Serum Soluble Fas Levels and Incidence of Liver Cancer in Nested Case–Control Study

Fas (CD95/Apo-1) is a cell surface receptor protein that can induce programmed cell death (apoptosis) through its cytosolic tail after binding to its specific ligand, Fas ligand (FasL/CD95L/CD178; refs. 1–3). Fas and FasL are crucial to homeostasis. Fas receptor is constitutively expressed in various organs, such as the heart, lungs, liver, and kidneys. The liver is particularly sensitive to Fas-mediated induction of apoptosis (4).

Soluble Fas (sFas) receptor is expressed in human cells, including hepatocytes, and can specifically inhibit Fas-mediated apoptosis (5). Cells obtain sFas in one of two ways: (i) by alternative splicing (6), or (ii) by matrix metalloproteinase-7 (MMP-7/matrilysin) cleaving Fas to generate sFas (7). Matrilysin can be produced by cancer cells and may contribute to an apoptosis-resistant phenotype, ultimately promoting immune escape (8). Thus, sFas might play an important role in tumor progression (1, 2, 9–13).

Cases with chronic hepatitis (CH), liver cirrhosis (LC), and hepatocellular carcinoma (HCC) caused by hepatitis C virus (HCV) show significantly increased levels of sFas compared with normal controls (14–16). Although the increase of sFas in HCC was also significantly higher than that in CH, positive hepatic expression of Fas antigen was significantly lower in HCC compared with CH. Expressions of sFas and hepatic Fas in HCV-induced liver disease might thus provide clues to the mechanisms of liver injury, fibrosis, and carcinogenesis caused by death receptors. Both the higher degree of hepatic fibrosis and the lower expression of hepatic Fas protein are correlated with increased incidence of HCC (16). In addition, sFas is associated with the severity of fibrosis (17).

Levels of sFas were found to be significantly higher and levels of soluble FasL were significantly lower in patients with HCC than in controls (15). Levels of sFas were higher in patients with multiple tumors than in those with a solitary mass. In all patients with a solitary mass and elevated sFas levels, surgical removal reduced their sFas to negative levels within 1 week (14). Messenger RNA for sFas was also found to be expressed in a vast majority of HCCs (84%; ref. 18). Another study found that sFas was supplied by normal hepatocytes and tumor-infiltrating mononuclear cells rather than by tumor cells (15). Carcinoma cells may thus eliminate Fas expression in themselves and force hepatocytes and mononuclear cells to produce sFas to achieve immune escape and facilitate dissemination (19).

Few epidemiologic studies have examined the relationship between serum sFas level and carcinogenesis. Serum sFas may not be a suitable marker for identifying women at increased risk of ovarian cancer (20). In a Japan Collaborative Cohort (JACC) study, the risk of total cancer mortality was increased according to sFas levels (21). High serum sFas level was associated with the incidence of gastric cancer in women (22). However, serum sFas levels were not associated with the risk of pancreatic cancer death (23). Nevertheless, no reports have clarified the relationship between serum sFas level and the incidence of liver cancer. We therefore investigated the relationship between sFas and the incidence of liver cancer in a case–control study nested in a prospective JACC Study.

We conducted a prospective case–control study nested in the JACC Study, which evaluated cancer risks associated with lifestyle factors. Details about the JACC Study have been provided elsewhere (24–26). In brief, a primary survey was undertaken between 1988 and 1990, during which time 110,585 healthy individuals (age range, 40–79 years) from 45 community areas throughout Japan received a general health checkup and were enrolled as a basic cohort. The subjects completed a questionnaire that included information about lifestyle factors, demographic characteristics, and medical histories. Almost 35% of cohort participants (39,242 participants) voluntarily provided blood samples, which were kept at −80°C until biochemical assays were performed.

Written informed consent was obtained from all subjects by having the study participants sign the cover of the questionnaire in the majority of study areas. However, it was obtained at the group level in a few areas because the concept of informed consent was not popularized during the 1980s in Japan. In that case, the municipality head gave the consent to participation representing the participants living in that area. The current study was conducted in accordance with International Ethical Guidelines for Biomedical Research Involving Human Subjects and Declaration of Helsinki. This study was approved by the human ethics review committee at Hokkaido University.

