Association between Diabetes Mellitus and Hepatocellular Carcinoma: Results of a Hospital- and Community-based Case-control Study

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1 Original Contribution Kurume Medical Journal, 50, 91-98, 2003 Association between Diabetes Mellitus and Hepatocellular Carcinoma: Results of a Hospital- and Community-based Case-control Study MICHIYO MATSUO Department of Public Health, Kurume University School of Medicine, Kurume , Japan Summary: The present study was performed to investigate the relationship between diabetes mellitus (DM) and primary hepatocellular carcinoma (HCC). Incident HCC cases were recruited in Kyushu, Japan. Ethnicity-, age-, gender-, residence-matched hospital controls and community controls were collected. Information on viral hepatitis B (HB5Ag) or viral hepatitis C infection, history of blood transfusion, past histories including DM, amount of drinking or smoking, and genotypes of alcohol metabolizing enzymes was collected. Associations between these items and HCC were analyzed multivariately by conditional logistic regression analysis. Two hundred and twenty two (177 males and 45 females) case-control sets were completed between July 1995 and June Since hospital controls turned out to be a biased one or those sampled from a DM-prone population, a multivariate analysis was performed for the HOC-community controls sets, and it yielded significantly elevated odds ratio (OR)s due to past histories of DM (2.522; 95% Confidence Interval (CI)= ), blood transfusion (1.747; ), and unit increment of alcohol consumption (1.358; ) for males. The same analyses of the HOC-community controls sets for females, revealed an elevated but not statistically significant OR due to past histories of DM (4.195; ). A multivariate analysis revealed that DM might be a risk factor for HOC. Key words diabetes mellitus, hepatitis B virus infection, hepatitis C virus infection, blood transfusion, alcohol consumption, cigarette smoking INTRODUCTION Established risk factors (RFs) of primary hepatocellular carcinoma (HCC) include viral hepatitis B or C infection [1] and some host factors including history of blood transfusion or past histories of liver cirrhosis. Associations between potential risk factors e.g. heavy drinking and/or smoking [2,3], diabetes mellitus (DM) [4-7], genotypes of alcohol dehydrogenase 2 (ADH2) or aldehyde dehydrogenase 2 (ALDH2) [8] and HCC have been reported, however, findings of DM are scarce in Japan where viral hepatitis C infection is the most prevalent and the strongest RF for Japanese HCC. The present study was performed to investigate the relationship between DM and HCC controlling for other potential RFs based on a well-designed hospital- and community- based case control study in an HCC prevalent area in the northern part of Kyushu, Japan. MATERIALS AND METHODS The study methods were similar to those used in our previous studies [9,10]. Briefly, incident HCC cases who were 1) Japanese, 2) aged 40-75, 3) resi- Received for publication August 27, 2003 Mail and reprint requests to: Michiyo Matsuo, Department of Public Health, Kurume University School of Medicine, 67 Asahi-machi, Kurume , Japan. Tel: Fax: Abbreviations: ADH2, alcohol dehydrogenase 2; ALDH2, aldehyde dehydrogenase 2; CCs, community controls; CI, confidence interval; DM, diabetes mellitus; HB5Ag, hepatitis B virus surface antigen; HCC, hepatocellular carcinoma; HCs, hospital controls; HCVAb, hepatitis C virus antibody; LC, liver cirrhosis; OR, odds ratio; RFs, risk factors.

