Hepatocellular carcinoma in patients with HCV

BaillieÁre's Clinical Gastroenterology Vol. 14, No. 2, pp. 327±339, 2000

doi:10.1053/bega.1999.0079, available online at http://www.idealibrary.com on

10 Hepatocellular carcinoma in patients with HCV Massimo Colombo

MD

Professor and Chairman Division of Internal Medicine, `Angela Maria e Antonio Migliavacca' Center for Liver Disease and the FIRC-University Unit for Liver Cancer, IRCCS Maggiore Hospital, University of Milan, Via Pace 9, 20122 Milan, Italy

In most Western countries hepatitis C virus (HCV) is a common risk factor for hepatocellular carcinoma (HCC). Many HCCs are multifocal in origin, but HCC may also grow as a single hepatic nodule for years before generating satellite or distant tumours. HCV may promote cancer through cirrhosis, which is often associated with HCV-related HCC, but it might also have oncogenic properties by interacting with cellular genes that regulate cell growth and di€erentiation. Treatment of patients with chronic hepatitis C using interferon might attenuate HCC risk, particularly in those who respond to therapy. Many patients whose cancer is detected early have been successfully treated by liver transplantation and have shown signi®cantly prolonged survival. This is less often achieved with hepatic resection or regional therapies, which may indeed destroy small tumours, without a€ecting the complications of portal hypertension. Screening remains the only realistic approach for improving the treatment of HCC patients, but its cost-e€ectiveness is uncertain. Key words: hepatitis C virus; hepatocellular carcinoma; cirrhosis; interferon; screening; liver transplantation.

INTRODUCTION The sequential development of chronic liver disease and hepatocellular carcinoma (HCC) seen in patients with chronic hepatitis C was a clue in the identi®cation of HCV as a risk factor for primary liver cell cancer.1±5 In 1989, with the advent of a serum assay for antibodies to HCV (anti-HCV) it became possible to measure the relevance of HCV in the aetiology of this tumour.6 Since HCC is a multi-step disease whose occurrence is linked to environmental factors, the great geographical variations in tumour incidence re¯ect the great variations in the levels of carcinogenic factors in the environment.7 Not unexpectedly, HCV-related HCCs are on the increase in many geographical areas as a consequence of an epidemic of community-acquired infections that occurred in the past.8,9 With the implementation of screening programmes for HCC, many patients with small, potentially treatable tumours have been detected and more information on the natural history of this tumour is now available, along with therapeutic options. 1521±6918/00/020327+13 $35.00/00

c 2000 Harcourt Publishers Ltd *

328 M. Colombo

EPIDEMIOLOGY The importance of the pathogenetic role of HCV in HCC varies worldwide. The rates of serum anti-HCV, a marker of past or ongoing infection with HCV, range from 6% to 75% in patients with HCC10±22 (Table 1), with most patients actually harbouring the virus at the time of sampling.23±25 The risk estimates for developing HCC are high (odds ratio (OR) ˆ 23.6) for HCV carriers, but there are important di€erences in di€erent geographical areas.26 The ORs for HCV in China, Korea and Africa, where hepatitis B virus (HBV) is highly endemic, are much lower than the ratios in Italy, Spain and Japan where HBV is less common. In some areas distinct populations of liver cancer patients exist, probably re¯ecting di€erent epidemiologies and modalities of infection with the hepatitis viruses. For example, in the Far East, chronic HBV carriers often originate from intrafamilial transmission of HBV, while HCV is likely to be transmitted later in life by iatrogenic spread.7 This explains why patients with HBVrelated cancer are younger than patients with HCV-related HCCs. Table 1. Prevalence of serum anti-HCV in patients with hepatocellular carcinoma.10±22 Geographical area

Serum anti-HCV in HCC patients (%)

Europe USA Far-East Africa

28±75 29±41 6±55 29±58

PATIENTS AT RISK OF DEVELOPING HCC Increasing age, male gender, severe and long-lasting hepatitis, high levels of serum alpha-fetoprotein (aFP), virus genotype 1b, older age at infection, alcohol abuse and HBV, have been associated with an increased risk of cancer in HCV carriers.27±34 Overall, liver disease severity seems to be the crucial risk factor for tumour development. In fact, whereas the annual risk of developing HCC was 0.4% for unselected HCV carriers with persistently high alanine aminotransferase (ALT) values27, it rose to 1.7% for those with a histological diagnosis of chronic active hepatitis28±31 and to 2.5% for those with compensated cirrhosis32±34 (Table 2). Cirrhosis plays a key role in human hepatocarcinogenesis probably in relation to long-lasting persistent regeneration and proliferation of liver cells, as suggested by the high risk of HCC found in patients with histological markers of increased liver cell proliferation.30,35±37 In Milan, HCC developed ®ve-fold more often in cirrhotics with high liver cell proliferation, measured using the proliferating nuclear antigen (PCNA) technique, compared to those with lower values of PCNA.37 The risk of HCC was also high in cirrhotic patients with ¯uctuating or persistently elevated serum levels of aFP38,39, and in cirrhotics with both HBV and HCV infections compared to those with either one or the other infection (27% versus 15% with hepatitis B surface antigen (HBsAg) and 10% with anti-HCV).40 The HCC risk seems to be particularly high in carriers who harbour cryptic infection with HBV, as suggested by the presence of serum anti-hepatitis B core antigen (antiHBC)11 or HBV-DNA in the liver cells.41 Unlike hepatitis B42, the course of chronic

Hepatocellular carcinoma in patients with HCV 329 Table 2. Prospective studies of hepatocellular carcinoma in patients with chronic HCV infection. Entry cirteria