In 24 of the 45 communities, the incidence of malignant neoplasms was followed, as population-based cancer registry systems were not established in 21 communities in the 1990s (26). Subjects were followed up from the primary survey, excluding participants with any history of malignant neoplasm at the primary survey. Participants (6,402 participants, 5.8%) who moved away from the original area were treated as dropouts from the current study, because deaths after moves could not be identified under the follow-up system used. The occurrence of liver cancer was confirmed from population-based tumor registries or by reviewing the records of local major hospitals. According to the International Statistical Classification of Diseases and Related Health Problems 10th Revision (27), we defined liver cancer as C22 (malignant neoplasm of the liver and intrahepatic bile ducts). Subjects diagnosed with liver cancer by 1997 were regarded as cases for the purposes of the current study. For each case, we selected 2 or 3 controls, matched for sex, age, and residential community. If 4 or more controls were available for the matching, we randomly selected 3. However, less than 3 controls were chosen in some cases depending on the selection criteria (28). The current analysis involved 86 cases and 249 control subjects. Cases contained 71 HCC (C22.0, 83%) and 6 intrahepatic bile duct carcinoma (C22.1, 7%).

Serum concentrations of sFas were assayed at a single laboratory (SRL, Tokyo, Japan) by enzyme-linked immune-adsorbent assay using commercially available kits (MBL, Nagoya, Japan) by trained staff blinded to case or control status in 1999 and 2000. Details of the assay for serum sFas level has been described elsewhere (29). Assays for both hepatitis B virus surface antigen and HCV antibody (3rd generation) were also performed in the SRL laboratory (30).

Proportions and mean values of starting point characteristics between cases and controls were evaluated using the χ² test or t test. Association between sFas and follow-up time interval among cases was assessed using one-way ANOVA. Results are presented as means ± SD. Values of P < 0.05 were considered to indicate statistical significance. Serum concentrations were divided into tertiles based on distributions of serum concentrations of controls by sex, with the first tertile used as a reference. In male subjects, sFas ranges for tertiles 1–3 were <0.9, 0.9–1.7, and >1.7 ng/mL, respectively. In female subjects, sFas ranges for tertiles 1–3 were <0.9, 0.9–1.9, and >1.9 ng/mL, respectively.

ORs for the incidence of liver cancer associated with serum sFas levels were assessed using conditional logistic regression. ORs were controlled for alcohol consumption (never, former, or current drinker, or missing), cigarette smoking status (never, former, or current smoker, or missing), body mass index (BMI, computed as weight in kilograms divided by the square of the height in meters; <18.5, 18.5–24.9, or ≥25.0 kg/m², or missing), and hepatitis viral infection. The statistical significance of trends across exposure tertiles was evaluated by including ordinal terms for each tertile of sFas and entering the variable as a continuous term in this model. Statistical tests for the interaction effects between selected variables (sex and age) and sFas on liver cancer risk were performed by adding an interaction term in the models. All P values and 95% confidence intervals (CI) presented were based on two-sided tests.

The data generated in this study are not publicly available due to not having permission from participants to open data set and rule of the JACC Study but are available upon reasonable request.

Table 1 shows the baseline characteristics of cases and controls. No significant differences in weight, height, BMI, or cigarette smoking were apparent between cases and controls. For alcohol intake, the percentage of never drinkers was higher in controls than in cases. For hepatitis viral infection, the percentage of positive individuals was higher in cases than in controls. When comparing all subjects, serum sFas levels were significantly higher in cases than in controls. In the control group, serum sFas levels were significantly higher in women (2.37 ± 0.78 ng/mL) than in men (2.16 ± 0.65 ng/mL; P = 0.022), so the sex-specific ranges were used for tertiles of sFas. sFas concentration was higher in control subjects with hepatitis viral infection than those without (P = 0.03, 2.55 ± 0.80 ng/mL and 2.19 ± 0.68 ng/mL, respectively). The follow-up time from blood draw to cancer diagnosis were divided to 3 groups, as follows; ≤3, > 3–≤ 6, and >6 years (numbers of case were 26, 27, and 33, respectively). sFas levels in liver cancer cases were not different among the follow-up time intervals (P = 0.257, 3.51 ± 1.46 ng/mL, 3.00 ± 1.30 ng/mL, and 3.00 ± 1.18 ng/mL, respectively).

Serum sFas was associated with the risk of liver cancer (P_trend < 0.001, Table 2). Moreover, subjects in the third tertile showed a higher risk compared with the first tertile (OR, 7.49; 95% CI, 3.22–17.41). The association was attenuated but still present after adjusting for BMI, alcohol consumption, smoking habit, and hepatitis viral infection (P_trend = 0.003). Participants in the third tertile displayed higher risk compared with the first tertile (OR, 3.53; 95% CI, 1.28–9.69).

