Clin Investig (1993) 71:49-53

Case Report Glinical

Investigator © Springer-Verlag 1993

Hepatitis C virus associated primary hepatocellular carcinoma in a noncirrhotic liver

W. Herr 1, G. Gerken 1, T. Poralla 1, S. Immenschuh 1, p. Schirmacher 2, K.W. Steegmiiller 3, H. Schwickert 4, and K.-H. Meyer zum Bfischenfelde 1 1 I. Medizinische Klinik und Poliklinik der Johannes Gutenberg-Universit/it Mainz z Institut fiir Pathologic der Johannes Gutenberg-Universit/it Mainz 3 Klinik und Poliklinik ffir Allgemein- und Abdominalchirurgie der Johannes Gutenberg-Universitfit Mainz e Institut ffir Klinische Strahlenkunde der Johannes Gutenberg-Universitfit Mainz

Summary. The case of a 71-year-old man with a primary hepatocellular carcinoma in a non-cirrhot- ic liver is reported. There were no risk factors of hepatocellular carcinoma (HCC)-like liver cir- rhosis, alcohol drinking, tobacco smoking, expo- sure to vinyl chloride, thorotrast, aflatoxin or ~l- antitrypsin deficiency. Serologically, the patient was positive for antibodies to the hepatitis B virus (anti-HBc, anti-HB~) and for anti-hepatitis C virus (HCV) antibodies. Virologically, positive and neg- ative strands of HCV RNA could be detected in the patient's serum and tumorous liver tissue by reverse transcription polymerase chain reaction as a sign of persistent HCV replication. Histologi- cally, the HCC was completely surrounded by liver tissue which showed the signs of nodular regenera- tive hyperplasia. Indeed, the mechanism of hepato- carcinogenesis remains to be clarified. However, this case supports the observation that HCC may also develop in patients with HCV infection with- out preexisting liver cirrhosis.

Key words" Hepatocellular carcinoma (HCC) - Non-cirrhotic liver - Hepatitis B virus (HBV) - Hepatitis C virus (HCV) - Nodular regenerative hyperplasia (NRH)

Primary hepatocellular carcinoma (HCC) is a high- ly malignant tumor with an extremely poor prog- nosis and an estimated incidence of 250000-1.2 million cases per year worldwide [23]. It is the sev- enth most common cancer in man [33]. The fre- quent coexistence of liver cirrhosis with HCC rais- es the possibility that cirrhosis may play an impor-

Abbreviations: HBV=hepa t i t i s B virus; HBsAg=hepat i t i s B surface antigen; HCC = hepatocellular carcinoma; HCV = hep- atitis C virus; N R H = nodular regenerative hyperplasia; PCR = polymerase chain reaction; RT = reverse transcription

tant role in the pathogenesis of this tumor [14]. The close relationship between cirrhosis and HCC was explained on the basis of chronic viral liver disease progressing to cirrhosis that in turn pre- disposes to hepatocarcinogenesis. Although these two pathologic conditions often occur together, they do not have parallel geographic distributions and differ between regions with high and low inci- dences of HCC. For example, in sub-Saharan Afri- ca and in many parts of southeast Asia, HCC oc- curs commonly and may exceed cirrhosis in inci- dence [13]. In contrast to these areas the incidence of HCC in Europe and the United States is rather low, and its association with a noncirrhotic liver is rare [30].

However, abundant epidemiological and bio- logical examinations show that chronic hepatitis B virus (HBV) infection is a major risk factor for the development of HCC associated with cirrhosis [2, 3]. An important mechanism by which HBV acts as a carcinogenic agent is by integration into the host cellular genome. The persistence of viral integration may cause genetic changes and epigen- etic phenomena [6, 10]. Recently, HBV DNA se- quences have been detected in the liver and serum of hepatitis B surface antigen (HBsAg) negative patients with chronic hepatitis and hepatocellular carcinoma [19, 24, 31]. Using polymerase chain reaction (PCR), HBV DNA and RNA sequences were found in the tumors of HBsAg-negative pa- tients with liver cancer who where from different geographic areas. Some of these patients had no liver cirrhosis or serological markers of HBV infec- tion such as antibodies to the surface and core antigens of HBV [24].

