the use of epidemiology, scientific data, and regulatory authority to determine risk factors in...

REGULATORY TOXICOLOGY AND PHARMACOLOGY 5,384-404 ( 1985)

The Use of Epidemiology, Scientific Data, and Regulatory Authority to Determine Risk Factors in Cancers of Some Organs

of the Digestive System

3. Liver Cancer’

D. JESSE WAGSTAFF

Epidemiology and Clinical Toxicology Unit, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, D. C. 20204

Received March 2. I985

INTRODUCTION

Primary liver cancer (PLC), although uncommon in developed countries, is one of the most common cancers in the world because of its high prevalence in parts of Africa and Asia (Blumberg and London, 1982). Liver cancer induced in animals by aflatoxin has been called the best opportunity to verify the extrapolation of animal cancer data to humans (Carlborg, 1979). The unique distribution of PLC among human population groups offers opportunities for the study of natural experiments and justifies the conclusion that this form of cancer is largely preventable and perhaps even curable.

This review was intended as a status report on the potential human hepatocarcinogenicity of aflatoxins as food contaminants; however, it became apparent that the subject should be placed in a larger context. Only selected references are cited but a bibliography of over 3500 entries is available from the National Technical Information Service (Wagstaff, 1984).

PLC is defined as cancer arising in any hepatic tissue and has been assigned Znter- national Classification of Diseases (ICD) code 155 (WHO, 197 1). The most commonly used classifications are those of Edmondson ( 1958) and the World Health Organization (Gibson and Sobin, 1978). Liver neoplasms are classified as benign or malignant and by histologic type. Other factors sometimes used in classification are biochemical tumor markers, antigens, and clinical course of the disease.

The most common type of liver cancer, hepatocellular carcinoma (HCC), arises in parenchymal cells (an older term, hepatoma, has largely been discarded). There are

’ This is the third in a series of papers on this subject in this journal. The second appeared in the September 1985 issue.

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RISK FACTORS IN CANCER OF DIGESTIVE SYSTEM ORGANS 385

several subtypes of HCC (Berman et al., 1980) one of the most important being the fibrolamellar variant (Craig et al., 1980). The second most common histologic type, cholangiocellular carcinoma (CCC), arises in intrahepatic biliary cells. Angiosarcoma is an uncommon but important type of PLC. In young children the most common liver cancer is hepatoblastoma. Cancers of other hepatic tissues are rare (Foster, 1982).

Survival after the first symptoms appear is usually a matter of months, and 5-year survival rates have been less than 5% (Axtell et al., 1976; Okuda et al., 1980); however, a few patients with well-documented, even metastasized, PLC have survived for many years (Gottfried et al., 1982; Lam et al., 1982a; Penalba et al., 1982). Some PLCs found incidentally during necropsy or other diagnostic procedures seem not to have caused illness or death (Thomson, 196 1; Shank et al., 1972a); these would not affect .martaIity statistics based on the primary cause of death but could alter evaluations of incidence data.

HISTORY OF LIVER CANCER

Liver cancer was first recognized in the mid- 1800s (Noeggerath, 1854) but the wide variety of cancers was only gradually appreciated. PLC was grouped with other visceral cancers in the ICD until 1955 (USHEW, 1960). Some of the best historic documen- tation comes from central and southern Africa. Explorers there such as Livingston found no cancer among native peoples (Oettle, 1964), and early medical missionaries and government medical officers found little cancer and often concluded that it was rare (Watkins-Pitchford, 1925). As hospitals were established it gradually became apparent that some cancers, especially liver cancer, were relatively common.

PLC probably existed at a high rate in some populations before the widespread use of synthetic chemicals and even before the use of industrial products. One of the best indications of this prevalence comes from the meticulous records of Albert Cooke, a medical missionary in early Uganda (Davies et al., 1964). The area of Uganda was not explored until 1862, and a rudimentary colonial government was set up in 1894. Few goods were imported because of the enormous expense and death loss of porters in head-carrying of goods from the East African coast. Cooke arrived in 1896 and kept records from the time of the opening of his hospital in 1897. The prevalence of PLC was not realized at first, but his records contained many cases from the turn of the century with symptoms, signs, and lesions identical with those of present-day PLC. From these records, Davies et al. (1964) concluded that the high level of PLC in Uganda has been relatively constant over most of the present century. Similarly, in the Philippines, PLC has been the most frequent cancer at necropsy since the turn of the century (Bulatao-Jayme et al., 1976).

A number of limitations must be recognized in interpreting human cancer data. First, some ill and dying people did not seek medical help (Mulligan, 1970); distrust, reliance on folk medicine, and other factors kept many away, and these situations still persist to some degree (Finau, 198 1). Among the general native population not in the employ of the colonists, the only patients seen were those who voluntarily presented themselves. People who became sick or old often moved from the catchment area near a hospital back to their home areas (Prates and Torres, 1965).

A second problem is that the segment of a population which is available to the reporting system may not represent the whole population. In some cultures females

386 D. JESSE WAGSTAFF

had to get permission from the head of the household before seeking medical attention (Prates and Tort-es, 1965). Elderly people in some areas were less apt to seek medical help than people of working age (Mulligan, 1970) but in other areas older men were more likely to seek medical help (Finau, 198 1). Providing subsidized medical care to segments of a population may affect the number of cases diagnosed (Mulligan, 1970).

