Assessment of Programmed Cell Death Ligand-1 Expression in Non-small Cell Lung Carcinomas by Immunohistochemistry

Abstract

Background:

Non-small cell lung carcinomas (NSCLCs) expressing programmed cell death ligand-1 (PD-L1), a transmembrane protein, are more likely to respond to immunotherapy drugs. All advanced NSCLC samples should be examined with PD-L1 immunohistochemistry (IHC). PD-L1 is now the only approved predictive biomarker of immunotherapy response in NSCLC patients. This study evaluates the expression of PD-L1 in NSCLC by IHC and assesses the morphological and clinical correlation of PD-L1 expression.

Materials and Methods:

All patients with histologically proven NSCLC with needle core biopsies and resected specimens were analyzed in the department of pathology. The correlation of PD-L1 expression with morphological and clinical parameters was analyzed.

Results:

On evaluating PD-L1 expression in 97 cases of NSCLC, 61.85% (n = 60) had positive PD-L1 staining. Among patients with a positive tumor proportion score, 22 had a history of smoking, 51 were ≥50 years old, 43 were male patients, 24 were in Stage IV, 44 had a history of chemotherapy or radiotherapy, 47 had adenocarcinoma, and 33 had Grade II tumors. The association of PD-L1 expression with various clinicopathological parameters such as gender, smoking status, histological type, histological grade, clinical tumor stage, previous treatment history, site and type of biopsy, necrosis, and peritumoral lymphoid response was not statistically significant.

Conclusion:

Our study indicated that PD-L1 expression was not associated with various clinicopathological variables in NSCLC. More research is needed to look into the probable factors that influence the PD-L1-positive distribution. Since expression of the PD-L1 protein is proven to be of therapeutic and prognostic significance, we recommend that testing for PD-L1 should be done at least in cases of advanced-stage NSCLC.

Introduction

Non-small cell lung carcinomas (NSCLCs) account for approximately 80%–85% of lung malignancies. The most common types of NSCLC are adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, but there are several other types that occur less frequently.^[1],[2] Surgery, chemotherapy (CT), radiotherapy (RT), and targeted therapy are among the treatment options for NSCLC, which are determined mostly by the cancer's stage.^[3] Immunotherapy is often used in conjunction with CT and, in rare cases, as first-line monotherapy. Tumor is tested for the programmed cell death ligand-1 (PD-L1) protein, and those tumors expressing PD-L1 are more likely to respond to corresponding immunotherapy drugs.^[4] The transmembrane protein PD-L1, also known as CD274/B7-H1, binds to its receptors programmed death-1 (PD-1) and B7-1. Binding of PD-L1 to its receptor, PD-1, activates downstream signaling in T cells, thus inhibiting its proliferation and cytotoxicity. Inhibition of PD-1 or PD-L1 will enhance T-cell response to cancer, which is the basis of immunotherapy. The Food and Drug Administration has approved several antibody-blocking PD-L1/PD-1 for cancer immunotherapy, which includes anti-PD-1 (pembrolizumab, nivolumab, and cemiplimab) and anti-PD-L1 agents (atezolizumab, avelumab, and durvalumab).^[5],[6] At present, pembrolizumab, nivolumab, durvalumab, and atezolizumab are approved for treating NSCLC.^[6],[7] All advanced NSCLC samples should be examined with PD-L1 immunohistochemistry (IHC) in a reflex manner, according to the guidelines of the National Comprehensive Cancer Network.^[3],[4] PD-L1 is now the only approved predictive biomarker of immunotherapy response in NSCLC patients. Four commercial antibodies, such as 22C3, 28-8, SP263, and SP142, are available to measure PD-L1 protein expression in formalin-fixed, paraffin-embedded (FFPE) lung tissue specimens.^{[8],[9],[10],[11]} This study evaluates the expression rate of PD-L1 in NSCLC with clinicopathological correlation in the Indian population.

Materials and Methods

Patients

This was a prospective observational study conducted at a quaternary care center. All patients with histologically proven non-small cell lung carcinomas (NSCLC) from biopsy and resected specimens were analyzed in the department of pathology. Specimens from primary and metastatic sites and those with at least 100 viable tumor cells that meet the adequacy criteria were included. The study duration was between November 2020 and March 2022.

