An Oxidative Stress Response Gene Model for the Prediction of Prognosis and Therapeutic Responses in Hepatocellular Carcinoma

BackgroundOxidative stress response genes are critical for the development and progression of hepatocellular carcinoma (HCC). Still, the predictive value for prognosis and treatment response of oxidative stress response genes needs further elucidation. MethodsWe obtained the transcriptomic data and corresponding clinicopathological information of HCC patients from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases. Oxidative stress response genes (OSRGs) were retrieved from the MSigDB database. LASSO Cox regression analysis was utilized to establish an integrated multi-gene signature in the TCGA cohort, and its prediction performance was validated in the ICGC cohort. The risk score of each patient ware determined by the multi-gene signature. The CIBERSORT algorithm was employed to evaluate the immune cell infiltration. Response rate to immune checkpoint inhibition (ICI) therapy was assessed using a TIDE platform. Tumor mutation burden was estimated using VarScan processed somatic mutation data. The drug activity data from the Cancer Genome Project and NCI-60 human cancer cell lines were used to predict sensitivity to chemotherapy. ResultsThe gene signature comprises G6PD, MT3, CBX2, CDKN2B, CCNA2, MAPT, EZH2, and SLC7A11. Patients with high risk scores had shorter overall survival. The risk score was identified as an independent prognostic marker. The immune cell infiltration patterns, response rates to immune checkpoint inhibition (ICI) therapy, and the estimated sensitivity of 89 chemotherapeutic drugs were associated with risk scores. Individual prognostic gene was also associated with the susceptibility of various FDA-approved drugs. ConclusionOur study indicates that an integrated transcriptomic analysis may provide a reliable molecular model that better predicts diagnosis and forecasts the response of ICI therapy and chemotherapy.

PD-L1 expression on circulating tumor cells and platelets in patients with metastatic breast cancer

Background Immune checkpoint inhibition is effective in several cancers. Expression of programmed death-ligand 1 (PD-L1) on circulating tumor or immune effector cells could provide insights into selection of patients for immune checkpoint inhibition. Methods Whole blood was collected at serial timepoints from metastatic breast cancer patients and healthy donors for circulating tumor cell (CTC) and platelet PD-L1 analysis with a phycoerythrin-labeled anti-human PD-L1 monoclonal antibody (Biolegend clone 29E.2A3) using the CellSearch® assay. CTC PD-L1 was considered positive if detected on at least 1% of the cells; platelet PD-L1 was considered positive if ≥100 platelets per CellSearch frame expressed PD-L1. Results A total of 207 specimens from 124 metastatic breast cancer patients were collected. 52/124 (42%) samples at timepoint-1 (at or close to time of progressive disease) had ≥5 CTC/7.5ml whole blood. Of those, 21 (40%) had positive CTC PD-L1. In addition, platelet PD-L1 expression was observed in 35/124 (28%) at timepoint-1. Platelet PD-L1 was not detected in more than 70 specimens from 12 healthy donors. Platelet PD-L1 was associated with ≥5 CTC/7.5ml whole blood (p = 0.0002), less likely in patients with higher red blood cell counts (OR = 0.72, p<0.001) and a history of smoking tobacco (OR = 0.76, p<0.001). Platelet PD-L1 staining was not associated with tumor marker status, recent procedures or treatments, platelet-affecting drugs, or CTC PD-L1 expression. Conclusion PD-L1 expression was found in metastatic breast cancer patients on both CTC and platelets in an independent fashion. Inter-patient platelet PD-L1 expression was highly heterogeneous suggesting that it is a biological event associated with cancer in some but not all patients. Taken together, our data suggest that CTC and platelet PD-L1 expression could play a role in predicting which patients should receive immune checkpoint inhibition and as a pharmacodynamics biomarker during treatment.

