Journal ofHepatology, 1992; 15: 225-236 @ 1992 Elsevier Science Publishers B.V. All rights reserved. 016&8278l92/$05.

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HEPAT 01002

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assimo Colombo Instituteof Medicine, Universityof Milan, Milan, Italy

Hepatocellular carcinoma (HCC) is a highly malignant tumor with an extremely poor prognosis and an estimated incidence of between 250 000 and 1.2 million cases per year, worldwide (1,2). It is the seventh most common cancer in man (1). In recent years, both molecular biology studies of woodchucks persistently infected with a hepadnavirus, and prospective investigations of patients with cirrhosis who are at risk of developing HCC, have greatly improved our understanding of the pathogenesis and natural history of this cancer. In particular, studies of viral DNA.integration have revealed that persistent infection by hepidnaviruses, including the human hepatitis B virus (HBVj~, may generate genetic mutations and epigenetic phenomena with carcinogenic effects (3). Sequential ultrasound investigations of patients at risk have clearly established that the natural history of HCC in many instance5 is an extension of the underlying cirrhosis (4), although the course of the tumor may vary in different geographical areas. In Oriental and southern European patients a longer time may elapse between diagnosis and death, than in southern African patients (5). In contrast with the plethora of data for HCC in patients with cirrhosis, there is less information about tumor development in non-cirrhotic livers (6-S). One serious gap is the limited knowledge concerning the risk factors for this tumor, so that prospective studies of high risk patients are extremely difYicult.

pidemidogy The geographical

distribution

of HCC has been de-

scribed in terms of age-adjusted incidence rates, mortality and relative frequencies, i.e., the percentage of each type of cancer in a total series (9). For the world areas with the highest risk of liver cancer, the information is based on a limited number of cancer registries. High-risk countries (more than 30 cases of HCC per 100 000 males per year) include most of the sub-Saharan Africa, Southeast Asia and the coastal areas of mainland China. In Europe, relatively high rates (more than 61100 000 males) are seen in certain areas of Switzerland, Italy, Romania, Spain, France and Poland. Rates are low (fewer than 2/100 000 males) in Norway, the U.K., The Netherlands, Ireland, North America and Australia. In all these countries, however, there is considerable internal variation. For countries with incidence data for at least two different intervals, time-trend analysis can be done. Preliminary evidence indicates that the time trends for HCC are increasing among males in the U.S.A., Canada, northern European countries and Japan, while they are decreasing in other populations such as black males in parts of southern Africa (9-11). One important point of these epidemiolog ical studies that must be kept in mind is that it is uncertain whemer these changes in tumor rates are true or artefactual, i.e., the result of increased sensitivity of the currently available diagnostic tools. However, the increased incidence of HCC observed in North America has also been associated with increased immigration from areas of high HCC endemicity. In the Osaka area, the death rates for HCC rose from 16.3/100 000 per year in 1966 to 34.2/100 000 per year in 1981-1982. Many data indicated that chronic liver disease unrelated to HBV has been on the rise and that the increase in HCC was the result of this (11).

Correspondence: N. Colombo, Professorof Medicine, Institute of Internal Medicine, Via Pace 9,20122 Milan, Italy