Recently, in patients with non-A, non-B hepati- tis the hepatitis C virus (HCV) has been identified as the infectious agent responsible for up to 90% of the HBsAg-negative posttransfusion hepatitis cases [8, 17]. Several recent reports showed that patients with HCC are frequently positive for anti- HCV antibodies [7, 26]. Moreover, current epide-

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Serum Liver cDNA O M (9 1 2 3 4 e

4 - - - - I -

r str. j i s,r. T

229 bp

Fig. 1. Detection of HCV RNA by RT- PCR [9]. + , HCV RNA positive serum control; cDNA-, HCV RNA negative se- rum control; 1, patient's serum, positive strand of HCV RNA; 2, patient's serum, negative strand of HCV RNA; 3, tumor- ous liver tissue, positive strand of HCV RNA; 4, tumorous liver tissue, negative strand of HCV RNA; - , water control of RT-PCR; M, reference molecular weight marker (123 bp ledder)

miological studies from Japan have shown a steady decline in the prevalence of HBsAg in patients with HCC [21]. However, from these observations it could be suggested that HCV infection may play a role in the carcinogenesis of HCC in Japan, but a causal relationship between chronic HCV hepati- tis and HCC has not been clearly established. In this report we present the case of a HB~Ag-negative patient with persistent HCV infection who devel- oped a HCC in a noncirrhotic liver with nodular regenerative hyperplasia (NRH).

Case report

The 71-year-old patient was admitted to our center because of slight cholestasis and a solitary inhomo- geneous tumor in the right lobe of the liver with a size of 5 x 4 x 3 cm seen by ultrasound examina- tion. At the time of admission the patient had no complaints. In his history there were infectious hepatitis, spotted fever, and dysentery in 1946. During a colonoscopy in September 1992 three po- lyps of the colon sigmoidium were removed. The histology of one of these polyps showed a non- invasive carcinoid tumor with a diameter of 2 cm. Later the patient had a mild arterial hypertension.

On physical examination we saw a patient in a good general condition and normal weight with- out any signs of a serious disease. The laboratory showed an erythrocyte sedimentation rate of 30/50. The serum glutamic pyruvic transaminase level at 32 U/l, gamma-glutamyltransferase at 58 U/l, and sGLDH at 8 U/1 were slightly increased. All other liver function parameters were in the normal range. The ~-fetoprotein level was elevated to 86 ng/ml. As a sign of resolved HBV infection antibodies to the HBV, i.e., anti-HBo and anti-HBs, were de-

tected in the patient's serum. HBV DNA was nega- tive in the patient's serum and tumorous liver tis- sue using PCR assay performed as recently pub- lished by our group [i 1, 24]. As markers of HCV infection anti-HCV antibodies were detected by second generation assay (anti-HCV ELISA, Ab- bott, Wiesbaden, FRG). For the detection of HCV RNA reverse transcription PCR (RT-PCR) was performed according to F6ray et al. [9]. We found HCV RNA in the patient's serum and tumorous liver tissue. Furthermore, negative strand of HCV RNA was also present in the patient's serum and tumorous liver tissue (Fig. 1 ) .

A computed tomography controlled puncture of the liver tumor was performed. The histology examination of the tumor tissue showed a moder- ately differentiated, trabecular hepatocellular car- cinoma (Fig. 2b). The surrounding nontumorous liver tissue showed no cirrhosis or significant fibro- sis but diffuse occupation by nodular regenerative hyperplasia (Fig. 2a). Only the portal tracts dis- played a mild round cell infiltration and an in- crease in connective tissue fibers as a sign of a discrete chronic hepatopathy.

Computed tomography angiography was per- formed to estimate the total size of the HCC (Fig. 3). The staging of the tumor showed no me- tastasis in other organs, and a right-sided hemihe- patectomy was therefore performed in our surgical clinic. During the postoperative course serum bili- rubin level raised up to 47 mg% without signs of an extrahepatic cholestasis or a cholestatic hepati- tis. Hyperbilirubinemia might be due to a drug- induced anesthesia complication during operation or an intermittent liver failure in the postoperative phase. However, it showed spontaneous remission to completely normal levels within 3 weeks. A se-

