Research Design and Participants
The Kailuan study is a prospective cohort study based on community population in Tangshan city, northern China (Trial Registration Number: ChiCTR-TNRC-11001489) [18,19]. Since 2006, the employees( ≥18 years, including the retired) of the Kailuan Group, Tangshan City, were invited to participate in biennial health check-up at 11 affiliated hospitals. The Kailuan Study was conducted to estimate the prevalence chronic disease, nutritional disorders and major risk factors for these diseases. The details of the study design and procedures are available elsewhere[18,19]. From 2006 to 2007, 101,510 participants completed the survey, which constituted Kailuan Study I. From 2008 to 2009, 2010 to 2011, both 25,337 adults and 10,519 adults formed the Kailuan Study II and Kailuan Study III, respectively. And all participants(137,366) underwent questionnaire survey, clinical and laboratory examinations.
In our current study, we excluded 463 subjects who had PLC and a history of malignant tumors at the baseline, excluded 1,830 and 490 subjects with missing information of TC and ALT, respectively. According to the adult standard of American College of Gastroenterology(ACG)[20], 19,611 subjects with abnormal data of ALT were excluded (male serum ALT >33U/L or female serum ALT >25U/L). A total of 114,972 individuals were finally included in the current analyses(Fig 1).This study was approved by Ethics Committee of Kailuan General Hospital and in compliance with the Declaration of Helsinki. Informed consent was obtained from the participants.
Assessment of Exposure Factor and Other Related Laboratory
At 7:00-9:00 a.m., the fasting (8h-12h) elbow venous blood of all participants was collected about 5 ml and placed in a vacuum tube that containing EDTA. The upper serum was taken after centrifugating for 10 minutes at 3000 rotations per minute at 24℃. The serum samples were assured to complete the detection within 4 hours. Serum TC and serum ALT were determined by professional laboratory physicians using an autoanalyzer(Hitachi 747; Hitachi, Tokyo, Japan) and strictly following the instructions of reagents. TC was measured enzymatically(CHOD-PAP) with an upper limit of detection of 20.68 mmol/L. ALT(ALT, in U/L) was measured by enzymatic rate methed. The upper limit of detection of ALT was 1000 U/L. Other biochemical parameters, including serum high-density lipoprotein cholesterol(HDL-C), triglyceride(TG), hemoglobin(HGB), fasting blood glucose(FBG), hypersensitive C-reactive protein(hs-CRP) were determined by automatic biochemical analyzer(Hitachi 747; Hitachi, Tokyo, Japan). All plasma samples were analyzed at the central laboratory of Kailuan General Hospital.
Assessment of Other Relevant Variables
On the day of physical examination, the trained medical and nursing personnel would assist the participants to fill in the questionnaires via face-to-face interviews. The information of the questionnaire included: age, gender, smoking habits, drinking status, physical activity, past medical history and so on(eg, Hypertension, diabetes mellitus, malignant tumors, etc.)[21,22]. Height and weight were measured by professionally trained staff. BMI was calculated as body weight(kg) divided by the square of height(m2). Hypertension was defined as systolic blood pressure ≥140mmHg, and/or diastolic blood pressure ≥90mmHg, or using antihypertensive medication. Diabetes was defined as FBG ≥7.0mmol/L or use of oral hypoglycemic agent. Smoking was defined as having smoked at least 1 cigarette per day on average for at least 1 year. Alcohol consumption was defined as having taken alcohol of 100mL/day(alcohol contents>50%) of alcohol for more than 1 year. Physical activity was defined as taking exercises more than four times a week, each time lasting at least 30 minutes[23]. Ultrasound diagnosis standard of fatty liver: Comparing the liver echogenicity with the kidney, the diffuse echo enhancement in liver, image of intrahepatic blood vessels and the diaphragm was blurry or invisible[24]. The diagnostic criterias of cirrhosis in ultrasound: the coarse tissue and nodularity in liver surface or parenchyma, with or without ascites and splenomegaly; or subjects with medical history of cirrhosis[24, 25].
Definition and Ascertainment of Outcome Events
During the period from participants' first physical examination to December 31, 2018, subjects which were first diagnosed with hepatocellular carcinoma, intrahepatic cholangiocarcinoma and other liver cancers with unclear types(excluding liver metastasis), we defined as PLC. Follow-up began at the first physical examination, and ended at occurrence of cancer, death, or December 31, 2018, whichever event came first. In our cohort, cancer events were confirmed via biennially health screening with face-to-face questionnaires and medical examinations. Additionally, medical records from Municipal Medical Service System(including medical insurance system and social security system) were checked yearly in detail to obtain outcome information of participants that may have been missed[8]. The outcome information was collected by professionally trained staff, and the CanReg 4.0 software that provided by the International Agency for Research on Cancer of the World Health Organization (IARC/WHO) was used to input and logically verify about new cases of LC. According to the International Classification of Diseases, Tenth Revision(ICD-10), and PLC was defined as C22.
Statistical analysis
Participants were divided into 4 groups according to TC(low level/ non-low level) and ALT(normal level/ normal-high level) status and using the lower quartile(P25) value(4.24 mmol/L) of TC and the upper quartile(P75) value(22 U/L) of ALT as a threshold, respectively. Low TC level was defined as TC less than its P25 value as “TC(-)” group; non-low TC level was defined as TC greater than or equal to its P25 value as “TC(+)” group. Normal ALT level was defined as ALT less than its P75 value as “ALT(-)” group; normal-high ALT level was defined as ALT greater than or equal to its P75 value as “ALT(+)” group[26,27]. Four groups were obtained as follows via cross-matching method: “TC (-) +ALT (+)”, “TC (-) +ALT (-)”, “TC (+) +ALT (+)” and “TC (+) +ALT (-)”. Quantitative data with normal distribution was expressed as mean±standard deviation, one-way analysis of variance was used for multiple comparison between groups. The measurement data with skewed distribution were described as M(P25-P75), the nonparametric Kruskal-Wallis test of variance was used for multiple comparison between groups. Categorical variables were described by percentage and compared using the Chi-square test. Incidence rates were calculated by dividing the number of events by person years of follow up in each group. To investigate the joint effect of TC and ALT for PLC, three dummy variables were included in the models, and “TC(+) + ALT(-)” with minimum incidence in all groups was used as reference group. The Cox proportional hazards model was used to estimate the hazard ratios(HRs) and 95% confidence intervals(CIs) for the separate and joint effect of TC and ALT on PLC. Furthermore, interactive additive model was constructed to further test the joint effect of TC and ALT for PLC risk. We calculated the relative excess risk due to interaction(RERI), proportion of disease attributable to interaction(AP), synergy index(SI) and P value for interaction [28,29].
As sensitivity analyses, we further excluded 2,488 HBsAg positive participants, 231 participants in cirrhosis, 31,567 fatty liver participants, 38 participants who took statins, 11,127 ALT ≥40U/L participants during follow-up, and 13 participants who occurred PLC within 1 year after entry to the cohort, respectively. And the Cox proportional hazards model was repeated again. The data management and all analyses were conducted using SAS statistical software, version 9.4(SAS Institute, Cary, NC). P < 0.05 was considered statistically significant for 2-sided tests.