RNA-SCOPE TECHNOLOGY AND IMMUNOHISTOCHEMISTRY REVEAL LOW EXPRESSION OF MUC5AC IN METASTATIC PANCREATIC PATIENTS: EPIGENETIC PITFALLS OR A BETTER DISEASE STATUS?

Background: Pancreatic ductal adenocarcinoma (PDAC) patients are in advanced stage of disease and most of them are metastatic (mPDAC) and are not candidate for surgery and clinical treatments remain a palliative. Identification of diagnostic and prognostic biomarkers for mPDAC have been investigated. MUC5AC was identified as high expressed mucin gene in PDAC compared with benign pancreatic pathologies. Its expression, in early pancreatic intraepithelial precursor lesions (PanIN), makes MUC5AC a potential diagnostic and prognostic biomarker. MUC5AC increases during cancer progression, but its role in mPDAC is not well establish. The goal of present study was to investigate MUC5AC expression in mPDAC patients. 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 as follows: normal pancreatic tissue (8) , hyperplasia of ducts (10), IPMN foci (10) and PDAC cases (30). To highlight the presence of both mRNA and protein of MUC5AC, we used a conventional brown color obtained by peroxidases reaction conjucated with antibodies and RNA probes. In order to verify the pancreatic origin of this lesions, all dots of TMA was staiend with AE1/AE3 antibody. IHCs and RST were performed according to automated protocol into a Bound III machine (Leica Microsystems). Positive and negative controls were used to validate the staining. Results: All TMA dots (100%) representing normal pancreata, pre neoplastic lesions, PDACs and mPDAC shwoed a strong positivity for AE1/AE3 antigen. While, looking at the MUC5AC immunostaining we found that its presence decreased in linear way according PDAC progression. The hyperplastic lesions had the highest positivity (8/10; 80%), folllowed by IPMN (7/10; 70%), PDAC (19/30; 66%) and mPDAC (23/52; 44%). We analysed at least 3 dots for each patients and we din’t found etherogenity for both MUC5AC and AE1/AE3 protein expressions. Indeed, we matched IHC and RST analyse for MUC5AC, in order to verify wether the presence of protein was accociated with presence of its RNA. The total of pathological lesions showing positivity for MUC5AC protein expression (49/102; 48%), dimostrated 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 pancreata. Conclusions: Genetic analyses revealed MUC5AC as most differently overexpressed mucin in PDAC. The expression of MUC5AC was examined in home-made TMA. Our IHC positivities for both MUC5AC and AE1/AE3 demostrated simalar results comparing them in front of the literature. However, we found a lower percentace (44%) of mPDAC positives for MUC5AC expression. This results were supported by the RNA-Scope technologies validating the presence of MUC5AC mRNA. Indeed, we can have a subcohort of mPDAC patients with a lower agrressiveness desease. 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 pro