Expression and clinical significance of programmed death ligand-1 evaluated by 22C3 antibody in pleural effusion metastatic non-small-cell lung cancer

INTRODUCTION

Lung cancer ranks as one of the most common cancers, leading in both incidence and mortality rates globally. In 2020, an estimated 2.2 million new cancer cases and 1.8 million deaths were attributed to lung cancer, making it the second most common cancer and the leading cause of cancer death. Lung cancer accounted for approximately 11.4% of all cancer diagnoses and 18.0% of cancer deaths.^[1] Non-small-cell lung cancer (NSCLC) represents 80–85% of lung cancer cases. Of these, 20% of newly diagnosed lung cancer cases are in stages I and II, and 30% are in stage III.^[2] After the diagnosis of NSCLC, only about 26% of patients survive for more than 5 years, and for advanced patients, the 5-year survival rate is only 6% with traditional chemotherapy.^[3]

Lung cancer stands as the most prevalent malignant tumor, with NSCLC constituting over 80% of cases.^[4] Due to its unique anatomical structure, tumors are predisposed to hematologic or lymphatic metastases. Moreover, mid to late-stage NSCLC frequently presents with malignant pleural fluid, resulting in a dismal clinical prognosis. Despite the successful integration of clinically targeted drugs, which has enhanced patients’ quality of life and moderately prolonged survival, individuals with intermediate and advanced NSCLC still encounter challenges such as restricted gene-targeted therapy options or drug resistance in later stages of treatment. Immunotherapy represents a burgeoning area in cancer research. Investigating the mechanisms of the tumor immune checkpoint (Programmed death receptor-1 [PD-1]/Programmed death ligand-1 [PD-L1])-related pathways holds promise for furnishing novel theoretical underpinnings for the immunotherapy of clinical NSCLC.^[5]

PD-1, also referred to as CD279, is a member of the B7/CD28 receptor superfamily. T-cell hybridoma assay conducted by Ishida et al. in 1992, utilizing the ablative hybridization technique, it earned the designation of the programmed death molecule.^[6] PD-L1, the principal ligand of PD-1, is commonly expressed on the cell membrane surfaces of various immune cells including T-cells, B-cells, NK T-cells, macrophages, as well as non-immune cells such as mesenchymal stem cells and cancer cells. Serving as an immunosuppressive agent, PD-L1 regulates its expression and is notably abundant on the surfaces of lymphohematopoietic or cancer cells, predominantly induced by interferon-γ. Current research underscores the pivotal role of the PD1/PD-L1 pathway in enabling tumor immune evasion.^[7] The active PD-1/PD-L1 axis within the tumor microenvironment facilitates immune escape by attenuating the anti-tumor immune response. Many tumors exhibit growth and progression correlated with this immune evasion mechanism mediated through the PD1/PD-L1 pathway.

Malignant serous effusion is a common complication of NSCLC, indicating advanced disease progression.^[8] Malignant pleural effusion can cause lung compression, leading to dyspnea and impaired respiratory function.^[9] The prognosis for patients with malignant pleural effusion is poor, significantly reducing survival time.^[10] Traditional treatment methods have limited efficacy against malignant serous effusion, necessitating the search for effective drug treatments to control disease progression and improve patient quality of life.^[11]

Immunotherapy has become a focal point in cancer research. Immunotherapies, particularly those involving PD-1 and PDL1 monoclonal antibodies, have become important treatment options for the first-line and subsequent treatment of NSCLC. Studies suggest that malignant pleural effusion creates a tumor tolerance environment complicated by immunosuppressive factors. Immunotherapy may benefit patients with malignant pleural effusion by stimulating tumor-specific immune responses in the pleural cavity to counteract the tumor tolerance environment.^[12] However, clinical research and application of immunotherapy in metastatic NSCLC patients with malignant pleural effusion remain limited. PD-L1 is currently the most valuable predictor of NSCLC immunotherapy response.

This study aims to detect the expression of PD-L1 in 149 cases of NSCLC malignant pleural effusion cell paraffin block samples using immunochemistry (IHC). It also seeks to analyze the relationship between PD-L1 expression and clinicopathological characteristics, anaplastic lymphoma kinase (ALK) expression, epidermal growth factor receptor (EGFR) mutation, and overall survival (OS) time of NSCLC patients. The goal is to provide a theoretical basis for the immunotherapy of metastatic NSCLC patients with pleural effusion.

