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IMPORTANCE: The safety of laparoscopic total gastrectomy (LTG) for the treatment of gastric cancer remains uncertain given the lack of high-level clinical evidence. OBJECTIVE: To compare the safety of LTG for clinical stage I gastric cancer with that of conventional open total gastrectomy (OTG). DESIGN, SETTING, AND PARTICIPANTS: The Chinese Laparoscopic Gastrointestinal Surgery Study (CLASS) Group CLASS02 study was a prospective, multicenter, open-label, noninferiority, randomized clinical trial that compared the safety of LTG vs OTG with lymphadenectomy for patients with clinical stage I gastric cancer. From January 2017 to September 2018, a total of 227 patients were enrolled. Final follow-up was in October 2018. INTERVENTIONS: Eligible patients were randomized to LTG (n = 113) or OTG (n = 114) by an interactive web response system. MAIN OUTCOMES AND MEASURES: The primary outcome was the morbidity and mortality within 30 days following surgeries between LTG and OTG with a noninferiority margin of 10%. The secondary outcomes were recovery courses and postoperative hospital stays. RESULTS: A total of 214 patients were analyzed for morbidity and mortality (105 patients in the LTG group and 109 patients in the OTG group). The mean (SD) age was 59.8 (9.4) years in the LTG group and 59.4 (9.2) years in the OTG group, and most were male (LTG group, 75 of 105 [71.4%]; OTG group, 80 of 109 [73.4%]). The overall morbidity and mortality rates were not significantly different between the groups (rate difference, −1.1%; 95% CI, −11.8% to 9.6%). Intraoperative complications occurred in 3 patients (2.9%) in the LTG group and 4 patients (3.7%) in the OTG group (rate difference, −0.8%; 95% CI, −6.5% to 4.9%). In addition, there was no significant difference in the overall postoperative complication rate of 18.1% in the LTG group and 17.4% in the OTG group (rate difference, 0.7%; 95% CI, −9.6% to 11.0%). One patient in the LTG group died from intra-abdominal bleeding secondary to splenic artery hemorrhage. However, there was no significant difference in mortality between the LTG group and the OTG group (rate difference, 1.0%; 95% CI, −2.5% to 5.2%), and the distribution of complication severity was similar between the 2 groups. CONCLUSIONS AND RELEVANCE: The results of the CLASS02 trial showed that the safety of LTG with lymphadenectomy by experienced surgeons for clinical stage I gastric cancer was comparable to that of OTG. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03007550

ObjectiveOur previous studies have identified CXCL8 as the crucial chemokine responsible for gastric cancer metastasis mediated by loss of RACK1. However, the regulatory effect of CXCL8 on immune surveillance in gastric cancer remains obscure.DesignFlow cytometry analyses were performed to examine major source of CXCL8 and phenotypes of immune cells in fresh tumour tissues from 76 patients with gastric cancer. Real-time PCR was performed to analyse CXCL8 mRNA level in gastric cancer tissues. For immunohistochemical analyses, a total of 420 patients with gastric cancer undergoing curative resection were enrolled. In vitro culture of fresh tumour tissue was performed to evaluate the potential therapeutic effect of blocking CXCL8 pathway in gastric cancer.ResultsIncreased level of CXCL8 indicates poor clinical outcome and tumour progression in patients with gastric cancer. In gastric cancer tissues, CXCL8 is predominantly secreted by macrophages and colony stimulating factor 2 (CSF-2) facilitates macrophage-derived CXCL8 secretion. High level of CXCL8 is associated with decreased CD8+T cells infiltration and Ki67+CD8+T cells proportion. Moreover, CXCL8 also inhibits CD8+T cells function by inducing the expression of PD-L1 on macrophages. Finally, we show that a small-molecule CXCR2 inhibitor, reparixin, drives the decreased programmed death-ligand 1 (PD-L1+) macrophages and promotes antitumour immunity. Accordingly, high levels of CXCL8+macrophages are positively correlated with poor prognosis in patients with gastric cancer.ConclusionsCXCL8 is predominantly secreted by macrophages and contributes to the immunosuppressive microenvironment by inducing PD-L1+macrophages in gastric cancer. CXCL8 inhibitors may drive antitumour response, providing potential therapeutic effects for patients with gastric cancer.

