Study population In the present hospital-based case-control study, we observed 80 patients with HCC who attended a tertiary care facility from November 2018 to July 2019. This facility is the largest postgraduate public medical university in Bangladesh, located in the capital city, Dhaka, and acts as a reference center for patients having unmanageable diseases. After matching the demographic characteristics of HCC patients, such as age, sex, income, and sociodemographic status, 101 control subjects were chosen. Among the patients visiting public hospitals in Bangladesh, most are the poor and lower-middle-income groups. Hence, we assumed that only this group of people represented the study population in this particular research study. This study was approved by the ethical committee of that particular tertiary hospital and was conducted according to the European Association for the Study of the Liver (EASL) clinical practice guidelines for the management of hepatocellular carcinoma. We clarified to the participants the purpose and procedure of the study in detail, their benefits and risks, and subsequently informed consents were obtained from both patients and controls.
Study design and sample size calculation A matched case-control design was used for this study. The sample size was calculated based on a conservative predictor such as intake of leafy vegetables and whether a low amount of intake compared to moderate intake increases the odds of HCC. Assuming a prevalence of the risk factor in the unexposed population to be 50%, to detect an odds ratio (OR) of at least 2.5 with 80% power with 95% confidence would require 162 subjects. The sample size was calculated using the R package epiR.
Patient selection The inclusion criteria for patient choice include both male and female patients with HCC regardless of etiology. The exclusion criteria included a) patients with additional cancer as well as HCC and b) patients with co-morbid conditions such as severe congestive cardiac failure (CCF), ischemic heart disease (IHD), chronic kidney disease (CKD), etc., and not fit for fineneedle aspiration cytology (FNAC).
Diagnosis procedure HCC has been diagnosed on the grounds of clinical and radiological characteristics (ultrasonography and computed tomography), followed by EASL clinical practice guidelines. The confirmation of HCC was done by cytopathology examination, collecting tissues through fineneedle aspiration cytology (FNAC) technique. Patients under 18 years of age were excluded.
Clinical and biochemical evaluation All patients were clinically assessed, and blood pressure level and Body Mass Index (BMI) were recorded. Patients having a BMI of >25 kg/m2 were marked as obese, and patients with a BMI of <25 kg/m2 were considered as non-obese. Patients’ blood samples were drawn under fasting conditions, and the accompanying tests, for example, complete blood count (CBC), albumin, total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), international normalized ratio (INR), and alpha-fetoprotein (AFP) were performed for diagnosis purpose. Barcelona Clinic Liver Cancer (BCLC) staging and Child-Pugh scores were determined from laboratory tests, and clinical features obtained from diagnostic reports. Patients were categorized into four BCLC stages, for example, Early-stage, A; Intermediate stage, B; Advanced stage, C; and Terminal stage, D. BCLC staging was determined by physician, and patients’ performance status, tumor size and number, Child-Pugh score and portal vein involvement were documented according to EASL guidelines.
Data collection We collected patients’ demographic, clinical, and biochemical information through an interview with a structured questionnaire. Demographic information, such as age, sex, education, earnings, food habits, HCC etiologies, and first presenting symptoms were collected. Diverse clinical and biochemical information such as serum levels of total bilirubin, albumin, INR, AFP, aspartate aminotransferase, alanine aminotransferase, presence of ascites, hepatic encephalopathy, hepatomegaly, splenomegaly, was collected from patient’s diagnostics reports. A liver radiologist deliberately looked into patients’ computed tomography (CT), and ultrasonography report and the size, area, number of tumor lesions, portal vein thrombosis were noted. Child-Pugh classification and BCLC staging of the patients were recorded.
Assessment of dietary habit The data on food consumption per capita were obtained by an interview-based, structured questionnaire. The survey incorporated the food habit pattern from both cases (n = 80) and controls (n = 101). Through a case-control statistical analysis, we explored the link between food habit patterns and HCC development. In the case of fruits and vegetables, seasonal consumption and the corresponding duration are subject to variation. The dietary items included 82 foods or food groups and were divided into 9 sections: i) rice (primary course); ii) bread, and roti (secondary course); iii) leafy vegetables (water spinach, pumpkin leaves, taro stem, Indian spinach, spinach, red amaranth, cauliflower, cabbage); iv) non-leafy vegetables (okra, tomato, balsam apple, eggplant, carrot, pumpkin, potatoes, sweet potatoes); v) meat and meatbased food items such as burger, sandwich; vi) fish (both river and ocean); vii) milk, tea, coffee, sugar, tea with condensed milk; viii) fruit (litchis, mangoes, jackfruits, blackberries, dates, guavas, pineapple, papayas, bananas, watermelon, coconuts, apples, grapes, oranges, tropical fruits, etc.); ix) sweets, rice-based desserts, and soft drinks. The selection of food items was based on foods regularly consumed by the Bangladeshi people. The standard serving size was obtained from the dietary guidelines from BIRDEM (Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine, and Metabolic Disorders). We focused on the above-mentioned food items to explore the relationship between these food groups and the advancement of HCC in the Bangladeshi population. The consumption rate among our casecontrol population of targeted food groups was transformed into g/day or ml/day. In the case of tea it was considered as cup/day. Consumption of specific food items of more than the suggested value is defined as “high intake.” In contrast, the consumption of specific food items of lower than the suggested value is defined as “low intake.” A recent report suggested an inverse association of tea intake with primary liver cancer; however, the preparation of tea in Bangladesh or South Asia is different compared to Western countries. In Bangladesh, condensed milk with sugar is commonly used to prepare the tea. Moreover, the tea leaves are usually continuously boiled for a long time. Hence, we investigated the primary or combined effect of tea drinking on the risk of HCC with or without the presence of other risk factors.
Statistical analysis Data management and statistical analysis were performed using R statistical software. Continuous variables were expressed as mean ± standard deviation, and categorical variables were presented as numbers and percentages or frequencies. The Chi-square (χ2) test with continuity correction was employed to find significant differences between groups. Crude and adjusted odds ratios were calculated using the multiple logistic regression model. For the regression strategy, we first performed bivariate analysis of the potential factors with the outcome variable of interest. If the bivariate results were significant at 20% level, we considered them in the regression model. In addition, certain variables were included in the model for their importance from demographic and clinical perspectives regardless of the results of the bivariate analysis. These included age, sex, diabetes status, and weight status (overweight or normal). The analysis was performed to assess the effect of risk factors on the likelihood of developing HCC. A p-value <0.05 was considered as statistically significant.