MATERIAL AND METHODS

RESULTS

Identification of cancer cells in malignant pleural fluid of NSCLC patients

Specimens sent for examination underwent pleural fluid exfoliation cytology smear, HE staining, and IHC detection of pleural fluid cell wax blocks. Tumors were diagnosed through microscopic examination using immune markers related to lung cancer [Figure 1 a-l and Table 1].

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Figure 1:

The expression of programmed death ligand-1 (PD-L1) in serous effusions of metastatic lung adenocarcinoma and metastatic lung squamous cell carcinoma. (a) Hematoxylin and eosin (HE) staining of non-small-cell lung cancer (NSCLC) (metastatic adenocarcinoma) in pleural effusion, ×20. (b) HE staining of NSCLC (metastatic adenocarcinoma) from pleural effusion cell block, ×20. (c) Immunochemical (IHC) staining for Epithelial cell adhesion molecule, displaying positive staining in the cell membrane or cytoplasm, ×20. (d) IHC staining for thyroid transcription factor 1, exhibiting positive staining in the nucleus, ×20. (e) IHC staining for Napsin A, revealing cytoplasmic granular positivity, ×20. (f) IHC staining demonstrating PD-L1 membrane positivity and high expression, ×40. (g) HE staining of NSCLC (metastatic squamous cell carcinoma) in pleural effusion, ×20. (h) HE staining of NSCLC cell blocks (metastatic squamous cell carcinoma) from pleural effusion, ×40. (i) IHC staining for Cytokeratin 5/6 (CK5/6), showing positive staining in the cell membrane or cytoplasm, ×20. (j) IHC staining for p63, displaying nuclear positivity, ×20. (k) IHC staining positive for p40 in the nucleus, ×20. (l) IHC staining of PD-L1, exhibiting cytoplasmic membrane positivity and low expression, ×20. Scale bars: 100 μm.

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Table 1: The main immunophenotypic results of different pathological types of serous cavity effusion metastatic non-small-cell lung cancer.

Pathological type	n	Ep-CAM	CK7	TTF1	NapsinA	CK5/6	P40	Calretinin
Metastatic lung adenocarcinoma	144	+	+	+	+	-	-	-
Metastatic lung squamous cell carcinoma	5	+	-	-	-	+	+	-
Positive rate (%)		100	96.6	96.6	96.6	3.4	3.4	0

TTF1: Thyroid transcription factor 1, Ep-CAM: Epithelial cell adhesion molecule

Positive expression of PD-L1 protein in malignant pleural fluid of NSCLC patients

Among the 149 cases of metastatic NSCLC in pleural effusion, PD-L1 staining was positive on the cell membrane. The positive expression rate was <1% in 17 cases [Figure 2a], between 1% and 49% in 29 cases [Figure 2b], and 50% or more in 17 cases [Figure 2c], resulting in an overall positive rate of 42.3%.

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Figure 2:

Different levels of programmed death ligand-1 (PD-L1) expression in serous cavity effusion metastatic lung adenocarcinoma. (a) PD-L1 expression (<1%) on immunochemical (IHC) staining in metastatic non-small-cell lung cancer (NSCLC) from pleural effusion, ×10. (b) PD-L1 expression on IHC staining in metastatic NSCLC from pleural effusion (1–49%), ×20. (c) PD-L1 expression (≥50%) in pleural effusion metastatic NSCLC, ×40. Scale bars: 100 μm.

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Relationship between PD-L1 expression in malignant pleural fluid of NSCLC and OS time of patients

Tracking the survival of 149 NSCLC patients with malignant pleural fluid revealed a significant disparity (P < 0.05) in median OS between those who were PD-L1 positive and those who were negative [Figure 3].

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Figure 3:

Relationship between programmed death ligand-1 (PDL1) expression in malignant pleural fluid of non-small-cell lung cancer and overall survival time of patients.

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DISCUSSION

The 2019 NSCLC Guidelines from the National Comprehensive Cancer Network moved PD-L1 testing from Class 2A to Class 1 recommendation.^[15] PD-L1 is the most valuable predictor for NSCLC immune response to treatment. However, clinical studies and applications in patients with metastatic NSCLC with malignant pleural effusion are still limited. The novelty and advantage of this study is to detect the expression of PD-L1-22C3 in wax samples of malignant pleural effusion cells of NSCLC by IHC method and its relationship with clinicopathological features, so as to provide a theoretical basis for immunotherapy of patients with metastatic NSCLC complicated with pleural effusion.

