Anti-PD-1 therapy has shown a promising clinical outcome in gastric cancer (GC). We evaluated the clinical significance of systemic immune-related gene expression in GC patients who underwent surgery.
The correlation between the preoperative PD-1, PD-L1, and CD8 mRNA levels in peripheral blood (PB) and clinicopathological factors, including survival, in 372 GC patients was evaluated using quantitative RT-PCR. PD-1- and PD-L1-expressing cells were identified by flow cytometric analysis.
The PD-1, PD-L1, and CD8 mRNA levels in GC patients were significantly higher than those in normal controls, respectively (all P < 0.0001). The levels of each gene were positively correlated with those of the other two genes (all P < 0.0001). 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 < 0.01, P < 0.05, and P < 0.05, respectively). Multivariate analysis showed that low PD-1 and high PD-L1 mRNA levels were independent poor prognostic factors for OS (PD-1: HR 2.38, 95% CI 1.27–4.78, P < 0.01; PD-L1: HR 1.81, 95% CI 1.15–2.78, P < 0.05). PD-1 and PD-L1 expression occurred on T cells (> 90%) and T cells or monocytes (> 70%), respectively.
The PD-1, PD-L1, and CD8 mRNA levels in preoperative PB reflected the anti-tumour immune response, and the low PD-1 and high PD-L1 mRNA levels in PB were independent poor prognostic markers in GC patients who underwent surgery.
Recently, programmed death 1 (PD-1)/PD-1 ligand (PD-L1) blocking agents have been tested in the clinical setting and have shown significant therapeutic promise for various cancers.1,2 In patients with GC, anti-PD-1 antibodies, such as pembrolizumab and nivolumab, demonstrated improved survival and a manageable safety profile.3,4 On the other hand, less than half of all patients respond to these immunotherapies. In addition, these agents are enormously expensive and potentially lead to severe immune-related adverse events.
The outstanding clinical benefit of blocking immune checkpoints urged us to evaluate the precise role of anti-tumour immunity, especially that regulated by immune-related genes, in the fate of cancer patients. Among multiple cancer types, a good response to PD-1/PD-L1-targeted therapy has been observed in patients with high PD-L1 expression on tumour cells or tumour-infiltrating immune cells.5,6,7,8 Although PD-L1 protein expression on tumour cells, as determined by immunohistochemical analysis, is widely recognized as a potential biomarker for disease progression and prediction of the therapeutic benefit of PD-1/PD-L1 targeted therapy in multiple cancers, including GC, the usefulness and accuracy of immunohistochemical analysis remains controversial.7,9,10 Moreover, immunohistochemical analysis is only useful for resected specimens from operable cancer patients or tissue biopsy specimens. Therefore, we focused on determining the significance of systemic immune-related gene (PD-1, PD-L1, and CD8) expression in peripheral blood (PB), which has attracted attention as liquid biopsy. Even if sampling from the tumour tissue itself is difficult, this approach is easier to perform, can be repeated at several time points, and provides a more systemic view of the immune status of cancer patients. Various immune-related genes, including PD-1, PD-L1, and CD8, in PB of cancer patients have been reported in diverse malignancies to be part of the “cancer-immunity cycle”.11
To better understand the systemic immunological response to GC to predict the fate of GC patients, we evaluated the clinical significance of the relationship between mRNA expression of immune-related genes (PD-1, PD-L1, and CD8) in preoperative PB and clinicopathological factors, including survival of GC patients who underwent surgery.
Patient Characteristics and Sample Collection
In our previous study, PB samples were collected immediately before surgery under general anaesthesia from Japanese GC patients who underwent surgery at the National Cancer Center Hospital, Tokyo, Japan between October 2002 and December 2004. Because we used these PB samples in our previous studies, the samples from 372 patients were available, including 338 patients who received curative surgery (R0) and 34 patients who received noncurative surgery (R1/2: 19/15 cases), and were used in this study to analyse PD-1, PD-L1 and CD8 mRNA expression.12,13,14 Method of getting PB is described elsewhere.12 For normal controls (NC), PB was collected from 23 healthy volunteers, and we did not use specific exclusion criteria except for the presence of malignancy. For flow cytometric analysis, peripheral blood mononuclear cells (PBMCs) were collected from 26 Japanese GC patients at Kyushu University Beppu Hospital, Beppu, Japan between May 2016 and November 2017. For normal controls (NC), PBMCs were collected from 7 healthy volunteers, and we did not use specific exclusion criteria except for the presence of malignancy. Stage classification and assessment of the resected specimens were performed in accordance with the Japanese classification of gastric carcinoma.15 The study was approved by the Ethics Committee of Kyushu University (Approval Number: 29-597) and National Cancer Center Hospital (Approval Number: 13-54, 18-35).
