We retrospectively enrolled 54 GC patients who had undergone curative gastrectomy at 103 Military Medical Hospital, Vietnam, between December 2018 and November 2020. All patients were diagnosed using pathological results of H&E staining specimens to identify gastric cancer and also underwent gastrectomy and scraping of regional nodes. These patients were not treated with chemoradiotherapy beforehand and were monitored after surgery. We selected patients with recurrent GC, metastatic cancer to the stomach, or a combination of these and other cancers. The Ethics Committee of 103 Military Medical Hospital approved all study procedures (code: 140/2016/IRB-MH103).

We collected data on patient features including tumor location, tumor size, gross appearance (based on the Bormann classification), histologic type (based on the Lauren classification), histopathology classification (based on the WHO classification of Tumors of the Digestive System, 201016), tumor differentiation, depth of invasion (T stage according to the 8^th edition of the UICC/AJCC TNM classification17), lymph node status, and vascular invasion.

Tumor specimens in a paraffin-embedded block were cut into 3 µm thick sections. We carried out immunohistochemical (IHC) staining for PD-L1 (clone 73-10, rabbit anti-human monoclonal antibody, 10 mg/ml, Leica, UK) on an automated slide stainer (Leica ST5010 Auto Stainer XL, Leica Biosystems), following manufacturer instructions. The samples were deparaffinized in xylene and rehydrated using a series of ethanol. Sections were incubated with the primary antibody for 30 minutes, then the secondary antibody of biotin-labeled anti-rabbit IgG was applied for 8 minutes. After washing in PBS, signals were visualized by incubation with polymer solution for 8 minutes and then 3-3’-diaminobenzidine (DAB) for 30 seconds. Slides were counterstained with hematoxylin for 6 minutes before mounting. After each step, wash through Bond-was solution two times in 2 minutes. Positive and negative controls were used for IHC reactions. Tonsils were used as the positive control, as PD-L1 should show strong staining in the crypt epithelium and weak to moderate staining of the follicular macrophages in the germinal centers.

We evaluated the expression of PD-L1 and calculated CPS following the protocol of PD-L1 IHC 22C3 pharmDx, which is FDA-approved for in vitro diagnostic use18. We randomly examined four fields on the tumor site slide of each specimen at 400X magnification. The expression of PD-L1 was recorded according to the percentage of tumor-stained cells per total viable tumor cells. We defined less than 1%, 1 — 49%, and ≥ 50% as weak, moderate, and strong expressions, respectively. For calculating CPS, we used the formula as follows:

CPS = PD-L1 staining cells*Total viable tumor cells X 100*(tumor cells, lymphocytes, macrophages)

Based on CPS, we divided samples into two groups: CPS < 1 and CPS ≥ 1.

After collecting sufficient information, the data for categorical variables was shown by frequency and percentage. We used a chi-square test to analyze correlations between PD-L1 expression, CPS, and clinicopathological features. We used the Kaplan-Meier method to construct the survival curves and a log-rank test for survival comparison. Statistical analysis was performed using SPSS software ver. 20.0 (IBM, Armonk, NY, USA). A p-value < 0.05 was considered statistically significant.

Table 1

Characteristics		(%)
Tumor location	Body	3 (5.5)
	Lesser curvature	17 (31.5)
	Pylorus	34 (63)
Tumor size	< 5cm	43 (79.6)
	≥ 5cm	11 (20.4)
Bormann classification	Polyp	3 (5.6)
	Fungating	6 (11.1)
	Ulcerous-infiltrative	37 (68.5)
	Diffuse-infiltrative	8 (14.8)
Lauren classification	Diffuse	33 (61.1)
	Intestinal	8 (14.8)
	Mixed	13 (24.1)
WHO classification	Tubular adenocarcinoma	40 (74.1)
	Mucinous adenocarcinoma	3 (5.6)
	Signet ring cell carcinoma	3 (5.6)
	Undifferentiated carcinoma	8 (14.8)
Tumor differentiation	High	5 (9.3)
	Mediated	22 (40.7)
	Poor	17 (31.5)
	Undifferentiated	10 (18.5)
Depth of invasion (T stage)	T1	2 (3.7)
	T2	11 (20.4)
	T3	22 (40.7)
	T4	19 (35.2)
Lymph node metastasis	Positive	27 (50)
	Negative	27 (50)
Vascular invasion	Positive	3 (5.6)
	Negative	51 (94.4)

n: number; WHO: World Health Organization

Table 2

Parameters		(%)
PD-L1 expression	Weak	42 (77.8)
	Moderate	11 (20.3)
	Strong	1 (1.9)
CPS	< 1	29 (53.7)
	≥ 1	25 (46.3)