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226 Risk

factors

Epidemiological observations clearly indicate that chemicals, hormones, viruses, metabolic diseases and cirrhosis are all risk factors for HCC. The effects of these factors, in turn, are likely to be modulated by characteristics such as the patient’s age, sex and race. In all populations, regardless of risk, rates of the incidence of HCC increase progressively with age, with a tendency to level-off in the older groups (9). In high-incidence areas, there is a shift toward the younger age groups. Men are more prone to develop HCC than women (9). In high-incidence areas the male-to-female risk ratio is around 4-8:1, whereas the male predominance is less marked in the low-risk populations of America and Europe. Despite many studies, it is still unclear whether the male preponderance of HCC reflects increased susceptibility to the tumor or greater exposure to the environmental factors associated with HCC. Studies of immigrants have provided unequivocal evidence that the risk of HCC in some ethnic groups largely depends on that population’s exposure to environmental factors. The rates of HCC are lower for the Chinese, Japanese and Filipino people born in the U.S.A. than for those who immigrated to the U.S.A. (9,12). The latter have lower rates than those living in their countries of origin. Better standards of hygiene in the U.S.A. than in Asian countries, resulting in reduced exposure to alimentary carcinogens and diminished intrafamilial spread of HBV, are likely explanations. AfIatoxin B1, the most toxic member of a family of difurano-coumarins produced as secondary metabolites by strains of Aspergil2u.sj7uw.s cd related fungi, has been associated with liver cancer (13). Humans can be exposed to afIatoxin by ingestion of contaminated foodstuffs or by products of animals fed with aflatoxin-contaminated cereals (14,15). There is strong evidence of carcinogenic effects of aflatoxin for many animal species (16). Such evidence for humans is less conclusive and it is based primarily on many population correlation studies and a few casecontrol studies (17-25). Militating against the pathogenic role of aflatoxin in humans is that the association between I-ICC and aflatoxin was not consistently recognized in studies where the biological markers of exposure to aflatoxin were used (26-30). Discrepancies are probably due not only to the lack of appropriate methods for assessing the past exposure of individuals, but also to confusion about the interplay of environmental factors that are not homogeneously distributed in the various geographical areas (31). An association between ethanol and human HCC is biologically plausible, since HCC has been observed in

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5-20% of all cases of micronodular cirrhosis and alcohol promotes cancer in organs such as oral cavity, pharynx, larynx and esophagus (32). Although ethanol has not been experimentally shown to be mutagenic, it might have effects on DNA metabolism that could be associated with carcinogenic activity (32). Acetaldehyde, the first metabolite of ethanol, can induce sister chromatid exchanges in hamster ovary cells grown in tissue cultures (33). Moreover, ethanol-fed animals have prolonged persistence of DNA adducts, which signals reduced capacity of the cell to repair DNA damage (32). The association of alcohol abuse with human HCC has been investigated in 22 studies involving more than 2700 patients: it was present in 14 studies (20,34-46) (relative risk, 1.3-12.0) but not in the remaining eight (47-54). Alcohol had a dosedependent effect, with approximately a 40% excess HCC risk entailed by heavy consumption. However, case-control studies in which drinking patterns are ascertained subsequent to HCC diagnosis could be biased by the patient’s tendency to under-report alcohol consumption. In areas where the prevalence of HBV carriers is low, alcohol may be an important factor for HCC: it does promote cirrhosis, the development and progression of which may act in conjunction with other environmental liver carcinogens. One study in Japan demonstrated that development of HCC occurred 10 years earlier in alcoholic patients who were HBsAg carriers compared to non-carriers (39 vs. 49) (34). The association between tobacco smoking and human HCC is also biologically plausible, since the liver is a natural target of the many potential carcinogens in tobacco and data exist for experimental liver carcinogenesis (55). However, there is no epidemiological evidence for a pathogenic role of tobacco smoking in human HCC. In fact, the existence of this association, especially in HBsAgnegative patients with HCC who had been long-term and heavy smokers, has been proven in only three of 20 studies performed so far (35-44,46-49,51,53,54,56,57). The available data do not support an interaction of tobacco with alcohol consumption. However, important methodological problems in the designs of these studies might explain some of the discrepancies. Several dietary constituents may be genotoxic after metabolic activation by the liver and, theoretically, promote HCC (58). Small amounts of these substances might cause enough initiation for tumors to occur as long as there is a concomitant condition to cause persistent liver cell proliferation. Thorotrast, a colloidal solution of thorium dioxide with the isotope radiothorium, which was formerly used as contrast medium in radiology, has been associated with development of HCC (59). Due to the long half-life of the isotope, hepatic tumors have developed after a latent pe-

