Improvement of sensitive and specific detection of circulating tumor cells using negative enrichment and immunostaining-FISH.

BACKGROUND

Circulating tumor cells (CTCs) provide an opportunity to obtain pivotal biological information required for the development of personalized medicine. However, the current assays of CTCs' detection face serious challenges regarding specificity and sensitivity.

METHODS

In this study, we developed a novel strategy that combined negative enrichment (NE), immunocytochemistry CD45 staining and fluorescence in situ hybridization (FISH) to identify, enumerate and characterize CTCs. CTCs were identified as DAPI+/CD45-/Chromosome multiploid. The assay was evaluated with different cancer cell lines including lung, breast, esophageal and gastric cancer. And then, the developed assay was applied in cancer patients to explore the possibility of clinical application and whether CTC number was related to clinicopathological factors.

RESULTS

The average recover rate of esophageal cancer cell line Eca-109 using negative enrichment was higher than 80% and the multiploid cells rate of four cancer cell lines were >96%, which demonstrate the NE-FISH platform is favorable for CTCs detection. CTCs count was significantly higher in lung cancer patients than healthy controls and benign lung disease with an area under ROC curve of 0.905 (95% confidence interval 0.866-0.944, P < .001). Using a cutoff value of 2 CTCs, the positive rate of detecting lung, gastric, breast and esophageal cancer patients were 71.33%, 86.21%, 76.77% and 78.35%, respectively. Besides, CTCs could be detected in stage I with the positive rate of 64.15% for lung cancer, 83.33% for gastric cancer, 78.95% for breast cancer and 68.18% for esophageal cancer, which may promote the early diagnose and influence the treatment decision for better management of those cancer in clinic.

CONCLUSIONS

Our study showed that CTCs could be detected in diverse cancers using the novel NE-FISH platform with high sensitivity and specificity. Therefore, analysis of CTCs with NE-FISH has a clear potential to improve the management of cancer patients in clinical use.