CPS: combined positive score; PD-L1: programmed death ligand 1

Table 3

Characteristics		PD-L1, n (%)		p	CPS, n (%)		p
	Weak	Moderate	Strong		< 1	≥ 1
Tumour location
Cardia	2 (66.7)	0 (0.0)	1 (33.3)		2 (66.7)	1 (33.3)	0.442
Lesser curvature	13 (76.5)	4 (23.5)	0 (0.0)	0.001	7 (41.2)	10 (58.8)
Polyrus	27 (79.4)	7 (20.6)	0 (0.0)		20 (58.8)	14 (41.2)
Tumour size
< 5cm	33 (76.7)	9 (20.9)	1 (2.3)	0.853	23 (53.5)	20 (46.5)	0.950
≥ 5cm	9 (81.8)	2 (18.2)	0 (0.0)		6 (54.5)	5 (45.5)
Bormann classification
Polyp type	2 (66.7)	1 (33.3)	0 (0.0)	0.473	2 (66.7)	1 (33.3)	0.178
Fungating	6 (100.0)	0 (0.0)	0 (0.0)		2 (33.3)	4 (66.7)
Ulcerous-infiltrative	26 (70.3)	10 (27.0)	1 (2.7)		23 (62.2)	14 (37.8)
Diffuse-infultrative	8 (100.0)	0 (0.0)	0 (0.0)		2 (25.0)	6 (75.0)
Lauren classification
Diffuse	26 (78.8)	6 (18.2)	1 (3.0)	0.759	18 (54.5)	15 (45.5)	0.950
Intestinal	7 (87.5)	1 (12.5)	0 (0.0)		4 (50.0)	4 (50.0)
Mixed	9 (69.2)	4 (30.8)	0 (0.0)		6 (50.0)	6 (50.0)
WHO classification
Tubular adenocarcinoma	30 (75.0)	9 (22.5)	1 (2.5)	0.903	20 (50.0)	20 (50.0)	0.383
Mucinous denocarcinoma	3 (100.0)	0 (0.0)	0 (0.0)		3 (100.0)	0 (0.0)
Signet ring cell carcinoma	3 (100.0)	0 (0.0)	0 (0.0)		2 (66.7)	1 (33.3)
Undifferentiated arcinoma	6 (75.0)	2 (25.0)	0 (0.0)		4 (50.0)	4 (50.0)
Tumour differentiation
High	5 (100.0)	0 (0.0)	0 (0.0)	0.586	3 (60.0)	2 (40.0)	0.986
Mediated	18 (81.8)	4 (18.2)	0 (0.0)		12 (54.5)	10 (45.5)
Poor	11 (64.7)	5 (29.4)	1 (5.9)		9 (52.9)	8 (47.1)
Undifferentiated	8 (80.0)	2 (20.0)	0 (0.0)		5 (50.0)	5 (50.0)
Depth of invasion (T stage)
T1	2 (100.0)	0 (0.0)	0 (0.0)	0.873	1 (50.0)	1 (50.0)	0.902
T2	9 (81.8)	2 (18.2)	0 (0.0)		6 (54.5)	5 (45.5)
T3	17 (77.3)	4 (18.2)	1 (4.5)		13 (59.1)	9 (40.9)
T4	14 (73.7)	5 (26.3)	0 (0.0)		9 (47.4)	10 (52.6)
Lymph node metastasis
Positive	22 (81.5)	4 (14.8)	1 (3.7)	0.384	13 (48.1)	14 (59.1)	0.413
Negative	20 (74.1)	7 (25.9)	0 (0.0)		16 (59.3)	11 (40.7)
Vascular invasion
Positive	1 (33.3)	2 (66.7)	0 (0.0)	0.122	1 (33.3)	2 (66.7)	0.467
Negative	41 (80.4)	9 (17.6)	1 (2.0)		28 (54.9)	23 (45.1)

CPS: combined positive score; n: number; PD-L1: programmed death ligand 1; WHO: World Health Organization.