At present, the four most commonly used PD-L1 antibodies in major international multi-center clinical trials are as follows: DAKO’s 22C3, 28–8, and Roche Diagnostics (Ventana)’s SP263 and SP142. Famous blue print plan phase 1 study of four antibodies (28–8, 22 c3, SP263, and SP142) consistency between the evaluated.^[16] The results showed that the three antibodies (28–8, 22C3, and SP263) had highly consistent staining effects on the expression of PDL1 in tumor cells, while the positive rate of SP142 was low. The PD-L1 assay of DAKO 22C3 is the earliest approved companion diagnosis for immunotherapy of NSCLC and has high reliability. Although a number of studies have shown high consistency among 22C3, 28–8, and SP263, there is insufficient prospective clinical evidence to support the feasibility of interoperability of antibody test results. In a consistent study of the expression of PD-L1 22C3 and 28–8 in 420 Japanese NSCLC surgical specimens: The expression of PD-L1 in the 22C3 detection group, at 25% and 50% cutoff values, was higher than that in the 28–8 group, and the evaluation of PD-L1 expression with 28–8 instead of 22C3 might miss some positive patients. The consistency of results of 28–8, SP263, and 22C3 was analyzed by comparing real-world data, and both 28–8 and SP263 were likely to miss some patients who tested positive for 22C3. Therefore, in this study, PD-L1-22C3 was used to detect the wax block samples of NSCLC malignant pleural effusion cells, which has unique advantages, and its expression results are relatively reliable.

PD-L1 expression is detected across various tumor types, including NSCLC, breast, gastric, liver, kidney cancer, multiple myeloma, glioma, and melanoma, while PD-1 is primarily expressed in the lymphocytes of tumor patients. Zheng et al.^[17] observed that PD-L1 was significantly upregulated in gastric cancer tissues compared to adjacent non-cancerous tissues, with its levels correlating with tumor size and differentiation, indicating a poorer prognosis. Lin et al.^[18] discovered that positive expressions of PD-1 and PD-L1 in lung cancer tissues were associated with tumor size, lymph node metastasis, and histopathological grade. In our study involving 149 NSCLC patients with malignant pleural fluid, we found that PD-L1-positive expression was detected in <1% of 17 patients, 1–49% in 29 patients, and 50% or more in 17 patients, yielding an overall positivity rate of 42.3%. The distribution of PD-L1-positive expression was correlated with tumor size and the occurrence of metastases to other organs (P < 0.05). In addition, PD-L1 expression showed correlations with tumor differentiation, lymph node metastasis, and metastases to other organs (P < 0.05). These findings suggest that PD-L1 contributes to the progression and development of NSCLC, with its expression linked to tumor advancement and unfavorable outcomes. Hence, PDL1 emerges as a critical molecular biomarker for assessing malignancy progression and prognostication in NSCLC. In our study involving 149 patients with malignant pleural effusion in NSCLC, the positive rate of PD-L1 expression was 42.3%. This aligns with existing literature reporting PD-L1-positive rates in NSCLC ranging from 39.9% to 53.1%.^[19,20] Yang et al^[21] demonstrated a correlation between PD-L1 expression and lymph node metastasis and TNM Classification of Malignant Tumors staging, consistent with our findings analyzing the relationship between PD-L1 expression and clinicopathological features. However, contrasting results from Chen et al.^[22] suggested no association between PD-L1 expression and gender, smoking status, lymph node metastasis, histological type, or differentiation. Therefore, our results warrant further validation through large-scale studies.

With the advancements in targeted therapy for NSCLC, molecular targeted treatments focusing on driver genes such as ALK and EGFR have reached clinical maturity. ALK is expressed not only in lung cancer but also in breast, cervical, and various other tumors.^[23] Wang et al.^[24] reported a positive expression rate of echinoderm microtubule-associated protein-like 4-ALK fusion protein of approximately 6.7% in lung adenocarcinoma, with a significant proportion of patients being under 60 years old. EGFR mutations are prevalent in female non-smoking NSCLC adenocarcinomas, and studies by Harrison et al^[25] revealed higher mutation rates of the EGFR gene in Asian populations compared to white and black populations. For cases exhibiting ALK fusion protein positivity alongside EGFR gene mutations, pertinent studies have demonstrated clinical benefits with treatment involving EGFR-tyrosine kinase inhibitor and crizotinib.^[26] In our investigation, we explored the correlation between PD-L1 expression and ALK expression as well as EGFR mutation. We found a positive correlation between PD-L1 and ALK expression (P < 0.05), while no significant correlation was observed between PD-L1 and EGFR mutation (P > 0.05). Understanding the interplay among genes can elucidate the mechanisms of drug resistance encountered during targeted therapy in clinical tumors. Furthermore, it can furnish a theoretical foundation for selecting targeted therapy drugs, determining their combinations, and implementing personalized treatment strategies. At present, multi-target synergistic therapeutic regimens and efficacy analyses for lung cancer patients are undergoing clinical studies for validation.

