The present study included liver cancer patients who underwent surgical treatment at Hanyang University Hospital (Seoul, Korea) between January 1991 and September 2013, and 166 patients with complete follow-up data were retrospectively enrolled. We excluded 17 cases with unavailable tumor tissue or who underwent preoperative treatment (embolization or ablation). Finally, 149 cases were included in the present study. To compare the ASAP1 expression between HCC and normal liver parenchyma, we additionally obtained 12 normal liver tissues. Two pathologists (HK and SP) reviewed all tissue slides and pathology reports used at diagnosis. We obtained tumor size, histological grade, vessel invasion, perineural invasion, tumor focality, proliferation index, and pathological stage. Edmondson and Steiner grading system was used to evaluate histological grade, and the pathological stage was based on the 8th edition of the American Joint Committee on Cancer (AJCC). The proliferation index was evaluated using IHC staining for Ki-67 protein. Medical records were reviewed to obtain clinical characteristics, including patient age, sex, etiological factors, underlying diseases, alpha-fetoprotein (AFP) level, Child-Pugh class, Barcelona Clinic Liver Cancer (BCLC) stage, and follow-up data. The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of the Hanyang University Hospital (IRB file No. HYUH 2015-12-017). The requirement for informed consent was waived.

A manual tissue microarrayer (Unitma, Seoul, Korea) and formalin-fixed paraffin-embedded tissues were used for tissue microarray (TMA) construction. We selected a representative portion of the tumor that contained almost no necrosis by light microscopy and obtained tissue cores (2.0 mm in diameter, 1 core per case) from the corresponding donor block. Then, the tissue cores were transferred to a recipient block consisting of 8 × 5 samples.

IHC staining for ASAP1 was performed with 4-μm-thick sections using the Benchmark XT automated staining system (Ventana Medical Systems, Tucson, AZ, United States). Heat-induced epitope retrieval is performed with CC1 Tris-EDTA buffer (Ventana Medical Systems, Tucson, AZ, United States) at 100°C for 80 min. Subsequent procedures were performed with OptiView DAB IHC Detection Kit (Ventana Medical Systems, Tucson, AZ, United States). For blocking endogenous peroxidase, OptiView Peroxidase Inhibitor was used, and Optiview DAB was used as Chromogen. Counterstaining was performed using modified Mayer’s hematoxylin (Hematoxylin II). The recombinant anti-ASAP1 antibody (ab125729, Abcam, Cambridge, UK; diluted 1:200) was used as the primary antibody. IHC staining for Ki-67 (M7240, Dako, Glostrup, Denmark) was performed using the Bond-Max automated immunostainer (Leica Biosystems, Wetzlar, Germany).

As reported in previous studies (10, 12, 14-16, 19), we considered cytoplasmic staining of the tumor cells to be positive. All IHC sections were evaluated using light microscopy by two pathologists (SB and SP) without access to clinical data. For semi-quantitative assessment, the immunoreactive score (IRS) was calculated as the product of staining intensity and proportion of positive cells. The intensity of staining was categorized as 0–3 (0: negative, 1: weak, 2: moderate, and 3: strong), and the proportion of staining was assigned as 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51–75%), and 4 (>75%). A receiver operating characteristics (ROC) curve analysis was used to determine the optimal cutoff value, and we divided the cases into low and high expression groups (IRS ≤4 and IRS >4, respectively).

Kaplan-Meier plotter (KM plotter, http://kmplot.com/analysis/) was used to predict the association between the mRNA expression level of ASAP1 and the survival of HCC patients. KM plotter is a web-based tool based on The Cancer Genome Atlas (TCGA) data and includes 364 HCC cases with available follow-up data (22). Using this tool, HCC cases were divided into two groups (high or low expression), and the effect of ASAP1 expression on the prognosis was estimated through survival analysis (23).

We used SPSS software version 25.0 (IBM, Armonk, NY, United States) for statistical analysis. ASAP1 expression in HCC and normal liver tissue was compared using the Mann-Whitney U test. The correlations between ASAP1 expression and clinicopathological characteristics were analyzed using Pearson’s chi-square (χ2) or Fisher’s exact test. Overall survival (OS) and recurrence-free survival (RFS) of HCC patients were evaluated using the Kaplan-Meier method with log-rank test. The Cox proportional hazards model was used to determine the significant prognostic factors. We used univariate analysis to determine the effect of individual parameters (ASAP1 expression and other clinicopathological factors) on OS or RFS. Then, multivariate analysis was used to analyze how prognostic factors jointly affect. A two-tailed p-value < 0.05 was considered statistically significant.

The median age of the patients was 55 years (range: 28–87 years), and the male/female ratio was 3.02:1. Among etiological factors, HBV (123 cases, 82.6%) was the most common, followed by alcohol intake (10 cases, 6.7%) and HCV (7 cases, 4.7%). Based on the Edmondson and Steiner grading system, 12 cases (8.1%) were grade 1, 54 cases (36.2%) were grade 2, 70 cases (47.0%) were grade 3, and 13 cases (8.7%) were grade 4. Vessel invasion was identified in 59 cases (39.6%). Most patients were stage I or II (126 cases, 84.6%) according to the 8th AJCC staging system. Among patients, 65 (43.6%) received only curative surgery, 5 (3.4%) received radiofrequency ablation, 68 (45.6%) received chemoembolization, 6 (4.0%) received systemic chemotherapy, and 5 (3.4%) received combined treatment. The clinicopathological characteristics of patients with HCC are summarized in Table 1.

We evaluated ASAP1 expression in 12 normal liver tissues and 149 HCC tissues. The mean IRS of ASAP1 expression was 2.50 (±1.88) in normal liver tissues and 6.26 (±3.16) in HCC tissues. The mean rank of IRS in HCC was significantly higher than that of normal liver tissues (p = 0.001, Mann-Whitney U test).

Representative photomicrographs of ASAP1 IHC are presented in Figure 1, and we presented raw data including IHC results and clinicopathological characteristics for each case as Supplementary Material. Among the 149 cases, 89 (59.7%) were classified as high ASAP1 expression. High ASAP1 expression was significantly associated with male patients (p = 0.018), higher histological grade (p = 0.013), vessel invasion (p = 0.021), and higher stage (p = 0.020). High ASAP1 expression was identified more frequently in cirrhotic HCC than in non-cirrhotic HCC, however, it was not statistically significant (p = 0.054). The correlations between ASAP1 expression and clinicopathological characteristics are summarized in Table 2.

Based on the Kaplan-Meier method, high ASAP1 expression was significantly associated with shorter OS and RFS (p = 0.041 and p = 0.008, respectively; Figures 2A,B) in 149 patients with HCC. Univariate analyses for OS and RFS showed ASAP1 expression as a significant prognostic factor (p = 0.042 and p = 0.009, respectively). Among other clinicopathological factors, higher histological grade (p = 0.008), vessel invasion (p < 0.001), and higher stage (p = 0.001) were associated with shorter OS. Higher histological grade (p = 0.003), vessel invasion (p < 0.001), perineural invasion (p = 0.010), and higher stage (p < 0.001) were associated with shorter RFS. In multivariate analyses, short OS and RFS were significantly associated with higher histological grade (p = 0.009 and p = 0.003, respectively) and higher AJCC stage (p = 0.003 and p < 0.001, respectively); however, high ASAP1 expression did not remain statistically significant (Table 3). Similar to the results of the present study, the web-based analysis using the KM plotter demonstrated that high mRNA expression of ASAP1 was associated with shorter OS (p = 0.001, FDR = 0.2; Figure 2C).