Figure 1

Figure 2

We summarize the pathological characteristics of patients in Table 1. Results demonstrated that tumor location was the most common site in pylorus with 63% prevalence, followed by curvature at 31.5% and 5% in the body. Tumor size < 5 cm accounted for 79.6% of patients, whereas tumor size of ≥ 5 cm accounted for 20.4%. Borman classification was ulcerous-infiltrative for 68.5% of patients, diffuse-infiltrative for 14.8%, fungating for 11.1%, and polypoid for 5.6%. Lauren classification was diffuse for 61%, intestinal for 14.8%, and mixed for 24.1%. The WHO classification was tubular adenocarcinoma for 74.1%, mucinous adenocarcinoma, signet-ring cell carcinoma for 5.6%, and undifferentiated carcinoma for 14.8%. The tumor differentiation was high at 9.3%, moderate at 40.7%, low at 31.5%, and undifferentiated at 18.5%. Depth of invasion was T1 at 3.7%, T2 at 20.4%, T3 at 40.7%, and T4 at 35.2%. Lymph node metastasis was 50% for both positive and negative. Vascular invasion was 5.6% for positive and 94.4% for negative.

IHC results for PD-L1 expression are shown in Figure 1 and summarized in Table 2 with rates of weak expression at 77.8%, moderate expression at 20.3%, and strong expression at 1.9%. The corresponding rankings of CPS < 1 accounted for 53.7%, while CPS ≥ 1 accounted for 46.3% of patients.

We summarize the relationship between PD-L1 expression and CPS and pathological characteristics of GC patients in Table 3. The results show no statistically significant relationship between PD-L1 expression and CPS and tumor size, Bormann classification, Lauren classification, WHO classification, tumor differentiation, depth of invasion, lymph node metastasis, or vascular invasion. Tumor location was the exception, being significantly related to PD-L1 expression (p = 0.001, chi-square test).

We evaluated PD-L1 expression and CPS with regard to overall survival in gastric cancer patients. This analysis defined samples with PD-L1 strong and moderate expression as positive and samples with weak expression as negative. Based on PD-L1 expression and CPS, overall survival is represented by the Kaplan–Meier curves in Figure 2. PD-L1-positive patients had lower overall survival than PD-L1-negative patients; the difference was statistically significant, with p = 0.001 (Figure 2 A). Patients with CPS ≥ 1 had lower overall survival than patients with CPS < 1; the difference was statistically significant, with p = 0.047 (Figure 2 B). The results showed a relationship between the expression of PD-L1 and CPS and the prognosis of gastric cancer patients.

In this study, we divided PD-L1 expression into weak, moderate, and strong rankings, with ratios of 77.8%, 20.3%, and 1.9%, respectively. For comparison with other studies, we defined samples with PD-L1 strong and moderate expression as positive, with a combined percentage of 22.2% presence in patients (Table 2). Our results are similar results found in studies by Tatsuro Tamura et al. (29.6%)14, Kawazoe et al. (24.8%)19, and Boger et al. (23.73%)20. Our figure is, however, higher than that shown in some studies, such as Kang et al. (15.38%)21 and Dai et al. (14.32%)22. Conversely, PD-L1 positivity was shown to be much higher in some other studies, such as those by Lin Zhang et al. (50.8%)23 or KEYNOTE-059 (57.1%)24. Despite many studies on PD-L1 expression, only a few authors accounted for both PD-L1 positivity and CPS in patients with GC. Some recent reports have suggested immunotherapy for patients with advanced gastric cancer and CPS ≥ 125, 26. CPS measures PD-L1 staining in both tumor and immune cells. In immune cells, PD-L1 expression is essential for inducing immune tolerance27. Therefore, instead of only assessing PD-L1 positivity in tumor cells, CPS calculation also measures the colorization of immune function cells, such as lymphocytes and macrophages. Thus, the CPS ≥ 1 ratio was higher than the PD-L1 positivity ratio in our study. Patients with CPS ≥ 1 in our study accounted for 46.3% of participants (Table 2), which is lower than the results found by Shitara et al. (67%)26 or Yamashita et al. (71.7%)28. The percentages of PD-L1 positivity and CPS ≥ 1 in our study differ from other studies and may depend on factors like patient cohort, genotype differences, histopathological characteristics, immunohistochemical staining methods used, or cut-off values for distinguishing between positive and negative PD-L1 expression.

