Impact of CKLF-like MARVEL Transmembrane Domain Containing 6 (CMTM6) Expression in Gastric Cancer

doi:10.21203/rs.3.rs-48504/v1

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Impact of CKLF-like MARVEL Transmembrane Domain Containing 6 (CMTM6) Expression in Gastric Cancer

https://doi.org/10.21203/rs.3.rs-48504/v1

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published 01 Jan, 2021

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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 CMTM and PD-L1 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=0.20). 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), and PD-L1 expression was positively correlated with PD-1 expression (p<0.05).

Conclusions: CMTM6 is associated with a poor prognosis and immunotolerance through PD-L1 in GC.

Oncology

Cancer Biology

Gastric cancer

CMTM6

PD-L1

Recent refinements in the investigation of tumor immunity and tumor microenvironments have led to an increasing number of advanced cancer treatments. Since the advent of the clinical use of immune-checkpoint inhibitors, the treatment strategies for advanced solid tumors have been changing. Blockade of the programmed cell death protein 1(PD-1)/ PD-L1: programmed cell death-ligand 1(PD-L1) interaction is crucial to the inhibitory axis in the suppression of tumor-specific T-cell responses. We previously reported that PD-1 and PD-L1 expression are significant prognostic factors in gastric cancer (GC) patients after curative resection (1).

CKLF-like MARVEL transmembrane domain containing 6 (CMTM6) belongs to the CMTM family (CMTM1–8) and is expressed at the cell membrane of various types of cells (2). Recently, CMTM6 was identified as a master regulator of the PD-L1 protein. CMTM6 binds PD-L1 and maintains its cell surface expression. In the absence of CMTM6, endocytosed PD-L1 is rerouted for lysosomal degradation (3,4). Only a few reports have investigated the clinical significance of CMTM6 expression in malignancies, including lung cancer and hepatocellular carcinoma (5–8).

We aimed to clarify the significance of CMTM6 expression and analyze the relationship between CMTM6 and PD-L1 expression in patients with gastric cancer (GC).

Patients

A total of 105 patients who had undergone surgical resection for stage II/III gastric cancer at Tokushima University Hospital between 2006 and 2012 were included in this study (stage II/III: 42/63). The study included 75 men and 30 women, with a mean age of 67.8 years (range, 38–95 years). Thirty-nine patients (37.1%) had undergone total gastrectomy, and 66 (62.9%) had undergone distal gastrectomy. The mean follow-up period 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 (2).

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) and mouse monoclonal antibody for PD-L1 (1:100; Abcam; ab20034) (1). 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 (Fig. 1). Twenty-two patients (38.3%) were CMTM6 positive. The criteria and expression rate of PD-L1 have been described previously (1).

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 χ² 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.

The characteristics of the CMTM6-positive and -negative groups are shown in Table 1. Regarding the clinicopathological variables, CMTM6 expression was not positively correlated with any clinicopathological factor investigated.

Table 1

Characteristics of CMTM-positive and CMTM6-negative patients
Variables	Positive (n = 65)	Negative (n = 40)	p Value
Age	68 ± 12	67 ± 14	0.64
Sex (Male/Female)	42/23	33/7	0.06
Differentiation (Dif/Undif)	34/31	18/22	0.53
Number of Lymph node mets, N (≤ 5/≥6)	35/30	25/15	0.38
Type	3/16/40/6	7/8/22/3	0.14
Location (U/M/L)	14/26/25	9/13/18	0.56
Tumor size (< 5/≥5 cm)	27/38	23/17	0.09
CEA (< 5/≥ 5 ng/ml)	55/10	36/4	0.43
CA19-9 (< 37 IU/ml /≥)	50/15	31/9	0.95
DG/TG	35/30	19/21	0.53
Operative time (min)	296 ± 80	300 ± 68	0.75

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) (Fig. 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 2).