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Fig. 2. A Nontumorous liver with nodular regenerative hyperplasia (NRH). Arrows, hyperplastic area consisting of more den- sely packed parenchymal cells slightly re- duced in size. H & E; original magnifica- tion x 100. Insert, detail from within a hyperplastic area showing more than one cell layer thick liver plates. H & E; origi- nal magnification × 400. B Moderately differentiated, partly trabecular and partly pseudoglandular hepatocellular carcinoma from the same patient. H & E; original magnification x 100

Fig. 3. The solitary tumor in the right lobe of the liver with a size of 5 x 4 x 3 cm seen by computed tomography angiogra- phy

vere reactivation of the viral liver disease could be excluded from these data presented. Further- more, the postoperative c~-fetoprotein level re- turned to the normal range. After 3 weeks of con- valescence the patient was discharged in a good condition. In a follow-up evaluation 6 months after operation there was no clinical sign of a tumor recidive in ultrasound examination of the liver and no increase in ~-fetoprotein level.

Discussion

This case report describes the development of a HCV-associated primary HCC in a noncirrhotic liver. Epidemiological observations clearly indicate liver cirrhosis, HBV- and HCV-infections, nu- trients, chemicals, metabolic diseases, and hor-

52

mones as risk factors for HCC. However, in this case three possible precursors of hepatocarcino- genesis should be taken into account: N R H and chronic viral hepatitis of both types B and C.

NRH is a rare lesion of the liver and is charac- terized by diffuse nodulation of the liver tissue con- sisting of small hepatocytes in the absence of signif- icant fibrosis [28]. The frequency of N R H ranges from 0.6% to 2.6% in autopsies performed in gen- eral hospital populations [32]. Various systemic diseases, for example collagen vascular diseases, endocrine or myeloproliferative disorders, as well as drugs and hepatotoxins are frequently associat- ed with N R H [29]. Some of them may play a role in the pathogenesis of this lesion. There is only little experimental, histologic, and clinical evidence that N R H might be a precursor of HCC. Liver cell dysplasia is often associated with NRH, and therefore N R H was suggested to be a preneoplastic lesion [16, 25]. Three reported associations of N R H with HCC exist [27, 28, 32]. In two of the cases N R H was confirmed before HCC developed [27, 28]. Altogether, N R H certainly cannot be con- sidered an obligatory precursor of HCC since nod- ular transformation is not usually found in the nonneoplastic parenchyma adjacent to HCC, even in carcinomas arising in noncirrhotic livers [28]. However, N R H could have been mistaken for cir- rhosis in some cases of HCC.

Patients with HCC and HBV markers in serum have a high rate of persistence of HBV DNA in the liver. However, this is also true for a large number of patients with the tumor and no HBV serological markers [24]. Likewise, a prospective study of HBsAg-negative patients showed that some of them developed liver cancer [1]. Therefore, in a patient positive for anti-HBc or anti-HBs in whom liver cancer develops, a decline in the ex- pression of the viral genome, possibly partly relat- ed to the age of the patient during the development of the tumor [15], as well as masking of HBsAg in immune complexes [4] may account for serolog- ic negativity for HB~Ag. Thus, in HBsAg-negative persons exposure to HBV and the persistence of HBV DNA sequences (integrated or free molecule) would also represent a risk factor for liver cancer. Recently, extrahepatic HBV persistence was found in peripheral blood mononuclear cells, which might be a reservoir for HBV [18, 22]. The role of the viral DNA, however, is still unknown. It is worth noting that HBV DNA sequences were identified in only a few patients with HCC that developed in a noncirrhotic liver [24]. There is a much lower rate of both HBV serologic markers and liver HBV DNA in patients with HCC devel-

oped on histologically normal livers as compared to that observed in HCC with liver cirrhosis [19].

Coinfection with other viruses must also be considered as a possible mechanism of hepatocar- cinogenesis. It is important that antibodies to HCV have been detected in up to 60%-70% of patients with primary liver cancer [7, 26]. If other causes of chronic liver disease or cirrhosis are missing, the infection with HCV may act as a cofactor by inducing necroinflammation, regeneration, and possibly malignant transformation to HCC. The discovery of a few HCC cases in anti-HCV-positive patients without cirrhosis, however, suggests that HCV may exert direct effects during long-lasting infection which may play a role in the pathogenesis of HCC [12]. In an animal model a case of HCC has been reported in a chimpanzee 7 years after inoculation with human plasma from a patient with non-A, non-B hepatitis [20].

