Impact of CKLF-like MARVEL transmembrane domain containing 6 (CMTM6) expression in gastric cancer

: Background : CKLF-like MARVEL transmembrane domain containing 6 (CMTM6) is the master regulator of programmed cell death-ligand 1 (PD-L1). We aimed to clarify the significance of CMTM6 expression in gastric cancer (GC). Methods : A total of 105 patients who had undergone curative surgical resection for stage II / III GC at Tokushima University Hospital were included in this study. The expression of CMTM6 was examined by immunohistochemistry. Additionally, the relationship of each expression level to several prognostic factors was examined using univariate and multivariate analyses. Results : CMTM6 was not positively correlated with any of the factors examined. The overall survival (OS) rates were significantly poorer in the CMTM6 high-expression group than in the CMTM low-expression group (5-year OS : 57.2% vs. 79.2%, respectively ; p < 0.05). Disease-free survival (DFS) was significantly poorer in the CMTM high-expression group than in the CMTM6 low-expression group (5-year DFS : 52.8% vs. 72.4%, respectively ; p < 0.05). Multivariate analysis confirmed CMTM6 expression as an independent prognostic factor in DFS ( p < 0.05). CMTM6 expression tended to be correlated with PD-L1 expression ( p = 0.07). Conclusions : CMTM6 is associated with a poor prognosis and immunotolerance through PD-L1 in GC. J. Med. Invest. 68 : 362-367, August, 2021

was 34 months (range, 7-87 months). Seventy-three patients had received adjuvant chemotherapy following standard guidelines. The characteristic factors were defined according to the 15th edition of the Japanese Classification of Gastric Carcinoma published by the Japanese Gastric Cancer Association (9). This study was authorized in advance by the Institutional Review Board of Tokushima University, and all the patients provided written informed consent (No. 2901). These patients were also included in our previous study (1).

Immunohistochemistry
Immunohistochemistry was performed as previously described (8). The tissue samples were formalin-fixed and paraffin-embedded. Serial sections were cut at 5-μm intervals, dewaxed, deparaffinized in xylene, and rehydrated through a series of graded alcohols. The samples were boiled for 20 min in a microwave oven in citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidases were blocked with 0.3% hydrogen peroxidase for 30 min, followed by incubation in 5% goat serum for 60 min to prevent nonspecific antigen binding. The slides were then incubated with primary antibodies overnight at 4°C. The following primary antibodies and dilutions were used : mouse monoclonal antibody for CMTM6 (1 : 100 ; Thermo Fisher Scientific ; PA5-34744). Secondary antibody binding was detected using the Envision Dual Link System-HRP (DAKO). A secondary peroxidase-labeled polymer conjugated to goat anti-mouse immunoglobulins was applied for 60 min. The sections were developed in 3,3ʹ-diaminobenzidine and counterstained with Mayer's hematoxylin. Each slide was dehydrated through graded alcohols and covered with a coverslip. The presence of positive cells on each slide was determined by a pathologist in a blinded manner. CMTM6 expression was predominantly located in the cytoplasm at the invasive front of the tumor. The staining intensity was graded as follows : 0 = no staining, 1+ = weak staining, 2+ = moderate staining and 3+ = strong staining. The proportion scores were as follows : 0, none ; 1, <10% ; 2, 10%-50% ; 3, 51%-80% ; 4, >80%. A total score greater than 4+ was defined as CMTM6-positive expression ( Figure 1). Sixty-five patients (61.9%) were CMTM6 positive. We previously evaluated PD-L1 expression in gastric cancer tissue (1). Correlation between the CMTM6 expression and PD-L1 expression were evaluated.

Statistical analysis
All statistical analyses were performed using JMP 8.0.1 statistical software (SAS, Cary, NC, USA). Continuous variables were compared using the Mann-Whitney U-test, and categorical data were tested using χ 2 test. Survival curves were calculated using the Kaplan-Meier method and were compared using the log-rank test. The prognostic potentials of the parameters were analyzed by univariate analysis. Relative risk and 95% confidence intervals (CI) were estimated using the Cox proportional hazards model with stepwise forward selection. Statistical significance was defined as p < 0.05.