Study methodology

The clinical details were obtained from the histopathology requisition forms and electronic hospital records. The specimens were fixed in 10% neutral-buffered formalin for at least 6 h for small biopsies and overnight fixation for large resection specimens and grossed accordingly. The sections were treated using standard techniques; 3–5 μ-thick sections were cut and stained with Harris Hematoxylin and Eosin stain. The resected specimens were reported according to the College of American Pathologists protocol. IHC was performed in some cases as deemed necessary. PD-L1 testing was done in cases in which it had been requested by the clinician. Routinely processed FFPE tissues were used for the VENTANA BenchMark ULTRA automatic slide stainer and the VENTANA OptiView Diaminobenzidine IHC Detection Kit. The VENTANA PD-L1 (SP263) antibody is a recombinant rabbit monoclonal antibody that is isolated from cell culture supernatant. This antibody produces membrane and/or cytoplasmic staining. In NSCLC, tumors were evaluated for tumor proportion score (TPS) using the SP263 clone, and this score was calculated by the number of PD-L1-positive tumor cells, divided by all tumor cells, and then multiplied by 100. Basically, it is the percentage of PD-L1-positive tumor cells, and only viable tumor cells are scored.^[12] ≥1% of tumor cells with membrane positivity at any intensity greater than background staining were considered positive. Isolated cytoplasmic tumor cell staining was ignored for scoring. Placental tissue was used as a control.

The study has been approved by the institutional review committee with IEC Application Number: ASH-DNB-034/11–20.

Statistical analysis

Data analysis was carried out by IBM SPSS for Windows Version 25.0 (Armonk, NY, USA: IBM Corp). All the continuous variables were represented as mean ± standard deviation. Nonnormally distributed variables were represented as medians. Categorical variables were represented as percentages. Chi-square test was used to compare categorical variables. All P < 0.05 were regarded as statistically significant.

Results

This study evaluated PD-L1 expression in 97 cases of NSCLC in both primary lung and metastatic sites. The prevalence of PD-L1 expression and its correlation to clinicopathological characteristics were investigated. Ninety-five cases were primary lung biopsies, and 2 cases were metastatic lymph node biopsies. Among the 97 cases, 86 were core needle biopsy specimens and 11 were resection specimens. Of the 97 cases received during the study period, 61.85% had positive PD-L1 staining and 38.15% had negative PD-L1 staining. Among cases with positive PD-L1 staining, 65% (n = 39) had TPS between 1% and 49% and 35% (n = 21) had TPS of ≥50%. The general age range was 28–94 years, with a mean age of 61.82 years. There were 73 male patients and 24 female patients. Among patients with a positive TPS, 22 had a history of smoking, 27 had never smoked, and in the remaining 11 cases, details on smoking status were not available. It was observed that among cases with positive PD-L1 staining, most of the cases were in Stage IV, followed by Stage II. Among cases who had received previous chemo or RT, 16 had positive and 4 had negative PD-L1 staining. From primary lung biopsies, 58 had positive and 37 had negative PD-L1 staining. Both cases with lymph node biopsies had positive PD-L1 staining. 47 cases (62.67%) of adenocarcinoma, 9 cases (52.94%) of squamous cell carcinoma, and 4 cases (80%) of adenosquamous carcinoma showed positive PD-L1 staining. Among cases of adenocarcinoma with acinar, lepidic, papillary, and solid patterns, 55%, 100%, 62.5%, and 78.26% had positive PD-L1 staining, respectively. There was no evidence of a statistically significant association between TPS score and various clinicopathological parameters such as age groups, gender, smoking status, clinical tumor stage, chemo or RT treatment status, site and type of biopsy, histologic tumor type of NSCLC, patterns of adenocarcinoma, histological tumor grade, presence or absence of necrosis, and peritumoral inflammatory response. Various clinicopathological parameters of NSCLC in association with negative and positive PD-L1 staining in each parameter are summarized in [Table 1]. Various patterns of PD-L1 expression and nonspecific staining patterns are shown in [Figure 1] and [Figure 2], respectively.

Table 1

Various clinicopathological parameters of non-small cell lung carcinomas in association with negative and positive programmed cell death ligand-1 staining in each parameter

Figure 1

SP263 assay staining showing various patterns of programmed cell death ligand-1 expression. (a) Partial membrane pattern of staining. (b) Complete membrane pattern of staining. (c) Partial membrane and cytoplasmic pattern of staining. (d) Complete membrane cytoplasmic pattern of staining (a-d: ×400)

Figure 2

SP263 assay staining showing various nonspecific staining patterns. (a) Negative programmed cell death ligand-1 staining in tumor cells with nonspecific punctate staining in immune cells. (b) Tumor with nonspecific cytoplasmic staining of programmed cell death ligand-1 (a and b: ×400)

Discussion

PD-L1-positive NSCLC represents an important subgroup that benefits from immune checkpoint inhibitors (ICIs). Based on the percentage of expression of PD-L1, the ICIs are added on as either first-line or second-line therapeutic agents. The most well-studied indicators, such as PD-L1 expression, tumor mutation burden, and microsatellite instability, have all emerged as prognostic biomarkers for NSCLC therapy decisions.^[13],[14] Clinical studies have shown that increased PD-L1 expression correlates with better treatment outcomes in NSCLC and that ICIs that target PD-1/PD-L1 receptors have improved NSCLC prognosis.^{[11],[15],[16]} PD-L1 testing is recommended by the National Cancer Comprehensive Network for patients with advanced NSCLC.^[17] The IHC assay is one of the most cost-effective and quick ways to screen for and detect PD-L1 expression. Only limited studies are available in India providing details on the prevalence of PD-L1-positive NSCLC, and this study adds some beneficial information.