Although all the available information argues against a DNA intermediate in the replicative cycle of HCV [5], thus excluding the possibility of inser- tional mutagenesis by viral integration into the host cellular genome, the possibility that HCV could still activate cellular genes important for cell growth regulation at a distance remains to be in- vestigated. The introduction of confirmatory tests for anti-HCV antibodies by immunoblot, the de- velopment of immunological methods to detect HCV-associated antigens in liver tissue, and the detection of minus-stranded RNA replicative inter- mediates by RT-PCR may allow a better compre- hension of the link between the persistence of HCV and hepatocarcinogenesis. In the same way, cohort epidemiological studies, better understanding of the mechanism of HCV infection and an animal model of HCV-related HCC are needed to estab- lish a cause-effect relationship between HCV and HCC.

Acknowledgements. The excellent technical assistance of Mrs. S. Jakobs and Mrs. S. Mies is gratefully acknowledged.

References

1. Beasle~¢ RP, Blumberg B, Papger H, Lin CC, Chien CS (1982) Hepatitis B virus and hepatocellular carcinoma. In: Okuda K, Mackay I, (eds) Heptocellular carcinoma. Gene- va: Union Internationale Contre Cancer, pp 60-93

2. Beasley RP, Hwang LY (1984) Hepatocellular carcinoma and hepatitis B virus. Semin Liver Dis 4:113 121

3. Br6chot C (i 987) Hepatitis B virus and hepatocellular carci- noma. J Hepatol 4:266-279

4. Brown SE, Howard CR, Steward MW, Ajdukiewicz AB, Whittle HC (1984) Hepatitis B surface antigen containing immune complexes occur in seronegative hepatocellular car- cinoma patients. Clin Exp Immunol 55 : 355-359

53

5. Choo Q-L, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359 362

6. Colombo M (1992) Hepatocellular carcinoma. J Hepatol 15: 225-236

7. Colombo M, Kuo G, Choo Q-L, Donato MF, Ninno ED, Tommasini MA, Diogurd N, Houghton M (1989) Preva- lence of antibodies of hepatitis C virus in Italian patients with hepatocellular carcinoma. Lancet II: 1006-1008

8. Esteban JI, Esteban R, Viladomiu L, Ldpez-Talavera JC, Gonzfilez A, Hernfindez JM, Roget M, Vargas V, Genesca J, Buti M (1989) Hepatitis C virus antibodies among risk groups in Spain. Lancet II : 294-297

9. F6ray C, Samuel D, Thiers V, Gigou M, Pichon F, Bismuth A, Reynes M, Maisonneuve P, Bismuth H, Br6chot C (1992) Reinfection of liver graft by hepatitis C virus after liver transplantation. J Clin Invest 89:1361-1365

10. Gerken G, Kremsdorf D, Capel F, Petit MA, Dauguet C, Manns MP, Meyer zum B/ischenfelde K-H, Br6chot C (1991) Hepatitis B defective virus with rearrangements in the preS gene during chronic HBV infection. Virology 183:555-565

11. Gerken G, Paterlini P, Manns M, Housset C, Terre S, Dienes H-P, Hess G, Gerlich WH, Berthelot P, Meyer zum Biischenf~lde K-H, Br~chot C (1991) Assay of hepatitis B virus DNA by polymerase chain reaction and its relation- ship to pre-S- and S-encoded viral surface antigens. Hepato- logy 13:158 166

12. Hasan F, Jeffers LJ, DeMedina M, Reddy KR, Parker T, Schiff ER, Houghton M, Choo QL, Kuo G (1990) Hepatitis C-associated hepatocellular carcinoma. Hepatology 12:589-591

13. Kew MC (1991) Hepatocellular carcinoma with and without cirrhosis. A comparison in southern African Blacks. Gas- troenterology 97 : 136 139

14. Kew MC, Popper H (1984) Relationship between hepatocel- lular carcinoma and cirrhosis. Semin Liver Dis 4:136-146

15. Kew MC, Rossouw E, Hodkinson J, Paterson A, Dusheiko GM, Whitcutt JN (1983) Hepatitis B virus status of south- ern African Blacks with hepatocellular carcinoma: compari- son between rural and urban patients. Hepatology 3 : 65-68