2 92 MATSUO dents in either Fukuoka or Saga Prefecture, and 4) with a confirmed diagnosis of HCC within 1 year prior to admission were identified through the records of Kurume University Hospital in Fukuoka between July, 1995 and June The hospital controls (HCs), who were not affected by chronic hepatitis or by hepatic cirrhosis, matched for age (within the same 5-year age class), gender, residence (prefecture) and time of hospitalization (within 3 months after a case interview) were recruited from inpatients of the two university-affiliated general hospitals in Kurume. One HC for each male HCC or 4 HCs for each female HCC were scheduled to be sampled, respectively. The community controls (CCs) who were Japanese residents of Kurume City without past histories of HCC, liver cirrhosis, or chronic hepatitis and age- and gender-matched to each case within ± 3 years, were randomly sampled from the roster of approximately 230,000 residents in October, of 1998 or During the 5 years between July, 1995 and June, 2000, 1,618 HCC patients were hospitalized in the institution. Of these, 1,385 were excluded mainly because the disease was not being primary HCC (463), they had been diagnosed 1 or more years prior to the time of admission (358), or they didn't match the age (189) or residence requirements (98). Eleven who fulfilled the inclusion criteria could not be matched with potential HCs within 3 months after being interviewed. The number of female HCs varied for each HCC-HC set, i.e. there were 4 sets composed of one HC, 5 sets of 2 HCs, 9 sets of 3 HCs or 27 sets of 4 HCs, respectively. Three HCC cases and several potential HCs declined to participate and 8% of 326 citizens sampled from the roster could not be contacted and 17% declined cooperation. Eventually, 222 (177 males and 45 females) sets were completed. Information on diseases on admission, e.g. HCC, DM, hepatitis B virus surface antigen (HBsAg) or anti-hepatitis C virus antibody (HCVAb) for HCC or HCs was obtained from the hospital records without applying any additional diagnostic procedure and information on other items including past history of DM as informed as such by a physician were obtained from HCC and HCs through structured interviews or from CCs through mailed questionnaires with verification by subsequent telephone calls. Information on blood transfusion obtained includes the years passed since the blood transfused. The cumulative amount of cigarette smoking (smoking amount) or the cumulative amount of alcohol consumption (drinking amount) was quantified by the number of pack-years or of the drink-years as in the previous study [10]. Nail clippings of HCC and HCs were sampled at the time of interview, and those of CCs were collected at home and mailed to the department laboratory in an envelope. ADH2 and ALDH2 genotyping using nail clippings was performed by the methods of Nishiyori et al [11,12]. Analytical methods used include chi-square tests on M X N tables by item, univariate analyses by item and multivariate analyses including multiplicative 1 St order interaction terms of DM and potential confounders for case control comparison based on a conditional logistic regression model. Each item was analyzed univariately to identify any association with TABLE 1. Age distribution of study subjects $ $ Collected between July, 1995 and October 2000 and age distributions shown were at the time of case identification

3 DM AND HEPATOCELLULAR CARCINOMA 93 HCC and statistically significant items were then analyzed multivariately by the hierarchy principle [13]. For the multivariate analyses, smoking or drinking amount was evaluated in terms of the trend of unit increment of each item. The SPSS software [14] was used for all of the analyses. P values less than 0.05 was regarded as statistically significant, otherwise, insignificant. This study was approved by the Ethics Committee of Kurume University School of Medicine. RESULTS Diagnosis of HCC for HCC cases was based on histological findings (male or M 38%; female or F 40%), angiographic results (M 45%; F 51 %) or other findings. Eighty four percent of male and 93% of female HCCs had liver cirrhosis. Primary diagnoses of HCs at the time of hospitalization include DM (M 22%; F 27%), peptic ulcer (M 12%; F 3%), stomach cancer (M 7%; F 5%), or various diseases. Age distribution with mean age of the study sub- TABLE 2. Item distribution of male subjects and odds ratios by univariate analysis & : Reference category &* : Items showing significant difference in proportion between cases and hospital controls &2 : Items showing significant difference in proportion among cases, hospital controls and community controls &3 : Items showing significant difference in proportion between hospital and community controls $ : including } $$ : Percentage are shown without preserving matching # : Odds ratio by univariate conditional logistic regression analysis ## : not analyzed because of biased data of hospital controls from the target population * :P<0.05