Our results showed no relationship between the expression levels of PD-L1, CPS, and histopathological characteristics in gastric cancer patients, except regarding tumor location, which was significantly related to the expression level of PD-L1 (p = 0.001, chi-square test, Table 3). Results from other studies suggest that PD-L1 overexpression is associated with depth of invasion (T stage)20, lymph node metastasis29, 30, and vascular invasion31. In contrast, there has not been any demonstrated association of PD-L1 expression with tumor location32, tumor size33, tumor differentiation34, Lauren classification22, or invasion the lymphatic system21. This suggests that the relationship between PD-L1 expression and pathological characteristics varies widely between studies.This study also showed that PD-L1 with weak expression had significantly more prolonged survival than PD-L1 with moderate and strong expression (p = 0.001, Figure 2 A). Other studies have shown similar results; patients with PD-L1 overexpression have been shown to have a shorter survival time and a worse prognosis than patients with weak or negative PD-L1 expression35, 36, 37. Our results show that patients with CPS < 1 had significantly greater prolonged survival than patients with CPS ≥ 1 (p = 0.047, Figure 2 B). This was similar to the results from Yamashita’s study28, which also showed that these patients had a significantly shorter overall survival than those with CPS < 1 (p = 0.017). CPS has better prognostic significance than TPS (Tumor Positive Score), suggesting that, in addition to PD-L1-stained cells, the presence of lymphocytes surrounding the tumors and in the stroma have a significant effect on the overall survival of the patient. Therefore, CPS is an essential indicator for immunotherapy. Schoemig-Markiefka et al. reported that the association between expression of PD-L1 and CPS was related to the assessment of responsiveness to PD-L1 inhibitor target therapy, in which CPS ≥ 1 increases the response to PD-L1 inhibitor38. The results of our study show that the combination of assessment of PD-L1 expression and CPS can be a factor in helping predict and consider immunotherapy in gastric cancer patients. We believe these findings will help us improve therapeutic strategies in future clinical trials for treatment with PD-1/PD-L1 inhibitors.

This study also had some limitations. The sample number was small, resulting in an analytical evaluation was not comprehensive. Additionally, the cut-off values for distinguishing between positive and negative PD-L1 expression still hold room for improvement.

Overexpression of PD-L1 and CPS ≥ 1 contribute to poor prognosis and reduced survival in gastric cancer patients. Our study indicates that CPS and PD-L1 expression are independent prognostic indicators, and that combination of these two indicators can improve strategies for targeting PD-1/PD-L1 inhibitors in therapy for gastric cancer patients.

AJCC: American Joint Committee on Cancer; CPS: Combined positive score; DAB: 3-3’-diaminobenzidine; FDA: Food and Drug Administration; GC: Gastric cancer; GC/GEJC: Gastric or gastroesophageal junction cancer; GLOBOCAN: Global Cancer Incidence, Mortality And Prevalence; IBM: International Business Machines Corporation; IHC: Immunohistochemical; NCCN: National Comprehensive Cancer Network; NY: New York; PBS: Phosphate buffered saline; PD-1: Programmed death 1; PD-L1: Programmed Death Ligand 1; SPSS: Statistical Package for the Social Sciences; TNM: Tumor-node-metastasis; UICC: Union for International Cancer Control; UK: United Kingdom; USA: United States of America

We would like to thank the patients for joining the study and gratefully acknowledge the support for equipment and techniques from the Leica Biosystems branch in Vietnam.

Conceived and designed the study: TND NMH. Patient characteristics data and sample collection: TND NMH DTT TDT NTL NKT PVT NBD NMH DTC. Analyzed and interpreted the data: TND NMH TDT NKT DTC. Contributed to drafting and writing the article: TND NMH DTT TDT DTC. Involved in all aspects of the work through correspondence: NMH DTC. All authors read and approved the final manuscript.

None.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

All patients provided informed consent for participation in the study. The Ethics Committee of 103 Military Medical Hospital (code: 140/2016/IRB-MH103) approved all study procedures.

Not applicable.

The authors declare that they have no competing interests.