Optimization of the Enrichment of Circulating Tumor Cells for Downstream Phenotypic Analysis in Patients with Non-Small Cell Lung Cancer Treated with Anti-PD-1 Immunotherapy

Abstract

The current study aimed at the optimization of circulating tumor cell (CTC) enrichment for downstream protein expression analyses in non-small cell lung cancer (NSCLC) to serve as a tool for the investigation of immune checkpoints in real time. Different enrichment approaches—ficoll density, erythrolysis, their combination with magnetic separation, ISET, and Parsortix—were compared in spiking experiments using the A549, H1975, and SKMES-1 NSCLC cell lines. The most efficient methods were tested in patients (n = 15) receiving immunotherapy targeting programmed cell death-1 (PD-1). Samples were immunofluorescently stained for a) cytokeratins (CK)/epithelial cell adhesion molecule (EpCAM)/leukocyte common antigen (CD45), and b) CK/programmed cell death ligand-1 (PD-L1)/ indoleamine-2,3-dioxygenase (IDO). Ficoll, ISET, and Parsortix presented the highest yields and compatibility with phenotypic analysis; however, at the patient level, they provided discordant CTC positivity (13%, 33%, and 60% of patients, respectively) and enriched for distinct CTC populations. IDO and PD-L1 were expressed in 44% and 33% and co-expressed in 19% of CTCs. CTC detection was associated with progressive disease (PD) (p = 0.006), reduced progression-free survival PFS (p = 0.007), and increased risk of relapse (hazard ratio; HR: 10.733; p = 0.026). IDO-positive CTCs were associated with shorter PFS (p = 0.039) and overall survival OS (p = 0.021) and increased risk of death (HR: 5.462; p = 0.039). The current study indicates that CTC analysis according to distinct immune checkpoints is feasible and may provide valuable biomarkers to monitor NSCLC patients treated with anti-PD-1 agents.

Antigen-agnostic microfluidics-based circulating tumor cell enrichment and downstream molecular characterization

“…Previous validation studies of the Parsortix PR1 system have shown similar recovery of epithelial cells [11,[18][19][20][21]. The imperfect capture efficiency can be partially attributed to deformation and capillary flow required by the platform resulting in lysis of some CTC.…”

Epithelial/Mesenchymal Characteristics and PD-L1 Co-Expression in CTCs of Metastatic Breast Cancer Patients Treated with Eribulin: Correlation with Clinical Outcome

“…PD-L1 expression levels on cancer and/or immune cells have been associated with clinical responses to anti-PD-L1/PD-1 therapy [ 20 ]. The expression of PD-L1 in CTCs, isolated using different approaches, has been extensively investigated and reported in lung cancer [ 21 , 22 ]. PD-L1 was also detected in CTCs from patients with melanoma, breast, prostate and gastrointestinal cancer [ 23 , 24 , 25 , 26 , 27 ].…”

Assessment of the Efficacy and Clinical Utility of Different Circulating Tumor Cell (CTC) Detection Assays in Patients with Chemotherapy-Naïve Advanced or Metastatic Non-Small Cell Lung Cancer (NSCLC)

“…These findings are in line with previous studies highlighting the necessity of the combined use of multiple markers in order to improve the sensitivity of CTC detection in NSCLC, which might be attributed to the high heterogeneity of CTCs identified in lung cancer [ 19 , 21 ]. In a recent comparative study from our group, different manual and automated enrichment approaches were shown to provide divergent CTC detection rates, and complementary clinical information for patients with advanced NSCLC treated with immunotherapy [ 29 ]. Moreover, a discordance between CellSearch and different detection approaches has been extensively reported in NSCLC [ 11 , 30 ].…”