Clinical design and patient selection: A prospective study was conducted in a cohort of patients with HCV- related cirrhosis followed up at the Division of Clinical Gastroenterology and Hepatology, Hospital das Clínicas of the Department of Gastroenterology, University of São Paulo School of Medicine(HCFMUSP), Brazil, a tertiary healthcare center, between 2011 and 2016. Inclusion criteria: HCV polymerase chain reaction RNA positivity for at least 6 months, and clinical or histopathological diagnosis of chronic HCV with a minimum baseline liver stiffness measument (LSM) of 12 kPa by TE. Exclusion criteria: patient under 18 years of age; hepatitis B virus or human immunodeficiency virus co-infection, significant and current alcohol intake (> 100 g/week),other chronic liver disease, non-cirrhotic portal hypertension, history of liver transplantation, or refusal to participate in the study.
Study protocol and variables evaluated: We collected the anthropometric and clinical data at inclusion to the study: sex, age, weight, height, body mass index (BMI), presence of diabetes mellitus, past alcohol ingestion and smoking status. Also, these patients were evaluated with serum biochemistry and scores, including: HCV genotype, alpha-fetoprotein (AFP), alanine aminotransferase (ALT), alkaline phosphatase (AP), aspartate aminotransferase (AST), total bilirubin (TB), gamma-glutamyltranspeptidase (γGT), platelet count, international normalized ratio (INR), albumin, urea, glucose and creatinine and Child-Pugh and MELD scores.
Patients were submitted to abdominal ultrasound examination (ACUSON S2000®, Siemens), with transducer 4V1 at the same time as TE evaluation, both performed by a skilled operator, at inclusion. The absence of focal suspected malignant liver lesions was also registered. We also included in the clinical evaluation the non-invasive liver fibrosis characterization by APRI and FIB-4. LSM and steatosis grade with Controlled attenuation parameter were both obtained using the FibroScan 402 device powered by VCTE (EchoSens, Paris, France), equipped with the standard M probe. TE examination was performed according to previous description.
After study inclusion, we systematically followed these patients every 6 months for HCC detection with US and serum AFP measurements, in accordance with routine institutional clinical practice. During the study period, we documented the HCV therapy and viral eradication. We also analyzed these variables in respect to the study outcome of HCC occurrence.
We followed the international guidelines to the diagnosis of HCC based on radiological criteria by multiphase contrast-enhanced magnetic resonance imaging (MRI) or computed tomography (CT) scan and/or liver biopsy.[17, 16]
Besides HCC development analysis, the following outcomes were provided: HCC stage in relation to Milan criteria, tumor therapy, and evolution to mortality.
Ethical considerations: The Ethics Committee of the HCFMUSP (number 6570) approved the study according to the ethical guidelines of the 1975 Declaration of Helsinki. We obtained from all participants the authorization of informed consent.
Statistical analysis: The Descriptive statistics (mean, standard deviation, minimum and maximum, and median values) were calculated. Univariate (continuous and binary with log rank) and multivariate Cox logistic regression analysis were performed, and hazard ratio (HR) for HCC occurrence was calculated, in addition to a 95% confidence interval (CI). To avoid collinearity among the significant variables mentioned on univariable analysis, we built models including LSM cutoff > 21.1 kPa in all of them, that was almost exclusive independently associated with HCC development. The routine liver function markers as MELD score and the fibrosis methods like FIB-4, APRI score and TE composed the final selected multivariable model. We used the Lausen's test to find the best cutoff point for HCC occurrence. In order to estimate the incidence of HCC and survival rate we applied the log rank test and Kaplan-Meier. Two-tailed test was used and a probability value of < 0.05 was considered significant. A biomedical statistician (João Italo França) using IBM Corp. Released 2010 conducted all statistical analyses (IBM SPSS Statistics for Windows Version 19.0).