362

ORIGINAL
Impact of CKLF-like MARVEL transmembrane domain
containing 6 (CMTM6) expression in gastric cancer
Masaaki Nishi, Mitsuo Shimada, Kozo Yoshikawa, Jun Higashijima, Takuya Tokunaga, Hideya Kashihara, Chie Takasu,
Shohei Eto, and Toshiaki Yoshimoto
Department of Surgery, University of Tokushima Graduate School, Tokushima, Japan

Abstract : 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 byimmunohistochemistry. 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 anyof 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-freesurvival (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 throughPD-L1 in GC. J. Med. Invest. 68 : 362-367, August, 2021
Keywords : Gastric cancer, CMTM6, PD-L1

BACKGROUND
Recent refinements in the investigation of tumor immunity
and tumor microenvironments have led to an increasing numberof advanced cancer treatments. Since the advent of the clinicaluse 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) andis expressed at the cell membrane of various types of cells (2).
Recently, CMTM6 was identified as a master regulator of thePD-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 AND METHODSPatients

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). Patients’ characteristics are shown in the Table
1. The study included 75 men and 30 women, with a mean ageof 67.8 years (range, 38–95 years). The mean follow-up period

Table 1. Patients’ characteristics
VariablesAgen = 105
68 ± 13Sex (Male / Female)

75 / 30

Differentiation (Dif  / Undif)

52 / 53

Number of Lymph node mets, N (≤ 5 / ≥ 6)

60 / 45

Type (1 / 2 / 3 / 4)

10 / 24 / 62 / 9

Location (U / M / L)

23 / 39 / 43

Tumor size (< 5 / ≥ 5 cm)

50 / 55

CEA (< 5 / ≥ 5 ng / ml)

91 / 14

CA19-9 (< 37 / ≥ 37 IU / ml)

81 / 24

DG / TG

54 / 51

Operative time (min)298 ± 76

DG : distal gastrectomy, TG : total gastrectomy
Abbreviations :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

Received for publication April 6, 2021 ; accepted August 22, 2021.
Address correspondence and reprint requests to Masaaki Nishi,
MD, PhD, F.A.C.S, Department of Surgery Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, 770-8503 Japan andFax : +81-88-631-9698.

The Journal of Medical Investigation Vol. 68 2021

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The Journal of Medical Investigation Vol. 68 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 the15th 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 aseries of graded alcohols. The samples were boiled for 20 min ina 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 werethen incubated with primary antibodies overnight at 4°C. The
following primary antibodies and dilutions were used : mousemonoclonal antibody for CMTM6 (1 : 100 ; Thermo Fisher Scientific ; PA5-34744). Secondary antibody binding was detectedusing 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 weredeveloped 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 blindedmanner. CMTM6 expression was predominantly located in thecytoplasm 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 definedas 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

Figure 1. Immunohistochemistry of CMTM6 in gastric cancer tissue.

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. Statisticalsignificance was defined as p < 0.05.

RESULTSThe characteristics of the CMTM6-positive and -negative
groups are shown in Table 2. Regarding the clinicopathologicalvariables, CMTM6 expression was not positively correlated withany clinicopathological factor investigated.
Table 2. Characteristics of CMTM6-positive and CMTM6-negative
patients

Positive(n = 65)

Negative(n = 40)

p Value

Age

68 ± 1267 ± 140.64

Sex (Male / Female)

42 / 23

33 / 70.06

Differentiation (Dif / Undif)

34 / 3118 / 220.53

Number of Lymph node mets,
N (≤ 5 / ≥ 6)

35 / 30

25/ / 150.38

Type (1 / 2 / 3 / 4)

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 / 170.09

CEA (< 5 / ≥ 5 ng/ml)

55 / 1036 / 4

0.43

CA19-9 (< 37 / ≥ 37 IU/ml)

50 / 1531 / 90.95

Variables

DG / TG

35 / 30

19 / 21

0.53

Operative time (min)296 ± 80300 ± 68

0.75

DG : distal gastrectomy, TG : total gastrectomy

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 (relativerisk : 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) (Figure2b). 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 forrecurrence (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).
CMTM6 expression tended to be positively correlated with
PD-L1 expression (p = 0.07) (Figure 4).

M. Nishi, et al. CMTM6 is a Prognostic Factor in GC

364
2a

2b

Figure 2. Kaplan–Meier analysis of OS and DFS for CMTM6 (a, b).

Table 3. Univariate and multivariate analysis for OS
Variables

Univariate

Multivariate

5-year OS rate (%)

p-Value

71.5 / 61.2

0.17

Sex (Male / Female)59.2 / 82.3

0.08

Dif / Undif

64.9 / 67.5

0.76

Age in years (< 70 / ≥ 70)

HR (95% CI)

p-Value

2.03 (0.67–6.13)

0.21

T 1–3 / 476.0 / 45.0

< 0.052.24 (0.98–5.13)

0.06

Number of Lymph node mets, N (≤ 5 / ≥ 6)

75.6 / 51.6

< 0.052.44 (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 / ≥ 37 IU / ml)74.2 / 45.0

< 0.052.47 (0.95–4.49)

0.11

DG / TG

68.8 / 63.0

0.68

CMTM6 (- / +)79.2 / 57.2

< 0.052.47 (0.97–6.21)

0.06

5-year OS rate (%)

p-ValueHR (95% CI)

p-Value

DG : distal gastrectomy, TG : total gastrectomy

Table 4. Univariate and multivariate analysis for DFS
Variables

Univariate

Multivariate

Age in years (< 70 / ≥ 70)

69.4 / 48.3

0.08

1.65 (0.78–3.47)

0.19

Sex (Male / Female)

52.6 / 75.6

0.08

0.62 (0.29–1.60)

0.32

Dif / Undif

60.3 / 59.6

0.73

T 1–3 / 463.9 / 51.1

0.13

Number of Lymph node mets, (≤5 / ≥6)

71.6 / 41.3

< 0.052.43 (1.16–5.10)< 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
1.79 (0.86–3.73)

0.12

2.22 (0.99–5.00)< 0.05

CA19-9 (< 37 / ≥ 37 IU/ml)

64.7 / 42.3

< 0.05

DG / TG

58.4 / 61.4

0.82

CMTM6 (- / + )72.4 / 52.8

< 0.05

DG : distal gastrectomy, TG : total gastrectomy

The Journal of Medical Investigation Vol. 68 August 2021
3a

365

3b

Figure 3. Kaplan–Meier analysis of OS and DFS for Survival for double CMTM6 and PD-L1 expression (a, b).

Figure 4. Correlation between CMTM6 and PD-L1.

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.
CMTM comprises eight subtypes (CMTM1–8). CMTM shows
broad-spectrum chemotactic activity and plays important rolesin 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 theeffectiveness 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 inboth 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

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M. Nishi, et al. CMTM6 is a Prognostic Factor in GC

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-associatedantigen-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 showedthat 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’ CONTRIBUTIONSMN analyzed and interpreted the patient data regarding thegastric 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 andapproved the final manuscript.

AVAILABILITY OF DATA AND MATERIALSThe 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-authorservices.edanzgroup.com/) for editing a draft of thismanuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATEAll procedures performed in studies involving human participants were completed in accordance with the ethical standardsof the institutional and / or national research committee andwith the 1964 declaration of Helsinki and its later amendments
or comparable ethical standards. Informed consent : Informed
consent was obtained from all individual participants includedin the study.

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