CMTM6 significantly relates to PD-L1 and predicts the prognosis of gastric cancer patients

Introduction

Gastric cancer is one of the most common malignant cancers of the digestive tract in the world (Globocan, 2018). The majority of gastric cancer (GC) patients in China are diagnosed in the advanced stages of the disease (Qiu et al., 2018; Liu et al., 2018). The 5-year survival rate for patients with advanced gastric cancer who receive comprehensive surgery-based treatment is less than 30% (Katai et al., 2018). GC is commonly treated using chemotherapy and anti-HER-2 targeted therapy, which have limited efficacies (Wagner et al., 2010). Immune checkpoint inhibitors (ICIs) are relatively new in the treatment and diagnosis of many cancers but they have demonstrated unprecedented clinical efficacy in multiple types of cancer and have good clinical application prospects in gastric cancer (Sharma & Allison, 2015; Fuchs et al., 2018; Shitara et al., 2018; Kang et al., 2017). However, immunotherapy is only effective in a small proportion of gastric cancers and the expression of PD-L1 on tumor cells or tumor-infiltrating immune cells does not accurately predict for the patient’s response to PD-1/PD-L1 inhibitors (Braun, Burke & Van Allen, 2016; Guan et al., 2017; Wang & Wu, 2017; Pardoll, 2012). Therefore, new immune-related therapeutic targets must be identified and their relationship with PD-L1 expression must be explored.

Two recent articles have identified CMTM6, which is a PD-L1 regulatory factor that plays an important role in inhibiting T-cell activation and anti-tumor responses. CMTM6 displays specificity for PD-L1 and promotes the expression of PD-L1 in tumor cells by T-cells. The depletion of CMTM6 decreases PD-L1 without compromising the cell surface expression of MHC Class I (Burr et al., 2017; Mezzadra et al., 2017). The role of CMTM6 in anti-tumor immunity and stabilization of the expression of PD-L1 has prompted us to explore its role in GC.

In this study, we evaluated the expression of CMTM6 and PD-L1 in 122 GC samples to investigate the prognostic success of CMTM6 and its relation to PD-L1 for GC patients.

Materials and Methods

Results

Expression of CMTM6 and PD-L1 in GC tissues

We detected the expression of CMTM6 and PD-L1in 122 gastric cancer tissues using an IHC assay. As shown in Fig. 1, CMTM6 is located mainly in the cell membrane and cytoplasm and the expression of PD-L1 was observed on the cell membrane. The low- and high-expressions of CMTM6 in gastric cancer tissues were 46.7% (57/122) and 53.3% (65/122), respectively, and the negative and positive expressions of PD-L1 were 35 (28.7%) and 87 (71.3%), respectively (Table 1).

The prognostic value of CMTM6 expression in patients with GC

The cut-off level for CMTM6 expression was determined using X-tile software (Fig. 2A). Kaplan–Meier survival analysis showed that patients with high CMTM6 expression had shorter overall survival (OS) than these with low expression (Fig. 2B, P < 0.001). Univariate and multivariate Cox proportional hazard regression analyses were used to evaluate the clinically-related factors associated with survival. CMTM6 expression was an independent risk factor for prognosis (Table 2, HR:2.221, CI% [1.36–3.628], P = 0.001).

Table 2:

The univariate and multivariate Cox proportional hazard regression analyses between the clinical related factors and survival in GC patients.

Variables	Univariate analysis	Multivariate analysis
	Wald χ2	HR (95%CI)	P
Age (year)	0.185		0.667
<60		Reference
≥60		1.119 [0.672–1.862]	0.667
Sex	0.206		0.650
Male		Reference
Female		0.881 [0.51–1.522]	0.650
Tumor size (cm)	4.82		0.090
≤3		Reference
3–6		1.805 [0.954–3.414]	0.069
≥6		1.088 [0.493–2.402]	0.834
TNM stage (AJCC)	0.514		0.773
I		Reference
II		0.887 [0.474–1.662]	0.709
III		1.136 [0.608–2.121]	0.689
Grade (AJCC)	0.436		0.509
I + II		Reference
III		1.188 [0.712–1.984]	0.509
Histology	2.599		0.107
Adenocarcinoma		Reference
Others		1.869 [0.874–3.997]	0.107
CMTM6	10.157		0.001
Low		Reference
High		2.221 [1.36–3.628]	0.001
PDL1	1.256		0.262
Negative		Reference
Positive		1.409 [0.774–2.566]	0.262

DOI: 10.7717/peerj.9536/table-2

Relationship between CMTM6 and PD-L1 in GC

Patients with positively expressed PD-L1 has a significantly shorter OS than those with negatively expressed PD-L1 (Fig. 3A) (P = 0.003). CMTM6 expression was significantly related to PD-L1 positivity in gastric cancer tissues (Fig. 3B) (r = 0.186, P = 0.041). Age, sex, tumor size, TNM stage, grade, histology, and CMTM6 expression were selected in the univariate and multivariate logistic regression analysis to further evaluate the relationship between the positive expression of PD-L1 and other clinically-related factors. The results showed that only CMTM6 expression was a risk factor of PD-L1 expression (Table 3) (OR:2.538, CI% [1.128–5.714], P = 0.024).