227 riod of about 2Q years from intravascular administration of Thorotrast. Although many cases of with Thorotrast administration have been associated with cirrhosis, tumors have also been found in non-cirrhotic livers 1 Characteristically, refracttile granules of Tborotrast are trapped in the phagocytes in the portal tracts. There are several reasons to suspect that exposure to exogenous steroidal sex hormones may enhance the risk Of HCC in humans. Estrogens, progestogens, androgens and oral contraceptives have been shown to increase hepatic tumor development in animals (60). Moreover, there have been several reports of HCC developing in patients who have been treated with androgenic or anabolic steroids or oral contraceptives (60). Seven case-control studies, most of which have been conducted in areas not highly endemic for HBV and HCC, revealed a positive correlation between use of combined oral contraceptives and HCC, with a relative risk of 3.8-13.5 (39,49,57,61-64). The risk of I-KC was increased primarily in long-term users of contraceptives, in the absence of any other factors known to be associated with liver cancer, such as cirrhosis V. Two studies that were limited to short-term usin endemic HBV areas gave negative results (47,65). The fact that the increase in HCC risk attributable to contraceptives was detectable only in areas not endemic for HCC suggests that oral contraceptives most likely enhance the tumor risk by a small absolute number. On the contrary, the association between HBV and WCC appears to be strong, specific and consistent (6667). The prevalence of HBsAg is common in the areas of the world in which there is also a high incidence of liver cancer. Liver cancer cases were found more often (lo-2Qg than in their controls, and the times) in carriers of risk of Hl3sAg carri loping HCC was substantially greater than the risk for non-carriers. In a prospective study of 22 707 Chinese men in Taiwan, Beasley found sAg-posi t ive that the incidence of the tumor in the population was 47YlOO 000 persons per year, compared to only 4.6 in the HBsAg-negative individuals (relative risk = 102) (68& Another study that led to the same conclusions was conducted in a British male population of blood donors (49). Recently, transcriptionally active HEW genomes were found in patients with Liver cancer who were negative for serum HBsAg, suggesting that the pathogenic role of this virus in HCC may be greater than indicated by conventional serology (70). bother important piece of evidence connecting HCC to HBV comes from the study of either naturally or experimentahy infected woodchucks. Ninety-seven percent of captive animals infected at birth with woodchuck hepatitis virus (wHV) and maintained in captivity developed HCC after 17-36 months of persistent infection (71). The time of life

when the infection occurs is importaut for subsequent development of the tumor. Infection early in life or during pregnancy often becomes persistent and frequently results in CC, probably acting in conjunction with tional family-related factors (72,73). Infection with in adults carries a far lower risk of a carrier state developuent HCC development. Thus, the oncoV seems to be particularly important in most Asian and African countries where high HSV endemicity is maintained by infection occurring early in hfe, but not in the VWesternworld and Japan where this tumor is often related to other factors such as alcohol and hepatitis C. Several types of evidence connect HCC with the RNA hepatitis C virus (HCV). In the last decade in Japan, the number of HCC associated with HBV has decreased by 50% while that of HBsAg-negative tumors in patients with history of blood transfusions has increased, suggesting that HCV has a leading role in the etiology of this tumor (II). Sequential development of cirrhosis and HCC has been observed in patients with transfusion-associated and community-acquired hepatitis (for review see Ref. 74). Development of chronic non-A, non-B hepatitis was also observed in one chimpanzee which was experimentally infected with human plasma, with subsequent development of WCC 6 years after the infection (75). Finally, a high prevalence of serum antibody to HCV has been observed in patients with HCC, particularly in the HBsAg-negative subgroup of patients (76-84). Interestingly, nearly all of the HCV-infected patients had cirrhosis, whereas a consistent number of them also had other risk factors, such as HBsAg and history of alcohol abuse, indicating that in many patients both cirrhosis and HCC may have multifactorial origins. In some patients, RNA sequences of HCV were isolated from the tumor tissue by the polymerase chain reaction ($5). Although these findings establish a strong link between chronic HCV infection and neoplastic transformation of the liver, it is unhkely that HCV plays a direct role in HCC, since no reverse transcriptase activity has been found in infected Liver tissue. One current opinion is that rather than having a direct oncogenic effect, HCV promotes HCC through cirrhosis which is per se an important risk factor for this tumor. HCC has also developed in patients with rare metabolic conditions: patients with porphyria cutanea tarda, genetic hemochromatosis (GH), tyrosinemia, hypercitrulhnemia and aI-antitrypsin deficiency are considered to be at high risk (5). Although almost all reported cases of HCC complicating GH have occurred in cirrhotic patients, more recently, liver cancers have also been found in patients with hemochromatosis without cirrhosis (86). These clinical