RESULTS
The characteristics of the CMTM6-positive and -negative groups are shown in Table 2. Regarding the clinicopathological variables, CMTM6 expression was not positively correlated with any clinicopathological factor investigated.
The overall survival (OS) rates were significantly poorer in the CMTM6-positive group than in the CMTM6-negative group (5-year OS : 57.2% vs. 79.2%, respectively ; p < 0.05) (Figure 2a). Univariate analysis identified T factor, the number of positive lymph node metastases, the CA19-9 level, and CMTM6 expression as significant prognostic factors for OS (p < 0.05). Multivariate analysis confirmed the number of positive lymph node metastases as an independent prognostic factor (relative risk : 2.44 ; p < 0.05). CMTM tended to be an independent prognostic factor (relative risk : 2.47 ; p < 0.06) ( Table 3).
Disease-free survival (DFS) was significantly poorer in the CMTM6-positive group than in the CMTM6-negative group (5-year DFS : 52.8% vs. 72.4%, respectively ; p < 0.05) ( Figure  2b). Univariate analysis identified that the number of positive lymph node metastases, CA19-9 level, and CMTM6 expression are significant prognostic factors for DFS (p < 0.05). Multivariate analysis confirmed that the number of positive lymph node metastases and CMTM6 expression are independent risk factors for recurrence (p < 0.05) ( Table 4). Additionally, both the CMTM6and PD-L1-positive expression groups (n = 20) had a poorer prognosis than the double-negative expression groups in OS (p < 0.05) ( Figure 3a) and DFS (p < 0.05) (Figure 3b).

DISCUSSION
The results of this study demonstrated that CMTM6 expression is a marker for poor prognosis in terms of both OS and DFS, and CMTM6 expression tended to correlate with PD-L1 expression in patients with stage II / III gastric cancer after curative resection.
The T-cell inhibitory capacity of PD-L1-expressing tumor cells is enhanced by CMTM6. CMTM6 shows significant specificity for PD-L1. CMTM6 is a therapeutic target that enhances the effectiveness of current immunotherapy : PD-L1 / PD-1 blocking therapies (3,4).
To our best knowledge, this is the first report concerning CMTM6 in GC. Only a few reports have investigated CMTM6 in other solid malignancies (5)(6)(7)(8). In lung adenocarcinoma, CMTM6 expression was positively correlated with PD-L1 in both the mRNA and protein levels. Furthermore, CMTM6 expression was positively correlated with immune-infiltrating cells, resting dendritic cells, eosinophils, M1 or M2 macrophages, neutrophils, and CD4 T cells (5). Kan demonstrated that CMTM6 could predict the clinical response to PD-1 inhibitors (6). In hepatocellular carcinoma, the survival time of the patients was different between the CMTM6-positive and -negative groups. Additionally, the downregulation of CMTM6 is related to distant metastasis (7).
CMTM6 controls PD-L1 expression (3,4). We previously reported PD-1 expression in gastric cancer patients and that PD-L1 expression or Indoleamine 2, 3-dioxygenase (IDO) correlated with a poor prognosis in gastric cancer after curative resection (1,15). Furthermore, PD-1 expression was correlated 3a 3b  with PD-L1 expression (1). The correlations between CMTM6 and PD-1, IDO, FoxP3, and TGFβ were investigated in the present study. However, no significant correlations were observed (data not shown). Immunogenic agents targeting T-cell immune checkpoints, such as PD-1, PD-L1, and cytotoxic T lymphocyte-associated antigen-4, are currently being applied in the treatment of several types of cancers (16)(17)(18). Nivolumab was adapted for unresectable or recurrent GC. However, the Attraction 2 trial revealed a median overall survival of 5.26 months and a 12-month overall survival rate of 26.2% in the nivolumab group (19). Koh showed that CMTM6 is an independent predictor of the response to PD-1 inhibitors. CMTM6 expression can be a promising predictor useful for therapeutic decision-making regarding PD-1 inhibitors. (20) In conclusion, CMTM6 expression is associated with a poor prognosis in patients with GC. Thus, CMTM6 expression may represent a useful new therapeutic target for GC treatment.

CONFLICTS OF INTEREST
The authors declare that they have no conflict of interest.

AUTHORS' CONTRIBUTIONS
MN analyzed and interpreted the patient data regarding the gastric cancer after curative resection. CT and HK performed the histological examination. MN and MS were major contributors in the writing of the manuscript. TT, KY, JH, SE and TY contributed to data interpretation. All authors read and approved the final manuscript.

AVAILABILITY OF DATA AND MATERIALS
The datasets used and / or analyzed during the current study are available from the corresponding author on reasonable request.

FUNDING
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.