ABSTRACTApolipoprotein B mRNA editing enzyme catalytic polypeptide 3B (APOBEC3B) plays an important role in tumor mutagenesis. However, its clinical significance in gastric cancer (GC) remains largely unknown. We enrolled a total of 482 GC patients from Zhongshan Hospital, Fudan University for immunohistochemistry (IHC) staining to evaluate the prognostic and predictive values of APOBEC3B. Genomic and phenotypic datasets from the Cancer Genome Atlas (TCGA) and Asian Cancer Research Group (ACRG) cohort were downloaded for external validation and complementary bioinformatic analysis. Fresh specimens of additional 60 patients from Zhongshan Hospital, Fudan University were collected to detect CD8+T cell phenotype with flow cytometry (FCM). The high expression of APOBEC3B indicated inferior overall survival (OS, P< .001 and P= .003) and disease-free survival (DFS, P< .001 and P< .001), yet superior therapeutic responsiveness to fluorouracil-based adjuvant chemotherapy (ACT) in TNM stage II patients. The tumor microenvironment (TME) of APOBEC3B-enriched tumors was characterized by reduced infiltration of tumor reactive CD8+T cells expressing both effector molecules and immune checkpoints. APOBEC3B highCD8+T cell highGC patients were most likely to benefit from ACT and PD-1 blockade. Our study demonstrates that APOBEC3B was an independent prognostic and predictive factor in GC. The potential interplay between APOBEC3B and CD8+T cells merited further investigations.

ABSTRACTAs an adverse survival prognosticator, chemokine (C-X-C motif) ligand 13 (CXCL13) has been studied in several types of malignancies. The secretion and physiological roles of CXCL13 in follicular helper T cells (TFH) cells have been well described, while the clinical significance of CD8+tumor-infiltrating lymphocytes (TILs)-associated CXCL13 remains unknown. This study aims to investigate the clinical significance of CXCL13+CD8+T cells in survival and chemotherapeutic responsiveness prediction in gastric cancer. In this study, 440 patients enrolled from Zhongshan Hospital with tumor microarray (TMA) specimens were randomly divided into testing set (n = 220) and validation set (n = 220) for analysis. CXCL13+CD8+T cells were detected by multicolor immunohistochemistry. Fresh tumor tissue samples from another 60 gastric cancer patients were collected to detect CXCL13+CD8+T cells functional status by flow cytometry (FCM). We found that high intratumoral CXCL13+CD8+T cells infiltration predicted poor overall survival and inferior chemotherapeutic responsiveness in gastric cancer. CXCL13+CD8+T cells were associated with immunoevasive contexture with increased regulatory T (Treg) cells and dysfunctional cytotoxic T lymphocytes (CTLs). Moreover, the combinational analysis of CXCL13+CD8+T cells and CD8+T cells infiltration stratified patients into distinct risk groups with different clinical outcomes and chemotherapeutic responsiveness. Conclusively, intratumoral CXCL13+CD8+T cells infiltration could be an independent prognostic and predictive marker for gastric cancer patients. CXCL13+CD8+T cells represented an exhausted CD8+T cell subset, and might be a potential immunotherapeutic target in gastric cancer.

ABSTRACTInterleukin-9 (IL-9) is a T cell cytokine that is associated with inflammation and allergy, but the expression level of IL-9 in gastric cancer and its clinical significance are less well established. Our study aims to uncover the critical role of IL-9 in the progression of gastric cancer. Here, a total of 453 patients with gastric cancer undergoing curative resection were enrolled for immunohistochemical analyses, and Kaplan-Meier analysis was conducted to compare overall survival of patients in different subgroups. We further investigated the correlation between IL-9 expression and functional status of intratumoral CD8+T cells by means of Flow cytometry. Moreover, in vitro study was preformed to further explore the influence of IL-9 on anti-tumor immunity. Results indicated that gastric cancer patients with high IL-9 expression showed improved overall survival and gained more benefit from 5-fluorouracil-based adjuvant chemotherapy (ACT). High IL-9 expression was associated with increased numbers and elevated function of intratumoral CD8+T cells. In vitro study revealed that recombinant human IL-9 (rhIL-9) exhibit anti-tumor activity via enhancing the function of intratumoral CD8+T cells. Moreover, we found rhIL-9 could augment the efficacy of Pembrolizumab in gastric cancer. In summary, these results suggest that IL-9 expression could act as an independent predictor for overall survival and ACT response and enhancing IL-9 signaling might represent an important therapeutic strategy in gastric cancer.

