Association Between Hepatitis C Virus and Hepatocellular Carcinoma Using Assays Based on Structural and Nonstructural Hepatitis C Virus Peptides1

Home , Hepatitis C, Hepatocellular carcinoma

[CANCER RESEARCH 52. 5364-5367. October I, 1992] Association between Hepatitis C Virus and Hepatocellular Carcinoma Using Assays Based on Structural and Nonstructural Hepatitis C Virus Peptides1

Xenophon Zavitsanos, Angelos Hatzakis, Evangelia Kaklamani,2 Anastasia Tzonou, Nektaria Toupadaki, Caryn Broeksma, JÃ¼rgenChrispeels, Hugo Troonen, Stephanos Hadziyannis, Chung-cheng Hsieh, Harvey Alter, and Dimitrios Trichopoulos Department of Hygiene and Epidemiology Â¡X.2.., A. H., E, Ã€'.,A, T., N. T., D. T.] and Academic Department of Medicine, Hippokration General Hospital [S. H.J, Athens University Medical School, Athens, Greece; Abbott GmbH Diagnostika, D-6200 Wiesbaden, Germany /C. B., J. C., H. T.]; Department of Epidemiology, Harvard School of Public Health, Boston, Massachusetts 021 IS {C-C. H., D. T.] and Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, NIH, Bethesda. Maryland 20892 [H. A.]

ABSTRACT rhosis, it has been suggested that non-A, non-B hepatitis may be associated with HCC (8, 9). Stored sera from 181 Greek patients with hepatocellular carcinoma The cloning of HCV and the development of an assay for (HCC), 35 patients with metastatic liver cancer, and 416 hospital con anti-HCV against the nonstructural HCV protein clOO (10, 11) trols with diagnoses other than malignant neoplasm or liver disease led to studies which have indicated that the prevalence of anti- were examined with first and second generation hepatitis C virus (HCV) enzyme immunoassays as well as with five HCV supplemental assays HCV among patients with HCC may be as high as 76% (12- based on structural and nonstructural HCV peptides. Second generation 31). However, the sensitivity and specificity of the anti-HCV HCV enzyme immunoassays were more sensitive than first generation assay based on clOO, especially for stored sera, have been ques assays. However, both assays had suboptimal specificity using the stan tioned on several lines of evidence (32). Therefore the preva dard reactivity criterion (absorbance of sample to cutoff > 1.0). Speci lence of anti-HCV in HCC and the extent of the HCV-HCC ficity was improved by centrifugation and by using a sample's optical association have not been adequately quantified. density to cutoff ratio >3.0 or supplemental assays; in this instance the In a large case-control study that was undertaken in Athens, prevalence of antibodies to HCV was 13.3% (24 of 181), 0 (0 of 35), and 1.4% (6 of 416) in HCC, metastatic liver cancer, and Greece, and was specifically designed to explore the viral etiol hospital controls, respectively. A similar estimation of prevalence of ogy of HCC (2, 3, 33), we have recently shown that only antibody to HCV in HCC (12.5% or 4 of 32) was obtained when the strongly anti-clOO reactive specimens are specifically associated recombinant immunoblot assay, second generation, was used to screen a with HCC (22). The recent advances in the serological diagno random sample of HCC patients. The relative risk linking HCV to HCC sis of HCV infection using recombinant and synthetic peptides was estimated as 10.4 (95% confidence interval, 4.2-26.0; /' < 0.0001). derived from structural and nonstructural regions of the HCV These data suggest that the prevalence of antibodies to HCV in HCC genome (34,35) provided the opportunity to further explore the using stored sera has been previously overestimated even though the specificity and strength of the association between HCV and evidence of a causal association of HCV with HCC persists. HCC.