No. patients

Persistently high transaminases Chronic hepatitis

Cirrhosis

Yearly HCC (%) HCC predictors

Study

385

0.4

Cirrhosis, Age, AFP

Tradati et al 199827

433 124 179 1500

1.2 1.7 1.2 1.4

Cirrhosis, Age, AFP Histological Severity Severe Irreg. Reg. Fibrosis, GGTP, BT, Albumin, Alcohol

Tsukuma et al 199328 Takano et al 199529 Shibata et al 199830 Ikeda et al 199831

188 163

2.2 2.5

246

2.2

Sex, Age, Alcohol, HBV BenvegnuÁ et al 199632 Virus type, Age, Sex, Bruno et al 199733 Alcohol Age, Sex, AFP Romeo et al 199834

hepatitis C may be aggravated by alcohol abuse. A prospective cohort study in Japan demonstrated that alcohol abusers with chronic hepatitis C, developed HCC more often than HCV carriers who were not alcohol abusers.43 Finally, the demonstration that HCV is a genetically heterogeneous virus44 led to speculation that a relationship might exist between virus heterogeneity and HCC risk. One prospective study demonstrated that cirrhotics infected by genotype 1b were at higher risk of HCC than similar patients infected by other genotypes.33 However, such a correlation between genotype 1b and HCC risk has not been shown by other prospective follow-up studies. In a study of 124 Japanese patients with chronic hepatitis C29, 13 patients developed HCC during a mean follow-up period of 73 months. The prevalence of genotype 1b and 2a/c was 78% and 22% respectively, compared to 75% and 18% of patients with chronic hepatitis C who did not develop HCC. In a cohort of 246 Italian patients with HCV-related cirrhosis who were prospectively followed up for more than 10 years34, 56 developed HCC, with a yearly incidence of 2.2%. The 1b:2a/c genotype ratio of 2:1 observed in the cohort at enrolment remained unchanged during follow-up in the patients who developed HCC or not, were alive or dead, and those who subsequently were lost. PATHOGENESIS Nucleotide sequences from HCV and virus-speci®c protein expression have been demonstrated in HCC tissue.23±25,45 However, the molecular basis of HCV-associated liver carcinogenesis is poorly understood and a variety of normal and abnormal HCV encoded proteins have been implicated. Carboxy-terminal deletion mutants of the HCV core protein may accumulate in the cell nuclei, suggesting that a putative nuclear targeting sequence is present within the core protein.46 Nuclear localization of the core protein has been repeatedly demonstrated47,48 in patients and its function questioned. The core protein is the fundamental unit for encapsidation of genomic viral RNA during the process of viral maturation that takes place in the cell cytoplasm, not the nucleus, and hence the role of nuclear core protein has attracted much interest. The ®nding that the core protein may have trans-regulatory functions on cellular and viral promoters and, in co-operation with H-ras transformed primary rat embryo®broblasts, on tumourigenic phenotype47,49,

330 M. Colombo

suggests that HCV core protein may be directly involved in liver carcinogenesis. Interestingly, there are also data showing that the expression of core protein inhibits cysplatin-mediated apoptosis in human cervical epithelial cells and apoptosis induced by the over expression of c-myc in Chinese hamster ovarian cells50, suggesting that this protein may also reduce apoptosis of defective cells in addition to promoting their growth. These observations suggest that HCV core protein may be a major factor in the development of HCC. On the other hand, during hepatitis C infection Fas antigen accumulates in liver tissue, along with in®ltrating mononuclear cells51, and, in experimental models, cells expressing HCV core protein are sensitized to Fas-mediated apoptosis.52 Thus, one may speculate that HCV core protein has a role in immunologically mediated liver cell injury and helps establish chronic infection by enhancing apoptosis. Other data suggest that HCV core protein may also have an important biological role in the promotion of cell growth by down-regulating human p53, thus weakening cellular tumour suppressor functions.53 HCC developed in mice transgenic for the HCV core gene, which developed hepatic steatosis early in life as a histological marker of chronic hepatitis C.54 Liver cancer developed in a stepwise fashion ± it ®rst appeared as an adenoma containing fat droplets in the cytoplasm, followed by a poorlydi€erentiated tumour within the adenoma, thus reproducing the `nodule in nodule' appearance of many human HCC cases. These data contrast with reports showing that transgenic mice expressing the full-length core protein showed no histological evidence of HCC, suggesting that HCV core protein may not be cytopathic for liver cells.55,56 Finally, there are data indicating that other regions of the HCV genome have oncogenic potential. A truncated NS3 protease transformed murine ®broblasts and caused HCC in immunode®cient male mice.57

NATURAL HISTORY Tumour growth pattern The natural history of HCC arising in HCV carriers is heterogeneous. Many tumours are multi-focal when they are ®rst detected58,59, particularly in patients with multiple aetiological factors for HCC compared to patients with a single aetiology (Table 3). There are data suggesting that multi-nodular HCC can also be secondary primary tumours, the risk of tumour secondaries correlating directly with the size of the primary tumour. In fact, microscopic invasion of hepatic vessels by tumour cells was more common with HCCs that were 2±5 cm in diameter than with tumours smaller than 2 cm (80% versus 30%), irrespective of whether the tumour had a ®brous capsule or not.60 Not only size, but also the growth pattern varies greatly from one tumour to another, contributing to the heterogeneity of the natural history of HCC. In most patients with small HCV-related tumours, the so-called encapsulated expanding type was frequent (450% of the cases)61,62, whereas it was rarely found in most South African patients with HBV-related tumours.63 In the latter patients, HCC more frequently grew by replacing adjacent hepatic parenchyma. The presence of one or another histological growth pattern may be clinically important, since it in¯uences the outcome of treatment, particularly hepatic resection. Expressed as tumour volume doubling-time, the growth rate of HCC ranged from 1 to 20 months, with a median of 6.64±66 These observations provided the rationale for suggesting a 6 month interval