A third problem is that demographic characteristics of the patients were often unknown. Patients gave false names and inaccurate ages (Davies et al., 1958) or attended different clinics during the course of their illness (Oettle, 1964).

A fourth problem beyond those of ill people not seeking medical help or not being accurately characterized in the records is that of inadequate diagnosis. Diagnosis should be based on both antemortem and postmortem examinations, including histologic verification; conclusions based only on clinical impressions or biopsy material can be misleading. Coady ( 1965) surveyed physicians in Ethiopia and found that liver disease was commonly diagnosed but PLC was only vaguely recognized with some respondents saying it was rare or nonexistent. At about the same time, in the medical department of a hospital in Addis Ababa, 20% of the deaths were due to PLC (Pavlica and Samuel, 1970). Failure to recognize PLC in Ethiopia was attributed to the lack of biopsy and necropsy capabilities. In Taiwan, only 3% of cancers in a biopsy series were PLC compared with 22% in a necropsy series (Yeh, 1966). PLC was the 17th most common cancer in a biopsy series in the Philippines but the first most common cancer in a necropsy series (Barrera et al., 1958). Among PLC cases found at necropsy in Sweden, less than one-fourth had been diagnosed antemortem (Axelsson, 1982). Low necropsy rates can result not only from a lack of facilities but also from cultural prohibitions (Finau, 198 1) or reliance on other diagnostic methods (Geller, 1983).

A fifth problem is that not all diagnoses are reported. Even when a necropsy is performed the result is not always recorded on the death certificate (USHEW, 1979).

Some of these problems were solved in studies of black African men recruited into organizations with adequate medical surveillance systems. Among black miners in South Africa, hundreds of cases of rapidly fatal PLC were diagnosed in young men who had been selected only months before for their apparent good health and ability to perform hard manual labor; over 90% of all diagnosed carcinomas were PLC (Ber- man, 1959). The miners were under constant medical surveillance, and any illness that hindered work was quickly noticed. A similar situation existed when native men were taken into the armed forces (Findlay, 1950).

All the biases to which human cancer statistics are liable could not make PLC appear rare among black Africans, whereas the question “how common?’ requires an estimate of incidence. This in turn requires population censuses, which are sometimes lacking in developing nations. Prates and Torres ( 1965) resorted to aerial pho- tographs and spot sampling to obtain an estimation of population in Mozambique. The first incidence survey on an illiterate population was started in the Bantu of Johannesburg in 1953 (Higginson and Oettle, 1960). Incidence data from around the world are published by the International Agency for Research on Cancer (IARC, 1982), and international cancer mortality rates are published by Segi et al. (1979). Even establishment of a cancer registry does not guarantee that all cases are found. In the Papua, New Guinea, tumor registry, the PLC incidence rose markedly when a medical officer conducted a study of liver disease (Powell and McGovern, 1974). IARC (1982) has published indices of reliability for cancer incidence data, which indicate that the reliability of data among different registries varies considerably.


DISTRIBUTION OF LIVER CANCER

Despite problems in case finding and population definition, a number of epidemiologic features of PLC have been rather consistently reported over wide geographic areas and over long periods of time.

In all population groups PLC incidence increases with age. For high incidence areas the increase is much steeper in young- to mid-adult life, i.e., during the working age period of 20 to 60 years of age. Most of the PLC incidence and mortality differences among population groups are accounted for by differences in the working age period. Black males under 40 years of age in Mozambique had a PLC rate 500 times greater than that of black males of the same age group in the United States, but the rate was only two times greater in corresponding groups over 65 years of age (Prates and Torres, 1965; Young et al., 1975). Incidence generally plateaus in old age and even declines slightly at the oldest period of life. It is not known whether the plateauing in old age is real or the result of less vigorous application of diagnostic procedures for an age group in which death is more accepted. In many areas of the world the average age of PLC patients is increasing (Bulatao-Jayme et al., 1976; Viranuvatti et al., 1980); at least part of this increase is due to increases in mean age of populations and part is due to improved medical surveillance (Sutnick and Puchkov, 1980).

The type of PLC varies with age. In children under the age of 2 years most cases of PLC are hepatoblastoma (Coronado-Perez and Angulo-Hernandez, 198 1). No sig- nificant geographic variation is apparent for the incidence of childhood PLC (Ohaki et al., 1983; Shabanov, 198 1). Childhood PLC data provide few etiologic clues other than some association with congenital defects (Shabanov, 198 1). Diseases of childhood, other than hepatitis B, do not seem to be associated with liver cancer later in life. Even conditions such as kwashiorkor or Indian childhood cirrhosis in which hepatic involvement is extensive are not correlated with PLC (Oettle, 1965; Nagasue, 1982). Only hepatic conditions that do not resolve seem to be followed by development of PLC. The fibrolamellar variant of HCC is more common in the adolescent and young- adult periods than at other ages (Craig et al., 1980; Goodman and Ishak, 1982) and survival is also greater for this subtype.

Race

The major racial groups among whom PLC is common are black Africans and Orientals (Oettle, 1964). It was once thought (Berman, 1940) that all dark-skinned races had higher PLC rates than whites but it is now known that there are some major exceptions. In the United States, PLC incidence among black females is similar to that of white females (Young et al., 1975). White males of some areas of Switzerland have higher PLC incidence than do blacks in cancer registry areas of the United States (IARC, 1982; Tuyns and Obradovic, 1975).