INTRODUCTION PATIENTS AND METHODS Hepatocellular carcinoma HCC3 is one of the most common cancers worldwide with an estimated 500,000 to 1 million new Subjects. The subjects of the present study have been described in cases/year and incidence rates varying from minimal to more previous publications that examined the role of hepatitis B virus and than 100 cases/100,000 persons/year (1). The tumor is espe other factors in the origin of HCC (3, 33). In the present analysis, study cially common in sub-Saharan Africa and certain regions of subjects were 181 patients with a final diagnosis of HCC (106 were histologically established whereas 75 were confirmed only by elevated Asia. There is substantial evidence that HCC is causally related a-fetoprotein values, >600 ^g/Hter) who were admitted between April to hepatitis B virus infection, aflatoxin consumption, ethanol intake, and possibly tobacco smoking (2-4). Several other fac 1976 and October 1984 to 9 major hospitals in Athens, Greece. Control subjects were 416 hospitalized patients with diagnoses other than ma tors may be involved in the etiology of HCC but the fraction of lignant neoplasms or liver disease. A second group of controls consisted HCC cases attributable to these factors is limited (5). Liver of 35 MLC patients who were, initially, suspected of having HCC but cirrhosis of any type, including cirrhosis associated with genetic later shown to have metastatic cancer (all with a known primary site). diseases such as hemochromatosis, Â«i-antitrypsin deficiency, Patients with MLC form a unique comparison group, since these pa and tyrosinemia, increases the risk of HCC development (6, 7). tients share with HCC patients a disease pattern that is similar in Since non-A, non-B hepatitis is one of the main causes of cir- severity and history of hospitalization. Moreover the use of MLC cases provides an unusual insight into the biological specificity of the anti- HCV assays (22, 36). Received 5/6/92: accepted 7/23/92. All subjects were Caucasians of Greek nationality and residence. The The costs of publication of this article were defrayed in part by the payment of three patient populations were similar with respect to years of schooling page charges. This article must therefore be hereby marked advertisement in accord (an indicator of socioeconomic status) and place of birth (urban, semi- ance with 18 U.S.C. Section 1734 solely to indicate this fact. urban, or rural), but their distributions by age and gender were different. ' This study was supported in part by a grant from the Greek Ministry of Health, Laboratory Methods. Sera were stored at â€”¿25Â°Cfor7 to 15 years. Athens. Greece. 2 To whom requests for reprints should be addressed, at Department of Hygiene However, the duration of sera storage was similar in the three groups. and Epidemiology. Athens University Medical School. M. Asias 75, Athens. 11527 Hepatitis B serology was done by radioimmunoassays in 1985 as re Greece. 3 The abbreviations used are: HCC, hepatocellular carcinoma; HCV. hepatitis C ported previously (3). Determination of anti-HCV was undertaken in virus; MLC, metastatic liver cancer; SOD, Superoxide dismutase; SHCVA, supple 1990 and repeated in 1991. Anti-HCV testing in 1990 was done by a mental HCV assays; RIBA-2, second generation recombinant immunoblot assay; first generation EIA based on the recombinant HCV peptide clOO, EIA, enzyme immunoassay(s): EIA-1. first generation EIA; EIA-2, second gener ation EIA; CKS, cytidine monophosphate-2-keto-3-deoxyoctonate synthetase; derived from the nonstructural (NS4) region of the putative HCV ge S/CO, absorbance of the sample to cutoff; anti-HCV, circulating antibodies to nome, expressed in yeast as a SOD fusion protein (Ortho HCV Anti HCV. body ELISA Test Systems, Raritan, NJ) (11, 22, 36). 5364

