A case±control study on family history of liver cancer as a risk factorfor hepatocellular carcinoma in North Italy

Francesco Donato1,*, Umberto Gelatti1, Roberta Chiesa1, Alberto Albertini1, Elena Bucella1,Paolo Bo�etta2, Alessandro Tagger3, Maria Lisa Ribero4, Giuseppe Portera3, Michele Fasola3 &Giuseppe Nardi1 for the Brescia HCC Study1Cattedra di Igiene, UniversitaÁ di Brescia, Via Valsabbina 19, 25123 Brescia, Italy; 2International Agency for Researchon Cancer, Lyon, France; 3Istituto di Virologia, UniversitaÁ di Milano, Milan, Italy; 4Istituto di Igiene, UniversitaÁ diMilano, Milan, Italy (*Author for correspondence)

Received 6 January 1999; accepted in revised form 22 April 1999

Key words: alcohol drinking, family history, hepatitis B virus, hepatitis C virus, hepatocellular carcinoma.

Abstract

Objectives: We carried out a case±control study to investigate the role of history of liver cancer in a ®rst-degreerelative as a risk factor for hepatocellular carcinoma (HCC).Methods: Two hundred eighty-seven HCC incident cases and 450 subjects una�ected by liver disease (controls) wereenrolled in the study. Family history of liver cancer and other malignancies and history of alcohol intake werecollected by face-to-face interview. Blood samples were analyzed for HBsAg, anti-HCV and HCV RNA positivity.Results: Family history of liver cancer was associated with HCC (odds ratio [OR] = 2.4; 95% con®dence interval[CI] = 1.2±4.7), whereas family history of other malignancies was not (OR = 1.0; 95% CI = 0.6±1.5). Anincreased OR for family history of liver cancer was found among subjects negative for the other risk factors(OR = 2.0; 95% CI = 0.6±6.9). A synergism of family history of liver cancer was also evident with hepatitis B andhepatitis C virus infection and with heavy alcohol intake.Conclusions: This study suggests a role of family history independent from and interacting with known risk factorsfor hepatocellular carcinoma.

Introduction

Hepatitis B virus (HBV), hepatitis C virus (HCV) andheavy alcohol intake have been de®nitely recognized asthe main causes of hepatocellular carcinoma (HCC)[1, 2], which is the most common type of primary livercancer in western countries. Family history of cancer is arisk factor for many human neoplasms, as has been

shown by population registry-based studies [3, 4]. Noprecise genetic locus has been associated with livercancer, though some markers of susceptibility thatmight modify the risk of developing HCC among peopleexposed to a¯atoxin or infected with HBV have beendescribed [5±7].Familial aggregations of liver cancer have been report-

ed, but they can be explained by common environmentalrisk factors of HCC, mainly HBV infection [8±10]. Somecase±control studies found an association between familyhistory of liver cancer and HCC [11±16]. However, mostof them did not account for possible confounding by themain risk factors of the disease, such as HBV, HCV,alcohol intake and a¯atoxins. Furthermore, while host±environment interactions seem relevant for the develop-ment of several human neoplasms [17], the interactionsbetween family history of liver cancer and environmentalrisk factors have not yet been investigated.

Participants in the Brescia HCC Study: L. Bettini and M.G. De

Tavonatti (I Medicina), G. Pelizzari (II Medicina), E. Radaeli and L.

Biasi (III Medicina), M. Puoti (II Malattie Infettive) F. Bonetti (I

Anatomia Patologica) and C. Leali (Dermatologia) at the Spedali

Civili di Brescia; N. Portolani and M. Ronconi (Cattedra di Chirurgia

Generale II), M. Ghirardi (Cattedra di Chirurgia Generale I), L.

Matricardi (Cattedra di Radiologia), M. Favret (Cattedra di Anatomia

Patologica) at the UniversitaÁ di Brescia; A. Salmi and G. Lanzani

(Medicina), A. Savio (Anatomia Patologica) at the Ospedale S. Orsola

di Brescia; C. Scarcella at ASL Brescia.

Cancer Causes and Control 10: 417±421, 1999. 417Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.

We performed a case±control study to investigate therole of history of liver cancer in a ®rst-degree relative asa risk factor for HCC when alone or combined withother risk factors for the disease. Some results concern-ing the role of HBV and HCV infections and heavyalcohol intake in a subset of the cases and controlsrecruited in this study have been reported [18].

