Patterns of PD-L1 expression on circulating tumor cells in Japanese patients with advanced lung cancer

Background: Immune-checkpoint blockage with anti-PD-1 antibodies is effective against human malignancies including lung cancer. Although the expression of PD-L1 on tumor cells is expected as a predictive biomarker, detection of PD-L1 expression level on tissues remains challenging due to its dynamic and unstable expression. Here, we isolated circulating tumor cells (CTCs) and then evaluated the expression of PD-L1 on them in patients with advanced non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Methods: Immuno-staining for PD-L1 was established using NSCLC cell lines H820, H441, A549 and H23, spiked in the peripheral blood obtained from healthy donors. For clinical evaluation, 3 ml of peripheral whole blood was collected from 41 advanced lung cancer patients prior to the initiation of chemotherapy. Cells were captured and stained by using MCA system (Hitachi Chemical Co., Ltd). CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. Results: Characteristics of 41 lung cancer patients enrolled in clinical study were as follows: median age 70 (range, 39 to 86); male 66 %; stage II/III/IV, 2/7/91 %; NSCLC/SCLC, 80/20 %. More than 5 CTCs were detected in 32 patients (median 21.5; range, 7 to 58), and PD-L1-positive CTCs were detected in 22 patients (median 4; range, 1 to 15). All the patients bearing PD-L1-positive CTCs also had PD-L1-negative CTCs concurrently. We found a total of 49 CTC clusters from 24 patients and most of clusters contained PD-L1-positive CTCs (44/49 clusters, 90%). On top of that, CTCs forming clusters were more likely to be PD-L1-positive than those detected as a single CTC (28/80, 35% versus 104/769, 14%, respectively. p = 0.001). We also found that CTC-interacting white blood cells were more intensively bound with clustered CTCs than with single CTCs (15/77, 19% versus 55/598, 9%, respectively. p = 0.016). Conclusions: Our data suggest that PD-L1 expression on CTCs have diverse intrapatient heterogeneity in advanced lung cancer. Further investigation is warranted to address its clinical significance in response to PD-1/PD-L1 blockade and to clarify the biology of the aggregated CTCs and those interacting with white blood cells.