287 Combined COX-2 inhibition with fish oil and aspirin as adjuncts to anti-PD-1 immunotherapy in metastatic melanoma

BackgroundOnly 30–40% of metastatic melanoma patients experience objective responses to first line anti-PD-1 immune checkpoint inhibition (αPD-1 ICI). Cyclooxygenase (COX-1/2) inhibition with aspirin (ASA) and other non-steroidal anti-inflammatory drugs has been associated with prolonged time to recurrence and improved responsiveness to ICI in human melanoma,1 with inhibition of myeloid-induced immunosuppression in the tumor microenvironment (TME) a purported mechanism.2 Similarly, dietary omega-3 fatty acids metabolized by COX-2 elicit downstream effects on T-cell differentiation akin to ASA administration, abrogating murine melanoma and human breast cancer progression. Mechanisms of ICI resistance remain unclear, and adjunct therapies look to bridge the gap from current response rates to cure.MethodsYUMM 1.7 melanoma cells were injected into flanks of C57-BL6/J mice. Mice were fed control diets or supplemented with omega-3 rich fish oil (FO) chow (10% weight/weight, 30%kcal/kcal), ASA in drinking water (ASA, LO – 300, MED – 600, HI - 1000 ug/mL), or the combination of these agents (COMBO, with ASA-MED) starting at day 7 post tumor implantation. Intraperitoneal αPD1 was administered every 3–4 days starting at day 12. Tumors were assessed for growth, harvested at day 32 (day 26 for ASA LO/HI), and characterized with flow cytometry. All significant results (p<0.05) assessed by 2-way ANOVA or t-test as appropriate.ResultsFO resulted in lesser tumor volume at day 32 in αPD-1 treated mice, while ASA-HI resulted in lesser tumor volume in mice not treated with αPD-1 but did not synergize with αPD-1. ASA-MED and COMBO groups trended towards decreased tumor size (p = 0.07 and 0.07 respectively) by day 32 in αPD-1 treated mice. FO and COMBO increased total CD3+ T-cells and monocytes (CD45+, CD19-, CD11b+, Ly6C+, Ly6G -) in the TME. FO increased PD-L1 + CD4+ T-cells, while COMBO increased total CD8+ T-cells and PD1+ CD8+ T-cells. ASA-HI increased monocytes and the proportion of PD-1+, CD8+ T-cells in the TME.ConclusionsMyeloid-induced suppression of T-cell function in tumors may contribute to immune checkpoint inhibition resistance. In the present study, both fish oil and aspirin altered melanoma tumor growth, with only fish oil synergizing with anti-PD-1 at the doses assessed. Both fish oil and aspirin augmented monocyte populations in the tumor microenvironment, with differential effects on T-cell populations. The partially synergistic mechanism between substrate-limited (FO) and pharmacologic (ASA) inhibition of cyclooxygenase-2 may provide a cost-effective avenue to combat immune escape in melanoma patients treated with anti-PD-1 immune checkpoint inhibition, requiring further investigation in humans.ReferencesWang SJ, et al. Effect of cyclo-oxygenase inhibitor use during checkpoint blockade immunotherapy in patients with metastatic melanoma and non-small cell lung cancer. J Immunother Cancer 2020;8(2).Zelenay S, et al. Cyclooxygenase-dependent tumor growth through evasion of immunity. Cell 2015;162(6):1257–70.

588 Caloric restriction sensitizes melanoma to anti-PD-1 immune checkpoint inhibition

BackgroundIndividual response to immune checkpoint inhibition (ICI) in patients with metastatic melanoma varies from 10–40% for monotherapy and 50–60% with combination therapy [1]. Identification of adjuncts to ICI to further bridge treatment response to cure is imperative. We focus on caloric restriction (CR) as an adjunct to anti-PD-1 ICI given its inexpensive nature, relative ease of application, and increased tolerability as compared to fasting.Methods12-week C57BL/6J mice were inoculated with Yale University Mouse Melanoma (YUMM 1.7), randomized into diet groups and further randomized into αPD-1 and control (IgG) groups on day 12 (D12). Full diet mice ad lib fed while CR mice were 40% calorically restricted based on average daily food intake. Tumors were measured every 3 days with digital calipers (q3d). αPD-1 or control was intraperitoneally injected starting D12 continuing q3d for 7 injections. Mice were sacrificed and tumors harvested on D31. RNA sequencing was performed on CD45+ CD3+ T cells.ResultsUnder CR conditions, mice treated with αPD-1 had significantly smaller tumor volumes compared to the full diet cohort treated with αPD-1 (D22, 271.15 m3 vs. 336.72 m3, p=0.031) and persisted to harvest (D31, 600.96 m3 vs. 1039.84 m3, p=0.034). A significant difference in tumor volumes between CR αPD-1 and CR control treated cohorts was observed starting at D28 (439.34 m3 vs. 667.63 m3, p=0.005) and persisted to harvest (D31, 600.96 m3 vs. 884.08 m3, p=0.009). However, no significant difference in tumor growth under full diet conditions in murine cohorts treated with αPD-1 or control or separately between CR and full diet cohorts treated with control was observed.On pathway enrichment analysis inflammatory response, cytokine-mediated, response to interferon-gamma, and cell proliferation pathways were downregulated in the CR + αPD-1 cohort. Notable genes found in these pathways include B-cell linker protein (BLNK), tyrosine-protein kinase Lyn (LYN), SYK (spleen tyrosine kinase), toll-like receptor (TLR) TLR4, TLR7, and TLR8.ConclusionsCaloric restriction significantly sensitizes YUMM 1.7 murine melanoma to anti-PD-1 therapy resulting in decreased tumor growth. We show significant modulation of tumor growth in a murine tumor cell line, which has previously demonstrated limited response to αPD-1. In the present study, caloric restriction may decrease inflammation via downregulation of cytokine and toll-like receptor mediated pathways. Furthermore, caloric restriction may reverse the immunosuppressive tumor microenvironment and provide an inexpensive means to increase treatment response to anti-PD-1 therapy.ReferencesWard WH, Farma JM. Cutaneous Melanoma: Etiology and Therapy. Brisbane (AU): Codon Publications. 2017; Chapter 8.Ethics ApprovalThis study was approved by the University Committee on Animal Resources (UCAR), UCAR-2018-014.