Materials and methods

All patients with HCC born in Italy, living in theprovince of Brescia, aged up to 75 years and admittedto the two main hospitals in the area were enrolled inthe study as cases. A total of 284 incident caseshospitalized between January 1996 and March 1998were enrolled, 210 of whom (73.9%) had a ®rstdiagnosis con®rmed by histology, the rest having beendiagnosed by cytology, ultrasound examination, orbecause they had alpha-fetoprotein serum levels above500 ng/ml. Among HCC cases, 259 (91.2%) had apersonal history of chronic liver disease, which wascirrhosis in most of them (84.2%); 464 subjects,admitted to the departments of Ophthalmology, Der-matology, Urology, Cardiology and General Medicineat the same hospitals and without clinically overt liverdisease or malignant neoplasms, were enrolled ascontrols. The inclusion criteria used for HCC caseswere also applied in control selection. Two controls foreach case were recruited at the same hospitals wherecases were admitted, according to age (�5 years), sexand date of admission. The project was approved by thelocal Ethics Committee and written informed consentwas obtained from all the patients.The cases and controls were interviewed at the

hospital using a standardized questionnaire, whichshowed a high reproducibility for history of alcoholdrinking when used in hospital settings [19]. Inassessing alcohol intake we considered only the periodof maximum consumption before the beginning of theliver disease, according to the average ethanol contentof wine (12% in volume), beer (5%) and spirits (40%).An intake greater than 80 g/day of ethanol wasregarded as ``heavy''. Lifetime tobacco smoking wasalso assessed. However, since no association was foundwith HCC, no data on smoking are shown. Subjectswere classi®ed as having a positive family history ofliver and other cancers if at least one of their parentsor siblings had su�ered or died from the disease. Sincefew subjects reported more than one relative a�ectedby liver cancer, family history of liver cancer wasanalyzed as a dichotomous variable. The mean numberof siblings did not di�er between a larger series of cases

(n = 5.31) and controls (n = 4.72) (p-value of thedi�erence: p = 0.3) so we did not adjust for family sizein the analysis.Blood samples were taken from all subjects by

venipuncture and aliquots were separated. Sera werestored at )80 °C until tested. The presence of HBVmarkers was assessed using commercial enzyme-linkedimmunosorbent assays (ELISA) (Abbott Labs, NorthChicago, IL, USA) and that of anti-HCV antibodiesusing third-generation ELISA (Abbott Labs) andrecombinant immunoblot assay (RIBA) (Ortho Diag-nostic Systems, Raritan, NJ). Total RNA extractedfrom 100 ll of serum using the guanidinium isothio-cyanate±phenol±chloroform method was ampli®ed byreverse transcriptase±polymerase chain reaction (RT-PCR) using nested primers of the 50 noncoding region.The odds ratios (OR) for each risk factor and their95% con®dence intervals (95% CI) were computed byunconditional logistic regression analysis using themaximum-likelihood method [20]. Sex, age, educationand area of residence (town of Brescia versus the restof the province) were included in the models aspossible confounders. The interaction between familyhistory and each other factor was assessed by comput-ing the synergy index S according to Rothman [21] asthe ratio of the relative risk of the joint exposure,minus one, divided by the sum of the relative risks ofthe two factors computed individually, minus two, theOR being used as an estimate of the relative risk. Avalue of S equal or similar to the unit indicates simpleadditivity, whereas a higher value suggests that thee�ect of the joint exposures is greater than the sum ofthe separate e�ects. All the analyses were performedusing the BMDP/Dynamic program for personal com-puter [22].

Results

Table 1 shows the distribution of subjects by sex, age,education and residence. No di�erence was evidentbetween cases and controls with regard to sex and age,whereas cases had on average fewer years of educationand a higher proportion with respect to controls livedin municipalities in the province other than Brescia.The distribution of subjects according to HCV and

HBV infection, alcohol intake and family history of livercancer and other neoplasms, and the corresponding ORvalues, are shown in Table 2. Both HBV and HCVinfections showed a strong association with HCC.Alcohol intake was signi®cantly associated with HCConly for values above 80 g/day. Family history of livercancer was associated with HCC (OR = 2.3; CI = 1.2±