4 94 MATSUO jects is shown in Table 1 at the time of case identification. Sixties were the most prevalent for both genders without significant difference among HCCs, HCs and CCs. There was no difference in proportion of the longest occupation for males between HCs and CCs (HC/CCs), and approximately 30% of the subjects belonged to the largest sub-category ( "category I") composed of craftsmen, miners, production process or construction workers. In contrast, the largest occupational sub-category for female CCs was housewife (33%). As shown in Tables 2 or 3, the past history of DM was significantly more prevalent among HCs than CCs in both genders (p<0.05), but there was no substantial difference in prevalence of past history of other diseases e.g. colorectal diseases or peptic ulcer between HC/CCs, and of parental history of hepatitis, liver cirrhosis or HCC between HC/CCs (not shown in tables). The prevalence of blood transfusion was more prevalent among HCs than CCs (p< 0.05) in males but not between female HC/CCs. The proportional differences between non-smokers, smok- TABLE 3. Item distribution of female subjects and odds ratios by univariate analysis & : Reference category &* : Items showing significant difference in proportion between cases and hospital controls &2 : Items showing significant difference in proportion among cases, hospital controls and community controls &3 : Items showing significant difference in proportion between hospital and community controls $ : including } $$ : Percentage are shown without preserving matching # : Odds ratio by univariate conditional logistic regression analysis ## : not analyzed because of biased data of hospital controls from the target population * : P<0.05

5 DM AND HEPATOCELLULAR CARCINOMA 95 ers or ex-smokers, or the compositional differences in smoking amount by the time of investigation in terms of pack-years and that of drinking amount by age 40 in drink-years were not statistically significant between HC/CCs of both genders. Among males, exdrinkers were more prevalent among HCs (p<0.05) than CCs. Comparison of HC/CCs also showed insignificant difference of the genotype proportion of ADH2 or ALDH2 in either gender. Among HCs of both genders, either HBsAg or HCVAb did not significantly associated with DM. Odds Ratios calculated by univariate conditional logistic regression analysis by item were shown in Tables 2 and 3. In comparison with HCs, some of the items i.e. HBsAg, HCVAb, cumulative amount of cigarette smoking or cumulative amount of drinking by age 40 showed significant ORs among either gender. Compared to CCs, male showed positive association between HCC and items, e.g. history of DM (2.667, P<0.05), history of blood transfusion over 10 years (2.966, P<0.05), or some category of smoking or drinking habit, but female did not show any significant OR. ADH2 or ALDH2 genotypes showed insignificant ORs compared with either HCs or CCs for both genders. Multivariate analysis for males compared with CCs yielded no significant interaction terms and a finally reserved model under the hierarchy principle was composed of several main effect terms including DM and some confounding terms. The results based on the final model revealed a significantly increased OR due to DM for males being adjusted by blood transfusion, cumulative amount of smoking, and drinking amount by age 40 as shown in Table 4. The comparable analysis for females showed increased OR for DM, however, it was not statistically significant. DISCUSSION The largest composition of the "category I" workers might not be directly comparable with the occupational compositions of residents aged 40 to 74 in Kurume City according to the 1995 national Population Census data as of October 1St, 1995 [15]. However, the 3 largest occupational categories were clerical and related workers, sales workers and " category I" workers for both controls and Kurume citizens of both genders. Only two national surveys reported point prevalence of probable DM, i.e. of strongly suspected DM based on HbA 1 c among Japanese aged over 40 being approximately 11.5% for male and 9.2% for female at 1997 [16], or of visiting-clinic rate due to DM obtained by interview for Japanese aged from 40 to 84 being approximately 4.4% at 1998 [17]. Although those study design were not the same as the present study, the prevalence of past history of DM observed by the HCs was higher than that of the target population and that observed by the CCs might be almost comparable to that of the target population. The HCCs of both genders in the present study TABLE 4. Odds ratios by multivariate conditional logistic regression analysis $ : 95% confidence interval of odds ratio & : Odds ratio for trend of the 3 categories && : Odds ratio for trend of unit increment, i.e. no, < 10 years, or 10 years * : P<O.05