Some tribal differences in PLC occurrence have been observed but they are difficult to evaluate. Berman ( 1935) found that the crude PLC incidence was six times greater among miners from the tribes of Mozambique than among miners from tribes of


South Africa. This tribal difference in the miners was confirmed over 30 years later (Geddes and Falkson, 1970); however, the difference may be related to something in the environment of Mozambique, because the rate for Shangaan tribesmen living in South Africa was not as high as that for members of the same tribe from Mozambique (Robertson et al., 1971).

Some racial groups, such as Chinese and Caucasians, tend to retain their PLC status after migration to a new land but other groups, such as blacks and Asian Indians, tend to assume a status similar to that of the surrounding people in the new land (Nagasue, 1982). Evaluation of data for migrant populations should take into account changes in life styles and changes in the accuracy of the diagnostic and reporting systems that accompany migrations. An example is PLC data for Indians; the incidence reported from India is among the lowest in the world but rates are much higher in Indians who have migrated to other countries. Hesitancy to seek medical help for cancer, low necropsy rates, and sudden shifts in reported incidence indicate that inadequate case finding in India might account for some of the differences (Sutnick and Puch- kov, 1980).

Orientals, particularly the Chinese, have tended to keep their high rates in some areas to which they have migrated, and in other areas their rates are midway between those of China and their new homeland. A study in Taiwan has demonstrated that Chinese men born in provinces north of the Yangtze River, i.e., the area of China in which intermarriage with descendants of the invading Mongols is greatest, have a lower risk of liver cancer than those born south of the Yangtze, where most of the people are descended from the pre-Mongol Han Dynasty (Beasley et al., 1982).

Marked racial differences are seen in the United States, with rates for Chinese, Japanese, and Filipinos nearly always being higher than those for whites (Nagasue, 1982). Recently a high PLC incidence was found among the Alaskan native population (Heyward et al., 1981).

Separation of cultural and genetic factors is difficult. Racial differences have been observed in the willingness to attend hospitals (Ahluwalia and Duguid, 1966) or to grant permission for necropsy (Marsden, 1955). Some human genetic traits have been thought to be associated with PLC, e.g., deficiency of LY- I-antitrypsin, but present information is meager and conclusions vary (Rubel et al., 1982). A simple recessive gene transmission of susceptibility of the PLC risk factor hepatitis B has been discounted (Stevens and Beasley, 1976) but efforts are continuing to determine if PLC susceptibility is inherited (Nakao et al., 1983).

Sex

PLC occurs more frequently in males than females in all parts of the world (IARC, 1982); even before puberty males are in the majority among PLC patients (Lack et al., 1982). Sex ratios (M/F) are elevated in areas with high PLC incidence (IARC, 1982). Erratic M/F ratios have been seen occasionally in small data sets and are probably due to chance (IARC, 1982; Joishy et al., 1982). For some types of PLC such as CCC, hepatoblastoma, and the fibrolamellar variant of HCC, low M/F ratios are often reported and sometimes female cases even outnumber the males (Berman et al., 1980; Ban-era and Dalmacio-Cruz, 1958). The M/F ratio tends to diminish in old age. In addition, there seems to be a sexual difference in malignancy of hepatic neoplasia. In


a survey of U. S. hospitals, 92% of liver neoplasms in males but only 42% of those in females were malignant (Vana et al., 1979).

Geography

PLC is distributed around the world in a unique pattern. The areas of highest incidence are the sub-Sahara1 region of Africa and the Orient. The areas of lowest incidence are the United States, Canada, Australia, New Zealand, India, and the northern and western portions of Europe. Intermediate incidence areas are Latin America and the southern and eastern portions of Europe (IARC, 1982). Within these three broad areas there is discontinuous distribution of PLC; e.g., in China the disease is more common in the southeastern coastal provinces (Armstrong, 1980).

Climate has been thought to be associated with PLC because the endemic areas of Africa and Asia have warm moist climates. However, the frequency is moderate or low in many warm moist regions such as India, Hawaii, and parts of Latin America (IARC, 1982) and is high in some cold areas (Heyward et al., 1981).

In most of Europe and the United States, PLC incidence is moderately lower in rural areas (Axelsson, 1982; IARC, 1982), but in Africa it is higher (Becker and Chat- gidakis, 1961; Kew et al., 1983).

Time Trends

PLC occurrence fluctuates markedly in some areas. A seasonal pattern has been observed in Africa (Francis and Smith, 1972; Sankale et al., 1970; Higginson and Oettle, 1957). These seasonal fluctuations were thought to be due to inaccessability of medical help during the wet season but data from patient interviews indicated that, regardless of any delay in seeking medical help, 60% of the PLC patients first noticed symptoms during the dry season, compared with 13% during an equal time span of the wet season (Okonkwo and Obionu, 198 1). Longer periods of decline, lasting 1 or 2 years, have been reported from West Africa (Denoix and Schlumberger, 1957) and Hong Kong (Ho et al., 1982). No such marked variations were seen for other cancer types such as lung cancer in the same data sources. These fluctuations raise the possibility that some factors related to weather or other cyclic phenomena may affect the final manifestation of PLC, although the basic carcinogenic process could have been progressing subclinically for many years.