Total RNA Extraction and First-Strand cDNA Synthesis
Samples transferred from Tokyo to Beppu remained frozen while in transit. Total RNA was extracted from PB as described elsewhere, and a reverse transcriptase reaction was performed as described elsewhere.16,17
Quantitative Real-Time RT-PCR
Quantitative real-time PCR (qRT-PCR) was performed to analyse the mRNA expression of PD-1, PD-L1, and CD8. We used glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as an internal control. The primer sequences of PD-1, PD-L1, CD8, and GAPDH are summarized in Table S1. The identities of the PCR products were confirmed by sequencing. Real-time monitoring of the PCR reactions was performed using the LightCycler 480 system II (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s instruction. The reaction mixture was prepared on ice and contained 1 μl of cDNA, 10 μl of LightCycler 480 SYBR Green I Master (Roche Diagnostics, Tokyo, Japan), and 400 nM of each primer. The final volume was adjusted to 20 μl with water, and qRT-PCR was performed with the following cycling conditions: initial denaturation at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at each temperature (Table S1) for 10 s, and extension at 72 °C for 10 s.
Data Analysis for qRT-PCR
The fit point method was employed to determine the cycle in which the log-linear signal was first distinguishable from the baseline, and this cycle number (threshold cycle) was used as the crossing point value. Serial dilutions of cDNA from TE-1 (human oesophageal squamous cell carcinoma cell line purchased from RIKEN, Saitama, Japan on February 3, 2015) and PBMCs (1 healthy volunteer) were prepared to obtain a standard curve. The concentration of each gene expression was calculated by plotting their crossing points against the standard curve and was divided by that of GAPDH expression of the same sample as an internal control.
Flow Cytometric Analysis
Flow cytometric analysis was performed to identify PD-1 and PD-L1-expressing cells in PBMCs obtained from 7 NC and 26 GC patients one day or more before surgery. PBMCs (1 × 105 cells) were suspended in PBS containing 2% FBS and were incubated for 45 min on ice with the appropriate dilution of antibodies. The antibodies used in this study were: PE anti-human PD-1 (clone EH12.2H7), PE anti-human PD-L1 (clone 29E.2A3), APC anti-human CD3 (clone OKT3), and PerCP/Cy5.5 anti-human CD14 (clone M5E2) from Sony Biotechnology (San Jose, CA). Control samples were stained with PE mouse IgG2b, κ isotype (clone MPC-11), APC mouse IgG2a, κ isotype (clone MOPC-173), PerCP/Cy5.5 mouse IgG2a, κ isotype (clone MOPC-173) from Sony Biotechnology. Dead cells were excluded from analysis by Live or Dye Fixable Viability Staining Kits (APC-Cy7, 32008-T) from Biotium (Hayward, CA). Stained cells were analysed on the Cell Sorter SH800 from Sony Biotechnology after gating of lymphocytes and monocytes.
To evaluate the sample size of normal controls for mRNA analysis, the statistical power was calculated using the “pwr.t2n.test” program included in the “pwr” package of R software (version 3.3.3) at the 5% significance level and large effect size (d = 0.8), and the value was 0.96. All statistical analyses were performed using the JMP software (SAS Institute, Cary, NC). For clinical analysis, patients were divided into two groups using the minimum P value approach—a comprehensive method that determines the optimal risk separation cutoff point in continuous gene expression measurements using the R software (version 3.4.1).18 Associations between variables were tested with Pearson’s Chi square test and Student’s t test or the Mann–Whitney U test. The association between mRNA expression of each gene was evaluated using Pearson’s correlation coefficient. Overall survival (OS) and the disease-free survival (DFS) rates were calculated by the Kaplan–Meier method and compared between the two groups with the log-rank test. The prognostic relevance of each variable was analysed univariately by log-rank tests. Multivariate analysis was performed using Cox’s proportional hazard model to evaluate the independent factors that were predictive of patient survival. Each predictor variable for which the P value was < 0.05 by univariate analysis was assessed for the presence of multicollinearity, and factors for which the correlation coefficients (Spearman) to the other factors were < 0.5 were included in multivariate analysis. A P value of < 0.05 was considered statistically significant.
