HCV RNA levels in hepatocellular carcinomas and adjacent non-tumorous livers

Abstract

To determine the antiviral effects of drugs targeted to hepatitis C virus (HCV) in chronic hepatitis patients, an accurate quantitative method with high sensitivity is needed. Reverse transcription nested polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of HCV sequences in clinical specimens. However, this method is not quantitative. For this purpose, a quantitative competitive assay was developed that combines RT and PCR followed by image analysis to quantify HCV RNA. This assay targets the highly conserved 5′ non-coding region of HCV and is based on the co-amplification of wild type HCV RNA with known amounts of mutant synthetic RNA. The mutant internal control used in these experiments differs from the wild type RNA by two nucleotide substitutions, which introduces an internal restriction enzyme site. In this report, this method was used to determine the levels of positive strand RNA in 11 HCV positive hepatocellular carcinomas (HCC) and compared these with adjacent non-tumorous liver tissue. To confirm that the difference in viral titers is not related to variations in the amount of RNA used in the assay, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was also assessed by competitive RT-PCR in all tissue extracts. Using this competitive assay it was determined that HCV RNA levels in the liver and tumor samples ranged from 10 3 to 10 6 molecules per μg of total RNA which is similar to previous reports. Interestingly, the amount of HCV in all the non-tumorous liver specimens were found to be significantly higher ( P<0.05) than the surrounding tumors, while the GAPDH mRNA levels were found to be similar in both liver and tumor. Competitive RT-PCR is a sensitive, accurate and reliable method to determine HCV titers in clinical specimens. Using this method it was determined that malignant tumor cells harbor less HCV as compared with the surrounding non-tumorous liver cells.