Abstract 1026: A novel detection system of circulating tumor cells based on size-dependent enrichment with immunofluorescent staining of CK8/18 in advanced non-small cell lung cancer
Authors: Sakai Tetsuya , Zenke Yoshitaka , Onishi Mami , Morita Eri , Yanagida Masatoshi , Shibata Yuji , Izumi Hiroki , Nosaki Kaname , Udagawa Hibiki , Matsumoto Shingo , Yoh Kiyotaka , Goto Koichi 
Published: June 2023

Abstract
Background: Detection of circulating tumor cells (CTCs) in the blood is a potentially noninvasive tool for cancer diagnosis, monitoring and prognosis evaluation. However, the CTC detection is difficult because of the rarity of CTCs, especially in non-small cell lung cancer (NSCLC), and the detection sensitivity of CTCs was reported to be less than 50% in NSCLC patients. Herein, we developed a novel CTC detection system and evaluated its performance in advanced NSCLC patients.
Methods: Whole blood samples were collected from stage IV or recurrence NSCLC patients. CTC detection was performed with a novel system (Sysmex Corporation, Hyogo, Japan) by using size-dependent enrichment for capturing CTCs and immunofluorescent staining reagents including anti-cytokeratin (CK, AE1/AE3 or CK8/18) (specific for epithelial cells), anti-CD45 (specific for leukocyte) and Hoechst (for nucleus). Fluorescence intensity and cell images of each marker were obtained by Molecular Imaging Flow Cytometry (MI-FCM). Circulating cells that were positive for Hoechst and anti-CK but negative for anti-CD45 were defined as CTCs. To confirm whether CTCs detected were derived from lung cancer origin, CTCs obtained from patients with EGFR mutations were subjected to single cell sorting, whole genome amplification and PCR analysis for the mutation detection.
Results: A total of 20 NSCLC patients (10 EGFR mutant, 10 EGFR wild-type) were enrolled in this study. Of these, CTCs were detected in 16 (80%) patients (number of CTCs, 1 to 66 in 4.5ml whole blood) when CK8/18 was used as epithelial cell staining, but in only 11 (55%) patients (1 to 6 CTCs) when using AE1/AE3. The detection sensitivity of CTCs based on CK8/18 was significantly higher compared to that based on AE1/AE3 in our novel system (80% vs. 50%, p = 0.03). The number of CTCs was significantly lower in EGFR mutation-positive patients than in EGFR wild-type patients (median number of CTCs, 3 vs. 22, p = 0.01). Furthermore, among 10 EGFR mutation-positive patients, EGFR mutations were detected in CTCs derived from 6 (60%) patients. All the mutation types detected in CTCs were matched with those identified in the corresponding primary tumors.
Conclusion: Our novel CTC detection system demonstrated that the detection rate of CTCs (80%) was higher than those in previous studies. Further studies are needed for the clinical application of this system.
Citation Format: Tetsuya Sakai, Yoshitaka Zenke, Mami Onishi, Eri Morita, Masatoshi Yanagida, Yuji Shibata, Hiroki Izumi, Kaname Nosaki, Hibiki Udagawa, Shingo Matsumoto, Kiyotaka Yoh, Koichi Goto. A novel detection system of circulating tumor cells based on size-dependent enrichment with immunofluorescent staining of CK8/18 in advanced non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1026.