11531 Background: Programmed death 1 (PD-1)/PD-1 ligand (PD-L1) blocking agents to gastric cancer (GC) in the clinical setting show significant therapeutic promise. However, since these agents are enormously expensive and potentially toxic, it is crucial to identify predictive biomarkers for detecting the best candidate who would benefit from these agents by less invasive and simpler method, such as liquid biopsy. Methods: Expression levels of genes coding for PD-1, PD-L1 and CD8 (CD8+ T cells are closely associated with cellular immune responses to tumors) were assessed in peripheral blood (PB) samples using quantitative RT-PCR. Samples were obtained from 407 GC patients (392 patients with neoadjuvant chemotherapy [NAC] and 15 patients without NAC) before surgery and 23 PB from normal controls (NC). Flow cytometric analysis was performed to identify PD-1-expressed cells in PB mononuclear cells. Results: PD-1, PD-L1 and CD8 mRNA levels of GC patients were significantly higher than those of NC: 4.2-, 3.0- and 6.1-fold increases, respectively (P < 0.0001, P = 0.0001 and P < 0.0001). PD-1 mRNA levels were significantly lower in GC patients with NAC than in GC patients without NAC (P < 0.01). GC patients with low PD-1, high PD-L1 and low CD8 mRNA levels had significantly poorer overall survival (OS) than those with high PD-1, low PD-L1 and high CD8 mRNA levels, respectively (P < 0.05, P < 0.05 and P < 0.05). Multivariate analysis showed that PD-1 low/ PD-L1 high mRNA levels was independent risk factors for OS (OR 2.15, 95%CI 1.29-3.45, P < 0.01). Flow cytometric analysis demonstrated the proportion of CD3 (T cell marker)-positive cells in the PD-1-positive fraction were 95.4 ± 6.9% in GC patients. Thus, most PD-1 protein expression occurred on T cells. Taken together, PD-1, PD-L1 and CD8 mRNAs in PB were overexpressed in GC patients, and PD-1 mRNA levels which was mostly expressed on T cells in protein levels in PB were decreased in GC patients with NAC. Furthermore, relative levels of PD-1, PD-L1 and CD8 were associated with prognosis, respectively. Conclusions: Preoperative PD-1, PD-L1 and CD8 mRNA levels in PB may reflect antitumor immune response, and PD-1 low/ PD-L1 high mRNA levels in PB are markers of poor prognosis in GC patients.
Flow cytometric analysis of the DNA content of gastric cancer