Background: The chemokine receptor CXCR4 and the transcription factor JUNB, expressed on a variety of tumor cells, seem to play an important role in the metastatic process. Since disseminated tumor cells (DTCs) in the bone marrow (BM) have been associated with worse outcomes, we evaluated the expression of CXCR4 and JUNB in DTCs of primary, nonmetastatic breast cancer (BC) patients before the onset of any systemic treatment. Methods: Bilateral BM (10 ml) aspirations of 39 hormone receptor (HR)-positive, HER2-negative BC patients were assessed for the presence of DTCs using the following combination of antibodies: pan-cytokeratin (A45-B/B3)/CXCR4/JUNB. An expression pattern of the examined proteins was created using confocal laser scanning microscopy, Image J software and BC cell lines. Results: CXCR4 was overexpressed in cancer cells and DTCs, with the following hierarchy of expression: SKBR3 > MCF7 > DTCs > MDA-MB231. Accordingly, the expression pattern of JUNB was: DTCs > MDA-MB231 > SKBR3 > MCF7. The mean intensity of CXCR4 (6411 ± 334) and JUNB (27725.64 ± 470) in DTCs was statistically higher compared with BM hematopoietic cells (2009 ± 456, p = 0.001; and 11112.89 ± 545, p = 0.001, respectively). The (CXCR4+JUNB+CK+) phenotype was the most frequently detected [90% (35/39)], followed by the (CXCR4–JUNB+CK+) phenotype [36% (14/39)]. However, (CXCR4+JUNB–CK+) tumor cells were found in only 5% (3/39) of patients. Those patients harboring DTCs with the (CXCR4+JUNB+CK+) phenotype revealed lower overall survival (Cox regression: p = 0.023). Conclusions: (CXCR4+JUNB+CK+)-expressing DTCs, detected frequently in the BM of BC patients, seem to identify a subgroup of patients at higher risk for relapse that may be considered for close follow up.
Upregulation of Vimentin (VIM), alpha-Tubulin (TUB) and Detyrosinated tubulin (GLU) in circulating tumor cells (CTCs) derived from breast cancer patients is related to poor prognosis. In the current study we evaluated for the first time, these cytoskeletal proteins in sixty Non-Small Cell Lung Cancer (NSCLC) patients’ CTCs (33 treatment-naïve and 27 pre-treated). Samples were isolated using the ISET platform and stained with a pancytokeratin (CK)/CD45/TUB, CK/GLU/VIM and CK/programmed death ligand 1 (PD-L1) combination of antibodies. Subsequently, slides were analyzed using confocal laser scanning microscopy. CTCs were detected in 86.7% of the patients. CTCs with TUB expression were identified in 65.4% (34/52) of the CK (+)-patients. GLU, VIM and PD-L1 were also evaluated. The frequency of the observed phenotypes was as follow: (CK+/GLU−/VIM−): 35.2%, (CK+/GLU+/VIM+): 63.0%, (CK+/GLU+/VIM−): 16.7%, (CK+/GLU−/VIM+): 72.2%, (CK+/PD-L1−): 75% and (CK+/PD-L1+): 55%. The OS was significantly decreased in patients with high GLU (3.8 vs. 7.9 months; p = 0.018) and/or high VIM (3.2 vs. 7.1 months; p = 0.029) expression in their CTCs. PD-L1 was also related to OS (3.4 vs. 7.21 months; p = 0.035). Moreover, TUB-high and TUB-low expression in CTCs inversely influenced patients’ OS as independent prognostic factors (p = 0.041 and p = 0.009). The current study revealed that TUB, GLU, VIM and PD-L1 were overexpressed in CTCs from NSCLC patients. Furthermore, the presence of GLU, VIM-positive and PD-L1 in CTCs is potentially related to patients’ outcomes.
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.
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