418 F. Donato et al.

4.5), whereas family history of other neoplasms was not(OR = 1.0; CI = 0.6±1.4). There was no di�erence inmean age at diagnosis between HCC cases who reporteda family history of liver cancer (64.2 years; SD = 7.6)and those who did not (63.8 years; SD = 7.4).No demographic characteristic or risk factor apart

from HBsAg was associated with a positive familyhistory of liver cancer: the proportion of HBsAgpositivity was 37.8% among HCC patients with afamily history of liver cancer and 21.6% among thosewithout (p = 0.03).The interactions between family history of liver cancer

and each of the other risk factors are shown in Table 3.The OR values for HCV RNA, HBsAg and heavyalcohol intake increased about two-fold in subjects witha family history of liver cancer as compared withsubjects without. A synergism with family history ofliver cancer was found for each factor, the S index beingequal to or greater than 2 for all of them. Twenty-nineHCC cases (10.2%) and 279 controls (60.1%) werenegative for HBsAg, HCV RNA and heavy alcoholintake; family history of liver cancer was found in four(13.8%) of the cases, a proportion no di�erent from thatobserved among cases positive for at least one factor (33of 255, 12.9%). An independent e�ect of family historyof liver cancer was apparent among subjects negative forHBsAg, HCV RNA or heavy alcohol intake, though itwas not statistically signi®cant due to the small numberof cases with a family history of liver cancer (four of 29,OR = 2.1; CI = 0.6±7.3).

Discussion

This study showed a two-fold OR increase for positivefamily history of liver cancer, con®rming the results ofprevious epidemiologic research [11±16]. Familial ag-gregations of liver cancer may be due to either environ-mental exposures shared by family members or togenetic factors.Among environmental factors, HBV infection is likely

to be one of the main factors responsible for familialaggregations of liver cancer. Indeed, HBV infection wasthemost important factor of familial clustering ofHCC inAlaska Natives [8], and a higher frequency of HBsAgpositivity was found among relatives of patients withchronic liver disease in Korea than those without [9]. Wefound that the OR for family history of liver cancer didnot decrease when excluding HBsAg positive subjectsfrom the analysis, thus suggesting that intrafamilial HBVtransmission was not responsible for the associationbetween HCC and family history of liver cancer. On theother hand, we found no association between familyhistory of liver cancer and HCV infection or heavyalcohol intake. The former ®nding is consistent with datasuggesting that intrafamilial transmission of the infection

Table 1. Demographic characteristics of cases and controls

Demographic

characteristics

Cases Controls

No. (%) No. (%)

Sex

Male 231 (81.3) 386 (83.2)

Female 53 (18.7) 78 (16.8)

Age (years)

40±49 12 (4.2) 26 (5.6)

50±59 56 (19.7) 72 (15.5)

60±69 133 (46.8) 219 (47.2)

70±75 83 (29.2) 147 (31.7)

Residence

Town of Brescia 136 (47.7) 318 (68.5)

Rest of the province 148 (52.3) 146 (31.5)

Education (years)

0±5 199 (70.1) 296 (63.8)

6±8 59 (20.8) 92 (19.8)

9+ 26 (9.1) 76 (16.4)

Total 284 464

Table 2. Distribution of cases and controls according to HCV and

HBV infection, history of alcohol intake, and history of liver cancer

and other neoplasms in a ®rst-degree relative: OR estimates (95%

con®dence interval, 95% CI) adjusted for sex, age, education and

residence, and the risk factors in the table, by unconditional logistic

regression

Risk factor Cases Controls OR (95% CI)

No. (%) No. (%)

HCV infection

Anti-HCV) 170 (59.9) 436 (94.0) Reference

Anti-HCV+ and HCV

RNA)6 (2.1) 11 (2.4) 1.3 (0.4±4.1)

Anti-HCV+ and HCV

RNA+

108 (38.0) 17 (3.7) 36.9 (19.7±69.0)

HBV infection

HBsAg) 217 (76.4) 443 (95.5) Reference

HBsAg+ 67 (23.6) 21 (4.5) 14.6 (7.8±27.5)

Alcohol intake (g/day)

0±40 76 (26.9) 180 (38.8) Reference

41±60 37 (13.1) 77 (16.6) 1.4 (0.7±2.9)

61±80 21 (7.4) 50 (10.8) 1.7 (0.7±4.0)

>80 149 (52.7) 157 (33.8) 7.1 (4.0±12.7)

Family history of liver cancer

No 247 (87.0) 436 (94.0) Reference

Yes 37 (13.0) 28 (6.0) 2.3 (1.2±4.5)

Family history of other neoplasms

No 181 (63.7) 281 (60.6) Reference

Yes 103 (36.3) 183 (39.4) 1.0 (0.6±1.4)

