SEMIQUANTIFICATION OF CIRCULATING HEPATOCELLULAR-CARCINOMA CELLS BY REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION

Hepatocellular carcinoma (HCC) is one of the most common and rapidly f atal malignancies worldwide. Treatment options are severely limited by the frequent presence of metastases. If hepatocyte-specific mRNAs are detected in the circulation, it is possible to infer the presence of circulating, presumably malignant, liver cells. If these can be quanti fied it is possible to predict the likelihood of haematogenous metasta sis. In this investigation, we have attempted to gain an index of the mass of circulating HCC cells (with reference to the number of hepatob lastoma cells) by measuring the amounts of PCR products for albumin (a lb) mRNA and cr-fetoprotein (afp) mRNA by reverse transcriptase polyme rase chain reaction (RT-PCR) and Southern blot analysis. For calibrati on, total RNA from 1-10(6) HepG2 cells was mixed with total RNA from 1 0(6) normal peripheral mononuclear cells. A linear relationship was de monstrated between the amount of alb-or afp PCR product and the level of HepG2 total RNA spiked. The assay is sensitive down to a detection level of one HepG2 cell. Alb mRNA was detected in 50% of 18 normal sub jects and afp mRNA in only two normal subjects. The alb mRNA cut-off l evel for the normal was exceeded by seven normal subjects and 34 out o f 64 HCC patients, and that for afp mRNA was exceeded by six HCC patie nts but none of the normal subjects. The level of alb mRNA detected wa s not linearly proportional to the amount of afp mRNA detected in peri pheral blood of the same patients, suggesting heterogeneous expression of alb and afp genes in different circulating tumour cells. In additi on, no significant linear association between the levels of afp mRNA a nd serum AFP was observed. Semiquantification of both mRNA markers for HCC cell detection may prove useful in prediction of metastases.