Abstract 780: Alpha tubulin and EMT-related molecules expressed in circulating tumor cells (CTCs) isolated from non-small cell lung cancer patients
Authors: Kallergi Galatea , Katsarou Spyridoula D. , Voumvouraki Anastasia , Stournaras Christos , Martin Stuart S. , Georgoulias Vassilis 
Published: September 2020

Abstract
Introduction: Vimentin (VIM) expression in CTCs is a poor prognostic factor for breast cancer patients. Furthermore, upregulation of alpha Tubulin (TUB) and Detyrosynated tubulin (GLU) in CTCs is related to patients' outcome. In the current study, we evaluated TUB, GLU and VIM in NSCLC cell lines and in patients' CTCs.
Methods: Four NSCLC cell lines were used for the evaluation of our method. In addition, 60 patients with metastatic NSCLC before the initiation of any line of treatment, were enrolled in this study. Thirty-three patients' samples were taken prior to the 1st line treatment and 27 patients were enrolled before the initiation of ≥2 lines of treatment. CTCs were isolated using ISET platform and stained with pancytokeratin (CK)/CD45/TUB and CK/GLU/VIM antibodies. Samples were analyzed using confocal laser scanning microscopy.
Results: In patients' samples, CK(+)/CD45(-) cells were observed in 90% (54 out of 60 patients). CTCs with high expression of TUB were detected in 62.96% (34/54) of the patients. Among the total isolated CTCs/patient, 30.57 % belonged to the (CK+TUB+CD45) phenotype. The rest of the CTCs presented with either low (31.2%) or no expression (24.8%) of TUB. Interestingly, 4.15% of the CTCs (only in advanced disease) belonged to (CK-TUB+CD45-) phenotype. The proportion of TUB low or negative CTCs was statistically higher in treatment-naïve compared to pre-treated patients (p= 0.004 and p=0.007, respectively) whereas the proportion of TUB high CTCs was increased in pre-treated compared to treatment-naïve patients (85.72% vs 64.52%), implying that these CTCs persist after chemotherapy. Further analysis revealed that patients with high TUB expression in their CTCs experienced shorter OS compared to patients with CTCs with low or no TUB expression (7 vs 14 months, respectively; p=0.023). GLU and VIM were also evaluated in the same cohort of patients. The frequency of the observed phenotypes were as follow: (CK-GLU-VIM-): 35.19% (19/54 patients), (CK+GLU+VIM+): 62.96% (34/54), (CK+GLU+VIM-): 16.67% (9/54) and (CK+GLU-VIM+): 72.22%. The last one was the most abundant subclone in treatment naïve patients 81.25% (26/32). Furthermore the most abundant phenotypes in advanced disease were the (CK+GLU-VIM-) 59.09% (13/22), (CK+GLU-VIM+) 59.09% (13/22) and (CK+ GLU+ VIM+): 54.55% (12/22). The expression of VIM in advanced patients was also related to poor OS (1 vs 10 months, p=0.010).
Conclusions: In agreement to our previous results in breast cancer patients, current study also revealed that alpha-Tubulin, GLU and Vimentin are overexpressed in CTCs from NSCLC patients. In addition, the presence of TUB and/or VIM-positive CTCs is potentially related to poor patients' outcome.
Citation Format: Galatea Kallergi, Spyridoula D. Katsarou, Anastasia Voumvouraki, Christos Stournaras, Stuart S. Martin, Vassilis Georgoulias. Alpha tubulin and EMT-related molecules expressed in circulating tumor cells (CTCs) isolated from non-small cell lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 780.