16. Knowles DM, Kaye GI, Godman GC (1975) Nodular re- generative hyperplasia. Gastroenterology 69:746-751

17. Kuo G, Choo Q-L, Alter H J, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, Dienstag JL, Alter M J, Stevens CE, Tegtmeier GE, Bonino F, Colombo M, Lee W-S, Kuo C, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M (1989) An assay for circulating antibodies to a major etiologic group of human non-A, non-B hepatitis. Science 244:36~364

18. Lamelin JP, Trepo C (1990) The hepatitis B virus and the peripheral blood mononuclear cells : a brief report. J Hepa- tol 10:120-124

19. Marcellin P, Thiers V, Degott C (1989) Hepatocellular carci- noma with normal adjacent liver: hepatitis B virus DNA status. J Hepatol 8:249-253

20. Muchmore E, Popper H, Peterson DA, Miller MF, Lieber-

man HM (1988) Non-A, non-B hepatitis-related hepatocel- lular carcinoma in a chimpanzee. J Med Primatol 17:235- 246

21. Okuda F, Fujimoto I, Hanai A, Urano Y (1987) Changing incidence of HCC in Japan. Cancer Res 47:4967-4972

22. Omata M (1990) Significance of extrahepatic replication of hepatitis B virus. Hepatology 12:364-365

23. Parkin DM, Sjernward J, Muir CS (1984) Estimates of the worldwide frequency of twelve major cancers. Bull WHO 62:163-182

24. Paterlini P, Gerken G, Nakajima E, Terre S, D'Errico A, Grigioni W, Nalpas B, Franco D, Wands J, Kew M, Pisi E, Tiollais P, Br6chot C (1990) Polymerase chain reaction to detect hepatitis B virus DNA and RNA sequences in primary liver cancers from patients negative for hepatitis B surface antigen. N Engl J Med 323:80-85

25. Roncalli M, Borzio M, De Biagi G, Servida E, Cantaboni A, Sironi M, Taccagni GL (1985) Liver cell dysplasia and hepatocellular carcinoma: a histological and immunohisto- chemical study. Histopathology 9:209-221

26. Simonetti RG, Cottone M, Craxi A, Pagliaro L, Rapicetta M, Chionne P, Costantino A (1989) Prevalence of anti- bodies to hepatitis C virus in hepatocellular carcinoma. Lancet II: 1338

27. Sogaard PE (1981) Nodular transformation of the liver, al- pha-fetoprotein and hepatocellular carcinoma. Human Pa- thol 12:1052 (letter)

28. Strohmeyer FW, Ishak KG (1981) Nodular transformation (nodular "regenerative" hyperplasia) of the liver. A clinico- pathological study of 30 cases. Human Pathol 12:60-71

29. Trauner M, Stepan KM, Resch M, Ebner F, Pristautz H, Klimpfinger M (1992) Diagnostic problems in nodular re- generative hyperplasia (nodular transformation) of the liver. Z Gastroenterol 30 : 187-194

30. Wands JR, Blum HE (1991) Primary hepatocellular carcino- ma. N Engl J Med 325:729 731

31. Wands JR, Fujita YK, Isselbacher KJ (1986) Identification and transmission of hepatitis B virus-related variants. Proc Natl Acad Sci USA 83:6608 6612

32. Wanless IR (1990) Micronodular transformation (nodular regenerative hyperplasia) of the liver: a report of 64 cases among 2500 autopsies and a new classification of benign hepatocellular nodules. Hepatology 11 : 787-797

33. Waterhouse J, Muir C, Shnmugaratnam K, Powell J (1982) Cancer incidence in five continents. IARC Scientific Publi- cations. Lyon, vol IV 42:714-715

Received: August 7, 1992 Returned for revision: September 28, 1992 Accepted: October 27, 1992

Prof. Dr. Dr. K.-H. Meyer zum Biischenfelde I. Medizinische Klinik und Poliklinik Johannes Gutenberg-Universit/it Mainz Langenbeckstrasse 1 W-6500 Mainz, Germany