Hepatocellular carcinoma in patients with HCV 331 Table 3. Number of HCC nodules detected by ultrasound scan and staging in 178 patients with prospectively detected hepatocellular carcinoma. Number of tumor nodules Risk Factor

Imaging

One

More than one

All cases

Anti-HCV

US Staging

61 (78%) 48 (62%)

17 (22%) 30 (38%)

78 (100%) 78

HBsAg

US Staging

14 (67%) 13 (62%)

7 (33%) 8 (38%)

21 (100%) 21

Alcohol

US Staging

19 (86%) 15 (68%)

3 (14%) 7 (32%)

22 (100%) 22

Multiple

US Staging

30 (60%) 22 (44%)

20 (40%)* 28 (56%)

50 (100%) 50

None

US Staging

5 (71%) 3 (43%)

2 (29%) 4 (57%)

7 (100%) 7

*US: Multiple vs Alcohol, p 5 0.03. Staging: Multiple vs anti-HCV, p ˆ 0.05; Multiple vs Alcohol, p ˆ 0 06.

for ultrasound screening of cirrhotic patients. However, some nodules had constant rates of growth during follow-up, while others either had a declining growth rate in the late phases of follow-up or, after an initial phase of resting, increased in volume exponentially. It is not clear whether HCV-related tumours are as aggressive as HBVrelated HCCs.67±69

Cause of death With the advent of non-aggressive local therapies for HCC, fewer patients are left untreated and therefore few are available for studying the natural history of HCC. In Spanish patients with HCC (most are HCV-related), intrahepatic tumour progression and hepatic failure were the leading causes of death.70

Patient survival Because of the great diversity of the tumour growth patterns, the predictive power of the initial size of the tumour is not absolute. The combined assessment of tumour size and functional status of the liver provide more accurate estimates of patients' survival. Of 73 untreated Italian patients with Child's A cirrhosis and a single HCC smaller than 5 cm in diameter, 26% survived for 3 years compared to 13% for 43 patients with Child's B cirrhosis.71 However, the natural history of many patients with multi-nodular tumours does not di€er substantially from that of patients with a single node. The 3 year survival of 48 such inoperable patients who had no constitutional symptoms, no vascular invasion by the tumour, no extrahepatic spread of the tumour and had high performance status, was 50%.70

332 M. Colombo

SCREENING OF PATIENTS AT RISK In two Consensus Conferences, HCV carriers with cirrhosis were identi®ed as target populations for screening for early HCCs.72,73 The number of patients with cirrhosis due to chronic hepatitis C who develop HCC each year, is three times greater than that of HCV carriers with histological evidence of chronic hepatitis without cirrhosis (Table 2). Thus, cirrhotics are the best target population in terms of cost-ecacy, with a preference for those with high liver cell proliferation. In the latter patients HCC risk is ®ve times greater than in cirrhotics with low liver cell proliferation values.37 Serum aFP has been extensively used for screening and surveillance of patients at risk of HCC, with serum levels of 400 ng/ml being very suggestive of HCC. However, between the range of 20 and 200 ng/ml, patients with chronic liver disease and false positive results due to hepatitis ¯ares outnumber those with HCC.38,73±75 Serum aFP in patients with HCC has greater proportions of atypical aFP that can be assessed using lectin anity electrophoresis coupled with antibody-anity blotting. In a prospective study, 33 patients with cirrhosis showed elevated atypical aFP levels 3±18 months before HCC was detected by ultrasound (US).39 To minimize the false results of aFP determinations, cirrhotics are being monitored by means of real-time US. Most HCCs can be detected as a hypo-dysechoic mass.38 Since HCC nodes are also present as isoechoic or hyperechoic masses, di€erential diagnosis with small haemangiomas or regenerative macronodules may be dicult. TREATMENT The lack of controlled studies comparing the ecacy of the available treatments makes the selection of therapeutic options dicult. Because of the substantial heterogeneity of survival between control groups, it is not possible to assess the e€ects of di€erent therapies by comparing the results of separate trials. Furthermore, reassessment of treatment outcomes on the basis of intention-to-treat analysis would probably yield less encouraging ®gures. Thus, treatment options have largely been selected according to empirical criteria, such as the presence or absence of cirrhosis, number and size of tumours, and degree of hepatic deterioration. Unfortunately, many tumours smaller than 2 cm are hypovascular and therefore escape detection with biphasic spiralcomputed tomography (CT) scans.76 For staging clinical status, the Child±Pugh scoring system provides good estimates of patient survival.77 PATIENTS WITH SMALL TUMOURS Orthotopic liver transplantation Orthotopic liver transplantation (OLT) eliminates both detectable and undetectable tumour nodules, together with all the pre-neoplastic lesions in the cirrhotic liver, and reduces the risk of morbidity and mortality from portal hypertension. One major obstacle to the interpretation of OLT results is the large di€erences between transplantation centres in terms of the time-lag between listing for surgery and operation. The best long-term survivors (90% at 5 years) were patients for whom HCC was not the primary indication for OLT but was discovered by chance as a minute nodule during examination of the explanted liver.78 In Milan, 48 consecutive patients with viral cirrhosis and a single 55 cm tumour, or fewer than three 53 cm nodes