Family history and liver cancer 419

is uncommon [23]. On the contrary, family aggregation ofheavy alcohol intake is not rare, since ®rst-degree relativeswith alcoholism have been found to be at a higher risk fordeveloping alcoholism than individuals without relativeswho are alcoholics [24, 25]. However, most of thesestudieswere carried out in theUSAor northernEuropeancountries, where the drinking pattern is substantiallydi�erent from that of wine-drinking countries such asItaly, thus preventing any conclusion on the existence offamilial aggregation of alcohol-related diseases in Italy.Some results of this study suggest that the association

between family history of liver cancer and HCC may beindependent of HBV and HCV infection and heavyalcohol intake, though the small number of subjects insome categories prohibits a ®rm conclusion from beingdrawn. However, we also observed a synergism betweenfamily history and each of the environmental factorsinvestigated. Genetic epidemiologic studies support thehypothesis that host-related factors may modulatecarcinogenic activity of the major determinants ofHCC. Indeed, a complex segregation analysis carriedout in eastern China suggests the existence of a recessiveallele, which could increase the risk of developing cancerin HBV chronically infected people by about ten-fold[26]. Genetic variations have also been found in detox-i®cation enzymes, such as epoxide hydrolase, glutatione-S-transferase M1 and N-acetyltransferase [5±7], and incytochrome P450 enzymes involved in metabolic acti-vation of procarcinogens [6], which may modulate therisk of HCC by a¯atoxin intake or HBV infection.

Recent ®ndings also suggest that de®ciency in theactivity level of DNA repair enzymes may contributetowards increasing the risk of developing HCC [27].The main limitation of this study is the use of self-

reports of family history of liver cancer by patientsinterviewed at the hospital. Most HCC cases had apersonal history of chronic liver disease and, therefore,they may have been more aware of the presence of liverdiseases in their relatives, resulting in over-reporting ofliver diseases among relatives by cases as compared withcontrols, and subsequent recall bias. The lack of associ-ation of HCC with family history of cancers in sites otherthan the liver found in this study may not exclude thepossibility of a recall bias. Unfortunately, few data are asyet available on this issue. In one study no relevant recallbias was found regarding self-reported data on familyhistory of cancer when comparing cases and controls formost types of cancer, but no data on liver cancer werereported either [28]. Some registry-based studies showed afamilial aggregation for most cancer sites but did notreport ®ndings on family history of liver cancer, due to therarity of this kind of cancer in the populations investi-gated [4, 28], though ®gures from the Swedish Databaseshoweda familial hazard ratio of about 1.5 for liver cancer(Hemminki, personal communication). However, it hasbeen demonstrated that speci®city di�erences in self-reports of cancer history between cases and controls havelittle impact on OR estimates when exposure prevalencein the study base is below 10%, as in this study, forreasonable sensitivity values among controls [29].

Table 3. Interaction of HCV and HBV infection and alcohol intake >80 g/day with family history of liver cancer in a ®rst-degree relative: OR

estimates (95% con®dence interval, 95% CI) adjusted for sex, age, education and residence and the risk factors in the table by unconditional

logistic regression

Risk factors History of liver cancer in a ®rst-degree relative Synergy index (S)

No Yes

Ca/co OR (95% CI) Ca/co OR (95% CI)

HCV infection

Anti-HCV), Anti-HCV+

and HCV RNA)152/420 Reference 24/27 2.3 (1.2±4.7)

HCV RNA+ 95/16 35.5 (18.8±67.2) 13/1 67.8 (8.3±556.0) 1.8

HBV infection

HBsAg) 194/416 Reference 23/27 2.0 (1.0±4.2)

HBsAg+ 53/20 12.4 (6.5±23.6) 14/1 70.1 (8.6±569.0) 5.6

Alcohol intake (g/day)

0±80 114/287 Reference 21/20 2.1 (0.9±5.2)

>80 133/149 5.3 (3.3±8.5) 16/8 13.4 (4.7±38.1) 2.3

Presence of HBsAg, HCV RNA, alcohol intake > 80 g/day

None of them 25/261 Reference 4/18 2.0 (0.6±6.7)

At least one 222/175 16.5 (10.0±27.3) 33/10 44.5 (18.8±105.0) 2.8

Ca/co = cases/controls

420 F. Donato et al.

In conclusion, this study shows that subjects withparents or siblings with liver cancer have a two-foldincrease in their risk of developing HCC, which isindependent of the main risk factors for the disease.Furthermore, the risk of HCC for family history of livercancer seems to be further enhanced in the presence ofHBV or HCV infection or heavy alcohol intake,suggesting a gene±environment interaction in the devel-opment of the disease.

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