Recently calculations made from international data indicated that PLC incidence was increasing nearly 4% per year in males and 7% in females (Saracci and Repetto, 1980), but again the question must be asked whether disease occurrence has actually changed or whether case finding and reporting have improved. PLC rates generally increase when biopsy or necropsy rates increase (Finau, 198 1; Axelsson, 1982). Even if the increases were found to be real it does not necessarily mean that exposure to etiologic factors has increased; it may be that competing causes of death have declined and people are living long enough for liver cancer to develop. However, rates are not continually rising in all areas. A dramatic decrease in PLC has occurred among black gold miners in South Africa, with the crude incidence down about 75% in less than 20 years and esophageal cancer replacing PLC as the most common cancer (Harington et al., 1983). Clemmesen (198 1) reported moderately increased incidences for males


but slight decreases for females in Denmark. Deichmann and MacDonald (1976) found no general sustained increase in PLC mortality in the United States although increases may have occurred in some local areas (Krain, 1973).

Socioeconomic Status

Low socioeconomic status has been associated with PLC. PLC mortality in the United States is inversely related to socioeconomic status (Hoover et al., 1975), and a similar pattern has been observed in Greece (Trichopoulos et al., 1982) and China (Armstrong, 1980). In Uganda, PLC was more frequent among poor migrant workers (Alpert et al., 1969) and in Nigeria almost all of the cases came from the poor class (Francis and Smith, 1972). PLC occurs with high frequency in some dry or cold areas where water for sanitation is lacking or the diet is restricted (Heyward et al., 1981; Atiyeh and Ali, 1980; Tuyns et al., 197 1). PLC more frequently occurs in areas of China where the culinary water supply is derived from stagnant surface water than in areas where the water is supplied from wells (Su, 1979).

PLC rates have fallen in black Africans as they have become westernized in such things as wearing of shoes, decreased tribal skin scarring, decreased family size, improved sanitation, and improved parasite control (Kew et al., 1983; Bersohn et al., 1974). In contrast, socioeconomic status was not related to PLC in a large study in Taiwan (Beasley, 1982); however, only civil servants participated in that study, thereby decreasing the socioeconomic differences.

Occupation

Workers in some occupations have an enhanced risk of PLC; in some cases the risk is for a specific cancer type such as angiosarcoma (Falk et al., 1981). Workers in the alcohol and certain chemical industries have an enhanced risk of PLC (Stemhagen et al., 1983). Socioeconomic and occupational factors seem to be intertwined (Adelstein, 1980). Differences in classifications of occupational groups must also be considered in interpreting the data. Agricultural workers in Sweden (Wiklund, 1983) had less PLC than the national levels, while in an occupational case-control study in New Jersey (Stemhagen et al., 1983) the risk of PLC was increased for farm laborers but not for farm owners and managers.

RISK FACTORS

Ajlatoxins

Aflatoxins are a group of mycotoxins produced by molds, particularly Aspergillus jlavus. The most studied and most potent aflatoxin is Br . Food products that are sometimes contaminated include corn, wheat, barley, oats, rye, rice, cottonseed, sorghum, copra, spices, condiments, tree nuts, peanuts, milk, cassava, and yams (WHO, 1979; Bababunmi et al., 1982; BulataoJayme et al., 1982; StololY, 1983). Stoloff (1983) has reported results of extensive surveys of aflatoxin residues in food products in the United States.


Aflatoxins are potent hepatocarcinogens in some species such as rainbow trout (Hendricks el al., 1980) and rats (Wogan and Newberne, 1967), but some other species are more resistant. Monkeys fed aflatoxins develop liver cancer only after several years (Adamson and Sieber, 1979). Mice are susceptible only when exposed intraperitoneally as neonates (Vesselinovitch et al., 1972). Chickens are affected by aspergillosis, a severe respiratory Aspergillus infestation, and by acute aflatoxicosis, but outbreaks of aflatoxin- induced liver cancer do not occur. Cattle are commonly exposed to moldy feeds and calves are susceptible to acute aflatoxicosis but PLC is rare; this rarity exists despite palpation and incision of bovine livers in meat inspection programs in which hepatic inflammatory and fibrotic lesions are commonly seen, as are also cancers of nonhepatic tissues.

The basis for the species selectivity in the toxicity and carcinogenicity of aflatoxins is differences in metabolism. Aflatoxins, like many other foreign compounds, are me- tabolized to substances which generally are more excretable. The rate at which each metabolite is produced and its toxicity depend on the animal species and the conditions of exposure; for example, the metabolite aflatoxicol is a much less potent carcinogen in rats than aflatoxin B1 but in trout the alcohol is nearly as potent as the parent compound (Schoenhard ef al., 1981). One metabolite, the 2,3-oxide (Swenson et al., 1974) reacts with guanine in DNA. Aflatoxin Bi binds to rat DNA to a greater extent than to mouse DNA (Essigmann et al., 1982), which correlates with the greater susceptibility of rats to aflatoxin carcinogenesis. One route of aflatoxin detoxication is glutathione conjugation; glutathione treatment reduces DNA binding by aflatoxin and even causes regression of HCC in rats (Novi et al., 1982; Novi, 1981; Neal et al., 198 1). The effects of somatic mutations produced by aflatoxin in Drosophila are re- versible (Fahmy and Fahmy, 1980). There is interest in applying principles from these animal studies to treatment of human HCC.