Hepatocellular carcinoma in patients with HCV 333

were treated by OLT.79 The 4-year actuarial survival was 92% for the 35 patients who were con®rmed at the operation as having met the selection criteria, compared to 60% for the 13 patients who did not, because they were found to have ancillary nodes. Although survival of transplanted patients seems to be largely in¯uenced by tumour size and number, there is no general agreement on the ideal tumour size that entails the least risk of recurrence, mostly because small tumour volume does not mean an early biological stage for all cases. Indeed, vascular invasion by the tumour can occur even in patients with small tumours and can be assessed only during the operation.78 The outcome of OLT may be in¯uenced by the recurrence of viral hepatitis, since infection of the graft may facilitate rejection and re-establish the oncogenic potential of the liver. The ecacy of interferon (IFN) and the nucleoside analogue ribavirin against HCV is under evaluation (see Chapter 9). Hepatic resection Since liver transplantation cannot be o€ered to all patients with cirrhosis who are found to harbour a small HCC, in many centres, limited hepatic resection (segmentectomy and subsegmentectomy) remains the primary therapeutic option for patients with HCC. Survival of patients undergoing hepatic resection is not only in¯uenced by the tumour size and invasiveness, but also by the functional status of the liver expressed as the Child±Pugh score. The 3 year cumulative survival was approximately 50% for patients with single tumours and Child's A status, 35% for patients with Child's B status and 0±12% for those with Child's C status.62,80 In patients with Child±Pugh A cirrhosis, portal hypertension is the most reliable predictor of survival after resection. None of the 14 surgically treated patients with an hepatic venous pressure gradient of less than 10 mmHg hepatic had unresolved hepatic decompensation, compared to 11 of the 15 patients with higher gradients.81 Thus, resection is de®nitely contraindicated for patients with decompensated cirrhosis or compensated patients with severe portal hypertension in view of the high operative risk and short life expectancy. There are no controlled data demonstrating that chemotherapy improves the survival of resected patients by eradicating occult nests of tumour cells.82 Also, there are no controlled studies comparing OLT and resection. In Paris83, resection and transplantation of cirrhotics with small liver tumours yielded the same medium-term results: 50% and 47% respectively at 3 years. However, the survival rates without recurrence were better after OLT than after resection (46% versus 27%), and in patients with small single or binodular tumours (53 cm), transplantation had much better outcomes than resection (83% versus 13%). PATIENTS WITH CIRRHOSIS NOT ELIGIBLE FOR SURGERY Advanced age, deteriorated liver function, large tumours, tumours localized in strategic positions or associated clinical conditions may contraindicate surgery and encourage treatment with less invasive local therapies. Percutaneous interstitial treatments Ultrasound guided interstitial treatments include tumour injection with absolute ethanol (PEI), 50% acetic acid or hot saline, or tumour thermoablation with radiofrequency, microwaves or laser. Once again, survival is largely in¯uenced by liver

334 M. Colombo

function, size and number of tumours. The 5 year survival of 293 Italian patients with Child's A cirrhosis and tumours of less than 5 cm diameter who were treated using PEI was 47%, compared to 29% for 149 patients with Child's B cirrhosis. Life expectancy of Child's A patients with a small tumour treated using PEI appeared to be as good as that of similar patients treated by hepatic resection, and was associated with a low risk of severe complications (1.7%) and mortality (0.1%). PEI is thought to be successful for small HCCs because these tumours are often hypovascular, and therefore trap the injected ethanol more eciently.76 Tumour disease recurred in virtually all treated patients, more often in those with high levels of serum aFP and those without a peritumoural capsule or with cirrhosis.84±86 In 60 randomly selected patients with tumours smaller than 3 cm and compensated cirrhosis, injection with 50% acetic acid was superior to ethanol in terms of 2 year cancer-free survival (92% versus 63%, P ˆ 0.02), being particularly active in patients with hypervascular tumours.87 The 3 year survival of patients treated using PEI was half that reported by previous studies in comparable patients for tumour size and liver function indicating that data on survival analysed by intention-to-treat yielded less encouraging ®gures than previously thought. In a prospective randomized study of 86 patients with compensated cirrhosis and small HCC, radiofrequency was superior to PEI in terms of complete tumour necrosis (90% versus 80%), and numbers of treatments (1.2% versus 4.8%), but it caused more complications (9.5% versus 0%).88 There are no guidelines on how to prevent tumour recurrence after percutaneous interstitial therapy. In a prospective controlled study, the risk of second primary tumours in 44 patients who were successfully treated by hepatic resection or PEI was reduced by long-term administration of polyprenoic acid.89 Transcatheter arterial chemo-embolization Transcatheter arterial chemo-embolization (TACE) of the proximal hepatic artery (conventional TACE) has been widely employed in Eastern and Western countries as an alternative to hepatic resection. This has now been improved as segmental or subsegmental TACE. The procedure is contraindicated for patients with venous tumour supply (hypovascular tumours), advanced liver deterioration, complete thrombosis of the portal vein trunk, renal failure or extra hepatic metastases. Randomized controlled studies of TACE in patients with unresectable HCC showed no bene®ts in terms of survival.90±92 Non-controlled studies of segmental TACE in Japanese patients with compensated cirrhosis and small HCC reported 4 year survivals comparable to those for similar patients treated using resection or PEI.93 Other treatments Systemic chemotherapy has been widely used to treat inoperable HCC, but the response rate was negligible whereas it caused fatal complications in several patients (25%) due to cardiotoxicity.94 In two large studies of 120 and 477 patients with inoperable HCC, but adequate liver function, treatment with the anti-oestrogen tamoxifen did not improve survival or quality of life, compared to controls.95,96 Perhaps, hormonal treatment of patients with inoperable HCC could be re®ned on the basis of the type of oestrogen receptor expressed by tumourous liver cells as indicated by the high response rate in patients with the wild-type oestrogen receptor.97

