Vimentin expression in circulating tumor cells (CTCs) associated with liver metastases predicts poor progression-free survival in patients with advanced lung cancer

Abstract

Objective: To investigate the presence of vimentin expression in CTCs and its clinical relevance in patients with advanced lung cancer.

Methods: Peripheral blood was obtained from 61 treatment-naive patients with advanced lung cancer. Subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) platform was applied to identify, enumerate and characterize CTCs based on cell size, aneuploidy of chromosome 8 (Chr8) and vimentin expression. Quantification and analysis of CTCs were performed on patients before chemotherapy administration and after two cycles of therapy.

Results: Before treatment, CTCs were detected in 60 (98.4%) patients, small cell CTCs (≤ 5 µm of WBCs) accounted for 52.8% of the absolute CTCs number, while 12 (19.7%) of the included patients had detectable vimentin-positive CTCs (vim⁺ CTCs). Liver metastases were reported in 7 (11.5%) patients and were significantly correlated to the presence of Vim⁺ CTCs (p = 0.002), with a high positivity rate of 71.4% (5/7). Vim⁺ CTCs were mostly in small cell size and Chr8 aneuploidy (77.0% and 82.05%, respectively). Baseline small cell CTCs ≥ 2/6 ml, triploid CTCs ≥ 2/6 ml, Vim⁺ CTCs ≥ 1/6 ml were found to significantly correlate with poor progression-free survival (PFS) (p = 0.017, p = 0.009 and p = 0.001, respectively). After adjusting for clinically significant factors, baseline Vim⁺ CTCs ≥ 1/6 ml was the only independent predictor of poor PFS [hazard ratio (HR):2.756, 95% confidence interval (CI): 1.239-6.131; p = 0.013].

Conclusions: This study demonstrates an important morphologic, karyotypic and phenotypic CTCs heterogeneity in advanced lung cancer patients. The majority of Vim⁺ CTCs are in small size and Chr8 aneuploidy. Baseline presence of Vim⁺ CTCs is correlated with liver metastases and may help predict poor PFS.

Keywords: Advanced lung cancer; Aneuploidy of Chr8; Cell size; Circulating tumor cells; Progression-free survival; Vimentin.

Fig. 1

Analysis of CTCs based on cell size and correlation of small cell CTCs with PFS. a Images of small and large CTCs in advanced lung cancer patients. Comparing to the size of WBCs (white arrow), an small (≤ 5um WBC) triploid CTCs as well as an large (≥ 5um WBC) multiploid CTCs are indicated by orange and red arrow. b Correlation of small cell CTCs with PFS. Pretreatment patients with small cell CTCs ≥ 2/6 ml had a PFS significantly shorter than patients with small cell CTCs < 2/6 ml (p = 0.017)

Fig. 2

Analysis of Chr 8 ploidy of CTCs and correlation of triploid CTCs with PFS. A Images of diverse Chr8 ploidy CTCs. a Haploidy, b diploidy, c triploidy, d tetraploidy, e multiploidy copies. B Proportion of diverse Chr8 ploidy CTCs in pretreatment patients. C Correlation of triploid CTCs with PFS. Baseline triploid CTCs ≥ 2/6 ml showed a much shorter PFS than patients with triploid CTCs < 2/6 ml (p = 0.009)

Fig. 3

Detection of vimentin expression in diverse Chr8 ploidy CTCs and correlation of Vim⁺ CTCs with PFS. A Vimentin expression in diverse Chr8 ploidy CTCs. a–e Vim⁺ CTCs with haploid, diploid, triploid, tetraploid, and multiploid Chr8, respectively. B Proportion of diverse Chr8 ploidy Vim⁺ CTCs in pretreatment patients. C Correlation of Vim⁺ CTCs with PFS. Patients with advanced lung cancer showing poor prognosis had positive detection of Vim⁺ CTCs (≥ 1/6 ml). Subgroup analysis showed the presence of Vim⁺ CTCs indicating worse prognosis in both SCLC and ADC patients