High expression of programmed death ligand-1 (PD-L1) on tumor cells (TC) and/or on tumor-infiltrating immune cells (IC) is associated with a high response rate in patients with advanced nonsmall-cell lung cancer (NSCLC) treated with PD-L1 inhibitors. The use of a PD-L1 immunohistochemical (IHC) test in determining the responsiveness to immunotherapy has raised the question of the reliability and reproducibility of its evaluation in lung biopsies compared with corresponding resected surgical specimens. PD-L1 expression in TC and IC was assessed in 160 patients with operable NSCLC on both whole surgical tissue sections and matched lung biopsies, by using a highly sensitive SP142 IHC assay. The specimens were scored as TC 0–3 and IC 0–3 based on increasing PD-L1 expression. PD-L1 expression was frequently discordant between surgical resected and matched biopsy specimens (the overall discordance rate = 48%; 95% confidence interval 4.64–13.24) and κ value was equal to 0.218 (poor agreement). In all cases, the biopsy specimens underestimated the PD-L1 status observed on the whole tissue sample. PD-L1-positive IC tumors were more common than PD-L1-positive TC tumors on resected specimens. The discrepancies were mainly related to the lack of a PD-L1-positive IC component in matched biopsies. Our results indicate relatively poor association of the PD-L1 expression in TC and IC between lung biopsies and corresponding resected tumors. Although these results need to be further validated in larger cohorts, they indicate that the daily routine evaluation of the PD-L1 expression in diagnostic biopsies can be misleading in defining the sensitivity to treatment with PD-L1 targeted therapy.

Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer. Migration of circulating tumor cells (CTCs) into the blood stream is an early event that occurs during carcinogenesis. We aimed to examine the presence of CTCs in complement to CT-scan in COPD patients without clinically detectable lung cancer as a first step to identify a new marker for early lung cancer diagnosis. The presence of CTCs was examined by an ISET filtration-enrichment technique, for 245 subjects without cancer, including 168 (68.6%) COPD patients, and 77 subjects without COPD (31.4%), including 42 control smokers and 35 non-smoking healthy individuals. CTCs were identified by cytomorphological analysis and characterized by studying their expression of epithelial and mesenchymal markers. COPD patients were monitored annually by low-dose spiral CT. CTCs were detected in 3% of COPD patients (5 out of 168 patients). The annual surveillance of the CTC-positive COPD patients by CT-scan screening detected lung nodules 1 to 4 years after CTC detection, leading to prompt surgical resection and histopathological diagnosis of early-stage lung cancer. Follow-up of the 5 patients by CT-scan and ISET 12 month after surgery showed no tumor recurrence. CTCs detected in COPD patients had a heterogeneous expression of epithelial and mesenchymal markers, which was similar to the corresponding lung tumor phenotype. No CTCs were detected in control smoking and non-smoking healthy individuals. CTCs can be detected in patients with COPD without clinically detectable lung cancer. Monitoring “sentinel” CTC-positive COPD patients may allow early diagnosis of lung cancer.

Purpose: Pathologic TNM staging is currently the best prognostic factor for non‐small cell lung carcinoma (NSCLC). However, even in early-stage NSCLC, the recurrence rates after surgery range from 25% to 50%. The preoperative detection of circulating tumor cells (CTC) could be useful to tailor new therapeutic strategies in NSCLC. We assessed the presence of CTC in NSCLC patients undergoing surgery, using cytologic analyses, after their isolation by size of epithelial tumor cells (ISET method). The presence and the number of CTCs were considered and correlated with clinicopathologic parameters including patient follow-up. Experimental Design: Of the 247 blood samples tested, 208 samples were from patients with resectable NSCLC and 39 from healthy subjects. The mean follow-up was 24 months. An image of detected cells with presumably nonhematologic features [initially defined as “circulating nonhematologic cells” (CNHC)] was recorded. The presence of CNHC was assessed blindly and independently by 10 cytopathologists, using cytologic criteria of malignancy on stained filters. The count of detected CNHCs was made for each filter. Results: One hundred two of 208 (49%) patients showed CNHCs corresponding to CNHC with malignant cytopathologic features in 76 of 208 (36%) cases. CNHCs were not detected in the control group. A level of 50 or more CNHCs corresponding to the third quartile was associated with shorter overall and disease-free-survival, independently of disease staging, and with a high risk of recurrence and death in early-stage I + II-resectable NSCLC. Conclusion: A high percentage of NSCLC patients show preoperative detection of CNHC by the ISET method. The presence and level of 50 or more CNHCs are associated with worse survival of patients with resectable NSCLC. Clin Cancer Res; 17(3); 505–13. ©2010 AACR.

Comparison of the efficacy of different enrichment methods for detection of circulating tumor cells (CTCs) before radical surgery is lacking in non‐small‐cell lung carcinoma (NSCLC) patients. Detection and enumeration of CTCs in 210 consecutive patients undergoing radical surgery for NSCLC were evaluated with the CellSearch Assay™ (CS), using the CellSearch Epithelial Cell Kit, and by the isolation by size of epithelial tumor (ISET) method, using double immunolabeling with anti‐cytokeratin and anti‐vimentin antibodies. CTCs were detected in 144 of 210 (69%) patients using CS and/or ISET and in 104 of 210 (50%) and 82 of 210 (39%) patients using ISET and CS, respectively. Using ISET, 23 of 210 (11%) patients had vimentin‐positive cells with cytological criteria of malignancy. Disease‐free survival (DFS) was worse for patients with CTCs compared to patients without CTCs detected by CS alone (p < 0.0001; log rank = 30.59) or by ISET alone (p < 0.0001; log rank = 33.07). The presence of CTCs detected by both CS and ISET correlated even better with shorter DFS at a univariate (p < 0.0001; log rank = 42.15) and multivariate level (HR, 1.235; 95% CI, 1.056–1.482; p < 0.001). CS and ISET are complementary methods for detection of CTCs in preoperative radical surgery for NSCLC. CTC detection in resectable NSCLC patients using CS and/or ISET could be a prognostic biomarker of great interest and may open up new avenues into improved therapeutic strategies for lung carcinoma patients.

Immunohistochemistry has become an essential ancillary examination for the identification and classification of carcinomas of unknown primary site (CUPs). Over the last decade, the diagnostic accuracy of organ- or tumour-specific immunomarkers and the clinical validation of effective immunohistochemical panels has improved significantly. When dealing with small sample sizes, diagnostic accuracy is crucial, particularly in the current era of targeted molecular and immune-based therapies. Effective systematic use of appropriate immunohistochemical panels enables accurate classification of most of the undifferentiated carcinomas as well as careful preservation of tissues for potential molecular or other ancillary tests. This review discusses the algorithmic approach to the diagnosis of CUPs using CK7 and CK20 staining patterns. It outlines the most frequently used tissue-specific antibodies, provides some pitfalls essential in avoiding potential diagnostic errors and discusses the complementary tools, such as molecular tumour profiling and mutation-specific antibodies, for the improvement of diagnosis and prediction of the treatment response.