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observations agree with studies which showed that iron can be mutagenic under appropriate experimental conditions. Patients with glycogenosis, type I and III, Wilson’s disease and hereditary fructose intolerance may also develop HCC, but are at substantially lower risk (5). The above analysis of the risk factors associated with HCC clearly indicates that patients with cirrhosis are a high risk population for HCC. This supports epidemiological observations that 30-40% of the patients who died of cirrhosis were found on autopsy to have HCC, and 90% of the patients with HCC have associated cirrhosis (87,88). In Taiwan, the risk of developing HCC was lOOO-times greater in cirrhotic male carriers of HBsAg than in noncirrhotic HBsAg-negative males (68). More recently, close follow-up studies of cirrhotic patients with highly sensitive and specific diagnostic methods, such as realtime ultrasound, have led to recognition of tumors at an early stage (4,89-91). The significance of the association of HCC and cirrhosis is still obscure and no single explanation accounts for the various forms this association takes in different parts of the world. In endemic areas, an etiology common to the two pathologic disorders, namely chronic infection with HBV or HCV, accounts for the vast majority of cases. In low endemicity areas, it seems likely that cirrhosis makes the hepatocytes more susceptible to environmental carcinogenic agents such as alcohol. in some patients HCC could be the inevitable consequence of long-standing hepatic disease, whilst in others it could be an independent response to a hepatic insult common to HCC and cirrhosis. A key factor in the pathogenesis of HCC development in patients with cirrhosis is liver cell regeneration. Cell regeneration during cirrhosis, unlike in normal livers, may be oncogenic since it is associated with abnormalhormone patterns, alteration in the liver array of parenchymal cells, altered production of growth factors and abnormal oncogene expressions (9,87,92).

Pathogenesis Classical studies by Farber with rat hepatocarcinogenesis models have led to the concept that the fundamental mechanism of hepatocel!ular carcinogenesis is clonal expansion of transformed normal liver cells (93). According to this hypothesis, liver cancer develops in a step-wise manner, through the phases of initiation, promotion and progression. During the long period of cancer development, the transformed liver cells become more basophilic, indicating active growth, followed by the appearance of focal proliferations of ‘nodules’ of altered hepatocytes. The appearance of additional altered foci of cells or ‘nodules within nodules’ has been taken as evidence of carcin-

ogenic progression within nodules. In addition to this proposed classic, ‘step-wise’ cellular pathway to HCC, recent evidence strongiy suggests another mechanism of hepatic carcinogenesis based on the proliferation of liver stem cells (94). According to this hypothesis, the predominant cellular pathway to HCC begins with proliferation of a population of stem cells called ‘oval cells’. In the experimental animal, electron microscopy and autoradiography studies have shown that oval cells, which originate in the portal tracts, are the first cells to proliferate l-3 days after carcinogen treatment and that these cells have the potential to differentiate either into basophilic, albumin-producing hepatocytes or into bile duct cells. Useful models for investigating these pathways to HCC are animals infected with hepadnaviruses, i.e., the woodchuck, the Peking duck and the ground squirrel. When infected persistently, these animals are continuously exposed to both a carcinogenic agent, the hepadnavirus, and a mitogenic stimulus, the accompanying chronic hepatitis (95). In appropriate experimental conditions, the accumulation of toxic quantities of HBsAg within the hepatocyte may be a sufficient stimulus for the liver to progress to hepatocelular necrosis and neoplasia (96). No single mechanism has been proposed for hepadnavirus-induced oncogenesis, and most data point against the existence of a viral transforming oncogene (97). One general mechanism by which the hepadnavirus acts as a carcinogenic agent is by integration into the host cellular genome. The persistence of viral integrations in animals and in man may cause genetic changes and epigenetic phenomena (97). The unstable nature of the cellular DNA into which the hepadnavirus has been integrated may cause such chromosome alterations as deletions of cellular DNA, transformation and duplication of viral and cellular DNA and the chromosomal translocation found in HCC. The gradual accumulation of these mutations in the cellular genome may predispose a cell to malignant transformation. More specific mechanisms of genetic damage (insertional mutagenesis, transactivation) have also been observed in man and animals. Integration of WHV DNA into the C-myc and/or N-myc genes was detected in a large fraction of HCCs from WHV carrier woodchucks (3,98). HBV DNA has been shown to integrate into a human cyclin A gene and retinoic acid receptor gene, both important in cell cycle control (93,100). There is much evidence that these virns integrations have created new genes with potential regulatory properties. It is more difficult to interpret the pathogenesis of HCC in livers with no viral insertioms. In these, hepadnaviruses may have caused cellular DNA mutations, which are no longer linked to viral DNA, by a ‘hit and run mechanism’.