Table 1 Patterns of reactivities of screening and supplemental HCV assays sample after adding the CKS-clOO was decreased >50% compared with Each of these test patterns was considered to confirm EIA reactivity, i.e., to the base-line absorbance (35). represent a "true positive" reaction The algorithm of the anti-HCV testing by EIA-1, EIA-2, and A- 1" or EIA-2 SHCVA in order for a specimen to be considered reactive is shown in after CKS-core CKS-33c sp65/67 sp75 inhibition Table 1. Briefly, any sample repeatedly reactive by EIA-1 or EIA-2 was Pattern1 centrifugationEIA+ EIA EIA EIA assayND tested for CKS-core, CKS-33c, and sp65/67. Every sample reactive to + + + ND CKS-33C (any combination with CKS-core and sp65/67) was consid 23 + + + - ND ND ered as confirmed reactive. Samples reactive with CKS-core (only) or + - + + ND ND sp65/67 (only) were further tested by the sp75 EIA or the CKS-clOO 4 ND++ + NDNDND 5 inhibition assay, respectively, in order to confirm anti-HCV reactivity. 6 + NDCKS-clOO Samples reactive to both CKS-core and sp65/67 were tested by the sp75 7 EIA and when this was reactive the specimen was considered confirmed gEl reactive. If sp75 was not reactive the specimens were further tested by " EIA-1 and EIA-2, first and second generation enzyme immunoassays devel the CKS-clOO inhibition assay and when the percentage of neutraliza oped by Abbott GmbH Diagnostica, respectively; EIA, enzyme immunoassay; ND, not done. tion was >50% the sample was considered confirmed reactive. A random sample of 32 HCC patients and 13 hospital controls was also tested by RIBA-2 (Chiron, Emeryville, CA) (34). Antibody reac Because of the uncertainties surrounding HCV serology (32), anti- tivity in RIBA-2 was assessed by comparison with a weak and strong HCV testing was repeated in 1991 using first and second generation IgG positive control, including a SOD negative control, and it was rated EIA from Abbott GmbH Diagnostica, Wiesbaden, Germany. EIA-1 as â€”¿,Â±,1+, 2+, 3+, and 4+. A positive band was rated 1+ or greater. (35) uses the recombinant HCV clOO solid phase antigen expressed in A sample was considered as reactive (+) when 1+ or greater reactivity yeast as a SOD fusion protein. EIA-2 is based on recombinant HCV existed in any of the two recombinant antigens and indeterminate when nonstructural peptides, derived from NS4 and NS3 (putative protease) an 1+ or greater reactivity was present in one recombinant antigen. regions of the HCV genome, and on a recombinant structural peptide Evaluation of RIBA-2 was done blindly to the EIA-1 or EIA-2 results. (putative core) derived from the 5' end region of the HCV. Accordingly, Statistical Analysis. The 2x2 and 2 x k tables were analyzed by the following antigens are coated onto a polystyrene bead of the HCV Fisher's exact and x2 tests, respectively (37). The association between EIA, second generation: (a) the c 100 (NS4) expressed in yeast as a SOD anti-HCV and hepatocellular carcinoma was based in the estimation of fusion protein; (b) the pHCV 31 expressed in Escherichia coli as a CKS the odds ratio. When cases are newly diagnosed the odds ratio is an fusion protein representing the peptide 33c (NS3) and parts of the clOO estimate of the rate ratio, which, in turn, is often referred to as the (NS4); and (c) the pHCV 34 (putative core) expressed in E. coli as a relative risk (38). CKS fusion protein; this protein has been also designated c22-3. The specimens were tested in duplicate by EIA-1 and EIA-2 and only repeatedly reactive samples were considered positive. In an attempt to RESULTS improve the specificity of testing by EIA-1 and EIA-2 the specimens Prevalence of anti-HCV by Screening and Supplemental Im were tested as crude sera as well as after centrifugation at 5000 rpm for 20 min. Every repeatedly reactive sample with either EIA-1 or EIA-2 munoassays. Table 2 shows the distribution of 181 patients after centrifugation was further tested by SHCVA (Abbott Labs, North with hepatocellular carcinoma, 35 patients with metastatic liver cancer, and 416 hospital controls by S/CO ratio in anti-HCV Chicago, IL). The following SHCVA were used: (a) a recombinant HCV peptide EIA based on the putative core HCV protein expressed in EIA-1 and anti-HCV EIA-2 performed before and after sera E. coli as CKS-fusion protein (CKS-core EIA); (b) a recombinant HCV centrifugation. It is evident that the majority of samples had peptide EIA based on the putative protease (NS3) protein (33c) ex S/CO ratios between 1.0 and 2.99 irrespective of the group or pressed in E. coli as a CKS fusion protein (CKS-33c EIA); (c) a syn the assay used. However, a S/CO ratio >3.0 clearly differenti thetic peptide EIA based on the combination of two synthetic peptides ated HCC patients from MLC and hospital controls using anti- sp65/67 from the c 100 (sp 65/67 EIA) (35); (d) a synthetic peptide EIA HCV EIA-1. The same S/CO ratio plus the centrifugation step based on the synthetic peptide sp75 from the putative core (sp75 EIA); and (c) a solution-blocking or inhibition assay based on a recombinant were necessary in order to eliminate some apparently nonspe cific results in the MLC group according to the anti-HCV HCV antigen expressed in E. coli as a CKS fusion protein containing 256 of the 363 amino acids of the clOO (CKS-clOO) (35). EIA-2. All centrifuged sera found reactive for anti-HCV (S/CO > AH supplemental EIA were tested in a protocol similar to that for the EIA-1 and EIA-2 according to the manufacturer's instructions. Every 1.0) with either EIA-1 or EIA-2 were tested with SHCVA. All sample was considered reactive when the S/CO ratio was > 1. The HCV sera found reactive with EIA-1 but not reactive with EIA-2 were inhibition assay was considered reactive when the absorbance of the confirmed as nonreactive. Table 3 shows the results of SHCVA

