Claudin18.2 in extrahepatic cholangiocarcinoma: prevalence and insights into the immune milieu

Background. Biliary cancers are an extremely heterogeneous group of neoplasms affecting the biliary tree (cholangiocarcinomas: CCA) or the gallbladder (gallbladder cancer: GBC). Among them, GBC, despite being considered a rare neoplasm, is the most frequent. Cholangiocarcinomas are conventionally subdivided into intra- (iCCA) and extra-hepatic (eCCA) cholangiocarcinomas, based on their anatomical localization. This is not a mere anatomic classification, considering that iCCAs present a molecular landscape that is completely different from that of eCCAs. Extrahepatic cholangiocarcinoma is further classified into perihilar (pCCA) and distal (dCCA) cholangiocarcinoma, even in this case, according to the anatomic site. Currently, the possibilities for targeted therapy in eCCA and GBC are very limited, and the only druggable target is represented by HER2 overexpression, affecting less than 20% of these neoplasms. Targeted therapy directed against Claudin 18.2 (CLDN18) represents a promising strategy in the management of solid tumours, and it is currently approved for CLDN18-positive gastric and gastroesophageal junction adenocarcinomas. The objective of this study is to evaluate CLDN18 expression in extrahepatic cholangiocarcinomas (eCCAs) and gallbladder cancers (GBCs), and to compare the immune response-related transcriptomic profile in tumours with different CLDN18 expression levels. Materials and methods. A total of 192 biliary tract cancers (BTCs) were collected. CLDN18 (clone 43-14A), together with HER2 expression, were assessed by immunohistochemistry. As regards HER2 status, it was determined according to the ASCO/CAP scoring criteria, while for CLDN18, tumours with a moderate to intense staining in ≥75% of cancer cells were considered positive. Gene expression profiling was performed using the Nanostring nCounter PanCancer IO 360 panel in a subgroup of cases, selected on the basis of CLDN18 expression. Accordingly, three groups were created: (a) negative: presenting any CLDN18 positivity, also in precursor lesions, which are frequently associated with these neoplasms (i.e. BilINs, IPNB), (b) neoplasms positive for CLDN18 in 40-74% of neoplastic cells, (c) neoplasms positive for CLDN18 in more than 75% of cells. Results. Our ‘real-world’ analysis revealed that 9.4% of eCCA and 6.7% of GBC presented with HER2 overexpression/ERBB2 amplification. In some cases, HER2 amplification is concomitant with CLDN18 expression. CLDN18 positivity was reported in 17.4% of eCCAs and in 14.1% of GBCs, in some cases, also with a heterogeneous expression pattern. CLDN18 positivity was also reported in the neoplasm-associated precursor lesions, such as BilIN, IPNB, PanIN, and ICPN. The transcriptomic profiling of a subgroup of CLDN18-positive eCCA, compared with negative ones, revealed the modulation of several pathways in the two cohorts, including the interferon signalling modulation. Among the most modulated transcripts, of note is the deregulation of the immune-repressor IDO1 in the CLDN18-positive subgroup, which is also associated with an increase in the TILs count in the CLDN18-positive subgroup. Conclusions. CLDN18 is expressed in a subset of eCCAs and GBCs, representing a potential therapeutic target, also in association with monoclonal antibodies targeting HER2, which in our cohort is expressed in less than 10% of eCCAs. Gene expression profiling highlights significant differences between pCCA and dCCA, which prompt us to (a) improve the number of investigated cases and to (b) further subdivide the investigated subgroup into pCCA and dCCA to better identify molecular changes associated with CLDN18 expression. Preliminary data suggest a different immunological behavior in the CLDN18-positive eCCA, and further immunohistochemical determinations are required to confirm the CLDN18-associated TILs increase.