PD-1, PD-L1, and CD8 mRNA Levels in PB in GC Patients
In all 372 patients, PD-1, PD-L1, and CD8 mRNA levels of GC patients were significantly higher than those of NC: 4.3-, 3.0-, and 6.2-fold increases, respectively (all P < 0.0001; Fig. 1a). The PD-1, PD-L1, and CD8 mRNA levels for all tumour stages in GC patients were significantly higher than those in NC (Fig. 1b). There was no significant difference in the PD-1, PD-L1, and CD8 mRNA levels among each tumour stage, except for the PD-L1 expression levels between stage I and IV (Fig. 1b). The PD-1 and CD8 mRNA levels were significantly lower in GC patients who received preoperative chemotherapy (n = 361, stage I/II/III/IV: 202/70/50/39 cases) than those who did not receive preoperative chemotherapy (n = 11, stage I/II/III/IV: 2/0/4/5 cases) (PD-1 and CD8: both P < 0.05) (Fig. S1). PD-1, PD-L1, and CD8 mRNA levels were positively correlated with mRNA levels of the other 2 genes (Fig. 1c); however, data regarding the proportion of T cells contained within each sample were not available.
Correlation Between the PD-1, PD-L1 and CD8 mRNA Levels in PB and Clinicopathological Factors Including Prognostic Outcome in GC Patients
High PD-L1 mRNA levels were significantly correlated with a larger size of tumour, advanced depth of tumour invasion, and less curability, whereas the mRNA expression levels of PD-1 and CD8 were not correlated with clinicopathological factors (Table S2). The OS rate of the 372 GC patients who underwent surgery and the DFS rate of the 338 GC patients who underwent curative surgery are shown in Fig. 2. The mean follow-up time after surgery was 7.1 years (range = 0.24–12.8 years) for OS and 6.9 years (range = 0–12.8 years) for DFS. GC patients with low PD-1, high PD-L1, and low CD8 mRNA levels had significantly poorer OS than those with high PD-1, low PD-L1, and high CD8 mRNA levels (P < 0.01, P < 0.05, and P < 0.05, respectively; Fig. 2a). In subgroup analyses for OS according to the tumour stage, the survival rates in stage II/III/IV GC patients with low PD-1 and low CD8 mRNA levels were significantly lower than those in stage II/III/IV GC patients with high PD-1 and high CD8 mRNA levels, whereas the mRNA levels of PD-1 and CD8 in stage I GC patients were not associated with OS (Fig. 2b). GC patients with low PD-1 and low CD8 mRNA levels had significantly poorer DFS than those with high PD-1 and high CD8 mRNA levels (both P < 0.05; Fig. 2c). Multivariate analysis showed that a low PD-1 mRNA level in PB was an independent poor prognostic factor for OS (HR 2.38, 95% CI 1.27–4.78, P < 0.01; Table 1) and DFS (HR 2.90, 95% CI 1.38–6.68, P < 0.01; Table 2) and that high PD-L1 expression was an independent poor prognostic factor for OS (HR 1.81, 95% CI 1.15–2.78, P < 0.05; Table 1). GC patients with high ratio of PD-L1/PD-1 mRNA levels had significantly poorer OS and DFS than those with low ratio of PD-L1/PD-1 mRNA levels (P < 0.01, P < 0.05; Fig. S2).
Localization of PD-1 and PD-L1 in PBMCs in GC Patients
To detect the localization of PD-1 and PD-L1 in PB, we performed flow cytometric analysis using markers of T cells (CD3) and monocytes (CD14) (Fig. 3a). The frequency of CD3 + cells in PBMCs of GC patients (mean ± SD: 47.0 ± 13.7%) was significantly lower than that of NC (59.7 ± 12.2%; P < 0.05), and the frequency of PD-L1 + cells in PBMCs of GC patients (13.4 ± 14.1%) was significantly higher than that of NC (2.1 ± 1.9%; P < 0.05; Fig. 3b). The frequencies of CD3 + cells in PD-1 + cells in PBMCs of NC and GC patients were 95.7 ± 5.6% and 93.8 ± 8.7%, respectively (Fig. 3c), suggesting that most PD-1 expression occurred on T cells at the protein level. On the other hand, the frequencies of CD3 + cells in PD-L1 + cells in GC patients (13.3 ± 9.8%) were statistically lower than those in NC (23.3 ± 11.6%; P < 0.05), while the frequencies of CD14 + cells in PD-L1 + cells in GC patients (59.4 ± 17.0%) tended to be higher than those in NC (46.9 ± 12.9%; P = 0.0808; Fig. 3c). Thus, more than 70% of PD-L1 expression occurred on T cells or monocytes in PBMCs at the protein level.