Aflatoxin seems to be a complete carcinogen capable of initiation and promotion of carcinogenesis, but exposures to multiple substances can result in various interactions that alter toxic and carcinogenic effects. A hepatitis virus enhanced induction of PLC by allatoxin in marmosets, and cruciferae vegetables fed to rats inhibited aflatoxin induction of PLC (Lin et al., 1974; Boyd et al., 1982). Although understanding of broad principles is increasing, it is still not possible to predict for each species the results of multiple exposures.

Hsieh et al. (1977) suggested that certain species, including humans and monkeys, which rapidly metabolize aflatoxins may be more susceptible to acute intoxication but relatively resistant to carcinogenic effects, whereas slow-metabolizing species such as the rat may be more prone to chronic or carcinogenic effects. In spite of the con- tention that aflatoxin-induced liver cancer is the best opportunity to verify extrapolation of animal cancer data to humans, extrapolation is still difficult. Humans are, in all probability, less susceptible to PLC induction by aflatoxin than some animals such as rats or trout.

Because of the ubiquity of molds, humans are exposed to aflatoxins. For example, milk samples from 84% of the bottling plants surveyed in the southeastern United States in 1977, when the corn crop of that area had unusually heavy aflatoxin contamination, contained aflatoxin M, which fortunately is much less carcinogenic than Bi (Stoloff, 1980). In some other situations the exposure to aflatoxins, mainly B, , has been sufficient to cause acute intoxication and death (Serck-Hanssen, 1970; Ngindu et al., 1982). In many outbreaks, of which there have been at least 22 (Linsell, 1980)


adequate samples were not available but the evidence justifies strong suspicion of aflatoxin intoxication. In one outbreak in India there were 397 cases and 106 deaths (Krishnamachari et al., 1975).

Several studies on the relationship of human intake of ahatoxin and PLC have given equivocal results. The most widely used design is the ecologic study in which aflatoxin intakes are sampled in populations of different geographic areas and the results correlated with PLC incidence in those areas. Adequate results require that foods eaten in each area are sampled and analyzed for aflatoxin in accord with a rigorous protocol. In addition, all PLC cases in each area must be found and the base population known. Logistical problems, especially in developing nations where most of the studies have been done, have forced compromises in study design or execution. All of the types of problems in interpreting human cancer data apply here. An additional problem is how to control for other possibly confounding factors such as hepatitis B. Such controlling has seldom been done and, even when done, has been for only a rather limited subset of the generally acknowledged set of risk factors. Combination of results from different studies to obtain more points on a dose-response plot does not resolve the problems but rather obscures them. The shape of the plots can be changed by altering the criteria for selecting studies from which data are plotted. Limiting selection to studies for which analytical methods are deemed acceptable is no more appropriate than limiting selection to those studies for which case finding is acceptable. Two factors to be considered are reliability of the data and the strength of the evidence that the correlation portrays a causal relationship.

The studies most widely cited and most often combined to form regression plots were performed in Mozambique (Van Rensburg et al., 1974), Swaziland (Peers et al., 1976), Kenya (Peers and Linsell, 1973), and Thailand (Shank et al., 1972a,b). In each of these nations except Mozambique, multiple areas were studied and there was a positive correlation of aflatoxin intake and PLC incidence. The same positive correlation held when results of the four studies were combined. First, the question of data reliability should be considered. A great deal of short-term migration of working age males seeking employment took place, at least in the African study areas. Nonavail- ability of people hindered both sampling aflatoxin intake and PLC case finding. The area in Mozambique and at least one area in Swaziland had in-migration of workers. In Kenya there was out-migration; 35% of randomly selected individuals were not available for sampling of their food. The male population over 15 years of age in the Kenyan study areas was 29% less than the female population of that age group. Ob- taining accurate information was so difficult that no age information was published for the PLC cases in the Kenya study.

Food samples were taken at the time of consumption but in some areas, particularly Thailand, only part of the diet was sampled. Snacks and foods in short supply such as meats were not sampled at the homes. This could have affected results because peanuts, a major aflatoxin source, were a favorite snack.

Case finding in all of the study areas was susceptible to the problems for human cancer data. In Kenya, the problem of diagnosis was so severe that necropsies were seldom done and only 54% of the cases were histologically confirmed. Diagnoses based on elevated a-fetoprotein (AFP) levels or clinical diagnoses together with death within 6 months were accepted as PLC cases despite the propensity of these methods for false-positive and false-negative diagnoses. In some other studies half or less of the PLC cases had elevated APP levels (Ayoola, 1980; Polterauer et al., 198 1). Difficulties


in case finding were so great in the Thailand study that incidence data in the rural study areas were actually taken from nearby urban areas (Peers et al., 1976). Little controlling was done; e.g., hepatitis B status was not determined in any of the four studies.

Since these four studies were published, a positive correlation of aflatoxin intake levels and PLC incidence has been observed in an ecologic study of seven areas in China (Armstrong, 1980). Insufficient details were published so that any logistical and interpretational problems can not be evaluated.

