A pilot study for cellular detection of circulating tumor cells and disseminated tumor cells of patients with hepatocellular carcinoma.

Abstract

e15038 Background: Circulating tumor cells (CTC) in peripheral blood (PB) and disseminated tumor cells (DTC) in bone marrow (BM) have been recently focused as predictive and prognostic markers in solid tumors. But hepatocellular carcinoma (HCC) usually lacks epithelial antigens, only a few reports described CTC or DTC at a cellular level. The aim of this study is to establish methodology for detecting CTC and DTC in HCC at a cellular level, and to determine to what degree DTC match the phenotype criterion of putative cancer stem cells. Methods: PB (n-17) and BM samples (n-12) were preoperatively collected from patients with HCC between October 2011 and December 2012. Mononuclear cells were collected by Ficoll gradient separation. CD45+ cell population was deleted through magnetic activated cell sorting system. Subsequently, double immunofluorescence for AFP, Arginase-1 or Hep-Par1 and CD45 was performed. PB samples from 20 healthy volunteers and BM samples from 3 patients with benign disease were used as controls. Our protocol has been approved by the ethical committee of Hokkaido University Hospital and written informed consent was obtained from all enrolled patients. Results: Positive rates of CTC and DTC were 70.5% (12/17) and 75.0% (9/12), respectively. No CTC or DTC was found in the control groups. Poorly differentiated HCC were found in 50.0% of CTC+ patients and 66.6% of DTC+ patients, whereas, 20% in CTC- patients and 0% of DTC- patients. There’re higher risk of recurrence in CTC+ or DTC+ patients, compared with that of CTC- or DTC- patients (33.3%, 55.5% vs 20%, 0%). Over 40% of DTC expressed putative stem cell marker CD133, although the median positive rate of CD133 in primary HCC was reported to be less than 1%. Conclusions: Our protocol might provide highly specific detection of CTC, DTC from HCC patients. Detection of CTC and DTC might closely correlate with histological grade of differentiation and recurrence rate.