The expressions of cancer stem cell markers and EMT- associated genes in peripheral blood during the perioperative period are related to postoperative recurrence of hepatocellular carcinoma

Abstract

Purpose: To investigate the correlations of messenger RNA (mRNA) expression levels of cancer stem cells (CSCs) markers and epithelial-mesenchymal transition (EMT)-associated genes in peripheral blood and their dynamic changes during the perioperative period with postoperative recurrence of hepatocellular carcinoma (HCC).Method: Between April 2011 and February 2012, a total of 84 consecutive patients who underwent curative resection (n=64) or liver transplantation (LT, n=17) for HCC were prospectively enrolled in this study. The mRNA expression levels of four surface markers for CSCs (K19, EpCAM, CD44, and CD90) and two EMT-associated genes (Snail and Twist) in peripheral blood and HCC tissues were evaluated using a real-time reverse transcription -polymerase chain reaction (RT-PCR). Peripheral blood samples from the study subjects were collected before operation, after operation and at 7 days after operation. These samples were also obtained from 23 control subjects. To evaluate the dynamic changes of these markers, the median changed value of each marker in control patients was used as the cutoff value to divide study subjects into elevation and no elevation groups. The cutoff values of K19, EpCAM, CD44 and CD90 were 1.99, 2.94, 1.02 and 0.26, respectively. The correlation of these markers who presented with postoperative recurrence of HCC was investigated in patients undergoing resection. The median follow-up after resection was 16 months (range, 1- 24 months).Results: The mRNA expression levels of K19 and EpCAM in preoperative peripheral blood were significantly higher in patients who underwent LT compared to patients who were treated with resection. In cirrhotic patients, the level of EpCAM mRNA was statistically higher in patients with Child-Pugh B and C than in patients with Child-Pugh A (p=0.009). Eighteen patients (34.9%) developed recurrence after curative resection. Although HCC tissues demonstrated higher levels of mRNA expression of K19 and CD44 in patients with recurrence than patients without recurrence, the peripheral blood mRNA levels of K19, EpCAM, CD44 and CD90 were not associated with postoperative recurrence. The elevation group of K19 in immediate postoperative peripheral blood tended to be associated with lower disease-free rate (p=0.082). The elevation of both K19 and CD44 was one of the independent poor prognostic factors for disease-free survival after curative resection (p=0.007). The increase in the expression of Twist or Snail mRNA in peripheral blood at postoperative seventh day was associated with lower disease-free survival rate (p=0.150). On analyzing the correlations between EMT and CSC markers, it was found that the patients with the elevation of K19 mRNA level and increased expression of EMT-associated genes (n=14) showed significantly lower disease-free survival rate after curative resection (p=0.003).Conclusion: The baseline levels of K19 and EpCAM in preoperative peripheral blood in patients with HCC were affected by the underlying liver status. If the elevation of K19 mRNA expression levels were combined with either the elevation of CD44 mRNA expression or increased expression of EMT-associated genes, it had significant correlations with postoperative recurrence and more likely represented true circulating CSCs in postoperative peripheral blood.