As HCC (C22.0) is a major type of liver cancers (C22), we assessed the relationship between serum sFas levels and risk of HCC (71 cases, 205 controls; Table 2). A high sFas level correlated with increased risk of HCC (P_trend < 0.001). Participants in the third tertile exhibited higher risk compared with the first tertile (OR, 8.63; 95% CI, 3.32–22.43). After adjustments, the association was diminished but still existing (P_trend = 0.005) and subjects in the third tertile had higher risk compared with the first tertile (OR, 3.97; 95% CI, 1.20–13.11).

To investigate the dependence on sex, we calculated ORs in subgroups (Table 3). In male subjects, serum sFas level correlated with risk of liver cancer (P_trend < 0.001). Participants in the third tertile displayed a higher risk compared with the first tertile (OR, 14.87; 95% CI, 4.12–57.73). After controlling for BMI, alcohol intake, tobacco smoking habit, and hepatitis viral infection, the association was attenuated but still present (P_trend = 0.003) and subjects in the third tertile exhibited higher risk compared with the first tertile (OR, 5.40; 95% CI, 1.29–22.54). Among female participants, serum sFas level was related to the risk of liver cancer (P_trend = 0.011). Participants in the third tertile showed higher risk compared with the first tertile (OR, 3.65; 95% CI, 1.17–11.36). After full adjustments, however, the results were not verified (P_trend = 0.372). The interaction effect between sex and sFas on liver cancer risk was assessed and no significant interaction were noted (P_interaction = 0.080). Then, we analyzed associations between serum sFas and the incidence of HCC by sex, the same trends were obtained (Supplementary Table S1).

To assess the dependence on age, ORs were analyzed in subgroups (Table 3). In non-elderly participants (< 65 years old), a high serum level of sFas correlated with an increased risk of liver cancer (P_trend < 0.001). Subjects in the third tertile displayed higher risk compared with the first tertile (OR, 6.74; 95% CI, 2.18–20.87). After controlling for BMI, drinking, smoking, and hepatitis viral infection, almost the same results were obtained, but were not statistically significant (P_trend = 0.088). In elderly individuals (≥ 65 years old), a high sFas level correlated with future risk of liver cancer (P_trend < 0.001). Participants in the third tertile had higher risk compared with the first tertile (OR, 8.56; 95% CI, 2.44–30.06). After full adjustments, the association was attenuated but still existed (P_trend = 0.008). Subjects in the third tertile displayed higher risk compared with the first tertile (OR, 6.08; 95% CI, 1.31–28.31). The interaction effect between age and sFas on liver cancer risk was assessed and no significant interactions were observed (P_interaction = 0.980). We then analyzed associations between sFas levels and HCC risk in two age groups, high sFas was related to liver cancer risk in both groups (Supplementary Table S1).

The association between sFas and liver cancer risk in subjects with or without hepatitis viral infection was assessed (Supplementary Table S2). Serum concentrations were divided into tertiles based on distributions of serum levels of controls by hepatitis viral infection status. In subjects with hepatitis viral infection, high sFas was related to liver cancer risk (P_trend < 0.001), and participants in the third tertile had higher risk compared with the first tertile (OR, 11.20; 95% CI, 2.98–41.70). However, in subject without hepatitis viral infection, sFas levels was not associate with liver cancer risk.

To exclude possible effects of latent cancers on sFas levels, we limited the analysis to participants followed up for over 3 years (60 cases, 173 controls; Table 2). A high serum sFas level correlated with increased risk of liver cancer (P_trend <0.001). Subjects in the third tertile exhibited higher risk compared with the first tertile (OR, 5.14; 95% CI, 2.08–12.74). After adjustments, the association was attenuated but still present (P_trend = 0.033) and participants in the third tertile had higher risk compared with the first tertile (OR, 2.94; 95% CI, 0.94–9.14). When we assessed on HCC cases only (Supplementary Table S1), the association after adjustments was still present (P_trend = 0.024) and participants in the third tertile had higher risk compared with the first tertile (OR, 3.59; 95% CI, 0.95–13.51).

The current study revealed that serum sFas level was associated with the risk of liver cancer. This is consistent with a previous report from the JACC Study that serum sFas was associated with overall cancer mortality (21). In addition, high serum sFas levels were associated with higher risk of gastric cancer developing in women (22). From the JACC Study, subjects developing liver cancer in the future displayed the highest mean serum levels of sFas among subjects with gastric, colorectal, liver, biliary tract, and lung cancers (21). Thus, sFas may show a particularly strong relationship with carcinogenesis in the liver. As sFas binds to FasL and averts Fas-induced apoptosis, high serum sFas levels seem to increase the risk of developing liver cancer.