In the immune system, which acts as a significant barrier to tumour formation and progression, CD8 + T cells are closely associated with cellular immune responses to tumours and can directly kill tumour cells that express the tumour antigen.2,19 On the other hand, PD-1 is upregulated on leukocytes during the normal course of a successful immune response to suppress excessive immune activation.2,10 In our study, despite the lack of significant differences in PD-1, PD-L1 and CD8 mRNA levels among tumour stages, low PD-1, and low CD8 and high PD-L1 mRNA levels were associated with negative clinical outcome, likely because the mRNA levels of these immune-related genes reflect anti-tumor immunity rather than cancer progression regardless of tumour stages. Our results showed that the PD-1 and CD8 mRNA levels in PB of GC patients were higher than those in NC, although the frequency of CD3 + cells in GC patients was lower than that of NC, which may be relevant to the antitumour response. In addition, the association between the lower mRNA levels of PD-1 and CD8, and preoperative chemotherapy and poorer prognosis in more advanced stages of GC, suggest an immunocompromised status of GC patients in these subgroups. Low PD-1 and low CD8 mRNA levels may reflect reduced T-cell activation, which promotes tumour growth and leads to poor prognosis, possibly resulting from the frailty associated with disease progression in cancer patients.20 Thus, we propose that PB analyses can be used to evaluate the systemic T-cell response to tumour and functional status of cellular immunity in GC patients. On the other hand, PD-L1 is expressed on multiple types of immune cells, such as T cells, dendritic cells, and macrophages, as well as tumour cells, and suppresses the activity of PD-1 + T cells.10 Our results showed that the PD-L1 mRNA level in PB and the frequency of PD-L1 + cells in PBMCs of GC patients were higher than those of NC. Furthermore, PD-L1 in PBMCs of GC patients was more expressed in monocytes than T cells compared with those of NC, which may associate the poorer prognosis of GC patients with a low lymphocyte-to-monocyte ratio.21,22 In addition, the PD-L1 mRNA level in PB was associated with the PD-1 and CD8 mRNA levels, and patients with higher PD-L1 mRNA levels had shorter survival, which is the consistent with the previous study.23 These findings may reflect the expansion of circulating tumour cells expressing PD-L1 in PB or the negative feedback of T-cell activation to suppress normal T-cell immunity, because PD-L1 is believed to exert negative signals on T cells by interacting with B7 on dendritic cells in lymph nodes during the priming phase.1 Because the balance between PD-1 and PD-L1 expression may be important for antitumour immune response, expression of these two genes were associated with each other, and the disruption of this balance, such as high ratio of PD-L1/PD-1 mRNA levels, ultimately leads to poor prognosis. In addition, considering PD-1 and PD-L1 expression on tumour cells, the gene expression profiles originated from circulating tumor cells in PB may provide more useful information, which should be investigated in the future.1,24
The current study has several limitations. First, in this study, we evaluated the 372 available GC PB samples from more than 800 consecutive GC PB samples that were collected between 2002 and 2004. The other samples could not be used because of depletion, which may cause sample selection bias. Second, the correlation between the PD-1, PD-L1, and CD8 mRNA levels in PB and those in the tumour tissue was lacking, because tumour tissue from the same patient cohort was not available. Third, because we did not evaluate the association between the PD-1, PD-L1, and CD8 mRNA levels and therapeutic benefit of using PD-1/PD-L1 blocking agents, further investigation will be required to confirm the candidates that respond to these agents. Finally, events other than GC (e.g., corticosteroids and anaesthetic agents) can affect the expression of immune-related genes.
Despite these limitations, our results show that preoperative PB analysis provides a noninvasive and simple strategy to evaluate the systemic immune status and postoperative prognosis of GC patients. In conclusion, preoperative PD-1, PD-L1, and CD8 mRNA levels in PB may reflect the antitumour immune response and low PD-1 and high PD-L1 mRNA levels in PB are independent poor prognostic markers in GC patients who underwent surgery.
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The authors thank all the patients that provided samples for the study. We thank Ms. Kazumi Oda, Ms. Michiko Kasagi, and Ms. Sachiko Sakuma for their technical assistance; Dr. Reiko Takahashi and Dr. Daisuke Oryoji for their advice for flow cytometric analysis; and members of the Department of Surgery, Kyushu University Beppu Hospital for technical assistance and discussion. This work was supported in part by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (C) (Grand 17K10593 to S.I.), Daiwa Securities Health Foundation (to S.I.), and Oita Cancer Research Foundation (to S.I.).
The authors declare no conflict of interest.
Electronic supplementary material
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mRNA expression of PD-1, PD-L1, and CD8 in peripheral blood of GC patients with or without preoperative chemotherapy. The median and fold increases of mRNA expression in patients with preoperative chemotherapy versus those without preoperative chemotherapy are shown. The P values were calculated by the Mann–Whitney U test (PDF 128 kb)
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Ito, S., Fukagawa, T., Noda, M. et al. Prognostic Impact of Immune-Related Gene Expression in Preoperative Peripheral Blood from Gastric Cancer Patients. Ann Surg Oncol 25, 3755–3763 (2018). https://doi.org/10.1245/s10434-018-6739-4
- Immune-related Genes
- Preoperative PB
- Immune-related Gene Expression
- Gastric Cancer
- Independent Poor Prognostic Marker