Purpose: Noninvasive diagnostics has been developed over the last decade and we have previously reported that CTCs can be utilized for evaluating molecular features of NSCLC. AXL, a receptor tyrosine kinase is linked to epithelial-to-mesenchymal transition (EMT) leading to cancer progression and regarded as a potential therapeutic target. However, many of current technologies rely on epithelial markers to detect CTCs and that makes it difficult to detect AXL-expressing CTCs. Here, we established the detection of AXL expression on CTCs using MCA system.
Methods: Preclinical experiments were performed using NSCLC cell lines H1299, PC9, and HCC827 and a breast cancer cell line MDA-MB231 with varying cytokeratin (CK) and AXL expression levels. The cells were spiked into 3 ml of peripheral blood from healthy donors, then enriched using MCA system, and detected by staining for CD45, DAPI, CK or vimentin (VM) with the addition staining for AXL. For clinical evaluation, 3ml of peripheral blood was collected from advanced NSCLC patients.
Results: DAPI-positive, CK or VM-positive, and CD45-negative cells were defined as CTCs. In spike-in experiments, when CK was used as a marker, AXL expression was detected in 5 and 17% of high CK-expressing HCC827 and PC9 cells, respectively and detected in 52 and 75% of low CK-expressing H1299 and MDA-MB231 cells, respectively. On the other hand, when VM was used as a marker, AXL expression was detected in 72 and 88% of high VM-expressing MDA-MB231 and H1299 cells, respectively whereas detected in 1 and 7% of PC9 and HCC827 cells with low VM expression, respectively. Twenty-four patients were enrolled in the clinical study. The patient characteristics were as follows: median age 69.5 years (range, 49-84); male 88%; stage III/IV, 25/75%; adenocarcinoma/ squamous cell carcinoma/ other, 63/33/4%. Both CK and VM staining in 17 patients, only CK in 6 patients, and only VM in 1 patient were performed. CK-positive single CTCs were detected in all patients (median, 4; range, 1-50) and AXL-expressing CK-positive single CTCs were detected in 26% of patients (median, 0; range, 0-1). On the other hand, VM-positive single CTCs were detected in 94% of patients (median, 6.5; range, 0-128) and AXL-expressing VM-positive single CTCs were detected in 89% of patients (median, 1; range, 0-106). Significantly more AXL-expressing single CTCs were detected in VM-positive than CK-positive (p < 0.001). Notably, all of identified CTC clusters were positive for only VM, not CK and AXL-expressing CTC clusters were detected in 33% of patients (median, 0; range, 0-22).
Conclusion: Our data indicate that incorporating VM staining is necessary to detect AXL-positive CTCs due to EMT. Further clinical evaluation is warranted for validating the method and the clinical significance of AXL-positive CTCs.
Citation Format: Mio Ikeda, Yasuhiro Koh, Shunsuke Teraoka, Jun Oyanagi, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Masayuki Higuchi, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Detection of AXL-expressing circulating tumor cells (CTCs) in non-small-cell lung cancer (NSCLC) patients using an automated microcavity array (MCA) system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 414.