Liquid-based cytology specimens for next-generation sequencing in lung adenocarcinoma: challenges and evaluation of targeted therapy.

BACKGROUND: To explore challenges of liquid-based cytology (LBC) specimens for next-generation sequencing (NGS) in lung adenocarcinoma and evaluate the efficacy of targeted therapy. METHODS: A retrospective analysis was conducted on the NGS test of 357 cases of advanced lung adenocarcinoma LBC specimens and compared with results of histological specimens to assess the consistency. The impact of tumor cellularity on NGS test results was evaluated. The utility of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) was collected. Clinical efficacy evaluation was performed and survival curve analysis was conducted using the Kaplan-Meier method. RESULTS: There were 275 TKI-naive and 82 TKI-treated specimens, the mutation rates of cancer-related genes detected in both groups were similar (86.2% vs. 86.6%). The EGFR mutation rate in the TKI treated group was higher than that in the TKI-naive group (69.5% > 54.9%, P = 0.019). There was no significant difference in the EGFR mutation frequency among different tumor cellularity in the TKI-naive group. However, in the TKI treated group, the frequency of EGFR sensitizing mutation and T790M resistance mutation in specimens with < 20% tumor cellularity was significantly lower than that in specimens with ≥ 20% tumor cellularity. Among 22 cases with matched histological specimens, 72.7% (16/22) of LBC specimens were completely consistent with results of histological specimens. Among 92 patients with EGFR-mutant lung adenocarcinoma treated with EGFR-TKIs in the two cohorts, 88 cases experienced progression, and the median progression-free survival (PFS) was 12.1 months. CONCLUSIONS: Cytological specimens are important sources for gene detection of advanced lung adenocarcinoma. When using LBC specimens for molecular testing, it is recommended to fully evaluate the tumor cellularity of the specimens.

Consistency Analysis of Programmed Death Ligand 1 Expression in Non-Small Cell Lung Cancer Between Pleural Effusion and Matched Primary Lung Cancer Tissues by Immunohistochemical Double Staining.

In clinical practice, programmed death ligand 1 (PD-L1) detection is prone to nonspecific staining due to the complex cellular composition of pleural effusion smears. In this study, diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) immunohistochemistry double staining was performed to investigate PD-L1 expression in tumor cells from malignant pleural effusion (MPE). MPE was considered as a metastasis in non-small cell lung cancer patients; thus, the heterogeneity between metastatic and primary lung cancer was revealed as well. Ninety paired specimens of MPE cell blocks and matched primary lung cancer tissues from non-small cell lung cancer patients were subjected to PD-L1 and thyroid transcription factor-1(TTF-1)/p63 immunohistochemistry double staining. Two experienced pathologists independently evaluated PD-L1 expression using 3 cutoffs (1%, 10%, and 50%). PD-L1 expression in MPE was strongly correlated with that in matched primary lung cancer tissues (R = 0.813; P < .001). Using a 4-tier scale (cutoffs: 1%, 10%, and 50%), the concordance was 71.1% (Cohen's κ = .534). Using a 2-tier scale, the concordance was 75.6% (1%, Cohen's κ = 0.53), 78.9% (10%, Cohen's κ = 0.574), and 95.6% (50%, Cohen's κ = 0.754). The rates of PD-L1 positivity in MPE (56.7%) were higher than that in lung tissues (32.2%). All 27 discordant cases had higher scores in MPE. The double-staining method provided superior identification of PD-L1-positive tumor cells on a background with nonspecific staining. In conclusion, PD-L1 expression was moderately concordant between metastatic MPE cell blocks and matched primary lung carcinoma tissues, with variability related to tumor heterogeneity. MPE should be considered to detect PD-L1 when histological specimens are unattainable, especially when PD-L1 expression is >50%. PD-L1 positivity rates were higher in MPE. Double staining can improve PD-L1 detection by reducing false-negative/positive results.