Table 3:

The univariate and multivariate logistic regression analysis between the clinical related risk factors and PD-L1 expression in GC patients.

Variables	Univariate analysis	Multivariate analysis
	Wald χ2	OR (95%CI)	P
Age (year)	1.348		0.246
<60		Reference
≥60		1.634 [0.713–3.741]	0.246
Sex	0.572		0.45
Male		Reference
Female		1.473 [0.54–4.017]	0.45
Tumor size (cm)	3.592		0.166
≤3		Reference
3–6		2.507 [0.946–6.645]	0.065
≥6		2.262 [0.695–7.358]	0.175
TNM stage (AJCC)	2.719		0.257
I		Reference
II		0.561 [0.2–1.568]	0.27
III		1.392 [0.421–4.597]	0.588
Grade (AJCC)	0.241		0.624
I + II		Reference
III		0.786 [0.3–2.059]	0.624
Histology	0.044		0.834
Adenocarcinoma		Reference
Others		0.862 [0.215–3.464]	0.834
CMTM6	5.063		0.024
Low		Reference
High		2.538 [1.128–5.714]	0.024

DOI: 10.7717/peerj.9536/table-3

Discussion

CMTM6 belongs to a family of proteins made up of eight members (CMTM1-4 on the 16q21-22 chromosomal region, CMTM5 on the 14q11 region and CMTM6-8 on the 3p22 region) that play a role in epigenetic regulation, embryonic development and tumorigenesis (Han et al., 2003; Sanchez-Pulido et al., 2002; Yafune et al., 2013). CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes where it prevents PD-L1 from being targeted for lysosome-mediated degradation (Burr et al., 2017). CMTM6 was identified as a major regulator of PD-L1, a key immunological checkpoint, and a potential therapeutic target for tumor cell immune evasion.

There have been reports on CMTM6 for its regulation of PD-L1 and its function at the genetic and cellular levels for different cell lines. However, the usefulness of CMTM6 in clinical cancer samples has yet to be determined. We reported the prognostic value of CMTM6 and its relationship to PD-L1 in GC for the first time in our study. High CMTM6 expression was associated with shorter overall survival (OS) versus low expression (P < 0.001). Multivariate analysis confirmed the prognostic value (HR:2.221, CI% [1.36–3.628], P = 0.001). The expression of CMTM6 was significantly related to the positive presence of PD-L1 in gastric cancer tissues (r = 0.186, P = 0.041). The expression of CMTM6 was the only independent risk factor of PD-L1 expression found by multivariate logistic regression analysis (OR:2.538, CI% [1.128–5.714], P = 0.024).

There are few studies reporting on CMTM6 expression and prognosis in other cancers. In primary pancreatic ductal adenocarcinoma (PDAC), a high CMTM6 expression was associated with shorter overall survival (OS). In the case of low expression, expression of CMTM6 also increased the prognostic value of PD-L1 expression (Mamessier et al., 2018). CMTM6 expression was associated with poor prognosis in hepatocellular carcinoma (HCC). The expression of CMTM6 was downregulated and was correlated with the AFP level and tumor metastasis of HCC patients (Zhu et al., 2019). CMTM6 overexpression was associated with poor prognosis in gliomas. CMTM6 is important for regulating T-cell activation and antitumor responses and is a promising target for developing immunotherapy of gliomas (Guan et al., 2018). A high CMTM6 expression was associated with reduced survival time and may be a strong indicator of poor prognosis in different cancer types.

CMTM6 maintains the stability of the PD-L1 cell surface expression and plays a key role in the survival of PD-L1 (Mezzadra et al., 2017). CMTM6 expression was positively correlated with PD-L1 in immunohistochemical and mRNA expression data of non-small cell lung cancer (Koh et al., 2019; Gao et al., 2019). Preventing CMTM6 from binding with PD-L1 may recover the existing immunosuppression response and serve as a promising target for immunotherapy. Zugazagoitia et al. found that high CMTM6 and PD-L1 co-expression in stromal cells, but not tumor cells, of lung cancer was significantly associated with longer overall survival in treated patients. However, this effect was not observed in the absence of immunotherapy (Zugazagoitia et al., 2019). Koh et al. (2019) also found that CMTM6 expression may be a promising tool for therapeutic decision-making regarding PD-1 inhibitors. Zhao et al. (2019) discovered a statistically significant interaction between CMTM6 and CD274 (PD-L1) indicating that CMTM4/6 may be a new therapeutic target for type I renal clear cell carcinoma (RCC) patients who are resistant to immune checkpoint blockade (ICB). CMTM6 expression was positively correlated with PD-L1 in GC, making it a promising immunotherapy target for GC.

This study also has certain limitations. Firstly, we used IHC to detect CMTM6 and PD-L1 expressions. However, this process is limited by positive criteria, the definition of cut-off values, and the specificity and reproducibility of the antibodies used. Secondly, due to some patients lacked the pathological data of Lauren classification, we did not perform a correlation analysis between CMTM6 and Laurent classification. Thirdly, because of the influence of various clinical factors, such as pathological type, tumor size, etc., TNM stage is not a risk factor affecting the prognosis of GC patients.

Conclusion

CMTM6 expression is significantly related to PD-L1 and may be a useful prognostic indicator and a specific therapeutic target for cancer immunotherapy for GC patients.

Supplemental Information