*Department of Urinary Surgery, The Affiliated Tumor Hospital, Harbin Medical University, Harbin †Department of General Surgery, Fourth Affiliated Hospital, Harbin Medical University, Harbin ‡Department of Pathology, Harbin Medical University, Harbin, China.
This study aimed to test the diagnostic utility of the total serum cell-free DNA (cfDNA) and DNA integrity index for detection of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC).
We initially evaluated the sodium iodide (NaI) method, Triton/Heat/Phenol (THP) protocol and QIAamp Kit for cfDNA extraction. Then cfDNA was isolated from the sera of 80 patients with HBV-related HCC, 80 patients with chronic HBV infection and 50 healthy subjects, and quantified by real-time quantitative polymerase chain reaction (qPCR) amplification of beta-actin genomic DNA fragments using two sets of primers of 100 and 400 bp. DNA integrity was calculated as the ratio of 400 bp to 100 bp β-actin fragments.
The THP approach was not only superior to the other two methods in terms of DNA quantity, but also was simpler, more rapid, and less costly. Serum DNA integrity in HCC patients was significantly higher than that in HBV patients or healthy controls. As for total cfDNA levels, although a significant difference was found between HCC patients and healthy individuals, no significant difference was found between HBV patients with and without HCC. DNA integrity was associated with tumour size, TNM stage, lymph node and distant metastasis. DNA integrity had a higher sensitivity and specificity in discriminating HCC from HBV patients than total DNA.
The THP method is preferred for extraction of cfDNA. DNA integrity is a promising molecular biomarker for detecting HCC in patients with chronic HBV infection; it reflects the progression and metastatic potential of the tumour.