Hepatocellular carcinoma in patients with HCV 335

PREVENTION A hepatitis C vaccine is not yet available for preventing HCV infection, which prediposes patients to liver cancer. The potential of IFN therapy for reducing HCC risk in patients with chronic HCV infection was prospectively assessed in three trials (Table 4), with con¯icting results.98,99 The retrospective study of patients with chronic hepatitis C or cirrhosis who were treated using IFN demonstrated a reduction in the risk of HCC in responders.101±103 However, when data were adjusted for biochemical and clinical variables at entry, the di€erences in HCC risk between treated and untreated patients disappeared.104 Secondary chemoprevention using acyclic retinoids has been attempted in HCV carriers with cirrhosis and HCC who were successfully treated by hepatic resection or PEI. In a 3 year study period, acyclic acid prevented second primary HCCs, compared with placebo, by 28%, and suppressed liver cell clones producing the lectin-reactive isoforms of aFP, known to be a serum marker of latent HCC.89,105 The clinical impact of these ®ndings, however, is weakened by such methodological problems as the assumption that tumour ablation using PEI was as ecient as that obtained after hepatic resection and that all cases of metachronous HCCs were correctly identi®ed. Table 4. Prospective studies of HCC incidence in patients with HCV-related cirrhosis treated by interferon. Nishiguchi* (1995)98

Mazzella (1996)99

Valla* (1999)100

45 45

114 53

47 52

IFN MU  mo.

66

3  12

3  12

Months of f-up

24±84

32

36

2 (4%) 17 (38%) RR ˆ 0.07 (0.01±0.53)

7 (6%) 13 (25%) P 5 0.02

5 (11%) 9 (17%) NS

No. Patients Treated Controls

No. of HCC Treated Controls

*Randomized controlled trials.

REFERENCES 1. Ayoola EA, Odelola HA & Johnson AOK. Primary liver cancer (PLC) after non-A, non-B hepatitis (NANBH) [letter]. Hepatology 1982; 2: 154. 2. Resnick RH, Stone K & Antonioli D. Primary hepatocellular carcinoma following non-A, non-B posttransfusion hepatitis. Digestive Diseases and Sciences 1983; 28: 908±911. * 3. Kiyosawa K, Akahana Y, Nagata A & Furuta S. Hepatocellular carcinoma after non-A, non-B posttransfusion hepatitis. American Journal of Gastroenterology 1984; 79: 777±781. 4. Gilliam JH, Geisinger KR & Richter JE. Primary hepatocellular carcinoma after chronic non-A, non-B post-transfusion hepatitis. Annals of Internal Medicine 1984; 101: 794±795. 5. Kiyosawa K, Sodeyama T, Tanaka E et al. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 1990; 12: 671±675. * 6. Kuo G, Choo QL, Alter HJ et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989; 244: 362±364.

336 M. Colombo 7. Bosch FX & Munoz N. Hepatocellular carcinoma in the world: epidemiologic questions. In Tabor E, Di Bisceglie AM & Purcell RH (eds) Etiology, Pathology and Treatment of Hepatocellular Carcinoma in North America, pp 35±54. Houston, TX: Gulf Publishing Company, 1991. 8. Okuda K. Hepatitis C virus and hepatocellular carcinoma. In Okuda K (ed.) Liver Cancer, pp 39±49. New York: Churchill Livingstone, 1997. 9. El-Serag HB & Mason AC. Rising incidence of hepatocellular carcinoma in the United States. New England Journal of Medicine 1999; 340: 745±750. 10. Bruix J, Barrera JM, Calvet X et al. Prevalence of antibodies to hepatitis C virus in Spanish patients with hepatocellular carcinoma and hepatic cirrhosis. Lancet 1989; ii: 1004±1006. * 11. Colombo M, Kuo G, Choo QL et al. Prevalence of antibodies to hepatitis C virus in Italian patients with hepatocellular carcinoma. Lancet 1989; ii: 1006±1008. 12. Chen DS, Kuo GC, Sung JL et al. Hepatitis C virus infection in an area hyperedemic for hepatitis B and chronic liver disease: the Taiwan experience. Journal of Infectious Diseases 1991; 162: 817±822. 13. Dazza MC, Meneses LV, Girard PM et al. Hepatitis C antibody and hepatocellular carcinoma [letter]. Lancet 1990; 335: 1216. 14. Hasan F, Je€ers LJ, De Medina M et al. Hepatitis C-associated hepatocellular carcinoma. Hepatology 1990; 12: 589±591. 15. Kaklamani E, Trichopoulos D, Tzonou A et al. Hepatitis B and C viruses and their interaction in the origin of hepatocellular carcinoma. Journal of the American Medical Association 1991; 265: 1974±1976. 16. Kew MC, Houghton M, Choo QL & Kuo G. Hepatitis C virus antibodies in Southern African blacks with hepatocellular carcinoma. Lancet 1990; 335: 873±874. 17. Nishioka K, Watanabe J, Furuta S et al. A high prevalence of antibody to the hepatitis C virus in patients with hepatocellular carcinoma in Japan. Cancer 1991; 67: 429±433. 18. Poynard T, Aubert A, Lazizi Y et al. Independent risk factors for hepatocellular carcinoma in French drinkers. Hepatology 1991; 13: 896±901. 19. Saito I, Miyamura T, Ohbayashi A et al. Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proceedings of the National Academy of Sciences of the USA 1990; 87: 6547±6549. 20. Srivantanakul P, Parkin DM, Khlat M et al. Liver cancer in Thailand. II. A case control study of hepatocellular carcinoma. International Journal of Cancer 1991; 48: 329±332. 21. Vargas V, Castells L & Esteban JI. High frequency of antibodies to hepatitis C virus among patients with hepatocellular carcinoma. Annals of Internal Medicine 1990; 112: 232±233. 22. Yu MC, Tong MJ, Coursaget P et al. Prevalence of hepatitis B and C viral markers in black and white patients with hepatocellular carcinoma in the United States. Journal of the National Cancer Institute 1990; 82: 1038±1041. 23. Ohkoshi S, Kato N, Kinoshita T et al. Detection of hepatitis C virus RNA in sera and liver tissues of non-A, non-B hepatitis patients using the polymerase chain reaction. Japanese Journal of Cancer Research 1990; 81: 862±865. 24. Shibata M, Takeda S, Kudo T et al. Minus strand RNA of hepatitis C virus in liver tissues. Journal of Hepatology 1991; 13: 379±380. 25. Tang L, Tanaka Y, Enomoto N et al. Detection of hepatitis C virus RNA in hepatocellular carcinoma by in situ hybridization. Cancer 1995; 76: 2211±2216. 26. Donato F, Bo€etta P & Puoti M. A meta-analysis of epidemiological studies on the combined e€ect of hepatitis B and C virus infections in causing hepatocellular carcinoma. International Journal of Cancer 1998; 75: 347±354. 27. Tradati F, Colombo M, Mannucci PM et al. A prospective multicenter study of hepatocellular carcinoma in Italian hemophiliacs with chronic hepatitis C. Blood 1998; 91: 1173±1177. * 28. Tsukuma H, Hiyama T, Tanaka S et al. Risk factors for hepatocellular carcinoma among patients with chronic liver disease. New England Journal of Medicine 1993; 328: 1797±1801. 29. Takano S, Yokosuka O, Imazeki F et al. Incidence of hepatocellular carcinoma in chronic hepatitis B and C: a prospective study of 251 patients. Hepatology 1995; 21: 650±655. * 30. Shibata M, Morizane T, Uchida T et al. Irregular regeneration of hepatocytes and risk of hepatocellular carcinoma in chronic hepatitis and cirrhosis with hepatitis-C-virus infection. Lancet 1998; 351: 1773±1777. 31. Ikeda K, Saitoh S, Suzuki Y et al. Disease progression and hepatocellular carcinogenesis in patients with chronic viral hepatitis: a prospective observation of 2215 patients. Journal of Hepatology 1998; 28: 930±938. 32. BenvegnuÁ L, Chemello L, Bernardinello E, Cavalletto L & Alberti A. Evidence for an association between etiology of cirrhosis and pattern of hepatocellular carcinoma (HCC) development [abstract]. Italian Journal of Gastroenterology and Hepatology 1998; 30 (supplement 1): A66. 33. Bruno S, Silini E, Crosignani A et al. Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study. Hepatology 1997; 25: 754±758.

