Molecular characterization of PDL1 status of circulating tumor cells (CTCs) isolated with a novel label-free inertial microfluidic system from patients (pts) with advanced cancers

Abstract

Background: The ClearCell® FX system (FX) is a novel label-free inertial microfluidic CTC isolation platform, in contrast to the EpCAM-based CELLSEARCH® system (CS). We hypothesise that label-free CTC capture will lead to more accurate assessment of CTCs, including PD-L1 expression, and capture CTCs that have undergone epithelial-mesenchymal transition, which is prevalent in advanced cancers. Methods: CellTrackerTM labelled EpCAM-high and EpCAM-low cell lines were spiked into healthy volunteer (HV) blood, prior to recovery on FX or CS platforms for initial validation studies. Blood samples were then obtained from pts with advanced non-small cell lung (NSCLC), prostate, ovarian, rectal and breast cancers for CTC isolation on FX and CS. CTCs captured on FX were assessed with 5-color immunofluorescence (IF) (CK, CD45, DAPI, PD-L1, as well as TTF-1 [lung adenocarcinoma], androgen receptor [AR; prostate cancer] or EpCAM [other cancers]). HV blood samples were assessed on FX and CS as controls. Results: FX and CS captured similar counts in EpCAM-high cell lines (FX 67% ± 11 vs CS 74% ± 10 [p = 0.11]). In contrast, higher cell counts were seen with EpCAM-low cell lines with FX vs CS [62% ± 8 vs 32% ± 9 [p < 0.0001]). Of 36 pts, CTC counts were higher with FX vs CS in 31 (86%) pts: 19/21 NSCLC, 7/10 prostate, 3/3 ovarian, 1/1 rectal and 1/1 breast cancer pts. No CTCs were detected in HV blood (N = 10) on FX and CS. 19/19 NSCLC, 8/9 prostate, 3/3 ovarian, 1/1 rectal and 1/1 breast cancer pts had ≥1 PD-L1+ CTCs. Heterogeneity in PD-L1 expression was observed. While 19/20 NSCLC pts had ≥1 PD-L1+ TTF-1+ CTCs, only 10 of these 19 pts had 100% PD-L1+ TTF-1+ CTCs. 5/10 prostate cancer pts had ≥1 PD-L1+ AR+ CTCs, but only 4 of these 5 pts had 100% PD-L1+ AR+ CTCs. All 3 ovarian cancer pts had ≥1 PD-L1+ EpCAM + CTCs, with no PD-L1- EpCAM+ CTCs detected. 1/1 rectal and 1/1 breast cancer pts had 100% PD-L1+ EpCAM+ CTCs. Conclusions: Higher CTC counts were isolated with FX vs CS in 86% of pts. CTC PD-L1 heterogeneity was observed and may in part explain differences in antitumor responses to PD-1/PD-L1 inhibitors. Clinical qualification of this 5-color IF PD-L1 CTC assay is ongoing in a PD-1 inhibitor NSCLC trial.