Patients and DNA extraction

Our study was approved by the ethics committee of Affiliated Nanhua Hospital, University of South China (approval No. 201812). We received written informed consent from the participants, and the participants agreed to the publication of the research data obtained from these collected samples. A total of 10 subjects were included in this study. Nine patients with primary hepatocellular carcinoma, aged 33–68 years, were enrolled from January 2019 to December 2019 in our hospital. The diagnostic criteria of primary liver cancer (meeting any of the following three criteria) was as follows: (1), Two typical imaging manifestations of liver cancer (ultrasound, CT, MRI or selective hepatic arteriography), with a lesion > two cm. (2), A typical imaging manifestation, lesions > two cm, AFP > 400 ng/ml. (3), Positive liver biopsy. Among the patients with primary liver cancer, 1 case was diagnosed by pathological biopsy, and the other 8 cases were diagnosed by clinical diagnosis. Data from two patients with liver metastases (one with gallbladder cancer and liver metastasis, one with rectal cancer and liver metastasis) were also collected. The diagnostic criteria were implemented according to the guidelines for the diagnosis and treatment of gallbladder cancer (2015 Edition) and the Chinese code for the clinical diagnosis and treatment of colorectal cancer. Patients with primary liver cancer complicated by other tumours (such as gastric cancer, lung cancer, cervical cancer, ovarian cancer and prostate cancer), human immunodeficiency virus infection or autoimmune liver disease, alcoholic liver disease, nonalcoholic fatty liver disease, history of other chronic liver diseases, and samples with haemolysis during the test were excluded. According to the inclusion and exclusion criteria, nine cases in the primary HCC group and two cases in the liver metastasis group were collected. However, one patient in the primary liver cancer group whose ctDNA was not qualified after blood extraction was excluded, while the rest qualified for study inclusion, leaving eight cases in the primary HCC group and two cases in the liver metastasis group. Five millilitres of venous blood was collected by a special nurse using an EDTA anticoagulant tube. After balancing, it was placed into a centrifuge (within half an hour of blood drawing) and centrifuged at room temperature for 5 min. After centrifugation, plasma was collected and stored in a two ml EP tube in an ultralow refrigerator at −80 °C until ctDNA was extracted. A GeneRead DNA FFPE Kit (Qiagen) was used to extract ctDNA from plasma following the instructions of the kit. Agarose gel electrophoresis was used to analyse the extent of DNA degradation and whether there was contamination with RNA or protein, and Qubit was used to quantify the DNA concentration (DNA samples with DNA concentrations above 20 ng/µl and 0.2 µg above 0.2 µg were used to build the database).