Hepatocellular carcinoma in patients with HCV 337 34. Romeo R, Rumi MG, Del Ninno E & Colombo M. Hepatitis C virus genotype 1b and risk of hepatocellular carcinoma [letter]. Hepatology 1997; 26: 1077. 35. Tarao K, Ohkawa S, Shimizu A et al. Signi®cance of hepatocellular proliferation in the development of hepatocellular carcinoma from anti-hepatitis C virus positive cirrhotic patients. Cancer 1994; 73: 1149±1154. 36. Borzio M, Trere D, Borzio F et al. Hepatocyte proliferation rate is a powerful parameter for predicting hepatocellular carcinoma development in liver cirrhosis. Journal of Clinical Pathology and Molecular Pathology 1998; 51: 96±101. 37. Donato MF, Arosio E, Balestrieri M et al. Increased risk of hepatocellular carcinoma (HCC) in cirrhotic patients with high proliferating cellular nuclear antigen index (PCNA-I) [abstract]. Italian Journal of Gastroenterology 1998; 30 (supplement 1): A66. * 38. Colombo M, de Franchis R, Del Ninno E et al. Hepatocellular carcinoma in Italian patients with cirrhosis. New England Journal of Medicine 1991; 325: 675±680. 39. Sato Y, Nakata K, Kato Y et al. Early recognition of hepatocellular carcinoma based on altered pro®les of alpha-fetoprotein. New England Journal of Medicine 1993; 328: 1802±1806. 40. BenvegnuÁ L, Fattovich G, Noventa V et al. Concurrent hepatitis B and C virus infection and risk of hepatocellular carcinoma in cirrhosis: a prospective study. Cancer 1994; 74: 2442±2448. 41. Cacciola I, Pollicino T, Squadrito G et al. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. New England Journal of Medicine 1999; 341: 22±26. 42. Austin H. The role of tobacco use and alcohol consumption in the etiology of hepatocellular carcinoma. In Tabor E, Di Bisceglie AM & Purcell RH (eds) Etiology, Pathology and Treatment of Hepatocellular Carcinoma in North America, pp 57±75. The Woodlands: Portfolio, 1991. 43. Yamauchi M, Nakahara M, Maezawa Y et al. Prevalence of hepatocellular carcinoma in patients with alcoholic cirrhosis and prior exposure to hepatitis C. American Journal of Gastroenterology 1993; 88: 39±43. 44. Simmonds P, Alberti A, Alter HJ et al. A proposed system for the nomenclature of hepatitis C viral genotypes. Hepatology 1994; 19: 1321±1324. 45. Rullier A, Le Bail B, Ghetti S et al. Immunohistochemical detection of HCV antigens in 23 hepatocellular carcinomas and surrounding cirrhotic tissue [abstract]. Italian Journal of Gastroenterology and Hepatology 1998; 30 (supplement 1): A66. 46. Ravaggi A, Natoli G, Primi D et al. Intracellular localization of full-length and truncated hepatitis C virus core protein expressed in mammalian cells. Journal of Hepatology 1994; 20: 833±836. 47. Shih CM, Lo SJ, Miyamura T et al. Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HUH-7 cells. Journal of Virology 1993; 67: 5823±5832. 48. Lo SY, Selby MJ & Ou JH. Interaction between hepatitis C virus core protein and E1 envelope protein. Journal of Virology 1998; 70: 5177±5182. 49. Ray RN, Lagging LM, Meyer K et al. Transcriptional regulation of cellular and viral promoters by the hepatitis C virus core protein. Virus Research 1996; 37: 209±220. 50. Ray RB, Lagging LM, Meyer K & Ray R. Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo ®broblasts to tumorigenic phenotype. Journal of Virology 1990; 70: 4438±4443. 51. Hiramatsu H, Hayashi N, Katayama K et al. Immunohistochemical detection of Fas antigen in liver tissue of patients with chronic hepatitis C. Hepatology 1994; 19: 1354±1359. 52. Ray RB, Meyer K & Ray R. Suppression of apoptotic cell death by hepatitis C virus core protein. Virology 1996; 226: 178±182. 53. Ray RB, Steel R, Meyer K & Ray R. Transcriptional repression of p53 promoter by hepatitis C virus core protein. Journal of Biological Chemistry 1997; 272: 10983±10986. * 54. Moriya K, Fujie H, Shintani Y et al. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nature Medicine 1998; 4: 1065±1067. 55. Kawamura T, Furusaka A, Koziel MJ et al. Transgenic expression of hepatitis C virus structural proteins in the mouse. Hepatology 1997; 25: 1014±1021. 56. Pasquinelli C, Shoenberger JM, Chung J et al. Hepatitis C virus core and E2 protein expression in transgenic mice. Hepatology 1997; 25: 719±727. 57. Sakamuro D, Furukawa T & Takenagi TJ. Hepatitis C virus non structural protein NS3 transforms NIH 3T3 cells. Journal of Virology 1995; 69: 3893±3896. 58. Takenaka K, Adachi E, Nishizaki T et al. Possible multicentric occurrence of hepatocellular carcinoma: a clinicopathological study. Hepatology 1994; 19: 889±894. 59. Fasani P, Sangiovanni A, De Fazio C et al. High prevalence of multinodular hepatocellular carcinoma in patients with cirrhosis due to multiple etiological factors. Hepatology 1999; 29: 1704±1707. 60. Ebara M, Ohto M & Kondo F. Strategy for early diagnosis of hepatocellular carcinoma (HCC). Annals of the Academy of Medicine of Singapore 1989; 18: 83±89.