6 96 MATSUO had slightly lower proportion of blood transfusion and HBsAg but had higher proportion of HCVAb compared to the findings of approximately 17,000 Japanese HCC collected from throughout Japan by the Japan Primary Liver Cancer Research Group between 1998 and 1999, i.e. 26.5%,18.2%, 70.1% for males, or 38.9%, 12.9%, 78.9% for females, respectively [18]. The prevalence of blood transfusion among HCs and CCs was larger than those observed by a mass screening for hepatic diseases conducted in Saga Prefecture, i.e % [19]. The prevalence of HBsAg or HCVAb among HCs were similar to those observed by the above mentioned mass screening, i.e % for positive HBsAg or % for positive HCVAb [19]. The composition of smoking status among HCs or CCs was fairly comparable to those obtained by the National Nutrition Survey in Japan at 1997 [20]. No other reports are available comparable to cumulative amount of alcohol consumption by age 40 among the target population. Accordingly, the HCs might be regarded as a biased control being deviated from the target population, particularly in prevalence of both past history of DM and blood transfusion. On the other hand, the general similarity between CCs and the target population in terms of items observed as above, e.g. occupation, past history of DM, blood transfusion, smoking or drinking habits, except HBsAg or HCVAb because of non-availability of the information might indicate that the CCs were less biased controls from the target population. In addition to that, the high proportion of agreement, 97.8%, about parental history of liver diseases, or a high intraclass correlation coefficient, 0.9 (P<0.05), about smoking status between the responses to the interview and the self administered questionnaire observed by a validation study [21] under the same study design as the present one do not seem that the reported proportion of past history of DM for the CCs is severely underestimated. Therefore, the HCs might be validly utilized to examine association between HCC and smoking or HBsAg or HCVAb, and the CCs might have sufficient validity to examine association between HCC and DM, smoking or other items except HB sag or HCVAb. Although the items HBsAg or HCVAb might be a confounder on the relation between DM and HCC because they might cause both HCC and type II DM [22-24], these items did not associate with the past history of DM among the present HCs of both genders. It might indirectly suggest that the present results obtained by the multivariate analysis based on CCs on the relation between DM and HCC was not confounded by HBsAg nor HCVAb status. Insignificant OR of developing HCC based on CCs for female might be partly due to the small sample size. The positive association between DM and HCC obtained by the present study is comparable to previous reports [6,7,25-28]. Although multivariate analysis revealed insignificant ORs due to smoking amount for both genders based on CCs, some categories of smoking amount was positively associated with HCC for males by univariate analysis based on either controls, which is consistent with a result from Greece [2] but is inconsistent with our previous findings [3]. The smaller sample size of the present study may partly account for the discrepancy. Cumulative consumption of alcohol by age 40 was utilized rather than that at the time of investigation because of its stronger correlation with HCC as shown in the previous study [9]. Multivariate analysis revealed significantly elevated ORs in terms of dose-response relationship with drinking, compared with either HCs or CCs among males, indicating an independent effect of alcohol on the development of HCC, similar to the results of previous studies. The present findings on drinking and HCC may be consistent with a report from Italy indicating that the effect of alcohol drinking is evident in the absence of hepatitis B or hepatitis C infection [29]. Association between ADH2 or ALDH2 and HCC was not statistically significant for either gender in the present study, which is consistent with a previous study among Japanese [8]. The etiological inference of DM on HCC may still have to be investigated. Although causal links between several risk factors for HCC, e.g. viral hepatitis B or C, blood transfusion, alcohol drinking, and liver cirrhosis (LC) may have several routes of progress, e.g. 1) blood transfusion viral hepatitis LC without DM HCC, 2) blood transfusion viral hepatitislc with DM HCC, or 3) alcohol PLC with or without DM HCC, sufficient quantitative data, e.g. cumulative incidence or ORs of HCC by route as above are not available, mainly because of the difficulty of conducting cohort studies by the above-mentioned routes of progress. Therefore, multivariate analysis of case-control data regarding a complicated web of causation tends to yield several apparent independent risk factors, as in the present study.

7 DM AND HEPATOCELLULAR CARCINOMA 97 Assuming that DM may be a risk factor for HCC, possible mechanisms may include several hypotheses: Patients with type II diabetes are characterized by insulin resistance and compensatory hyperinsulinemia. Insulin can bind not onlly to insulin receptors but also to insulin-like growth factor receptors and therefore the elevated levels of insulin may stimulate hepatic cell proliferation or depress apoptosis which may promote HCC development [30-34]. The liver of patients with diabetes mellitus may undergo fatty change with progression to cirrhosis by toxic fatty acids. These alterations may also stimulate liver cell proliferation. A cohort study of viral hepatitis carriers utilizing various biological markers would yield more findings on the mechanisms. ACKNOWLEDGMENTS: This work was partly supported by a Grant-in Aid for Scientific Research ( ) from the Ministry of Education Culture, Sports, Science and Technology. 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