ABSTRACTPodoplanin (PDPN) has been proved to have significant immunoregulatory effects in several types of malignancies and is considered to be a novel immune checkpoint molecule. However, the clinical significance of PDPN and its potential influence on immune contexture in gastric cancer remain obscure. Here, we aimed to investigate the clinical outcomes and immunoregulatory role of tumor-infiltrating PDPN+cells (tPDPNs) in gastric cancer. A total of 454 tumor tissue microarray specimens and 68 fresh tumor tissues of gastric cancer patients from Zhongshan Hospital, and transcriptional data of 293 gastric cancer patients from The Cancer Genome Atlas were included. We demonstrated that tPDPNs high subgroup experienced worse overall survival and disease-free survival, and indicated inferior therapeutic responsiveness to fluorouracil-based adjuvant chemotherapy (ACT) in gastric cancer. The abundance of tPDPNs was correlated with an immunoevasive contexture characterized by pro-tumor macrophage and dysfunctional CD8+T cell infiltration. Moreover, dysfunctional CD8+T cells in tPDPNs high subgroup exhibited decreased interferon-γ, granzyme B and perforin-1 expression yet elevated programmed cell death-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) expression. Stratification of gastric cancer patients into different risk groups based on tPDPNs and CD8+T cells showed distinct prognosis, responsiveness to ACT and molecular characteristics. This study revealed that the abundance of tPDPNs could identify an immunoevasive contexture and might be applied as an independent predictor for poor prognosis and suboptimal ACT responsiveness. Thus, we recommended tPDPNs as a promising therapeutic target in gastric cancer.

ABSTRACTOur previous study has identified intratumoral CD103+CD8+T cells as a favorable prognostic factor in gastric cancer. However, the significance of CD103+CD4+T cells in gastric cancer hasn’t yet been elucidated. Here, we aimed to investigate the clinical significance and phenotype characteristics of intratumoral CD103+CD4+T cells in gastric cancer. In our study, 469 formalin-fixed and paraffin-embedded samples and 24 fresh tissue specimens of patients with gastric cancer from Zhongshan Hospital were included. We manifested that intratumoral CD103+CD4+T cells in gastric cancer predicted poor overall survival and inferior responsiveness to fluorouracil-based ACT. The density and phenotypic characteristics of CD103+CD4+T cells in gastric cancer were detected by immunohistochemistry and flow cytometry, which showed that CD103+CD4+T cells exhibited an immunosuppressive phenotype and higher retention capacity in tumor tissues. Furthermore, increased CD103+CD4+T cells contributed to CD8+T cell dysfunction with decreased granzyme B (GZMB), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and perforin (PRF-1) expression in gastric cancer. Overall, this study revealed that intratumoral CD103+CD4+T cell infiltration defined immunoevasive contexture and predicted poor prognosis and inferior responsiveness to fluorouracil-based ACT. Therefore, we recommended that CD103+CD4+T cells might be a potential immunotherapeutic target for gastric cancer.

ABSTRACTImmune molecules, which have been found to be important in tumor microenvironment, seem prospective in tumor therapy, but they are still not effective enough to use in clinical practice. C-C motif chemokine 22 (CCL22) exists in various malignancies and correlates with migration of regulatory T cells, but its clinical significance in gastric cancer is still unclear. In this study, a combined data set of 466 patients with gastric cancer after surgical resection, comprised of a discovery (n = 319) and a validation data set (n = 147), was enrolled. CCL22 expression was assessed by immunohistochemical staining and we evaluated prognostic values of CCL22 staining and clinical outcomes with use of Kaplan-Meier curve and Multivariate Cox regression analysis. Positive CCL22 expression predicted adverse overall survival independent of traditional pathological grade. Multivariate analysis defined CCL22 and TNM stage as two independent prognostic factors for overall survival. Besides, in patients with TNM stage II/III disease, the rate of overall survival was higher among patients with CCL22-positive tumors who were treated with 5-fluorouracil based adjuvant chemotherapy than that among those who were not (P= 0.012, P< 0.001 and P< 0.001, in discovery, validation and combined data set). But for these with CCL22-negative tumors, whether to undergo adjuvant chemotherapy showed no statistical significance (P= 0.595, P= 0.085 and P = 0.252, respectively). To conclude, CCL22 was identified as an independent adverse prognostic immunobiomarker for patients with gastric cancer after surgery, which is associated with tumor-infiltrating immunocytes and could be incorporated into TNM staging system to redefine a high-risk subgroup who were more likely to benefit from 5-fluorouracil based adjuvant chemotherapy.