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transactivational potential in the X region of their genome, indicating that hybrid proteins encoded by these genes can influence the expression of cellular nuclear protooncogenes, which in turn influence growth and differentiation of infected hepatocytes (3). A second transcriptional transdctivator has recently been identified in the pre S,/S region of an integrated HBV-DNA which was cloned from an HCC (101). The existence of multiple oncogenes is quite consistent with the complexity of hepatocarcinogenesis in hepadnavirus carriers, as reflected by the multiple genetic changes present in the tumor cells. Studies in transgenic mice containing liver targeted oncongenes, have clearly shown that oncogenes function synergistically to induce KCC. Activated n7.s protooncogenes have been detected in a large percentage of HCCs in mice and man (3,97). The role of growth factors in the patbogenesis of HCC is obscure. Significant changes in the expression of insulin-like growth factor-11 (IGF-II) in the precancerous WHV livers have been observed and related to the growth of precancerous nodules (3,97). Several lmes of evidence indicate that HCC may also be the result of the inactivation of tumor suppressor genes. HBV integrations, deletions, duplications and rearrangements of the genes on chromosome 1lp, which contains a tumor suppressor gene, were observed in buman HCCs (102). Using restriction fragment length polymorphisms to study chromosome alteration in HCCs from HBV carriers, a high frequency of loss of alleles from chromosome llp in HCC DNAs were detected (3,97). More recently, point mutations in the suppressor ~53 gene (mutational hotspots) have been identified in some HCCs from Chinese and South African patients living in areas endemic for HBV and aflatoxin (103,104). Gene mutations consisted preferentially in guanin to thymiclm substitutions witbin clustering at codon 249. Tbe presence of this hotspot in HCC implies that there is a selective advantage for these specific mutations that could be etiopathogenetitally important. In fact, in appropriate experimental conditions, aflatoxin Br, may cause similar mutations and HBV, which is also implicated in HCC in Africa and China, could interact through a virally induced protein with the specific mutant ~53.

acroscopic and mlcrnseopic classification Liver cancer may develop in a nodular, massive or diifuse fashion (Eggel’s classification). Histologically, KCC can be divided into four types: trabecular, pseudoglandular, compact and scirrhous. The trabecular type is composed of tissue strands of various cell thickness separated

by blood-filled sinusoids. The pseudoglandular type is composed of gland-like structures of varying sizes. The compact tumor has a basically trabecular strnctnre but the tumor cells proliferate to solid and medullary, with modest amounts of stroma. e scirrbous type consists of strands of tumor cells snrronnded by large amounts of fibrous interstitial tissue. HCC is also defined cytologically as: (i) pleomorphic, when there is a marked range of differences in both cellular and nuclear size and in the shape and staining of the nuclei; and (ii) clear-cell tumors, composed of cells with clear cytoplasm. Finally, the degree of atypia of the tumor cells is classified according to criteria of Edmonson and Steiner (106): grade 1, highly diierentiated tumor cells resembling normal hepatocytes; grade 2, slight but definite atypism; grade 3, prominent nuclear atypism and pleomorphism, with the appearance of multinucleate giant cells; grade 4, uniformly composed of anaplastic tumor cells with very scanty cytoplasm. It is not nncommon for two or more of the above histological and cytologic& characteristics to be present in the same tumor. It is, therefore, recommended that the predominant histological type and degree of cell atypia shown by the KCC be indicated in all reports.