338 M. Colombo 61. Gozzetto G, Belli L, Capussotti L et al. Liver resection for hepatocellular carcinoma in cirrhotic patients. Italian Journal of Gastroenterology 1992; 24: 105±110. 62. Franco D, Capussotti L, Smadja C et al. Resection of hepatocellular carcinomas. Results in 72 European patients with cirrhosis. Gastroenterology 1990; 98: 733±738. 63. Anthony PP. Primary carcinoma of the liver. A study of 282 cases in Ugandan Africans. Journal of Pathology 1973; 110: 37±48. 64. Okazaki N, Yoshino M, Yoshida T et al. Evaluation of the prognosis for small hepatocellular carcinoma based on tumor volume doubling time. A preliminary report. Cancer 1989; 63: 2207±2210. * 65. Ebara M, Ohto M, Shinagawa T et al. Natural history of minute hepatocellular carcinoma smaller than three centimeters complicating cirrhosis. A study in 22 patients. Gastroenterology 1986; 90: 289±298. 66. Barbara L, Benzi G, Gaiani S et al. Natural history of small untreated hepatocellular carcinoma in cirrhosis: a multivariate analysis of prognostic factors of tumor growth rate and patient survival. Hepatology 1992; 16: 132±137. 67. Okuda H, Obata H, Motoike Y & Hisamitsu T. Clinicopathological features of hepatocellular carcinoma ± comparison of hepatitis B seropositive and seronegative patients. Hepatogastroenterology 1984; 31: 64±68. 68. Shijo H, Okazaki M, Kogaemaru F et al. In¯uence of hepatitis B virus infection and age on mode of growth of hepatocellular carcinoma. Cancer 1991; 67: 2626±2632. 69. Ikeda K, Saitoh S, Tsubota A et al. Risk factors for tumor recurrence and prognosis after curative resection of hepatocellular carcinoma. Cancer 1993; 71: 19±25. * 70. Llovet JM, Bustamante J, Castells A et al. Natural history of untreated non-surgical hepatocellular carcinoma: rationale for the design and evaluation of therapeutic trials. Hepatology 1999; 29: 62±67. 71. Livraghi T, Bolondi L, Buscarini L et al. No treatment, resection and ethanol injection in hepatocellular carcinoma: a retrospective analysis of survival in 391 patients with cirrhosis. Journal of Hepatology 1995; 22: 522±526. 72. McMahon BJ & London T. Workshop on screening for hepatocellular carcinoma. Journal of the National Cancer Institute 1991; 83: 916±919. 73. Colombo M. Early diagnosis of hepatocellular carcinoma in Italy. A summary of a Consensus Development Conference held in Milan, 16 November 1990 by the Italian Association for the Study of the Liver (AISF). Journal of Hepatology 1992; 14: 401±403. 74. Oka H, Kurioka N, Kim K et al. Prospective study of early detection of hepatocellular carcinoma in patients with cirrhosis. Hepatology 1990; 12: 680±687. 75. Cottone M, Turri M, Caltagirone M et al. Screening for hepatocelluolar carcinoma in patients with Child's A cirrhosis: an 8 year prospective study by ultrasound and alphafetoprotein. Journal of Hepatology 1994; 21: 1029±1034. 76. Toyoda H, Kumuda T, Nakano S et al. The signi®cance of tumor vascularity as a predictor of long-term prognosis in patients with small hepatocellular carcinoma treated by percutaneous ethanol injection. Journal of Hepatology 1997; 26: 1055±1062. 77. Child CG III. The Liver and Portal Hypertension. Philadelphia: Saunders, 1964. 78. Iwatsuki S, Starzl TE, Sheahan DG et al. Hepatic resection versus transplantation for hepatocellular carcinoma. Annals of Surgery 1991; 214: 221±229. * 79. Mazzaferro V, Regalia E, Doci R et al. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. New England Journal of Medicine 1996; 334: 693±699. 80. Nagasue N & Yukaya H. Liver resection for hepatocellular carcinoma: results from 150 consecutive patients. Cancer Chemotherapy Pharmacology 1989; 23 (supplement 1): S78±S82. 81. Bruix J, Castells A, Bosch J et al. Surgical resection of hepatocellular carcinoma in cirrhotic patients: prognostic value of preoperative portal pressure. Gastroenterology 1996; 111: 1018±1023. 82. Harada T, Shigemura T, Kodama S et al. Hepatic resection is not enough for hepatocellular carcinoma. A follow-up study of 92 patients. American Journal of Gastroenterology 1992; 14: 245±250. 83. Bismuth H, Chiche L, Adam R et al. Liver resection versus transplantation for hepatocellular carcinoma in cirrhotic patients. Annals of Surgery 1993; 218: 145±151. 84. Livraghi T, Goldberg SN, Lazzaroni S et al. Small hepatocellular carcinoma: treatment with radiofrequency ablation versus ethanol injection. Radiology 1999; 210: 655±661. 85. Castellano L, Calandra M, Del Vecchio Blanco & de Sio I. Predictive factors of survival and intrahepatic recurrence of hepatocellular carcinoma in cirrhosis after percutaneous ethanol injection: analysis of 71 patients. Journal of Hepatology 1997; 27: 862±870. 86. Pompili M, Rapaccini GL, de Luca F et al. Risk factors for intrahepatic recurrence of hepatocellular carcinoma in cirrhotic patients treated by percutaneous ethanol injection. Cancer 1997; 79: 1501±1508. 87. Ohnishi K, Yoshioka H, Ito S & Fujiwara K. Prospective randomized controlled trial comparing percutaneous acetic acid injection and percutaneous ethanol injection for small hepatocellular carcinoma. Hepatology 1998; 27: 67±72.