ABSTRACTPurpose: Tumor-infiltrating γδT cells (γδTILs) have different prognostic value and functions among various cancers. The aim of the present study was to evaluate the effect of γδTILs in gastric cancer.Patients and methods: A discovery set (n = 190) and a validation set (n = 273) were involved in this study. Patients with TNM II and III disease were used to predict response to 5-fluorouracil (5-FU)-based adjuvant chemotherapy (ACT) in both sets. γδTILs were defined as intense (γδT cells≥ 5/HPF) versus nonintense (γδT cells<5/HPF). Kaplan-Meier curve was plotted to analysis survival. Hazard ratio (HR) and 95%CI associated with γδTILs were evaluated by multivariable Cox models.Findings: The prognostic value of γδTILs in the discovery set (HR, 0.193; 95%CI, 0.097–0.383; P<0.001) was confirmed in the validation set (HR, 0.442; 95%CI, 0.251–0.779; P = 0.005) for overall survival (OS). Patients whose tumors with γδT cells≥ 5/HPF could benefit from ACT, with a reduced risk of compromised survival compared with those with γδT cells<5/HPF (HR, 0.086; 95%CI, 0.023–0.327; P<0.001 in discovery set; and HR, 0.077; 95%CI, 0.023–0.256; P<0.001 in validation set).Conclusion: The present study shows that intense γδT cells infiltration is an independent prognostic factor in patients with gastric cancer and is predictive of a survival benefit from adjuvant chemotherapy in patients with TNM II and III disease.

IMPORTANCE: Loss of O6-methylguanine-DNA methyltransferase (MGMT) protein expression has been reported in several malignant tumors and predicts dismal survival outcomes. In gastric cancer, existing studies on this topic are limited and the association between MGMT and fluorouracil-based adjuvant chemotherapy remains obscure. OBJECTIVE: To investigate the postoperative prognostic significance of MGMT in patients with resectable gastric cancer and its responsiveness to fluorouracil-based adjuvant chemotherapy. DESIGN, SETTING, AND PARTICIPANTS: This study recruited 496 patients with resectable gastric cancer who underwent radical gastrectomy between August 1, 2007, and December 30, 2008, at Zhongshan Hospital at Fudan University in Shanghai, China. However, 468 of 496 patients had comprehensive information about chemotherapy, clinicopathological data, and survival outcomes for complete analysis. In this study, we used tissue microarrays (Shanghai Outdo Biotech Co, Ltd), and 15 dots of the 468 patients were lost after immunohistochemistry. Additionally, 8 patients of TMN stage IV were excluded. Consequently, 445 patients were included in this study. Patients were randomly divided into a discovery data set (n = 200) and a validation data set (n = 245), and the range of follow-up time was from 2 to 76 months for the discovery group and 2 to 79 months for the validation group. The immunoreactivity for MGMT in cancer cells was reviewed under a light microscope by 2 pathologists who were blinded to the clinicopathological data. The association of MGMT expression with clinicopathological characteristics and measures and prognosis was inspected. Data and specimens were collected from patients from the date of surgery to April 25, 2014. Data analysis took place from May 9, 2016, to July 15, 2016. MAIN OUTCOMES AND MEASURES: Estimates of overall survival on the basis of MGMT expression and hazard ratio (HR) for estimates of overall mortality risk. RESULTS: Of the 445 patients included in the study, 315 (70.8%) were men, and the mean (SD) age of all patients was 60 (12) years. Positive expression of MGMT indicated better overall survival for patients with stage II or III gastric cancer in both the discovery data set (HR, 0.52; 95% CI, 0.32-0.84; P = .003) and the validation data set (HR, 0.63; 95% CI, 0.43-0.93; P = .01). Multivariate analysis identified MGMT expression and TNM stage as 2 independent prognostic factors for overall survival. In stage II disease, the benefit from fluorouracil-based adjuvant chemotherapy was superior among MGMT-positive patients (HR, 0.35; 95% CI, 0.13-0.95; P = .007 for interaction) compared with MGMT-negative patients. CONCLUSIONS AND RELEVANCE: Positive expression of MGMT in gastric cancer was identified as an independent, favorable prognostic factor. Incorporating MGMT expression into the current TNM staging system could lead to better prognostic accuracy. These findings should be confirmed within the framework of randomized clinical trials associated with genomic DNA sequencing studies.