[ RESEARCH 51. 6179-6179. November 15. 1991] Chromosome Abnormalities in Peripheral Blood Cells of Hepatitis B Chronic Carriers'

Daniela Simon,2 Thomas London, Hie-Won L. Hann,3 and Barbara B. Knowles

The Wistar Institute of Anatomy and [D. S.. B. B. K.j, Philadelphia. Pennsylvania 19104, and Fox Chase Cancer Center [T. L., H-W. L. H.], Philadelphia, Pennsylvania 19111


Cytogenetic analysis of chromosome spreads from periph Source of Material. Blood samples, obtained from 40 indi eral blood cells of hepatitis B virus (HBV) chronic carriers and HBV- viduals, were screened for HBV carrier status at the Liver Cancer negative individuals of the same ethnic origin revealed a significantly Prevention Center at the Fox Chase Center, Philadelphia, PA. This center seeks to serve as a registry and early warning center for those at higher incidence of chromosome breaks and other mitotic aberrations in risk for the development of liver tumors. The 40 participants in the the HBV chronic carriers. The highest incidence of chromosome breaks study are immigrants from east Asian countries, including Korea, was found in chronic carriers who evidenced circulating HBV. Such an China, Taiwan, and Vietnam and are between 18 and 58 yr of age. association between HBV and these genetic lesions assumes importance Thirty-one were classified as HBV carriers based on persistence for in of the known correlation between HBV chronic carrier status and more than 6 mo of seropositivity for HBsAg. It is likely that all of the the high risk of hepatocellular , where a mutagenic effect carriers were infected in their native countries during infancy or child of HBV cannot be excluded. hood. Nine individuals were HBsAg negative and HBV-DNA negative and were considered free of current HBV infection; one of these nine had anti-HBs, and another had anti-HBc, indicating their past acute INTRODUCTION infection with HBV (Table 1). Of the 31 HBV carriers. 13 were HBV4 infection and development of the chronic carrier state and 18 were male; of the 9 uninfected persons, 4 were female and 5 were male. None of the 40 subjects had a history of exposure to is strongly correlated with a high incidence of PHC (1, 2). radiation or mutagenic chemicals, all were regular tea drinkers, and of Although HBV integrates in the host , none of the HBV those who were smokers none smoked more than one-half package per products has proven to have direct transforming potential, day. Seven of the HBV chronic carriers reported a close relative who no common site of HBV integration in the multiple tumors had succumbed with hepatocellular carcinoma. Serum Assays. Each serum sample was tested for the presence of examined has been identified (3), and no underlying molecular major HBV antigens and antibodies (HBsAg, anti-HBs, HBeAg, anti- defect common to each PHC has been documented. The role HBe, and antibodies to HBcAg), using commercial enzyme-linked of HBV in PHC still remains enigmatic. One possible effect of immunoassays (Abbott Laboratories, North Chicago, IL) according to HBV on the infected cells is induction of random . the manufacturer's instructions. All positive tests were repeated and This possibility has not been investigated because of the limited confirmed using the manufacturer's confirmation procedures. experimental systems available. We previously found chromo Chromosome Analyses. Chromosome analysis was performed, with out knowledge of the HBV status or clinical history of the subjects, by some aberrations in metaphase chromosomes from peripheral standard methods after a 72-h incubation of whole blood at 37°Cin blood cells of an HBV chronic carrier with hepatocellular DMEM.10 and phytohemagglutinin (0.1 Mg/ml; Wellcome Diagnos carcinoma (4). In light of the results of others who reported tics, Dartford, England). Between 20 and 40 metaphase chromosome that HBV could infect and replicate in populations of lymphoid spreads from each individual were analyzed by the same worker and cells (for review, see Refs. 5 to 13), we embarked on a pilot scored for chromosome breaks and other mitotic aberrations, such as study to determine the level of chromosome abnormalities in pulverization or premature chromosome condensation, premature cen peripheral blood cells from a group of Asian immigrants, some tromere divisions, triradials, telomeric breaks and minute chromo of whom were HBV chronic carriers. We now report differences somes, endoreduplication, and hyperploidy (Fig. 1). Chromosome prep arations were analyzed after in 1% Giemsa or after treatment in the proportion of chromosome and other mitotic aberrations by G- and C-banding techniques (14, 15). within this population. HBV chronic carriers have a signifi DNA Extraction and Southern Blot Analysis. Peripheral blood cells cantly higher incidence of abnormalities; those who contain were separated from plasma by centrifugation (1500 x g, 10 min) and detectable circulating virions have the highest proportion of washed 3 times in DMEM.10. Both plasma and cells were treated with metaphase chromosome spreads with chromosome breaks. proteinase K (200 ¿ig/ml)in a solution of 0.01 M Tris-HCl (pH 7.4), 0.001 M EDTA, 0.1 M NaCl, and 0.5% sodium dodecyl sulfate for 18 These data suggest that HBV infection might have a more h at 37°C.DNA was extracted by the standard phenol:chloroform generalized effect on chromosome stability than previously procedure, precipitated with ethanol, and solubilized in 0.01 M Tris, recognized. 0.001 M EDTA, pH 7.4. Cellular DNA was digested with //indlll, according to the manufacturer's specifications (New England BioLabs, Received 7/26/91; accepted 9/10/91. Beverly, MA), separated on a 0.8% agarose gel, and blotted onto Nytran The costs of publication of this article were defrayed in part by the payment (16). Filters were hybridized with a '2P-labeled nick-translated full- of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. length HBV insert (17), purified from pBR322 sequences by electro- ' This work was supported by USPHS Grants CAI0815. CA37225, CA21124 phoresis and elution. (B. B. K. and D. S.), and CA40737 and CA43217 (T. L. and H. L. H.). 2To whom requests for reprints should be addressed. Statistical Analysis. The frequency of the various chromosome ab ' Present address: Department of Medicine, Thomas Jefferson University normalities in each individual was calculated, groups made, ranks School of Medicine. Philadelphia. PA 19107. assigned, and groups compared by the Mann-Whitney U test, using a *The abbreviations used are: HBV, hepatitis B virus; PHC, primary hepato correction for ties (18). The significance of the presence or absence of cellular carcinoma; HBsAg, hepatitis B virus surface antigen; anti-HBs, antibody abnormalities between groups were tested by the Fisher exact probabil to hepatitis B virus surface antigen; anti-HBc, antibody to hepatitis B virus core ity test (18). For purposes of statistical analysis, chromosome breaks antigen; HBcAg, hepatitis B virus core antigen; HBeAg. hepatitis B virus e antigen; anti-HBe, antibody to hepatitis B virus e antigen: DMEM.10, Dulbecco's were considered together as possible evidence of direct genetic damage, modified minimal essential medium containing 10^ fetal bovine serum. while all of the other mitotic abnormalities were grouped together. 6176