Serum sFas level was reportedly increased in patients with CH, LC, and HCC compared with controls (16) and also correlated with the stage of HCV-positive liver disease (31). In this cohort, sFas was higher in control with hepatitis viral infection than those without. As we performed analyses after adjusting for hepatitis B/C virus infection and major type (83%) of liver cancer in this cohort was HCC, those results might not contradict our finding that high sFas levels reflect the risk of developing liver cancer.

The association between sFas level and risk of liver cancer was clear in men, but weaker in woman. Several factors may contribute to such sex differences. Among control subjects in this cohort, sFas was significantly higher in females (P = 0.022). The relationship between sFas level and risk of developing liver cancer might thus have chance to become stronger in men. A previous result from the JACC Study was that serum sFas levels were significantly higher in smokers and past smokers than in never smokers (32). In this cohort, few men were never smokers, while most women were never smokers. Indeed, the highest sFas value was 3.35 ± 1.51 ng/mL in male case with smoking/past-smoking group and the lowest one was 1.91 ± 0.45 ng/mL in male control with nonsmoking (Supplementary Table S3). Four female groups were values between them. Thus, a second possibility is that smoking habits might have affected the results.

Another previous result of the JACC Study was that obesity and dyslipidemia were associated with significantly higher sFas levels in men, but not in women. Furthermore, the number of metabolic syndrome components showed a positive association with risk prevalence in men, but not in women (33). Given the recent increase in liver cancer derived from nonalcoholic steatohepatitis, higher sFas levels may have been a factor in the higher risk of liver cancer in men than in women.

One advantage of this study was that samples were from a large-scale study with 110,585 subjects. However, several limitations to the current study need to be considered. First, serum concentrations of sFas were measured only once, in the baseline survey. We therefore could not investigate sequential changes in association with tumorigenesis. Another limitation was that some data about BMI, drinking, and smoking were missing from the JACC Study, which used a self-administered questionnaire format (24–26). A third limitation was that we measured only sFas, not other Fas-associated molecules, including soluble FasL. A fourth limitation was the small number of liver cancer cases, in particular in the subgroup analyses. A fifth limitation was a relatively short period of follow-up, which limited the ability to examine whether the association between sFas and liver cancer risk would change over time.

In conclusion, high serum sFas level appears to be related to future risk of liver cancer.

A. Tamakoshi reports grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and grants from the Ministry of Health, Labor and Welfare, Health and Labor Sciences of Japan during the conduct of the study. No disclosures were reported by the other authors.

Y. Adachi: Investigation, writing–original draft. M. Nojima: Resources, data curation, methodology. M. Mori: Supervision, project administration, writing–review and editing. T. Kubo: Investigation, writing-review and editing. N. Akutsu: Investigation, writing-review and editing. Y. Sasaki: Investigation, writing-review and editing. H. Nakase: Supervision, writing-review and editing. Y. Lin: Visualization, methodology, writing-review and editing. Y. Kurozawa: Resources, project administration, writing-review and editing. K. Wakai: Supervision, project administration, writing-review and editing. A. Tamakoshi: Resources, supervision, funding acquisition, project administration.

This work was supported by Grants-in-Aid for Scientific Research on Priority Areas of Cancer; and Grants-in-Aid for Scientific Research on Priority Areas of Cancer Epidemiology from MEXT [MonbuKagaku-sho; nos. 20390156 (to A. Tamakoshi) and 26293138 (to A. Tamakoshi and K. Wakai)] and JSPS KAKENHI Grant Number JP 16H06277 (to A. Tamakoshi, K. Wakai, and M. Mori) and 21K08801 (to Y. Adachi and Y.Sasaki). The other members of the JACC Study are Yoshihiro Kaneko, Ichiro Tsuji, Yosikazu Nakamura, Hiroyasu Iso, Kazumasa Yamagishi, Haruo Mikami, Michiko Kurosawa, Yoshiharu Hoshiyama, Naohito Tanabe, Koji Tamakoshi, Masahiko Ando, Koji Suzuki, Shuji Hashimoto, Hiroshi Yatsuya, Shogo Kikuchi, Yasuhiko Wada, Satoe Okabayashi, Kotaro Ozasa, Kazuya Mikami, Kiyomi Sakata, Yoshihisa Fujino, and Akira Shibata.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.