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The relationship between PD-L1 expression and clinicopathological characteristics and prognosis of Vietnamese gastric cancer patients

Tran Ngoc Dung 1
Nguyen Mai Hanh 1
Dang Thai Tra 1
Truong Dinh Tien 1
Nguyen Thuy Linh 1
Nguyen Khac Tuyen 1
Pham Van Thinh 1
Do Nhu Binh 2
Nguyen Manh Hung 1
Dang Thanh Chung 1, * ORCID logo

  1. Department of Pathology and Forensic Medicine, 103 Military Medical Hospital, Vietnam Military Medical University, Ha Noi, Viet Nam
  2. Division of Military Science, 103 Military Medical Hospital, Vietnam Military Medical University, Ha Noi, Viet Nam
Correspondence to: Dang Thanh Chung, Department of Pathology and Forensic Medicine, 103 Military Medical Hospital, Vietnam Military Medical University, Ha Noi, Viet Nam. ORCID: https://orcid.org/0000-0003-2376-1665. Email: [email protected].
Volume & Issue: Vol. 9 No. 7 (2022) | Page No.: 5130-5139 | DOI: 10.15419/bmrat.v9i7.748
Published: 2022-07-30

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Copyright The Author(s) 2024. This article is published with open access by BioMedPress. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0) which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. 

Abstract

Background & objectives: Programmed Death Ligand 1 (PD-L1) is a putative biomarker response to an immune checkpoint blockade that is related to poor outcomes as well as treatment strategy of numerous carcinomas, including gastric cancer. However, there is still a lack of other biomarkers that can predict patient prognosis in clinical settings. For this reason, we investigated PD-L1 expression and Combined Positive Score (CPS), as well its association with histopathological characteristics in gastric cancer patients.

Methods: We collected clinicopathological data from 54 patients with gastric cancer who underwent surgery at 103 Military Medical Hospital, Vietnam, from December 2018 to November 2020. Immunohistochemistry staining of the tumor specimens for PD-L1 expression and CPS were evaluated and accessed relationship to patient characteristics and overall survival.

Results: PD-L1 with strong and moderate expression was 1.9% and 20.3%, and CPS >= 1 was 46.3%. PD-L1 expression and CPS had no statistical relationship with histopathological characteristics, with the exception that tumor location, which was significantly related to PD-L1 expression (p = 0.001). PD-L1 positivity and CPS1 were related to worse overall survival of gastric cancer patients.

Conclusions: Our data indicated that PD-L1 and CPS are independent prognostic markers and indicators for use in targeted therapy for gastric cancer patients.

Keywords: combined positive score gastric cancer histopathological characteristics PD-L1 expression

Introduction

Gastric cancer (GC) is the fifth most common cancer globally1. Most cases have been reported in Eastern Asia, particularly in China, Japan, and South Korea2. According to the 2020 GLOBOCAN, gastric cancer was ranked as the fourth most common cancer in Vietnam after liver, lung, and breast cancers, with an average number of new cases of 17,906, with the 5-year prevalence rate at 24.64 per 100,0003. The causes of gastric cancer are still unclear, but some factors have been shown to increase risk. The most common risk factor is the microbe Helicobacter pylori, which contributes to cancer development4. Diet and lifestyle risk factors include obesity, a diet high in sodium and low in vegetables, smoked foods, smoking, and alcohol consumption. The cancer can also develop from some stomach diseases, such as gastroesophageal reflux, chronic gastritis, and stomach polyps. Hereditary factors also increase risk of contracting gastric cancer5.

