Abstract
Background:
Immunotherapy currently stands as a novel treatment option, specifically in cases of advanced non-small cell lung carcinoma (NSCLC). Expression of programmed death ligand-1 (PD-L1) in tumor cells forms the mainstay for the use of anti-PD-L1 monoclonal antibodies in the treatment of NSCLC.
Aims:
The objectives of the study were to assess utility of cell blocks for testing of PD-L1 in adenocarcinoma lung and to compare the expression of PD-L1 in cell blocks and the corresponding biopsy specimens.
Materials and Methods:
The current study was a prospective case series that included 20 cases of NSCLC–adenocarcinoma lung. Cases included in the study had biopsies performed from lung masses, along with which cell blocks were prepared from fine needle aspiration cytology (FNAC) samples. Testing for PD-L1 was done using the monoclonal PD-L1 antibody, SP-263 clone on the Ventana Benchmark XT system. PD-L1 expression was assessed only in the tumor cells, and cases with >1% expression, cytoplasmic or membranous, in tumor cells were categorized as positive.
Results:
PD-L1 expression was identified in the biopsy samples of tumor cells of 20% of cases (n = 4/20). In the corresponding cell blocks, PD-L1 expression was identified in the tumor cells of 15% of cases (n = 3/20). Sensitivity and specificity of cell blocks were 75% and 100%, respectively. Positive and negative predictive values were 100% and 94.12%, respectively.
Conclusion:
PD-L1 testing has both predictive and prognostic implications. PD-L1 testing in cell block samples is a potential alternative, specifically in cases where biopsy tissue is minimal or unavailable.
Keywords: Adenocarcinoma lung, cell blocks, immunotherapy, PD-L1 testing
INTRODUCTION
The approach to the treatment and molecular diagnostics of non-small cell lung carcinoma (NSCLC)–adenocarcinoma phenotype has progressed significantly in the last decade.[1,2] Immunotherapy currently stands as a novel treatment option specifically in cases of advanced, particularly the NSCLC–adenocarcinoma phenotype.[2,3] The expression of programmed death ligand-1 (PD-L1) in the tumor cells forms the mainstay for the use of anti-PD-L1 monoclonal antibodies in the treatment of NSCLC. The selection of cases for specific anti-PD-L1 monoclonal antibodies is largely dependent on the expression of PD-L1 in the neoplastic/tumor cells.[1-3] The outstanding result of therapies with medications that inhibit the interaction of PD-L1 with programmed death protein 1 (PD1) has revolutionized prognostic treatment regimes. PD-L1 testing is performed by immunohistochemistry (IHC) in histology samples.[4] In cases of advanced and metastatic disease, wherein cytology forms the mainstay in diagnosis, the testing for PD-L1 is being done in cytologic material. The validation of the expression of PD-L1 in cytologic specimens is currently under evaluation.[5,6] Among the few studies that have been published in literature, the utilization of cytologic material for PD-L1 testing revealed encouraging results; however, the temporal and spatial heterogeneity of specimens needs to be addressed and further validated.[7]
The current study was undertaken with the objectives to assess the utility of cell blocks for testing of PD-L1 in adenocarcinoma lung and to compare the expression of PD-L1 in cell blocks and the corresponding biopsy specimens.
MATERIALS AND METHODS
The current study was a prospective case series that included 20 cases of NSCLC–adenocarcinoma lung. The study protocol was approved by the institutional ethical committee. The cases included in the study had biopsies performed from intrathoracic lung masses, along with which cell blocks were prepared from fine needle aspiration cytology (FNAC) samples under computed tomography (CT) or ultrasonography (USG) guidance.
Preparation of cell blocks
Cell blocks were prepared by expressing the contents of aspirated specimen onto a glass slide, which allowed the specimen to dry/clot. The material was scraped off the slides and wrapped in tissue paper. The specimen was placed in a histology cassette, and neutral buffered formalin was added. This was followed by processing in the automatic tissue processor.
Histopathologic diagnosis with the aid of IHC
In the current study, only those cases were included that had diagnostic biopsies performed from intrathoracic lung masses, and cell blocks were prepared from FNAC samples. Written informed consent was taken from all the cases included in the current study. The biopsies were evaluated, and basic panel of IHC, including napsin-A, thyroid transcription factor-1 (TTF-1), and markers for squamous differentiation, namely, p-40, was used.
Testing for PD-L1
Only those cases in which the histologic diagnosis was confirmed as NSCLC–adenocarcinoma and adequate material was present in both the biopsy and cell block samples (a minimum of 50 well-preserved tumor cells) were taken up for PD-L1 testing. The testing for PD-L1 was done using the monoclonal PD-L1 antibody, SP-263 clone from Ventana Medical Systems, and the Ventana Benchmark XT system using the OptiView DAB detection kit. A positive control, namely, human placental tissue, and a negative control (by omitting the primary antibody) were run with every batch.
Assessment of PD-L1
In the present study, PD-L1 expression was assessed only in the tumor cells, and cases with >1% expression membranous expression for PD-L1 in tumor cells were categorized as PD-L1 positive.
Statistical analysis
Statistical analysis was done using the Statistical Package for the Social Sciences (SPSS) software version 16. The frequency of PD-L1 expression was assessed in tumor cells in both histologic and the corresponding cytology (cell block) samples. Sensitivity, specificity, positive predictive value, and negative predictive value of cell blocks versus biopsies were calculated.
