Study population
We carried out a hospital-based case-control study. The study base was the population living in the province of Brescia and born in Italy. The cases were recruited consecutively and prospectively in two Divisions (General Surgery and Hepatology) of the main General Hospital of the province. 96% of the eligible cases agreed to participate in the study. Overall, we enrolled 102 patients with a first diagnosis of HCC (incident cases) in 2015-2018, before they underwent any treatment for the disease. The diagnosis of HCC was based on computerised tomography and was confirmed by histological analyses in 63 cases.
We selected 102 control subjects in the same age range and gender as the cases and without history of cancer or hepatic, endocrine or autoimmune diseases, who were admitted to the same hospital at the same time as the cases. When a subject suitable for the study according to the inclusion criteria was identified, he/she was invited to participate, and, in case of refusal, another individual was chosen and invited to participate. Overall, about 70% of the eligible subjects participated in the study. The enrolled controls were hospitalized for traumatic causes, vascular surgery or other minor surgery.
Both cases and controls provided a fasting blood sample for laboratory determination and were interviewed at the hospital on demographic variables, weight and height, residential and occupational history, smoking habit and alcohol intake by the research personnel. Since a rapid weight loss may determine an increase of PCB serum levels (19, 20), the HCC cases were also questioned about the quantity of, and time at, weight loss in the past.
Total alcohol intake was computed according to the average ethanol content of wine (12 percent by volume), beer (5 percent) and spirits (40 percent) and the frequency and quantity of alcoholic beverages consumed in the past. We considered the intake claimed by the subject during the decade in his/her lifetime with the highest consumption (“peak”), which had provided valuable results for evaluating the dose-effect relationship between alcohol intake and HCC occurrence in previous case-control studies carried out in this area (21). Heavy alcohol intake was defined as consumption of 60 or more grams of ethanol per day for at least 10 years.
The Ethics Committees of the main hospitals in the area and of the Local Health Authority of Brescia approved the project, and each participant provided a written informed consent.
Laboratory analyses
The serum samples were stored at -80° until and analyzed within 6 months since withdrawal. We investigated the following 33 PCB congeners, according to WHO classification (22): 28, 31, 52, 74, 77, 81, 99, 101, 105, 114, 118, 123, 126, 128, 138, 146, 153, 156, 157, 167, 169, 170, 172, 177, 180, 183, 187, 189, 194, 196, 201, 203, 206 and 209.
The PCB analysis was conducted following a previously defined analytical method (23), using an Agilent Technologies 6890N gas chromatograph coupled with an Agilent Technologies MSD 5973 (electron impact ionization, mass filter: quadrupole). A PONA column (Agilent Technologies; 50m x 0.20 mm ID) was used for chromatographic separation with carrier gas Helium. A 2 mL injection at 250 °C was performed by a 7683 Series Injector (Agilent Technologies) in splitless mode with a salinized injection liner (Agilent Technologies; 4mm, 78.5 x 6.5 OD).
The limit of quantification (10 times the signal-to-noise ratio peaks) varied among PCBs but was generally less than 0.1 ng/ml for each congener.
PCB analysis was performed at the Laboratory of Occupational Hygiene and Toxicology, Brescia University, Italy. The Lab participated in inter-comparison programmes for toxicological analyses in biological materials (Institute for Occupational and Social medicine of the university of Erlangen-Nuremberg, D-91054 Erlasngen, Germany), and fulfilled the requirements for congeners 28, 52, 101, 138, 153,180 in occupational and environmental medical fields.
Total PCB serum concentration was calculated as the sum of the 33 PCB congeners. Since PCB concentration is influenced by the amount of serum lipids, the ratio of PCB concentration to the total lipid levels was computed (lipid-adjusted PCB concentration) and expressed as ng/g lipid. We calculated the total lipid concentration from cholesterol and triglyceride levels using the formula proposed by Phillips et al. (24): total serum lipid (g/L) = 2.27 * serum cholesterol (g/L) + triglycerides (g/L) + 0.623.
We also investigated the three main risk factors for HCC in this area, i.e. HBV and HCV infection and alcohol intake (10), as potential risk modifiers of the possible association between PCB exposure and HCC. The presence of HBV and HCV infections was evaluated by testing sera for HBsAg and anti-HCV, respectively, using commercial immunoassays (EIA).
Statistical analysis
The distributions of total PCBs and each congener serum levels were examined using common statistical techniques for exploratory analysis. Due to the asymmetric non-normal distribution of PCB values, the median, range and 90th percentile are reported. The differences in PCB concentration between HCC cases and controls were evaluated using non-parametric methods, particularly the Mann-Whitney test for impaired data. The odds ratios (ORs) for HCC were calculated using the tertiles of serum PCB concentrations in the controls as the cut-offs and adjusting for sex, age, residence, education and presence of one or more risk factors for HCC using multiple logistic regression. The confidence intervals were computed at the 95% level. Furthermore, a test for linear trend was performed using the Wald test on the coefficients based on PCBs as continuous variables.
All the statistical tests were two-sided with a threshold of p=0.05 for refusing the null hypothesis. The statistical analyses were performed using the STATA software for personal computer (Stata Statistical Software release 14.0; Stata Corporation, College Station, Texas).