The time-lag between exposure to carcinogenic factors and tnmor development can vary from a few years to many decades. In a retrospective study of 231 patients with transfusion-associaited hepatitis, Kiyosawa et al. (80) demonstrated that the mean times between transfusion and the development of chronic hepatitis, cirrhosis and HCC were lo,21 and 29 years, respectively. In many patients, KCC grows slowly within the liver without causing any significant clinical signs or symptoms and patients with small KCC may only occasionally complain of abdominal pain or malaise. Signs and symptoms of more advanced disease include hepatosplenomegaly , jaundice, ascites, weight loss and fever (5). In many African patients, KCC may present with hepatosplenomegaly and jaundice, as a result of its accelerated growth rate (107). Kemoperitoneum, caused by spontaneous rupture of the tumor, is a rare event in Caucasian patients, but occurs more frequently in Oriental and African patients. Several endocrine and paraneoplastic manifestations may accompany development of the tumor. Erythrocytosis, probably due to increased erythropoietin production, occurs in S-10% of the cases. Episodes of hypoglycemia are not uncommon, attributable to production by the tumor, of a large molecular weight pro-IGF-II. In the terminal stages of tbe disease when the tumor is massive, hypoglycemia is

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due to hepatic glucose production which is insufficient to meet the needs of the tissues and large tumor. SYmPtomatic hypercdcemia is most uncommon and is usually associated either with bone metastase or ectopic parathormone secretion (5). Whether HCC originates from one or more than one clone of transformed liver cells is a matter of controversy. Studies of HBV-DNA integration patterns in HCC recurring after surgery of a primary HBsAg-positive tumor showed that in some patients HCC may grow either as a single monoclonal node or as a polyclonal disease (108). The discovery of solitary HCC nodes in variable proportions of patients subjected to routine periodic follow-up examinations with US scans suggests that in many patients HCC may have a long-lasting subclinical incubation period during which it may grow as a solitary mass. There are important regional differences in the growth patterns of HCC. The so-called encapsulated expanding type is frequent in the Oriental (4), Italian (109) and French (110) patients with small tumors, whereas it is rarely found in North America (111) and Africa (107). This type of tumor is often relatively well-differentiated and is associated with cirrhosis. Whether the fibrous tissue surrounding the tumor node is a true capsule or condensed cirrhotic stroma is controversial (112). In a minority of the patients, these small HCC tend to grow either by replacing the adjacent hepatic parenchyma or by mixing with it. The presence of one or another histological growth pattern is clinically important since it may influence the prognosis after resection (109-114). The growth rate of asymptomatic HCC varies greatly. When expressed as tumor volume doubling time, the tumor growth rate ranges from 1 to I9 months, with a median of 6, and this may be correlated directly with the patient’s survival time (4,89-91,113,114). It is not clear why the growth rates of HCC vary so greatly from patient to patient. Sequential studies of untreated patients have shown different growth kinetics of single HCC nodes (4,90,91,113). Some tumors have constant rates of growth during follow-up, others either have a declining growth rate in the late phase of follow-up or, after an initial phase of no growth, increase exponentially in volume. thus, the wide range in tumor volume doubling times that has been reported in the literature reflects the existence of different patterns of tumor growth. It has been said, but not universally accepted, that the sonographic patterns of HCC may change as tumor size increases. During tumor growth, the low echo pattern which is characteristic of smaher tumors may be progressively replaced by high or mixed echo patterns (89). The latter are rather common in advanced tumor disease, i.e., when the tumor has grown massively.