There is great diversity in the expression of PD-L1 in NSCLC due to the use of varied scoring methods and cutoff levels. This study used TPS, which is used in the current practice. Variable levels of PD-L1 expression have been reported in clinical series ranging from 7.4% to 72.7%.^[11] In the various clinical trials analyzed by Yu et al., the prevalence of PD-L1 expression in patients with NSCLC ranged from 24% to 60%.^[18] Domadia et al. reported 47% PD-L1 expression in Indian patients with NSCLC (n = 134) using the clone SP263.^[19] In our study, the percentage of cases with positive PD-L1 expression was 61.85, and the prevalence was higher compared to other studies in India. The use of different antibody clones may explain the differences in the expression of PD-L1 in NSCLC patients in different studies.

The majority of the patients with positive PD-L1 expression were in the age group of ≥50, which is similar to the other studies in the literature.^{[20],[21],[22]} In a comprehensive literature search by Pan et al., it was found that there was no difference in PD-L1 expression with gender status (P = 0.39), similar to our study (P = 0.448).^[23] The percentage of cases with positive PD-L1 staining among smokers was 37.7% and 33.10% in studies by Kumar et al. and Pawelczyk et al., respectively.^[4],[20] In our study, the percentage of cases with a positive PD-L1 score among smokers (62.85%) was higher compared to these studies. Pawelczyk et al. reported that among PD-L1-positive cases, 32.26% were in clinical Stage I, 36.17% were in Stage II, and 31.56% were in Stages III and IV.^[4] In the present study, 8.33% were in clinical Stage I, 33.3% were in Stage II, and 58.33% were in Stages III and IV. In both of these studies, PD-L1 expression was increased in the advanced stages of NSCLC, which is comparable. This study reveals that the majority of the cases with positive PD-L1 staining were post-CT or RT. However, in a study by Lee et al.,^[22] most of the patients with preoperative CT or RT had negative PD-L1 staining. CT or targeted therapy has been reported to induce PD-L1 expression. It is recommended to collect fresh samples and evaluate PD-L1 expression after other modes of therapy and before the administration of immunotherapy.

In contrast to the present study, there was a statistically significant difference associated with the type of sample in the study by Vigliar et al., and cytological samples were frequently negative for PD-L1 expression.^[21] In our study, there were no cytology samples. In both studies, the rate of negative staining was higher in resection samples than in biopsy samples. In this study, there was no significant association between different histological types and PD-L1 expression (P = 0.479), and similar results were observed in a study by Vigliar et al.^[21] In these two studies, adenocarcinomas had higher expression of PD-L1. In this study, we looked at the degree of PD-L1 expression in adenocarcinomas with different patterns. Expression of PD-L1 was observed more in a solid pattern, which was around 78.2% (18/23 cases), which is similar to what was observed in the study by Lee et al.^[22] This study observed that associations with positive PD-L1 expression were predominantly in Grade II tumors. Similar findings were reported by Kumar et al., Pawelczyk et al., and Lee et al.^{[4],[20],[22]}

Most of the cases with positive PD-L1 staining had a moderate lymphoid response. Many studies have found a positive association between increased TILs and PD-L1 expression in tumor cells.^{[14],[24],[25]} Since PD-L1 expression can display intratumoral or intertumoral heterogeneity, it is crucial to understand how the sampling method and sites affect PD-L1 expression. PD-L1 testing is hampered by the heterogeneity of PD-L1 expression.

This study has certain limitations, such as a limited sample size, no correlation with clinical response, and no follow-up. This precludes us from reaching far-fetching conclusions.

Conclusion

Our study indicated that PD-L1 expression did not correlate with any unique clinic pathological variables in NSCLC. In the literature, studies on the correlation between PD-L1 protein expression and clinic pathological parameters frequently yield inconsistent results. More research is needed to look into the probable factors that influence the PD-L1-positive distribution and multicentric studies can be done on the Indian population to arrive at a better conclusion. Updated recommendations are necessary to achieve improved standardization of PD-L1 testing in clinical practice. We suggest that testing for PD-L1 can be done at least in instances of advanced-stage NSCLC, as PD-L1 expression has been found to be of therapeutic and prognostic significance.