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Novel biomarkers for hepatocellular carcinoma (HCC) surveillance in patients with cirrhosis are urgently needed. We previously identified osteopontin (OPN) as a promising biomarker for the early detection of HCC. This study is to further validate the performance of OPN and identify fatty acids (FA) that could improve OPN's performance in HCC risk assessment in patients with cirrhosis. To that end, we selected 103 patients with cirrhosis under surveillance. Among them, 40 patients developed HCC during follow-up. We investigated in these 103 patients, the association between HCC incidence and prediagnostic serum levels of AFP, OPN, and 46 FAs. OPN performance was higher than AFP in detecting prediagnosis HCCs and the combination with AFP further improved OPN's performance. For patients with a diagnosis of HCC within 18 months of follow-up (HCC < 18 months), AUC for OPN + AFP was 0.77. Abundance of 11 FAs [four long-chain saturated FAs (SFA), four n-3 poly-unsaturated FAs (PUFA), and three n-6 PUFAs] were statistically different between patients who developed HCC and those who did not. Abundance changes correlated with time to diagnosis for the PUFAs, but not for the SFAs. Adding arachidic acid (20:0) and n-3 docosapentaenoic acid (22:5n3) to OPN and AFP improved the discriminatory performance (AUC = 0.83). AUC for this panel reached 0.87 for HCC < 18 months (82% sensitivity at 81% specificity). In conclusion, we identified a panel of 4 markers with strong performances that could have significant utility in HCC early detection in patients with cirrhosis under surveillance.Prevention Relevance:This study identified a panel of 4 biomarkers that identifies with high performance patients with cirrhosis at high risk for HCC. This panel could have utility in HCC early detection in patients with cirrhosis under surveillance.

Novel biomarkers for hepatocellular carcinoma (HCC) surveillance in patients with cirrhosis are urgently needed. We previously identified osteopontin (OPN) as a promising biomarker for the early detection of HCC. This study is to further validate the performance of OPN and identify fatty acids (FA) that could improve OPN's performance in HCC risk assessment in patients with cirrhosis. To that end, we selected 103 patients with cirrhosis under surveillance. Among them, 40 patients developed HCC during follow-up. We investigated in these 103 patients, the association between HCC incidence and prediagnostic serum levels of AFP, OPN, and 46 FAs. OPN performance was higher than AFP in detecting prediagnosis HCCs and the combination with AFP further improved OPN's performance. For patients with a diagnosis of HCC within 18 months of follow-up (HCC < 18 months), AUC for OPN + AFP was 0.77. Abundance of 11 FAs [four long-chain saturated FAs (SFA), four n-3 poly-unsaturated FAs (PUFA), and three n-6 PUFAs] were statistically different between patients who developed HCC and those who did not. Abundance changes correlated with time to diagnosis for the PUFAs, but not for the SFAs. Adding arachidic acid (20:0) and n-3 docosapentaenoic acid (22:5n3) to OPN and AFP improved the discriminatory performance (AUC = 0.83). AUC for this panel reached 0.87 for HCC < 18 months (82% sensitivity at 81% specificity). In conclusion, we identified a panel of 4 markers with strong performances that could have significant utility in HCC early detection in patients with cirrhosis under surveillance. Prevention Relevance: This study identified a panel of 4 biomarkers that identifies with high performance patients with cirrhosis at high risk for HCC. This panel could have utility in HCC early detection in patients with cirrhosis under surveillance.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease and ranges from isolated steatosis to NASH. To determine whether circulating fatty acids could serve as diagnostic markers of NAFLD severity and whether specific fatty acids could contribute to the pathogenesis of NASH, we analyzed two independent NAFLD patient cohorts and used the methionine- and choline-deficient diet (MCD) NASH mouse model. We identified six fatty acids that could serve as non-invasive markers of NASH in patients with NAFLD. Serum levels of 15:0, 17:0 and 16:1n7t negatively correlated with NAFLD activity scores and hepatocyte ballooning scores, while 18:1n7c serum levels strongly correlated with fibrosis stage and liver inflammation. Serum levels of 15:0 and 17:0 also negatively correlated with fasting glucose and AST, while 16:1n7c and 18:1n7c levels positively correlated with AST and ferritin, respectively. Inclusion of demographic and clinical parameters improved the performance of the fatty acid panels in detecting NASH in NAFLD patients. The panel [15:0, 16:1n7t, 18:1n7c, 22:5n3, age, ferritin and APRI] predicted intermediate or advanced fibrosis in NAFLD patients, with 82% sensitivity at 90% specificity [AUROC = 0.92]. 15:0 and 18:1n7c were further selected for functional studies in vivo. Mice treated with 15:0-supplemented MCD diet showed reduced AST levels and hepatic infiltration of ceroid-laden macrophages compared to MCD-treated mice, suggesting that 15:0 deficiency contributes to liver injury in NASH. In contrast, 18:1n7c-supplemented MCD diet didn't affect liver pathology. In conclusion, 15:0 may serve as a promising biomarker or therapeutic target in NASH, opening avenues for the integration of diagnosis and treatment.