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In many patients, serum levels of alphafetoprotein (AFP) seem to parallel tumor growth (115,116). Obviously, this is true only for AFP-secreting tumors, which represent 40-70% of the total HCCs. In massively grown HCC, the serum levels of AFP often exceed the cut-off value of 400 @ml. For some patients with cirrhosis, the pattern of serum AFP during follow-up may predict when HCC will develop (117). HCC may spread outside the liver, but only in the late phase of the disease metastases are clinically evident. The most common sites of extrahepatic metastases are the regional lymphnodes, the lung, the adrenals and the skeleton (118). The survival times for patients with this tumor are inversely correlated with the degree of hepatic replacement by the tumor and the severity of the liver impairment (115,118). A prospective study of Oriental and Italian patients with cirrhosis and early detected HCC has further demonstrated the long-lasting, slow growth of HCC in these racial groups. In fact, the survival of 22 Japanese patients with cirrhosis and HCC less than 3 cm was 91% at 1 year, 55% at 2 years and 13% at 3 years (89). The survival of 12 corresponding Italian patients with Child’s A cirrhosis ranged from 7 to 36 months, with a 2-year survival of 44% (90). Cancer is considered to be the cause of death of patients with HCC who died with advanced tumors or from cachexia and deterioration of the general condition rapidly progressive jaundice without developing (115,118). In a series of 659 patients with HCC, hepatic failure and gastrointestinal bleeding were the leading causes of death (39% and 23%, respectively), whereas cancer was the cause of death in only 70 (18%) (118). In reports of the cause of death for untreated patients with HCC who had been followed prospectively, hepatic failure and hemorrhage were the leading causes (89,90). Numerous epidemiological, laboratory and clinical parameters have been shown to be of prognostic value for patients with HCC (116,118-120). It is unanimously agreed that liver function tests, alone or in combination with ascites and encephalopathy, are the best prognostic indicators for these patients. Instead, the predictive value of such variables as sex, age, serum AFP and alcohol consumption is debated. Male sex and advanced age were found by some to be correlated with a poor prognosis, but not by others (120). There is also debate about whether the histological features of HCC are of prognostic value (121). Some studies have demonstrated a correlation between the patient’s survival and cytological grading of the tumor, and between doubling time of tumor volume and patient survival (91). Prognosis is said to be slightly better for the rare form of fibrolamellar carcinoma than for other forms of HCC (122). This tumor develops in young people (5-35 years)

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of both sexes and is not associated with cirrhosis. istologically, clumps of tumor cells are interspersed with bands of mature fibrous tissue. Serum APP is frequently normal and markers of serum hepatitis are rarely detected.

Real-time ultrasound plus thin-needle liver biopsy are the more sensitive and specific routine diagnostic means for HCC, with an estimated accuracy of 90% (100% for large tumors) (109). The false-negative results with this procedure are mainly for patients with tumors less than 2 cm in diameter and early neoplastic changes of the liver cells which can be recognized only by very experienced pathologists. HCC cannot be diagnosed with confidence on the basis of increased serum levels of APP only. In fact, this marker rarely exceeds the diagnostic level of 400 @ml in patients with small tumors (4,90), and there are tumors such as testicular tumors which may metastasize to the liver and have very high APP levels (5). In patients with small HCC and severe coagulopathy, tumor cells for diagnosis can also be obtained by needle aspiration. This approach is safe, since very thin needles are used, but may give numerous false positive and false negative results (117,123). The computerized tomography (CT) scan is as sensitive as ultrasound for detecting tumors greater than 3 cm in diameter (117,124). It is more difficult to identify tumors smaller than 3 cm by conventional CT (125). CT after hepatic arteriography with lipiodol is said to be ideal for detecting small HCCs which would escape detection by conventional CT. In fact, the identification of hepatic lesions as small as 3 mm in diameter is possible by the strong liver/lesion contrast iodized oil injection. There may be a problem, however, with false positives due to non-specific clusters of lipiodol in regenerative nodules or connective tissue. The diagnostic utility of nuclear magnetic resonance in this area is still under investigation (126).

Earlydiagnosis

Early diagnosis of HCC is possible by identifying individuals with recognizable risk factors for this tumor, and certain characteristics of the disease such as the long phase of incubation and the tendency to grow as a solitary mass in the liver. Although strategies for HCC screening should be modified according to the local epidemiological situation, there are general rules for the design of such studies. These rules take into account whether populations of individuals or groups of patients are being investi-

