S. Ito1, T. Fukagawa2, T. Sato3, T. Masuda1, Y. Kuroda1, H. Eguchi1, M. Sasako4, K. Mimori1 1Kyushu University Beppu Hospital,Department Of Surgery,Beppu, OITA, Japan 2National Cancer Center Hospital,Gastric Surgery Division,Tokyo, , Japan 3Kyushu University,Medical Institute Of Bioregulation,Fukuoka, Fukuoka, Japan 4Hyogo College Of Medicine,Department Of Surgery,Nishinomiya, HYOGO, Japan
Introduction: Liquid biopsy, which is less invasive and simpler method than tissue biopsy, in cancer patients has received enormous attention because of its significant clinical implications. We have reported the relationship between various cancer-related genes expression in preoperative peripheral blood (PB) and bone marrow (BM), and patient prognosis in gastric cancer (GC) patients who underwent surgery. In the current study, we investigated which marker shows the strongest prognostic marker in GC patients.
Methods: mRNA levels of oncogenic genes (BMI-1, JUN, CDC42, PLD1, FOS, FOSB, S1PR1), tumor suppressor genes (FBXW7, MIR-34c), cancer stem cell marker (CD44), circulating tumor cell marker (PLS-3) and immune related genes (PD-1, PD-L1, CD8) in PB and BM samples from 415 GC patients before surgery were investigated by quantitative RT-PCR. For survival analysis, cases were divided into two groups using minimum P value approach based on each gene expression level. Flow cytometric analysis was performed to identify PD-1-expressed cells in peripheral blood mononuclear cells.
Results: Multivariate analysis showed that mRNA levels (high/low) of FOS (HR 0.52, P < 0.05), CD44 (HR 2.57, P < 0.05), PD-1 (HR 0.41, P < 0.001), PD-L1 (HR 1.91, P < 0.01) and CD8 (HR 0.54, P < 0.05) in PB sample, and mRNA levels (high/low) of BMI-1 (HR 0.37, P < 0.05), CDC42 (HR 4.12, P < 0.01), PLD1 (HR 0.50, P < 0.05), FOS (HR 0.51, P < 0.05), S1PR1 (HR 0.23, P < 0.01), FBXW7 (HR 0.49, P < 0.05), CD44 (HR 0.35, P < 0.01), PD-1 (HR 1.75, P < 0.01) and PD-L1 (HR 1.56, P < 0.05) in BM sample were independent factors for overall survival (OS). The strongest independent factor for OS was mRNA levels of PD-1 in PB. PD-1 mRNA levels in PB of GC patients were significantly higher than those of healthy volunteers (n=23); 4.2-fold increase was observed (P < 0.0001). PD-1 mRNA levels in PB of GC patients with neoadjuvant chemotherapy (NAC) (n=15) were significantly lower than those in GC patients without NAC (n=392) (P < 0.01). The proportion of CD3-positive cells (CD4+ and CD8+ T cells) in PD-1-positive cells was 95.4 ± 6.9% in GC patients, suggesting that most PD-1-expressed cells were T cells. Taken together, PD-1 mRNA levels were most associated with survival among cancer- and immune-related genes examined. Since PD-1 is reported to mainly express on activated CD4+ T cells and CD8+ T cells which are closely associated with immune response to tumor, our findings that PD-1 mRNA levels which mostly express on T cells in protein levels in PB were overexpressed in GC patients, and were decreased in patients with NAC, suggest that PD-1 mRNA levels in GC patients may reflect antitumor immune response and immunocompromised condition with NAC.
Conclusions: The strongest prognostic factor was preoperative PD-1 mRNA levels in PB for GC patients who underwent surgery, and may reflect antitumor immune response.