The detection of PD-L1 expression on liquid-based cytology in pleural effusion of lung adenocarcinoma and its prognostic evaluation: Between paired liquid-based cytology and cell block samples.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression levels measured by immunohistochemistry have been proven to predict the outcome of immunotherapy in lung adenocarcinoma (LUAD). However, data on PD-L1 expression on liquid-based cytology (LBC) in malignant pleural effusion (MPE) is scarce. METHODS: This study cohort included 60 cases with MPE suffering from LUAD. PD-L1 SP263 assay was used for immunocytochemistry (ICC) on LBC and matched cell block (CB) to validate ICC protocols on LBC slides. Clinical outcomes were analyzed based on immunotherapy and PD-L1 tumor proportion scores (TPS) on LBC slides and CBs. RESULTS: PD-L1 expression with TPS ≥1% was lower in LBCs than in CBs (33 of 60 [55.0%] vs. 35 of 60 [58.3%]; p = .687). Even with the TPS ≥50% threshold, PD-L1 expression was lower in LBCs (10 of 60 [16.7%] vs. 15 of 60 [25%]; p = .125). Epidermal growth factor receptor (EGFR) exon 20 mutation, tumor cell proportion, and pleural fluid neutrophil-to-lymphocyte ratio were related to PD-L1 expression on CBs (p = .013, p = 0.022, and p = .011), respectively. Patients with subsequent immune checkpoint inhibitor therapy remained a better prognostic in subgroups of PD-L1 positive expression on LBC slides (TPS ≥1%, p = .041). CONCLUSIONS: LBC specimens had comparable performance to CBs in PD-L1 assessment and predicting treatment response to PD-L1-defined therapy.

Immunocytochemical detection by a fully automated immunostainer for assisting subclassification of pulmonary tumors on ThinPrep slides in real-life clinical samples: A single-institution experience.

INTRODUCTION: This study aims to investigate the feasibility and reliability of ThinPrep slides in detecting the subclassification of lung cancer and develop a process for immunocytochemistry (ICC) with optimized staining steps of an automated immunostainer. METHODS: Cytomorphology and ancillary ICC by automated immunostainer on ThinPrep slides were performed to subclassify 271 cytology cases of pulmonary tumor, which were stained with 2 or more of the following antibodies: p40, p63, thyroid transcription factor-1 (TTF-1), Napsin A, synaptophysin (Syn), and CD56. RESULTS: The accuracy of cytological subtyping was improved from 67.2% to 92.7% (p < .0001) after ICC. The accuracy of cytomorphology combined with ICC results for lung squamous-cell carcinoma (LUSC), lung adenocarcinomas (LUAD), and small cell carcinoma (SCLC) was 89.5% (51 of 57), 97.8% (90 of 92), and 98.8% (85 of 86), respectively. The sensitivity and specificity of 6 antibodies were as follows: p63 (91.2%, 90.4%) and p40 (84.2%, 95.1%) for LUSC, TTF-1(95.6%, 64.6%) and Napsin A (89.7%, 96.7%) for LUAD and Syn (90.7%, 60.0%) and CD56 (97.7%, 50.0%) for SCLC, respectively. P40 expression on ThinPrep slides had the highest agreement (κ = 0.881) with immunohistochemistry (IHC) results, followed by p63 (κ = 0.873), Napsin A (κ = 0.795), TTF-1 (κ = 0.713), CD56 (κ = 0.576), and Syn (κ = 0.491). CONCLUSION: The result of ancillary ICC on ThinPrep slides by fully automated immunostainer was in good agreement with the gold standard in pulmonary tumors subtype and immunoreactivity, objectively achieving accurate subtyping in cytology.

[Correlation between the Expression of PD-L1 in Pleural Effusion of Lung Adenocarcinoma and the Clinicopathological Features and Molecular Changes].

BACKGROUND: Pembrolizumab, an immunotherapy for advanced non-small cell lung cancer (NSCLC), needs to predict treatment response based on test results including immunohistochemistry (IHC), which detects the expression of programmed death ligand 1 (PD-L1). The aim of this study was to evaluate the feasibility of immunocytochemistry (ICC) in NSCLC cytology to detect PD-L1 and to investigate the correlation between PD-L1 expression and clinical pathology and molecular features. METHODS: Sixty cases of lung adenocarcinoma pleural cytology were collected and PD-L1 sp263 reagent was used for immunocytochemical staining according to the manufacturer's instructions. Next-generation sequencing (NGS) was performed on pleural cytology specimens to explore its correlation. RESULTS: Of the 60 cases of lung adenocarcinoma pleural effusion cell block, 35 cases were positive for PD-L1 expression, and the positive expression rate was 58.3%. The positive rate of PD-L1 expression in the specimens of our hospital was 33.3%, and there was no significant difference between the cytological specimens and the histological specimens (P>0.05). Of the 60 cytological specimens, 26 were tested for NGS, and 15 (57.7%) were found to have epidermal growth factor receptor (EGFR) mutations. No correlation was found between PD-L1 expression and EGFR mutation. The positive expression rate of PD-L1 were not correlated with the age and gender in the study population (P>0.05). CONCLUSIONS: When no surgical specimens are available, pleural cytology cell block specimens can be used for immunocytochemical detection of PD-L1, and the results are feasible.