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Table 1 Clinical status of individuals within the population investigated 10 i Antibody to HBV DNA" HBsAg HBeAg HBsAg HBeAg HBcAg HBV chronic 31 31 17 18 carriers HBV negative 9 0 0 3 " Southern blot hybridization. TJ * All individuals with circulating HBV contained HBeAg. 6 - "D C

RESULTS 4 - The chromosome analyses were performed and evaluated without knowledge of the patient's history. The incidence of 2- chromosome breaks and other mitotic aberrations was observed (Fig. 1). Correlation of the chromosome analyses with the clinical status of the individuals revealed a significantly higher frequency of metaphase chromosome spreads containing ab normalities in HBV chronic carriers than in the HBV-negative 0 001 01 02 03 04 05 06 07 08 09 1 Frequency of abnormal individuals (Fig. 2). Sixteen of the HBV chronic carriers exhib Fig. 2. Frequency of chromosome abnormalities in HBV-negative (•)and ited a higher frequency of metaphases containing abnormalities HBV chronic carriers (O). For each individual the number of metaphase chro than any of the HBV-negative individuals investigated. On mosome spreads containing one or more abnormalities was tabulated and divided average, more than 20% of the cells analyzed from the HBV by the total number of metaphase spreads analyzed to yield the frequency shown. chronic carriers contained abnormalities (Table 2). When the frequencies of all aberrations were ranked and the groups at 3.2 kilobases, a pattern observed during active viral replica compared by the Mann-Whitney U test, a highly significant tion. Analysis of the metaphase spreads from the HBV chronic difference was found between the HBV chronic carrier and carrier population revealed that the incidence of chromosome HBV-negative individuals (z = 2.73; P = 0.003). Partitioning breaks was significantly higher in the peripheral blood cells of the abnormalities revealed a highly significant difference be those HBV chronic carriers with detectable circulating HBV tween these two populations in the incidence of chromosome DNA than in the remaining chronic carriers (Table 3, Fisher's breaks (Fisher's exact probability = 0.002) and also other exact probability = 0.03). We found no significant difference in chromosome abnormalities (Mann-Whitney U test; z = 3.2, P the frequency of the other mitotic aberrations in chronic carriers = 0.0007). Although there was an elevated level of all categories with or without circulating levels of HBV (z = 0.541 ; P = 0.29). of abnormalities, the most obvious finding was that of single Thus, the presence of a high level of circulating virions corre minute chromosomes in multiple copies in numerous meta lates with a relatively high frequency of direct chromosome phase spreads. damage but not with an increased frequency of other mitotic Eight of the 31 HBV chronic carriers contained readily abnormalities in the metaphase chromosome spreads. detectable levels of HBV DNA in their plasma by Southern blot analyses. Fig. 3 shows a representative Southern blot DISCUSSION analysis of DNA extracted from the plasma and peripheral blood cells of two carriers. The HBV genome was detected in In the present study, we describe a highly significant differ the plasma as either a discrete 3.2-kilobase band representing ence in the frequency of mitotic abnormalities in metaphase the intact HBV genome or as a smear of hybridization beginning spreads of peripheral blood cells from HBV chronic carriers