More recently, in a Philippine case-control study (Bulatao-Jayme et al., 1982), the risk of PLC was estimated to be 18 times greater for those with high aflatoxin intake than for those with low intake. Alcohol drinking interacted with aflatoxin to increase the PLC risk to 35-fold. The case-control design circumvents the problem of finding all cases but the issues of determining aflatoxin exposure and controlling for confounding factors remain. In the Philippine study, aflatoxin was not determined in foods at the time of consumption nor was the sampling protocol described. The cal- culated aflatoxin levels in some foods, particularly cassava, were far higher than in most other reports (Okonkwo and Obionu, 198 1; Nwokolo and Okonkwo, 1978). Hepatitis B status was not determined even though hepatitis B virus (HBV) infection is common in the Philippines and a close relationship of HBV and PLC has been documented there (Lingao et al., 1981). The hospital control subjects did not pay their hospital bills, presumably because they lacked money, compared with 33% of the cases who did pay their bills, indicating a probability that the cases were in an economic class equal to or greater than the controls. Despite this, the PLC cases drank more alcohol, ate more cheap food (perhaps using money to buy alcohol rather than food), and moved their residences more often than the controls. Thus the cases and controls seemed to be from different social classes. This difference and the questions about the accuracy of exposure data limit interpretation.

Aflatoxins have been found in sera of PLC patients (Onyemelukwe et al., 1982). Because of the rapid clearance of aflatoxin from serum, 50-90% in 24 hr, the timing of exposure and sample collection is important to interpretation. It remains to be seen whether high aflatoxin levels in HCC cases are due to high exposure or to aberrant xenobiotic metabolism.

In contrast to the above reports of a positive correlation, in several other studies no correlation of PLC and aflatoxin has been observed. Besrat and Gebre ( 198 1) found less than 30 ppb aflatoxin in the major staple foods of Ethiopia and concluded that aflatoxin may not be the major cause for the high prevalence of HCC and other liver diseases in that country. The report of a recent case-control study of HCC in Hong Kong (Lam et al., 1982b) contained no indication of a difference in consumption of food types that have aflatoxin contamination; hepatitis B status and several other factors were determined but aflatoxin levels were taken from published results of market samples. The frequency of aflatoxin contamination of foods was so low that the difference in exposure of the cases and controls may not have been adequate to resolve the issue. Aflatoxin was not found in food samples from villages of Alaska with a high HCC incidence (A. P. Lanier, 1984, personal communication). In the 9 years since the large Indian outbreak of acute aflatoxicosis, no increased incidence of PLC from that area has been recorded. In Taiwan, illness and deaths have been related to aflatoxin-contaminated food (Tung and Ling, 1968), yet in a large prospective study of PLC conducted more recently, there was no evidence of aflatoxin involvement


(Beasley, 1982). In addition, no correlation of aflatoxin and PLC mortality has been found in the United States (Stoloff, 1983; Wagstaff and Cordle, 1980).

Thus the evidence regarding human hepatocarcinogenicity of aflatoxins is mixed. Even if conclusive evidence is not forthcoming, these chemicals are still acutely toxic and should be controlled. However, the evidence at present does not support the claim that prevention of aflatoxin contamination is the best hope of controlling liver cancer (Linsell, 198 1). The issue of human hepatocarcinogenesis by aflatoxins remains un- decided.

Hepatitis B

Chronic hepatitis following HBV infection is related to PLC. Blumberg and London ( 1982) even concluded that HBV is involved in nearly all HCC in humans. Hepatitis A virus infection seems not to result in PLC (Tabor et al., 1980). Fewer data are available for non-A, non-B hepatitis but there is speculation that it may also cause PLC (Resnick et al., 1983). HBV alone may not be a sufficient cause for PLC (Beasley, 1982; Gibson et al., 1980); there seems to be no single agent around the world that interacts with HBV if in fact interaction is even necessary.

The evidence for an association of PLC and HBV comes from several types of data. One of these is the ecologic association of PLC and HBV in diverse regions throughout the world (Blumberg and London, 1982; Nagasue, 1982), with a few exceptions such as Greenland (Skinhoj et al., 1978). A second data type is the case-control studies comparing prevalence of HBV serologic markers in PLC cases and controls. In those studies in which sensitive methods have been used, there is an association of PLC and HBV. In some of these studies, blood donors were used for the control group and they may not represent the non-PLC portion of the population (Trichopoulos et al., 1982). The most advanced epidemiologic studies of the association are of the prospective type. Beasley and his co-workers (Beasley et al., 1982; Beasley, 1982) have reported on a continuing follow-up study of 22,707 Chinese males in Taiwan and found that after 2-4 years the risk of developing PLC was 390 times greater in hepatitis B surface antigen (HBsAg) carriers than in those negative for this antigen. In a somewhat different approach, samples from a bank of frozen sera that had been collected several years earlier were found to be positive for HBsAg in 10 of 16 PLC patients but in none of 48 controls (Nomura et al., 1982). Similarly, frozen sera from Alaskan Eskimos were positive for HBV markers in 9 of 11 HCC cases (Heyward et al., 1982). Thus it has been established that HBV infection preceded clinical appearance of PLC.

Another line of evidence is somewhat unusual. HBsAg carriage in some groups is passed vertically from infected mothers to their children. The children of carrier fathers are no more apt to become carriers than children of noncarrier fathers (Blumberg and London, 1982; Beasley, 1982; Stevens et al., 197 5). PLC occurs at a higher rate among children of carrier mothers than among children of carrier fathers (Beasley, 1982). Thus the familial pattern of PLC matches that of passage of the virus.