Hepatocellular carcinoma in patients with HCV 339 88. Livraghi T, Giorgio T, Martin G et al. Hepatocellular carcinoma in cirrhosis in 746 patients: long-term results of percutaneous ethanol injection. Radiology 1995; 197: 101±108. 89. Muto Y, Moriwaki H, Ninomiya M et al. Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma. New England Journal of Medicine 1996; 334: 1561±1567. 90. Pelletier G, Roche A, Ink O et al. A randomized trial of hepatic arterial chemoembolization in patients with unresectable hepatocellular carcinoma. Journal of Hepatology 1990; 11: 181±184. 91. Groupe d'Etude et de Traitment du Carcinome HeÂpatocellulaire. A comparison of lipiodol chemoembolization and conservative treatment for unresectable hepatocellular carcinoma. New England Journal of Medicine 1995; 332: 1256±1261. 92. Pelletier G, Ducreux M, Gay F et al. Treatment of unresectable hepatocellular carcinoma with lipiodol chemoembolization: a multicenter randomized trial. Journal of Hepatology 1998; 28: 129±134. 93. Matsui O, Kodoya M, Yoshikawa J et al. Small hepatocellular carcinoma: treatment with subsegmental transcatheter arterial embolization. Radiology 1993; 188: 79±83. 94. Lai CL, Wu PC, Chan GCB, Lok ASF & Lin HJ. Doxorubicin versus no antitumor therapy in inoperable hepatocellular carcinoma. A prospective randomized trial. Cancer 1988; 62: 479±483. 95. Castells A, Bruix J, Bru C et al. Treatment of hepatocellular carcinoma with tamoxifen: a double-blind placebo-controlled trial in 120 patients. Gastroenterology 1995; 109: 917±922. 96. CLIP Group. Tamoxifen in treatment of hepatocellular carcinoma: a randomised controlled trial. Lancet 1998; 352: 17±20. 97. Villa E, Camellini L, Dugani A et al. Variant estrogen receptor messenger RNA species detected in human primary hepatocellular carcinoma. Cancer Research 1995; 55: 498±500. 98. Nishiguchi S, Kuroki T, Nakatani S et al. Randomised trial of e€ects of interferon-a on incidence of hepatocellular carcinoma in chronic active hepatitis C with cirrhosis. Lancet 1995; 346: 1051±1055. 99. Mazzella G, Accogli E, Sottili S et al. Alpha interferon treatment may prevent hepatocellular carcinoma in HCV-related liver cirrhosis. Journal of Hepatology 1996; 24: 141±147. 100. Valla DC, Chevallier M, Marcellin P et al. Treatment of hepatitis C virus-related cirrhosis: a randomized, controlled trial of interferon alfa-2b versus no treatment. Hepatology 1999; 29: 1870±1875. 101. Kasahara A, Hayashi N, Mochizuki K et al. Risk factors for hepatocellular carcinoma and its incidence after interferon treatment in patient with chronic hepatitis C. Hepatology 1998; 27: 1394±1402. 102. Ikeda K, Saitoh S, Arase Y et al. E€ect of interferon therapy on hepatocellular carcinogenesis in patients with chronic hepatitis type C: a long-term observation study of 1346 patients using statistical bias correction with proportional hazard analysis. Hepatology 1999; 29: 1124±1130. 103. International Interferon-a Hepatocellular Carcinoma Study Group. E€ect of interferon-a on progression of cirrhosis to hepatocellular carcinoma: a retrospective cohort study. Lancet 1998; 351: 1535±1539. 104. Fattovich G, Giustina G, Degos F et al. E€ectiveness of interferon alfa on incidence of hepatocellular carcinoma and decompensation in cirrhosis type C. Journal of Hepatology 1997; 27: 201±205. 105. Muto Y, Moriwaki H & Shiratori Y. Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma. Digestion 1998; 59 (S2): 89±91.