* %&>*. **

Fig. 1. Representative chromosome aberrations in %.L- metaphase spreads in this study. J, normal; /».breaksand ^** 7* gaps (arrows); c, premature chromosome condensation «M . "*« - * or pulverization (arrow); d, triradial formation (arrow): e, endoreduplication;/ minute chromosomes (arrow); g, y; g / v/^X/V H premature division; and h, hyperploidy. Jff'i&' 4)>jk&?-t\ •¿K^.< €¿mm& f^* ^i''>V 'Jf.1 <. y^» 6177

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. CHROMOSOME ABNORMALITIES IN PERIPHERAL BLOOD CELLS Table 2 Chromosome aberrations in metaphase spreads from peripheral blood cells ofHBV carrier and HBV-negative individuals No. of metaphase chromosome spreads containing mitotic aberrations"

Individuals/cells Breaks PCC PCD Triradial Endo Minutes Hyperploidy HBV chronic carriers HBV negative31/966 9/20034121 03052 014 01151212 0 " Chromosome breaks, premature chromosome condensation (PCC), premature centromere division (PCD), triradial chromosome formation, endoreduplication of all chromosome (endo), telomeric breaks or minute chromosomes, and hyperploidy. when compared with those from peripheral blood cells of HBV- Table 3 Frequency of chromosome abnormalities observed in peripheral blood cells of HBV chronic carriers with and without high levels of circulating HBV negative individuals. The number of minute chromosomes A" Breaks Other in the HBV chronic carriers is quite striking, although their abnormalities aberrations origin and significance are currently unclear. The only previous report of minute chromosomes in peripheral blood cells of n Mean Range Mean Range Mean Range HBV chronic carriers seemingly normal individuals was from a population of South without circulating American Indians (19) whose use of hallucinogenic drugs was HBV considered a possible generative factor. However, the appear HBV chronic carriers 80.2110.1970-1.00-0.40.0230.046°0-0.0-0.111250.2200.1490-1.00-0.33 with circulating ance of minute chromosomes proved ephemeral and the cause, HBV23 be it substance abuse or exposure to a toxic or an infectious * Chromosomes of peripheral blood cells from HBV chronic carriers with agent, was never established (20). detectable circulating HBV were more likely to contain breaks than those from chronic carriers without circulating HBV (Fisher's exact probability = 0.03). The correlation between an increased frequency of chromo some aberrations and the HBV carrier state and the fact that those chronic carriers with detectable HBV in the plasma of HBV integration or replication. In hepatoma cells integrated exhibited the highest frequency of chromosome breaks suggest HBV sequences are frequently associated with rearrangements that HBV may have a role in their induction. Chromosome of the host flanking sequences detectable at the molecular level lesions, such as the fragmentation that occurs upon measles (e.g., see Ref. 23). virus infection, have long been associated with virus-infected Chromosome studies of normal or HBV-infected hepatocytes cells (21). In the only published study of metaphase chromo have not been reported. However, even though HBV replicates some spreads of peripheral blood cells of patients in the acute in some cells of hematopoietic origin (5-13), the hepatocyte is phase of infection with hepatitis A and B , an elevated the major reservoir of HBV infection. Typically many years of level of chromosome aberrations and of sister ex persistent HBV infection precede hepatocellular carcinoma, a changes was revealed (22). We now show that chronic HBV during which chromosome damage may occur and mu infection is also associated with chromosome abnormalities. tations accumulate. Liver damage by the antiviral immune Chromosome damage may represent a direct or indirect effect response and compensatory regenerative proliferation may set the stage for selection of those mutations which deregulate liver growth, thus providing a basis for hepatocellular carcinogenesis. 1234 REFERENCES —¿23.1 1. Beasley, R. P. Hepatitis B virus: the major etiology of hepatocellular carci noma. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. Chromosome Abnormalities in Peripheral Blood Cells of Hepatitis B Virus Chronic Carriers

Daniela Simon, Thomas London, Hie-Won L. Hann, et al.

Cancer Res 1991;51:6176-6179.

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