Background: NSCLC exhibits intratumor heterogeneity, with subpopulations of cells undergoing epithelial-mesenchymal transition. Such CTCs from NSCLC pts may be missed by the EpCAM-based CELLSEARCH® system (CS). The label-free ClearCell® FX inertial microfluidic system (FX) isolates CTCs based on size and inertia and may lead to more accurate CTC capture. PD-1/PD-L1 inhibitors have shown benefit in PD-L1+ NSCLC pts, but responses are still seen in PD-L1- pts, suggesting limitations in tumor PD-L1 testing. Also, PD-L1 testing on CTCs may be more practical than tumor rebiopsies and may provide insights into cancer heterogeneity.

Methods: FX was validated with EpCAM-high and EpCAM-low cancer cell lines labeled with CellTracker dyes spiked into healthy volunteer (HV) blood for repeatability and reliability. Enriched cells were detected with the automated Bioview Duet imaging system for recovery (%). Identical spiking studies were conducted on CS for comparison. Antibodies for 5-color immunofluorescence (IF) (CK, CD45, DAPI, TTF1 [to detect lung adenocarcinoma cells], PD-L1) were optimized on EpCAM-high and EpCAM-low cell lines. 8ml of blood from NSCLC pts were enriched with FX for 5-color IF characterization. 7.5ml of blood from the same pts taken at identical timepoints were enumerated with CS for comparison. Blood was obtained from HV for CTC enumeration on FX and CS as controls.

Results: Cell recovery of EpCAM-high cancer cell lines using FX produced similar counts to CS, e.g. lung NCI-H2066 cells: FX 64.9%±4.5 vs CS 74.4%±9.2 (p = 0.05); lung H1975 cells: FX 74.1%±11.9 vs CS 74.7%±10.7 (p = 0.91). In contrast, for EpCAM-low cells, a significant difference in cell recovery between FX and CS was seen, e.g. lung A549 cells: FX 59.3%±5.8 vs CS 26.1%±7.6 (p<0.0001); and prostate PC3 cells: FX 68.6%±7.4 vs CS 35.5%±6.9 (p<0.0001). CTCs were detected in 19/21 pts with progressing stage IV NSCLC (adenocarcinoma) (mean 57; range 4-304) using FX, vs 7/21 pts (mean 7; range 1-38) using CS. CTC counts were higher with FX vs CS in 19/21 (90%) NSCLC pts, p = 0.012. No CTCs were detected in HV with FX (n = 10) and CS (n = 5). After FX enrichment, CTCs from 17/21 NSCLC pts were available for 5-color IF testing. Of these 17 pts, 15 had TTF1+ CTCs. All 15 pts with TTF1+ CTCs had ≥1 PD-L1+ CTC. 6/15 (40%) pts only had PD-L1+ TTF1+ CTCs, with no PD-L1- TTF1+ CTCs. PD-L1 CTC heterogeneity was seen in the other 9/15 pts, with co-existing PD-L1+ and PD-L1- TTF1+ CTCs. 6 of these 9 pts had more PD-L1+ TTF1+ CTCs than PD-L1- TTF1+ CTCs.

Conclusions: FX resulted in consistently high cell recovery rates regardless of EpCAM status. Higher CTC counts were isolated with FX vs CS in 90% of NSCLC pts. 40% of NSCLC adenocarcinoma pts had PD-L1+ TTF1+ CTCs, but PD-L1 CTC heterogeneity was seen in other pts, which may in part explain differences in responses to PD-1/PD-L1 inhibitors in NSCLC.

Citation Format: Zai Ahmad, Jen Fraser-Fish, Rajiv Kumar, Bernadette Ebbs, Gemma Fowler, Penny Flohr, Mateus Crespo, Sanjay Popat, Jaishree Bhosle, Udai Banerji, Mary O’Brien, Johann S. de Bono, Timothy A. Yap. Characterization of PD-L1 expression on circulating tumor cells (CTCs) isolated with a label-free inertial microfluidic system from advanced non-small cell lung cancer patients (NSCLC pts). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2243.