Abstract 4150: MDM4 expression is increased in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein

Introduction: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of hepatocellular carcinoma (HCC) that preferentially affects young adults with no underlying liver disease. Overall patient outcome is poor due to the lack of effective treatment options. Recent studies have shown a 400 kb pair deletion resulting in DNJAB1-PRKACA fusion transcript and protein in majority of FL-HCC tumors. The exact oncogenic mechanism of this fusion protein is yet to be elucidated. In our previous studies we had noted upregulation of p53 and phosphorylated p53 in tumor samples compared to normal liver tissue without expected p53-induced apoptosis. Since MDM4 (a.k.a. MDMX or HDMX), a known negative regulator of p53, is overexpressed in various human cancers, we sought to examine its role in dysregulation of p53 signaling pathway in FL-HCC containing PRKACA-DNAJB1 fusion. Methods: We analyzed 7 FL-HCC tumors containing DNAJB1-PRKACA fusion protein and 5 non-neoplastic samples. Immunohistochemistry (IHC) on formalin-fixed and paraffin-embedded sections was performed using primary anti-MDM4 antibody. Diaminobenzidine (DAB) substrate kit was utilized to detect the signal and the slides were counterstained with hematoxylin. The nuclear MDM4 expression was assessed using immune reactive score (IRS) in a blinded manner. The IRS gives a range of 0-12 as a product of multiplication between positive cells proportion score (0-4) and staining intensity score (0-3). We also analyzed transcript levels of MDM4 from tumor and non-neoplastic liver samples on 4/7 patients. Reactions were performed in duplicates and MDM4 was normalized to GAPDH. Results: We found that the percentage of positive MDM4 cells ranged from 0 to 80 in normal liver samples (5/7 patients) and 10 to 100 in tumor samples (7/7 patients). The IRS score of MDM4 ranged from 0 to 6 in non-neoplastic liver tissue and 2 to 12 in tumor tissue. Statistically, the IRS score of MDM4 protein expression was significantly upregulated in tumor samples in comparison to non-neoplastic samples (P=0.0098). Additionally, the MDM4 transcript levels in 2/4 tumors examined were increased by 2- and 180-fold, respectively, compared to non-neoplastic liver tissue. Conclusion: The overexpression of MDM4 protein in FL-HCC patients with PRKACA-DNAJB1 suggests that the p53 effector function of DNA repair or induction of apoptosis may be dysregulated by increased MDM4 expression. Further studies targeted at blocking MDM4 inhibition of p5