The concentration of PLC in lower socioeconomic classes matches the pattern for HBV distribution (Trichopoulos et al., 1982). In some groups a number of factors suggest mechanisms for transmission of HBV which could occur more frequently among the lower socioeconomic class. HBV markers are found in blood and some mucous membrane secretions of infected persons. Poor sanitation would increase


chances of contact with infected material. Tribal scarring (Kew et al., 1973), mosquitoes (Prince et al., 1972), and bedbugs (Jupp et al., 1983) are possible mechanisms for transmitting HBV from the blood of an infected person to a susceptible person.

Because of the long development period of PLC, the etiologic role of HBV, which is blood borne, is difficult to separate from that of aflatoxin, which is food borne. Often these factors are confounded: those who must eat mold-contaminated food are often also subject to poor sanitation. Interaction of the two factors has been speculated by a mechanism of immunosuppression by aflatoxin, permitting the HBV carrier state to develop (Lutwick, 1979).

Evidence of an HBV-PLC link also comes from natural field outbreaks of liver cancer in woodchucks and ducks infected with viruses similar to human HBV (Sum- mers et al., 1978). A current hope is that evidence from human HBV vaccination programs will indicate a decrease in PLC incidence in vaccinated groups (Zucker- man, 1982).

Alcohol

Often PLC patients have a history of heavy alcohol consumption but the exact role of alcohol in the pathogenesis is not defined. Alcohol alone is not an animal carcinogen but it can enhance the potency of carcinogens (Tuyns, 1979). Alcohol is not essential in all PLC development, since the disease is endemic in Moslem areas where alcohol is proscribed. PLC is more prevalent among alcoholic cirrhotics than among noncir- rhotics (Stemhagen et al., 1983; Hakulinen et al., 1974). Some authors have concluded that alcohol played no role for blacks in Africa (Oettle, 1965; Steiner, 196 1; Gelfand et al., 1972). The reasons for this conclusion were the lack of alcoholic cirrhosis in black African PLC patients and no obvious difference of alcohol intake reported by PLC patients compared to patients with other diagnoses (Gelfand et al., 1972). Some Africans consume large amounts of alcoholic beverages, with intakes up to 60 liters per week (Prates, 1957); these beverages are usually made with secret herbal additives (Lovelace and Nyathi, 1977). Alpert et al. ( 1969) found that the proportion of alcohol drinkers was higher among black African PLC cases than among hospital controls but there was no difference in alcohol intake between PLC cases and neighborhood controls; they commented that the PLC patients and hospital controls were from different neighborhoods. Black Africans are acquiring the problems of alcoholic cirrhosis as they become urbanized (Kew, 198 1).

Other Factors

Other factors that have been suspected of being associated with PLC include smoking, contraceptive and therapeutic steroids, membranous obstruction of the inferior vena cava (MOVC), hepatic parasites, vinyl chloride, Thorotrast, arsenicals, poisonous plants, liver cirrhosis and other diseases that chronically affect the liver.

An increased risk of PLC among smokers has been demonstrated in a case-control study of HBsAg-negative Greeks (Trichopoulos et al., 1980) and this was confirmed in a case-control study in Hong Kong (Lam et a/., 1982b). No increased risk for smoking was found in a case-control study in New Jersey (Stemhagen et al., 1983) but hepatitis status was not determined.


Concern has been expressed in recent years that contraceptive steroids may cause liver cancer (Christopherson and Mays, 1977). An association of oral contraceptive use and benign liver tumors has been demonstrated (Rooks et al., 1979). Although a number of cases of liver cancer in oral contraceptive users have been reported (Shar and Kew, 1982), no controlled studies have been done to compare disease rate in users and nonusers. Goodman and Ishak (1982) concluded that an apparent association of contraceptive steroids and HCC is probably coincidental HCC has occurred in people treated with anabolic steroids (Farrell et al., 1975); the disease sometimes re- gresses when the steroid therapy is stopped, indicating that it may be a less malignant variant of HCC.

MOVC, possibly of congenital origin, has been associated with HCC in case series from South Africa (Simson, 1982) and Japan (Nakamura, 1982). Passive chronic hepatic congestion occurs in MOVC and probably precedes development of HCC.

Several parasites affecting the liver have been postulated to cause liver cancer (Flavell, 1981). Infestation by the liver flukes Clonorchis sinensis or Opistorchis viverrini is found in many patients with CCC in parts of Asia such as northeast Thailand. Definitive epidemiologic studies confirming the association have not been reported.

Hepatic angiosarcoma has been shown in epidemiologic studies to be associated with manufacture of vinyl chloride (Infante, 198 1; Apfeldorf and Infante, 198 1; Beau- mont and Breslow, 198 1). Reduction of occupational exposure has been followed by a decline in the number of angiosarcoma cases (Jones, 198 1; Tamburro and Greenberg, 1980). Chemicals related to vinyl chloride which are animal carcinogens (Apfeldorf and Infante, 198 1) include vinylidene chloride, trichloroethylene, perchloroethylene, carbon tetrachloride, ethylene dibromide, and epichlorohydrin. Epidemiologic studies show an increased risk for epichlorohydrin in dry-cleaning workers and those handling degreasing solvents (Apfeldorf and Infante, 198 1).

PLC and a number of other cancer types have been diagnosed in patients treated with Thorotrast, a radioactive material formerly used in radiography (Stover, 1983).