Cancer immunotherapy has shown promising results in recent years. In some types of cancer, interaction between programmed death-ligand 1 (PD-L1) in cancer cells and programmed death-1 (PD-1) in T cells inhibits T cell function, thus helping to prevent the cancer cells from evading the immune system6. Currently, there are several approaches to locking interaction between PD-L1 and PD-1, such as the use of gene therapy or antagonistic monoclonal antibodies. Previously, a study by Su et al. used gene therapy to reprogram T cells by eliminating the PD-1 gene using the CRISPR-Cas9 technique; the results showed a significant reduction of PD-1 expression but did not affect the viability of T cells, suggesting a new strategy for cancer treatment7. Alternatively, the use of monoclonal antibodies to block immune checkpoint molecules has been shown to be a promising therapeutic strategy against several types of cancer8, 9 and the immune regulatory PD-1/PD-L1 axis has been used for immunotherapy for gastric cancer10. The National Comprehensive Cancer Network (NCCN) guidelines suggest that immunotherapeutic strategies be applied to patients with advanced-stage GC11. Some clinical trials have indicated that monoclonal antibodies targeting PD-L1 or its receptor PD-1 inhibit the PD-1/PD-L1 signaling pathway and enhance T-cell function, which improves outcomes in patients with GC, particularly in the advanced stage12, 13, 14. In a trial from CheckMate 032 (GC/GEJC cohort), there was a strong association between the combined assessment of PD-L1 expression with the CPS and the effectiveness of anti-PD-1 therapy15.

Beyond assessment PD-L1 expression and CPS, there are currently no biomarkers for evaluating the clinical efficacy of anti-PD-1/PD-L1 immunotherapy. In Vietnam, the evaluation of PD-L1 expression and CPS in gastric cancer patients is limited, thus constraining the access to anti-PD-1/PD-L1 therapy. Additionally, determining the association between PD-L1 expression and pathological features is essential but still controversial and not well-elucidated. Therefore, we investigate the relationship between PD-L1 expression with clinicopathological characteristics and the survival of Vietnamese patients with gastric cancer.

Methods

Patient cohort

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).

Patient characteristics

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 edition of the UICC/AJCC TNM classification17), lymph node status, and vascular invasion.

PD-L1 Immunohistochemistry

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.

Evaluation of PD-L1 expression and CPS scoring

We evaluated the expression of PD-L1 and calculated CPS following the protocol of PD-L1 IHC 22C3 pharmDx, which is FDA-approved for 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:

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

Statistical analysis

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

Clinicopathological features of gastric cancer

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)

Table 2

PD-L1 expression and CPS calculation

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)

Table 3

Correlation of PDL1 expression with clinicopathological charecteristics

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)

Figure 1

Immunohistochemical staining for PD-L1 expression in gastric cancer tissue. A: PD-L1 negative. C-D: The expression of PD-L1 in gastric cancer according to the intensity weak (B), moderate (C), and strong (at x400 magnification).

Figure 2

Kaplan-Meier survival curve for overall survival after gastrectomy. A: The overall survival time (OS) of patients with positive PD-L1 was significantly shorter than patients with negative PD-L1 (p = 0.001). B: OS of patients with CPS ≥ 1 was significantly shorter than patients with CPS <1 (p = 0.047).

Results

Patients' clinicopathologic data and relationship with PD-L1 expression

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).

Impact of PD-L1 expression and CPS on the survival of patients with gastric cancer

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 2A). Patients with CPS ≥ 1 had lower overall survival than patients with CPS < 1; the difference was statistically significant, with p = 0.047 (Figure 2B). The results showed a relationship between the expression of PD-L1 and CPS and the prognosis of gastric cancer patients.

Discussion

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 . (29.6%)14, Kawazoe (24.8%)19, and Boger . (23.73%)20. Our figure is, however, higher than that shown in some studies, such as Kang . (15.38%)21 and Dai (14.32%)22. Conversely, PD-L1 positivity was shown to be much higher in some other studies, such as those by Lin Zhang . (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 (67%)26 or Yamashita . (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 2A). 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 2B). 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 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.

Conclusions

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.

Abbreviations

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

Acknowledgments

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.

Author’s contributions

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.

Funding

None.

Availability of data and materials

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

Ethics approval and consent to participate

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.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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