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.

Maria A Papadaki,Afroditi I Sotiriou,Konstantinos Rounis,Despoina Aggouraki,Christina Vasilopoulou,Dimitrios Mavroudis,Sofia Agelaki,Christina A Apostolopoulou,Panormitis G Tsoulfas,Maria Filika

doi:10.3390/cancers12061556

Maria A Papadaki, Afroditi I Sotiriou + Show 8 more

Open Access

https://doi.org/10.3390/cancers12061556

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Journal: Cancers	Publication Date: Jun 12, 2020
Citations: 29	License type: CC BY 4.0

Affiliation: University of Crete, University Hospital of Heraklion

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.

Highlights

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths for both men and women worldwide [1]
The current study demonstrates for the first time that IDO is expressed on circulating tumor cells (CTCs) and suggests that IDO+ CTCs and the IDO+/programmed cell death ligand-1 (PD-L1)- subset may have prognostic relevance in patients with non‐small cell lung cancer (NSCLC) treated with anti-programmed cell death-1 (PD-1) immunotherapy
A series of manual and automated CTC-enrichment approaches were compared for their efficiency to enrich CTCs and for their compatibility for downstream protein expression analysis in NSCLC

Summary

Introduction

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths for both men and women worldwide [1]. Immunotherapy with immune checkpoint inhibitors (ICIs) targeting programmed cell death-1 (PD-1) or programmed cell death ligand-1 (PD-L1) has revolutionized the treatment of advanced NSCLC [3,4]. ICIs represent a standard treatment for the majority of NSCLC patients. Indoleamine-2,3-dioxygenase (IDO) has been shown to promote tumor evasion from both the innate and adaptive immune response and to be associated with resistance to anti-PD-1 treatment [8,9,10]. Preclinical findings and evidence from NSCLC tissue suggest that PD-L1 and IDO represent distinct, nonoverlapping routes to immune evasion and highlight their combined targeting using anti-PD-1/PD-L1 and anti-IDO inhibitors as a promising therapeutic strategy [11,12,13,14]

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