Table 2

Univariate and multivariate analysis for OS
Variables	Univariate		Multivariate
Variables	5-year OS rate (%)	p-Value	HR (95% CI)	p-Value
Age in years (< 70/≥70)	71.5/61.2	0.17
Sex (Male/Female)	59.2/ 2.3	0.08	2.03 (0.67–6.13)	0.21
Dif/Undif	64.9/67.5	0.76
T 1–3/4	76.0/45.0	< 0.05	2.24 (0.98–5.13)	0.06
Number of Lymph node mets, N (≤ 5/≥6)	75.6/51.6	< 0.05	2.44 (1.05–5.65)	< 0.05
fStage II / III	75.4/60.4	0.18
Tumor size (< 5/≥5 cm)	70.2/61.7	0.59
CEA (< 5/≥5 ng/ml)	68.9/58.4	0.19
CA19-9 (< 37 IU/ml /≥)	74.2/45.0	< 0.05	2.47 (0.95–4.49)	0.11
DG/TG	68.8/63.0	0.68
CMTM6 (-/+)	79.2/57.2	< 0.05	2.47 (0.97–6.21)	0.06

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) (Fig. 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 3). Additionally, both the CMTM6- and PD-L1-positive expression groups (n = 16) had a poorer prognosis than the double-negative expression groups in OS (p = 0.05) (Fig. 3a) and DFS (p = 0.05) (Fig. 3b).

Table 3

Univariate and multivariate analysis for DFS
Variable	Univariate		Multivariate
Variable	5-years DFS rate (%)	p-Value	HR (95% CI)	p-Value
Age in years (< 70/≥70)	69.4/48.3	0.08
Sex (Male/Female)	52.6/75.6	0.08	1.87 (0.74–4.71)	0.18
Dif/Undif	60.3/59.6	0.73
T 1–3/4	63.9/51.1	0.13
Number of Lymph node mets, (≤ 5/≥6)	71.6/41.3	< 0.05	2.20 (1.07–4.52)	< 0.05
fStage II/III	68.1/53.7	0.14
Tumor size (< 5/≥5 cm)	65.5/49.9	0.18
CEA (< 5/≥5 ng/ml)	61.1/53.9	0.24
CA19-9 (< 37 IU/ml /≥)	64.7/42.3	< 0.05	1.99 (0.96–4.09)	0.06
DG/TG	58.4/61.4	0.82
CMTM6 (-/+ )	72.4/52.8	< 0.05	2.34 (1.04–5.26)	< 0.05

CMTM6 expression tended to be positively correlated with PD-L1 expression (p = 0.07) (Fig. 3).

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. This report is the first to clarify the relationships between CMTM6 and PD-L1 expression in GC.

CMTM comprises eight subtypes (CMTM1–8). CMTM shows broad-spectrum chemotactic activity and plays important roles in the hematopoietic, immune, cardiovascular, and male reproductive systems (10–14). CMTM6 was identified as a master regulator of PD-L1 in 2017. CMTM6 directly or indirectly modifies one of the lysines in the PD-L1 cytoplasmic domain(3,4). 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 (5–8). 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 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 (16–18). 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.

CKLF-like MARVEL transmembrane domain containing 6: CMTM6, PD-1: programmed cell death protein 1, PD-L1: programmed cell death-ligand 1, GC: gastric cancer, OS: overall survival, DFS: disease-free survival

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.

Acknowledgements

We thank Nicole Okoh, PhD, from Edanz Group (https://en-author　services.edanzgroup.com/) for editing a draft of this manuscript.

Ethics approval and consent to participate

All procedures performed in studies involving human participants were completed in accordance with the ethical standards of the institutional and/ or national research committee and with the 1964 declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent: Informed consent was obtained from all individual participants included in the study.

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Journal Publication

published 01 Jan, 2021

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Impact of CKLF-like MARVEL Transmembrane Domain Containing 6 (CMTM6) Expression in Gastric Cancer

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Journal Publication

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Abstract

Figures

Background

Patients And Methods

Patients

Immunohistochemistry

Statistical analysis

Results

Discussion

Abbreviations

Declarations

References

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Journal Publication

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