A total of 324 patients of at least 20 years of age who regularly visited Shinshu University Hospital in Matsumoto, Japan, for HBV infection management in the year 2018 (January 1, 2018, to December 31, 2018) were retrospectively targeted. Patients exhibiting other causes of chronic liver disease, such as alcoholic liver disease, non-alcoholic liver disease, primary biliary cholangitis, or autoimmune hepatitis, were not considered. The study participant selection flowchart is depicted in Figure 1. After the exclusion of 44 cases (2 with acute hepatitis B virus infection and 42 without stored DNA or serum), 280 patients with chronic HBV infection were enrolled in the analysis. The racial background of all patients was uniformly Japanese.
Total bilirubin, alanine aminotransferase, and other relevant biochemical tests were performed using standard methods. HBsAg, HBeAb, and MAC-2 binding protein glycosylation isomer (M2BPGi; Sysmex Co., Kobe, Japan) were measured with a HISCL-5000 (Sysmex Co., Kobe, Japan) system using stocked serum. M2BPGi readings of <1.00 COI, ≥1.00 COI and <3.00 COI, and ≥3.00 COI were judged as negative, 1+, and 2+, respectively.
Determination of liver cirrhosis
Patients with liver cirrhosis were judged as those with histologically proven liver cirrhosis and/or characteristic clinical signs of advanced liver disease by imaging studies, including ultrasonography, computed tomography, and magnetic resonance imaging, in the year 2018.
Determination of HCC
HCC patients were defined as having HCC in the prior quarter century (i.e., between 1993 and 2018) or complicating HCC in 2018. HCC was diagnosed by imaging characteristics, arterial hypervascularity, and venous or delayed phase washout by contrast-enhanced dynamic computed tomography and/or magnetic resonance imaging when a nodular lesion was detected by ultrasonography or a tumor marker was elevated.
Definition of NUC freedom
Among the 133 patients with prior or ongoing NUC treatment, 20 patients had successfully discontinued therapy by the end of 2018 and were defined as NUC free.
HLA class I and KIR genotyping
Whole-genomic DNA was extracted from whole blood samples from all participants using QuickGene-800 assays (Fujifilm, Tokyo, Japan). HLA-Bw4, HLA-C1, and HLA-C2 genotyping 27 as well as KIR genotyping 28 were performed using the polymerase chain reaction with sequence-specific primers. HLA and KIR typing were used to stratify patients into groups according to predicted KIR-ligand interactions and binding affinities. The KIR/HLA pairs of interest were KIR2DL1/2DS1/2DS4-HLA-C2, 2DL2/3/2DS2-HLA-C1, and 3DL1/3DS1-HLA-Bw4. All genotyping was blinded to clinical variables.
Statistical analysis and data visualization were carried out using StatFlex ver. 7.0.11 software (Artech Co., Ltd., Osaka, Japan). Continuous variables were compared using the Mann–Whitney U test. Categorical variables were evaluated by Pearson’s chi-squared test, Fisher’s exact test, or Yates' continuity correction, as appropriate. A P-value of <0.05 was considered statistically significant.