Analysis of α-fetoprotein mRNA in peripheral blood: detection and semi-quantitation by reverse transcription polymerase chain reaction

Abstract

The presence of α-fetoprotein (AFP) mRNA in mononuclear cells of the peripheral blood was examined in patients with hepatocellular carcinoma (HCC), liver cirrhosis, chronic hepatitis and normal volunteers using nested reverse transcription polymerase chain reaction (RT-PCR). Then, the AFP mRNA levels were quantitatively analyzed in AFP mRNA-positive patients with HCC. AFP mRNA was detected in 23 of 38 (60.5%) cases with HCC, 2 of 12 (16.7%) cases with cirrhosis, and 1 of 9 (11.1%) cases with chronic hepatitis, respectively. AFP mRNA was not demonstrated in six healthy volunteers. The frequency of positive cases in TNM stage III–IV (16/21, 76.2%) was significantly higher than that in stage I–II (7/17, 41.2%)( P<0.05). AFP mRNA was detected more frequently in patients with positive levels of serum protein induced by vitamin K antagonist II (PIVKA-II) (≥40 mAU/ml, 18/25, 72.0%) than in those with negative level (40 mAU/ml, 5/13, 38.5%) ( P<0.05). However, there was no significant correlation between detectable rate of AFP mRNA and serum AFP level. The frequency of AFP mRNA positive result was not closely associated with L. culinaris agglutinin A, a reactive α-fetoprotein (AFP L3). On the other hand, the expression levels of AFP mRNA were measured in 23 AFP mRNA-positive patients with HCC. Quantitative analysis of AFP mRNA was performed by comparing the amplification kinetic curve of AFP mRNA with that of β-actin mRNA as an internal control. The AFP mRNA levels were expressed as Δ cycle values by this method. Δ cycle values were significantly increased in patients with HCC in stages III–IV (4.7±2.4) compared with patients with HCC in stages I–II (−1.3±7.3) ( P<0.01). These results suggested that the detection and quantitation of AFP mRNA in peripheral blood may indicate the hematogenous spreading of tumor cells in patients with HCC.