Table 2 Comparison of first and second generation EIA reactivity among HCC patients, MLC patients, and controls according to S/CO ratio

GroupHCC conditionsAnti-HCV n (%) (70.2) centrifugationAnti-HCVEIA-1 before MLC n (%) 35416181 2 (5.7) 33 (94.3) 0 (0.0) Controls n(%)HCC 121(29.1)47 288(69.2)115 7(1.7)19(10.5)

n (%) (26.0) (63.5) centrifugationAnti-HCVEIA-1 after MLC n (%) 35 2 (5.7) 33 (94.3) 0 (0.0) Controls n(%)HCC 416181 179(43.0)13 228(54.8)138 9(2.2)30(16.6)

n(%)MLC (7.2) (76.2) centrifugationAnti-HCVEIA-2 before n (%) 35 0 (0.0) 31 (88.6) 4(11.4) Controls n(%)HCC 416181 85(20.4)52 320(76.9)105 11(2.6)24(13.3)

n (%) (28.7) (58.0) EIA-2 after centrifugation MLC n (%) 35 0 (0.0) 35(100.0) 0 (0.0) Controls n (%)N181 416Â£0.9934(18.8)183(44.0)1.0-2.99127 224 (53.8)S/CO2:3.020(11.0)9 (2.2)Test 5365

Table 3 Confirmation of anti-HCV reactivity in HCC patients, MLC patients, of supplemental assays with excellent performance in other and controls according to S/CO ratio settings (35, 46) was not entirely satisfactory in this study. On toanti-HCVEIA-2 the basis of this study the use of anti-HCV supplemental assays in stored sera may be limited to samples, screened by EIA-2, aftercentrifugationS/CO supplementalHCV confirmedanti-HCVn/T assaysn58184428792406Prevalenceofwith S/CO ratios >3.0. GroupHCCMLCControlsHCCMLCControlsHCCMLCControlsReactiven1293523332>2.0 (%)58/181 The association of anti-HCV with HCC has been examined (32.0)18/35(51.4)44/416(10.6)28/181earlier in several studies (12-31). On the basis of the present study it appears that the anti-HCV prevalence in HCC cases and the proportion of HCC cases attributable to HCV may be (15.5)7/35 inflated in many of the studies. This was also suggested by other 81724>3.0 (20.0)9/416 (2.2)24/181 authors (47), on the basis of a very small study. This conclusion does not exclude the possibility that a substantial part of anti- (13.3)0/356/416 09Confirmedby HCV prevalence variation among HCC cases may be due to (1.4) other reasons such as geographical, racial, or other differences. In conclusion, the present analysis undertaken with second for all sera found reactive with anti-HCV EIA-2 after centrifu generation screening assays and a battery of supplemental tests, gation, by the level of S/CO ratio. It is evident that the use of provides insight into the sensitivity and specificity of the cur SHCVA has not completely resolved the problem of discrimi rently available means of detecting chronic HCV infection and nating specific from nonspecific anti-HCV reactivity in levels of suggests that the relative risk linking anti-HCV to HCC is S/CO ratios from 1.0 to 2.99 where anti-HCV reactive samples elevated but not overwhelmingly so. Case-control studies incor appeared to occur more frequently in MLC than in HCC. How porating recent advances in the diagnosis of chronic HCV in ever, the use of a S/CO ratio >3.0 was sufficient not only fection with polymerase chain reaction may further refine the to differentiate anti-HCV status in HCC and MLC but also parameters of the association by allowing a distinction between to generate perfect agreement of EIA-2 and SHCVA in HCC active and nonactive (past) HCV infection (48, 49). cases. 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Xenophon Zavitsanos, Angelos Hatzakis, Evangelia Kaklamani, et al.

Cancer Res 1992;52:5364-5367.

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