H status. From a molecular standpoint the genomic profile of GBASC closely resembles that of conventional GBC, with overlapping prevalence of driver mutations such as TP53 (64%), KRAS (16%), and CDKN2A (20%) and an enrichment of mutations in genes associated with the PI3K pathway, such as PIK3CA (44%) and PTEN (24%). Three cases (12%) also exhibited mutations in the HNF1A gene, which has not been previously linked to GBC but may play a role in oncogenesis due to its tumor-suppressive properties and involvement in gallstones formation. Further studies are needed to better understand the molecular basis of the aggressive behavior of GBASC, to draw comparisons between the molecular features of the different histotypes, to confirm the pathogenic role of HNF1A mutations and to test the potential role of druggable gene alterations of this tumour in therapeutic settings.