In recent years, immunotherapy has heralded a revolutionary breakthrough in cancer treatment. At present, a variety of PD-1/PD-L1 inhibitors, including nivolumab, atezolizumab, pembrolizumab, and durvalumab, among others, have emerged, with some obtaining approval for first-line treatment in NSCLC. The previous studies have demonstrated significant benefits for patients with high PD-L1 expression on tumor cells in terms of median OS, 1-year survival rate, and objective response rate following anti-PD-1/PD-L1 immunotherapy. The assessment of PD-L1 expression levels through IHC stands as the primary method to determine the suitability of NSCLC for immune checkpoint inhibitor treatment and the potential benefits derived from such therapy.^[27] This underscores PDL1 as a pivotal target for immunotherapy in NSCLC. Trials such as CheckMate-227 and CheckMate-9LA, evaluating dual immunotherapy, have showcased a 6-year OS rate of 22% in patients with stage IV or recurrent NSCLC and PD-L1 ≥1% who received first-line dual immunotherapy. Notably, the complete response rate or partial response rate of 15% of patients exceeds 80%, while the 6-year OS rate can soar to 59%. Even patients with PD-L1 <1% exhibit improved OS benefits compared to traditional chemotherapy.^[28] These results underscore the significance of different PD-L1 expression levels in influencing the efficacy of immunotherapy. In our study involving 149 NSCLC patients with malignant pleural effusion, the positive expression rate of PD-L1 was <1% in 17 cases, 1–49% in 29 cases, and more than 50% in 17 cases, yielding a total positive rate of 42.3%. Importantly, there was a significant difference in OS between the PD-L1 protein positive expression group and the PD-L1 protein negative expression group (P < 0.05). IHC assessment of PD-L1 antibody in malignant pleural effusion of advanced NSCLC patients offers a theoretical foundation for guiding clinical tumor immunotherapy.

The existing body of research underscores the pivotal role of PD-L1 in the development and progression of NSCLC, with its expression being associated with advanced disease stages, adverse prognoses, and impacts on patient OS. IHC analysis of PD-L1 in pleural fluid wax blocks presents as a straightforward, rapid, minimally invasive, and consistent method for evaluating patients with advanced NSCLC and malignant pleural fluid. Moreover, PD-L1 expression in patients with intermediate to advanced NSCLC and malignant pleural fluid holds substantial clinical significance for refining treatment strategies and monitoring prognosis.

It is important to note that this study is limited by its single-center design and the relatively small sample size of collected cases. Consequently, certain findings, such as the positive correlation between ALK expression and PD-L1 expression, may lack robustness due to the limited number of cases. Moving forward, we aim to conduct large-scale, high-quality studies to detect PD-L1, ALK, and delve further into the tumor microenvironment and related pathways. Such endeavors are anticipated to unveil the intricate interaction between gene expression and the tumor immune microenvironment, ultimately furnishing more effective treatment strategies for NSCLC patients and addressing their survival needs comprehensively.

SUMMARY

In our study, we found that the positive rate of PD-L1 expression (42.3%) was closely associated with tumor differentiation, lymph node metastasis, and distant organ metastasis. These factors significantly influence NSCLC invasion, metastasis, and OS. IHC evaluation of PD-L1 in malignant pleural effusion provides a theoretical basis for the application of immunotherapy in metastatic NSCLC patients with pleural effusion and aids in predicting patient prognosis. In the future, large-sample studies are expected to unravel the intricate interaction between these gene expressions and the tumor immune microenvironment, thereby providing more effective treatment strategies for NSCLC patients. The IHC assessment of PD-L1 in malignant pleural effusions provides a valuable foundation for implementing immunotherapy in the treatment of metastatic NSCLC patients with pleural effusion. This diagnostic approach is minimally invasive, straightforward, and rapid, making it particularly significant for individuals whose primary symptom is malignant pleural effusion.

AVAILABILITY OF DATA AND MATERIALS

Source data and reagents are available from the first author on reasonable request.