231

gated (117). In population-based studies, only a few individuals have identifiable risk factors, most of them are asymptomatic, and the screening intervals are standardized. This is net the case for clinic-based studies, where participants may have more than one risk factor for HCC, are often symptomatic and where screening intervals are not standardized. Recently, in a consensus development conference held in Anchorage (Alaska) (127), chronic HBsAg carriers, carriers with cirrhosis or a family history of I-ICC, and patients with rare metabolic diseases were identified as high risk patients for I-ICC and, therefore, as candidates for periodic screening. It was rccommended that healthy carriers of HBsAg have yearly determinations of serum APP and patients with cirrhosis be screened every 6 months by abdominal ultrasound and serum APP. In a prospective study carried out in Alaskan natives (128) 16 tumors were identified in a population of 1400 HBsAg carriers who had been tested annually for serum AFP. In a prospective study of 236 Chinese patients with cirrhosis (4), 20 patients with HCC were identified by ultrasound, some detected at an early enough stage to allow successful hepatic resection. However, even with the evidence that screening high risk individuals may increase the yield of potentially operable tumors, no firm conclusions have yet been reached that these policies reduce mortality rates for HCC. The theoretical reasons for this include the high rate of tumor recurrence after hepatic resection (129.130) the high rate of second primary tumors in the liver, and the coexistence of cirrhosis, which may reduce the number of patients eligible for surgery and increase postoperative mortality.

neoplastic lesions The recognition of preneoplastic lesions in patients at risk might improve chances of treatment for many patients with HCC. Small cell type dysplasia seems to be a likely candidate, especially when it is present as nodules (131). Macroregenerative nodules, formerly called adenomatous hyperplasia, have also been associated with an increased risk of liver cancer in patients with cirrhosis (132). In some instances, differential diagnosis between premalignant lesions and a well-differentiated tumor is diicult by routine histology (133). Studies with monoclonal antibody Ki67 and bromodeoxyuridine have been promising in that they allow detection of abnormal cell proliferation indices in liver samples from patients with ‘borderline’ lesions who later developed HCC (134,135).

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232 Treatment

Future directions

There is no evidence that mortality from HCC can be reduced by any of the available treatments. Hepatic resection is the primary therapeutic option, although it is often restricted by the coexistence of cirrhosis and the degree of liver replacement by the tumor. In rncontrolled studies, 30-50% of selected cirrhotic patients with small HCC survived 3 years after hepatic resection, although many of them had signs of disease recurrence (109,110, 129,130,136-139). Survival rates were better for patients with encapsuiated tumors and fibrolamellar HCC than for patients with infiltrating HCC. The rate of survival at 3 years was similar for patients with HCC who had been treated by orthotopic liver transplantation (140-142). The subgroup of patients with tumors detected by chance and those with tumor-free hepatic lymphnodes have had the longest life expectancy. Doxorubicin and its analogues given at 75-100 mg/m* of body surface every 3 weeks has had only palliative effects and in only a minority of the patients (143). The efficacy of therapeutic approaches such as intra-arterial chemo-embolization (144) and intratumor ethanol injection (145) is under evaluation.

Most investigators agree that our understanding of the pathogenesis and natural history of HCC could be improved if multicenter studies using large numbers of clinically homogenous patients could be performed. To achieve this objective, communication within the scientific community must be improved, with standardization of terminology and investigational strategies as the first step. To elucidate the pathogenic role of single risk factors and to outweigh the effects of agents versus associated liver disease, these multi-center studies should be focused on patients segregated by risk. The approach to the pathogenesis of the tumor in patient populations in whom multiple risk factors may interact is more complex. For this, epidemiological studies of selected populations of the Third World might be helpful, particularly if cancer registration is developed and newly developed markers for exposure to viral and chemical factors are employed. More effort should be put into clarifying whether screening programs for early detection of HCC are indeed costeffective, at least in terms of yield of potentially treatable tumors. Finally, in the wake of the growing but anedoctal information suggesting that new therapeutic modalities such as percutaneous ethanol injection, intraarterial chemoembolization and orthotopic liver trans&ntation may be effective in the treatment of selected patients, many agree that randomized, controlled trials should be performed in appropriate clinical settings.

Prevention

The only realistic way to reduce mortality due to HCC is prevention. Eradication of the HBV carrier state is now feasible with programs of universal vaccination of newborns. Some of these have already been started in endemic areas (146). A simiiar strategy is not available for controlling the spread of HCV, another leading cause of cirrhosis, but will be feasible once a vaccine is developed. Educational programs and new regulations can help to decrease alcoholism, which accounts for one-third of all cases of cirrhosis worldwide. Contamination by mycotoxins can be reduced by improved agricultural methods, storage and transport of grain.

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Acknowledgements

The author is indebted to Dr. Angelo Sangiovanni and Dr. Giovanni Covini for their constant support in preparing this manuscript.

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