Epigenetic expression of MUC5AC in metastatic pancreatic patients using RNA-scope technology and immunohistochemistry

Background and Objectives: Pancreatic ductal adenocarcinoma (PDAC) are often diagnosed in advanced stage of disease and most of them are metastatic (mPDAC). MUC5AC increases during cancer progression, but its role in mPDAC is not well establish. The aim of the present study was to investigate MUC5AC expression in mPDAC patients. Materials and Methods: Immunohistochemistry (IHC) and RNA-Scope technology (RST, ACD company) were used to detect both protein and mRNA expression of MUC5AC, respectively. Biomarkers detections were performed on home-made tissue microarrays (TMAs) representing mPDAC patients (52), plus other entities (8 normal pancreatic tissue, 10 hyperplasia of ducts, 10 IPMN foci and 30 PDAC cases). To highlight the presence of both mRNA and protein of MUC5AC, we used a conventional brown color obtained by peroxidases reaction conjugated with antibodies and RNA probes. In order to verify the pancreatic origin of this lesions, all dots of TMA were staiend with AE1/AE3 antibody. IHCs and RST were performed according to automated protocol into a Bound III machine (Leica Microsystems). Results: All TMA dots showed a strong positivity for AE1/AE3 antigen.The presence of MUC5AC decreased in linear way according PDAC progression. The hyperplastic lesions had the highest positivity (8/10; 80%), followed by IPMN (7/10; 70%), PDAC (19/30; 66%) and mPDAC (23/52; 44%). Indeed, we matched IHC and RST analyses for MUC5AC, in order to verify whether the presence of protein was associated with presence of its RNA. The total of pathological lesions showing positivity for MUC5AC protein expression (49/102; 48%), demonstrated to have a partial or total positivity for mRNA expression of MUC5AC in 92% of cases (45/49). Lesions having mRNA positivity without its protein expression were not observed. Neither protein and mRNA of MUC5AC were detected in normal pancreas. Conclusion: Genetic analyses revealed MUC5AC as most differently overexpressed mucin in PDAC. IHC positivity for both MUC5AC and AE1/AE3 was similar to results in literature. However, we found a lower percentage (44%) of mPDAC positives for MUC5AC expression. Indeed, we can have a subcohort of mPDAC patients with a lower aggressiveness disease. It should be remarkable that, to rich the diagnostic and prognostic performance of MUC5AC gene; additional studies combining microRNA (miRNA) by In Situ Hybridization analyses (IHS), with the methodologies used in this study are needed in order to clarify the epigenetic mechanism in PDAC metastatic process.