Arsenicals were suspected of causing hepatic angiosarcoma after a clustering of cases of this rare cancer was observed among wine makers exposed to arsenical her- bicides and among patients treated with Fowler’s solution, i.e., 1% potassium arsenite (Falk et al., 1981).

People in many developing nations are exposed to foods, drinks, and folk medicines made from a wide variety of cultivated and wild plants (Prates, 1957). Rural women in some cultural groups eat more wild plants than do men (Fleuret, 1979). Plants such as senecios are animal carcinogens but there is no clear evidence that they cause human liver cancer (Powell and McGovern, 1974; Schoental, 1982). Cycads are an example of a plant suspected of human hepatocarcinogenesis but the association has not been confirmed by studies in South Africa and New Guinea (Powell and McGovern, 1974; Kew et al., 1977). Thus there is no strong indication that poisonous plant substances other than mycotoxins are human hepatocarcinogens.

Cirrhosis is present in most livers with HCC but is less common in other types of liver cancer (Okuda et al., 1980). The risk of PLC among cirrhotics is high (Prates and Torres, 1965; Nakamura et al., 1982) particularly among HBV-carrier cirrhotics. In some areas, e.g., Thailand, the proportion of HCC cases with cirrhosis is increasing (Viranuvatti et al., 1980); one possible reason is that cirrhotics who are treated with drugs such as prednisolone may live long enough to develop PLC (Jenkins et al., 1981). In Western nations the cirrhosis type is usually alcoholic and is diagnosed


before appearance of the tumor, but among black Africans the cirrhosis type is generally posthepatitic and is discovered simultaneously with the tumor (Kew, 198 1). A possible role of cirrhosis in hepatocarcinogenesis is promotion by chronic regeneration of liver cells, but other possibilities are liver dysfunction and inflammation. Cirrhosis seems not to be an absolute requirement for development of PLC. Most PLC case series, even those in the endemic areas, contain cases that are noncirrhotic and some without fibrosis.

Other disease states such as hemochromatosis and certain nutrient imbalances (Shi- kata, 1976; Newbeme and Schrager, 1983) may be related to PLC but their etiologic role in human PLC is unknown. Any conditions that result in chronic liver impairment should be considered as potential risk factors. Over two-thirds of clinically normal men in PLC endemic areas in Africa and New Guinea were shown in separate studies to have liver impairment (Bersohn et al., 1954; Arter et al., 1968).

DISCUSSION

In summary, PLC is a disease concentrated in young to middle-aged adult males who have had periods of poor sanitation, malnutrition, immoderate life style, or chronic but often subclinical liver impairment. Many of the risk factors have this same pattern and are often intertwined. There is strong evidence that HBV plays some etiologic role for PLC in some population groups but the evidence is mixed for the other risk factors. For alcohol and smoking, there is good evidence for exposure of individuals before onset of the disease but such evidence is lacking for aflatoxin.

An important factor in understanding and controlling PLC is to determine whether it is a single disease with only one cause, an infinite spectrum of diseases, or a finite set of diseases with specific causes. The growing array of syndromes and epidemiologic patterns being defined in liver cancer patients argues against the single disease theory. Although the microscopic lesion of PLC is similar among population groups, there are several differences in the way the disease presents in tumor size, survival time, cirrhosis type, sex ratio, and mean age (Thomson, 1961; Joishy et al., 1982; Tricho- poulos et al., 1982). In animals there are many chemical, microbial, and genetic causes of liver cancer, and there may be multiple causes of human liver cancer. The definite pattern of liver cancer lesions in cellular structures, tissues, species, and human population groups argues against the infinite spectrum theory. Therefore it is reasonable to think that a limited number of cause and effect chains of events lead to liver cancer. Trichopoulos et al. ( 1982) presented data indicating that there is more than one epidemiologic group of liver cancer cases. Among Greek PLC cases, those which were HBsAg positive and those which were HBsAg negative differed in several aspects, including rural residence, socioeconomic status, cirrhosis, and survival. Because no studies have adequately determined the status of all major risk factors in the same individuals, it is not yet possible to apportion the total number of PLC cases among the risk factors.

Four recommendations are made. First, epidemiologic studies of PLC should determine all the major risk factors in the same individuals for better separation of the various syndromes and epidemiologic patterns. Second, reliable methods should be made available for determining irreversible tissue binding of aflatoxin in fresh and stored human tissue. Third, decisions regarding control of aflatoxin or any of the risk factors, based on extrapolation from animal data, should consider species variations,


published human incidence and mortality data, and contribution of other risk factors. It also should be kept in mind that in old age, when most PLC deaths occur in Western nations, the incidences in most groups become similar, indicating that perhaps no change, aflatoxin control or otherwise, will greatly alter the results. And fourth, general health recommendations should be followed including personal hygiene, safe culinary water, adequate diets containing fresh fruit and vegetables, moderate alcohol intake, curtailed smoking, and recommended vaccinations. It is questionable how effective any one step directed only against PLC would be if taken in the absence of all the other steps. HBV vaccination alone without other improvements to prevent chronic liver impairment may not fully control the disease. Likewise, vigorous efforts to min- imize aflatoxin levels in commercial foods without improvement of conditions which force people to eat deteriorated foods may not have a great effect on PLC incidence.

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