Our lab has obtained strong in vivo evidence from multiple murine tumor models that the production of methylthioadenosine (MTA) by methylthioadenosine phosphorylase (MTAP) deficient tumors potently attenuates anti-tumor immune responses, validating its role as a potent immunosuppressive molecule. We demonstrate that administration of an engineered human MTA degrading enzyme therapeutic reverses the deleterious effects of MTA on lymphocytes in vitro, and in vivo drastically impedes the growth of MTAP deficient tumors including MTAP-/- leukemia (L1210), MTAP low colon carcinoma (CT26) and an engineered MTAP-/- B16-F10 melanoma. As a single agent our therapeutic candidate elicits a large fraction of complete and durable responses in animals with established tumors, correlating with significant increases in the populations of effector T cells in both the tumor and tumor draining lymph node. We also note that enzyme treatment displays synergism in combination with existing immune checkpoint inhibitors. Despite the remarkable success of immune checkpoint inhibitors such as anti-PD1 and anti-CTLA-4 antibodies, a large fraction of tumors do not respond to immunotherapy due to additional suppressive mechanisms such as adenosinergic signaling. Approximately 15% of all cancers exhibit homozygous MTAP deletions resulting in accumulation and secretion of the adenosine (ADO) analog, MTA. Patients with cancer MTAP deletions show reduced tumor infiltrating lymphocytes (TILs) and poor overall survival in multiple tumor types. Recent work has even suggested that MTAP loss may confer resistance to antibody immune checkpoint inhibitors. Although MTA is reported to be an ADO receptor agonist, purinergic signaling alone does not explain the distinct immune suppression associated with increased MTA. In vitro, MTA (but not ADO) strongly inhibits the proliferation and viability of both CD4+ and especially CD8+ murine T cells. Similarly, treatment with ADO receptor antagonists does not restore proliferation or reverse the cytotoxicity of MTA. Only exogenous administration of MTA degrading enzymes was able to reverse these effects, indicating that MTA impacts the viability and proliferation of T cells by a mechanism independent of ADO receptor signaling. Quantitative proteomic data suggests instead that MTA acts, at least in part, via the inhibition of methyltransferases, chromatin remodeling and upregulation of P53/apoptotic pathways in lymphocytes. Collectively our data suggest that enzymatic depletion of MTA represents a unique opportunity for a biomarker driven (namely, tumor MTAP status) immunotherapeutic modality. The evidence that the consequence of MTAP deletion acts to suppress immune effector cells and promote tumor tolerance through the buildup of MTA now suggests a clear mechanism for why this is one of the more common gene deletions observed in cancer. Citation Format: Donjeta Gjuka, George Georgiou, Everett M Stone. The frequent tumor deletion of MTAP is a newly recognized potent immune checkpoint that is effectively reversed by a human methylthioadenosine degrading drug candidate [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A18. doi:10.1158/1535-7163.TARG-19-LB-A18