RESULTS
Clinicopathological features
The current study included 20 cases of NSCLC–adenocarcinoma phenotype in which testing for PD-L1 was done on both cell blocks and the corresponding biopsy samples [Figure 1a and b]. The study included 20% (n = 4) females and 80% (n = 16) males with an M: F ratio of 4:1. The age range of the patients varied from 27 to 78 years, with a mean age of 54.6 years. Majority of the cases had multiple clinical symptoms. The clinical symptoms included breathlessness in 80% cases (n = 16/20), cough in 75% cases (n = 15/20), hemoptysis in 65% cases (n = 13/20), and weight loss in 55% cases (n = 11/20). Among the 20 cases, 45% cases (n = 9/20) were nonsmokers and 55% cases (n = 11/20) were smokers [Table 1].
Figure 1.
(a) Cell block prepared from FNAC of intrathoracic mass. (b) Corresponding biopsy from the intrathoracic mass composed of tumor cells arranged in acini. The tumor cells have high nucleo-cytoplasmic ratio with moderate amount of amphophilic cytoplasm and vesicular nuclei. (c) PD-L1 expression in tumor cells in the cell block prepared from FNAC of intrathoracic mass. (d) Corresponding biopsy from the intrathoracic mass with PD-L1 expression in tumor cells. (a and b = H and E, ×200; c and d = 3,3′Diaminobenzidine, ×200). FNAC = fine needle aspiration cytology, H and E = hematoxylin and eosin, PD-L1 = programmed death ligand-1
Table 1.
The clinicopathological characteristics of the study group
| Clinicopathological features | Parameters | Number/percentage |
|---|---|---|
| Gender | Male | 16 (80%) |
| Female | 4 (20%) | |
| Age range | Up to 30 years | 1 (5%) |
| 31–40 years | 3 (15%) | |
| 41–50 years | 4 (20%) | |
| 51–60 years | 8 (40%) | |
| 61–70 years | 2 (10%) | |
| >70 years | 2 (10%) | |
| Smoking history | Present | 11 (55%) |
| Absent | 9 (45%) | |
| Clinical features | Breathlessness | 16 (80%) |
| Cough | 15 (75%) | |
| Hemoptysis | 13 (65%) | |
| Weight loss | 11 (55%) |
PD-L1 expression
PD-L1 expression was identified in the biopsy samples in 20% cases (n = 4/20) in the tumour cells. In the corresponding cell blocks PD-L1 expression was identified in 15% cases in the tumour cells (n = 3/20).[Figure 1 a-d].
Comparison of cell block versus biopsy for PD-L1 testing
The sensitivity and specificity of cell blocks versus biopsies for identification of PD-L1 expression was 75% and 100% respectively. The positive and negative predictive values were 100% and 94.12% respectively.
DISCUSSION
The introduction of monoclonal antibodies targeting immune checkpoints and the use of PD-L1 inhibitors have drastically changed the treatment landscape of NSCLC. This mandates the testing for these monoclonal antibodies, in particular, PD-L1. PD-L1 testing is routinely performed on histology samples. However, in cases of advanced metastatic disease or cases of recurrent disease or in patients with comorbidities, performing a biopsy is, at times, not feasible. NSCLC is frequently diagnosed in cytology samples. The testing of PD-L1 in cytology samples may prove to be a rapid and cost-effective tool.[1,5,7]
PD-L1 immunocytochemistry that is performed on cytological smears is challenging to standardize. The major reason is that cytological smears are usually prepared in alcohol-based fixatives and the cells in cytological smears are more dispersed. Therefore, the interpretation of PD-L1 immunostaining results is difficult and subjective and cell blocks that are fixed and processed using formalin as a fixative are easier to interpret.[5-7]
In the current study, PD-L1 expression was identified in the histology samples of 20% of cases, while 15% of cases in the corresponding cell block/cytology samples expressed PD-L1. The sensitivity and specificity of cell blocks versus biopsies were 75% and 100%, respectively, while the positive and negative predictive values were 100% and 94.12%, respectively.
In the study conducted by Chauhan et al.,[8] the authors performed testing for PD-L1 using the SP-263 clone in both the histology and the corresponding cytology samples of 40 cases. The authors concluded that 82.5% of cases showed concordant results for PD-L1 expression in cytology and histology samples, and stated that PD-L1 testing in cytology samples is feasible.
In the study conducted by Ambrosini-Spaltro et al.,[7] the authors concluded that PD-L1 expression among various materials did not bear any statistically significant difference. The authors inferred that PD-L1 testing can be performed in cytology samples, but the results should be interpreted with caution and repeat testing should be done in cases with inconsistent staining.
In the study conducted by Wang et al.,[9] the authors reported a slightly higher percentage expression of PD-L1 in cell block samples when compared to the histology samples. Heymann et al.[10] concluded from their study that PD-L1 expression was higher in cell block specimens when compared to biopsies or surgical resection specimens. Torous et al.[11] observed a similar distribution of PD-L1 expression between cell block and surgical pathology specimens in their study.
In the current study, there was one case with discrepant expression of PD-L1 in cell block versus biopsy. PD-L1 immunoexpression within the tumor is heterogeneous. Different intensities of staining for PD-L1 have been observed within the same tumor. This tumor heterogeneity can be attributed to the cause of discrepancy and discordance between the cytology and histology samples. The other factors like presence of necrosis, inflammatory debris, and macrophages in cytology samples may contribute to discordance between the cytology and histology samples.[12-14]
The limitations of the current study include the sample size of the current study that is relatively small to derive conclusive results. There is no follow-up/survival data of the patients, and treatment response is also not monitored, though this was not a part of the objectives of the study.
CONCLUSION
PD-L1 testing has both predictive and prognostic implications in lung carcinoma. Cytology samples serve as a rapid, cost-effective, and accurate sample for testing of molecular and immunotherapy markers. PD-L1 testing in cell block samples is a potential alternative, specifically in cases where biopsy tissue is minimal or unavailable.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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