Annals of Oncology 3: 783-791, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Special article Nutrition and cancer: Background and rationale of the European Prospective Investigation into Cancer and Nutrition (EPIC) E. Riboli Programme of Nutrition and Cancer, Unit of Analytical Epidemiology, International Agency for Research on Cancer, Lyon, France *

Summary. The development of epidemiological research on diet and cancer during the last decades is discussed. Apart from some consistent data on the protection against several cancers conferred by a diet rich in fruits and vegetables, the results provided by retrospective case-control studies on diet and cancer remain contradictory on many important issues, and primarily on the relation between diet and breast cancer. The rationale is presented for setting up large prospective cohort studies combining epidemiological and laboratory methods in order to expand the presently limited knowledge of the role of nutrition and related factors in cancer etiology. The main features of the European Prospective Investigation

into Cancer and Nutrition are outlined. The study is being carried out in seven European countries and it will collect data on diet, other lifestyle and environmental factors, anthropometry as well as biological samples in a cohort of about 400,000 healthy European adults. The subjects will be followed up to investigate the incidence of and mortality from cancer in relation to epidemiological data and biochemical markers

Introduction and overview of research on diet and cancer

cancer cases occurring in developed countries which are attributable to known and uncontroversially identified carcinogens, it is difficult to come up with more than 40% to 60% for men and even less for women. This obviously includes tobacco, which in Europe accounts for 20%-30% of all cancers in men and 5%10% in women, depending on the population under consideration. In view of this low proportion of total cancers for which at least one clearly defined and preventable environmental cause has been identified, it can be deduced arithmetically that we have not yet pinpointed any environmental carcinogen which can explain more than a fairly small fraction of the most common cancers occurring in developed countries, namely, cancers of the breast, prostate, stomach, colon and rectum (the only exception being lung cancer and tobacco). Certainly the list of reproductive, hormonal, anthropometric, dietary and other lifestyle factors suspected of being involved in the etiology of these cancers is quite long but, with few exceptions, epidemiological and laboratory studies have provided quite contradictory and controversial results.

The hypothesis that diet is related to the occurrence of a substantial proportion of cancers is today widely accepted, although it received very little notice as recently as 15 or 20 years go. Despite important experimental work carried out from the end of the 1930s onward, which demonstrated the effect of dietary changes on the yield of tumours in laboratory animals [1, 2], in the 1950s to 1970s the main lines of research in experimental carcinogenesis as well as in epidemiology lay in identifying environmental carcinogens, particularly chemical and physical agents. The prevailing cultural and methodological attitude in epidemiology was influenced by the discovery of 'strong' carcinogens such as tobacco smoking and some industrial chemicals and drugs. In almost all cases, identification of the carcinogen was facilitated by the (very) high risk among the subjects exposed and it was relatively easy to establish who was exposed (often at high doses) and who was not. This explains why epidemiological studies based on relatively simple methods of measuring exposure have been able to identify successfully quite a long list of environmental agents including radiation, industrial processes, drugs and personal habits which are causally related to various types of cancer (see Table 1) [3]. However, when estimating the proportion of total * See page 790 for members of the collaborative group.

Key words: biomarkers, cancer, cohort, diet, epidemiology, nutrition

Recently, Walter Willett expressed the sense of frustration of those who have invested a great deal of time and resources in the search for the causes of breast cancer by declaring: 'We probably won't find a single, major cause of breast cancer as we did for smoking and lung cancer.... I am not sure that we will find that simple lifestyle changes alone will substantially reduce breast cancer rates' (cited by Pollack [4]). About 10 years ago, estimates attributing to diet

784 Table 1. Preventable causes of cancer, suspected risk factors and proportion of cancers due to unknown factors in developed countries'1 % of total cancers

Cancer sites B

A

Oral cavity and pharynx Oesophagus

C

Known, preventable risk factors

Preventable proportion

Suspected risk factors

Further preventable proportion b

Proportion probably due to unknown, unsuspected risk factors

Alcohol & tobacco

60-80%

Low consumption of fruit and vegetables

10%

10-30%

Alcohol & tobacco

75%

Diet poor in fruit, vegetables and fresh meat products

10-20%

= 10%

Low consumDtion of fruit and vegetables, high consumption of salt and salted foods; Helicobacter pilori infection

50-60%

40-50%

Low consumption of fruit and vegetables, high intake of animal fats and/or meat

30-40%

60-70%

-

85-90%

Stomach

Colon-rectum

Liver

Alcohol Hepatitis B

10-15% Very small

Pancreas

Tobacco smoking

30%

Low consumption of fruit and vegetables

10-20%

40-50%

Larynx

Tobacco & alcohol Occupational exposures

85% 10%

Low consumption of fruit and vegetables

10% (?)

5-10%

Lung

Tobacco smoking

6()-90% c

Low consumption of fruit and vegetables

5-10% (?)

2-5%

Occupational exposures. including radiation Air pollution Environmental tobacco smoke

10% Uncertain Uncertain11

Skin (melanoma)

Sun bathing

>4()%

Uncertain

Skin (non-melanoma)

Sun bathing

High

Uncertain

Breast'

Overweight after menopause

10%

Cervix

Tobacco smoking Sexually transmitted infections

Uncertain 50%

Endometriume

Overweight Post-menopausal unopposed oestrogens

25% 40%E

Diet, excess energy intake, fats

Uncertain

40%

Ovary el

—

Uncertain

Very high

-

— -

Diary products (lactose?)

Prostate

Western life style"

Uncertain

Very high

Bladder

Tobacco smoking Certain occupational exposures

30-70% 10-20%

Some dietary factors High-fat diet, low consumption of fruit and vegetables

Kidney

Tobacco smoking

30-40%

Ochratoxins

Uncertain

60%

Leukemia

Radiation

Uncertain Uncertain

Tobacco

Uncertain

Very high

Benzene

High intake of fat and foods of animal origin

0-20%

70-80°/. 50%

20%

* This table is derived from Table 42 in Reference 3, modified by the author. b

Very approximate estimate. 60-80% in women, 80-90% in men. d Exposure to environmental tobacco smoke can cause lung cancer in non-smokers, but estimates of RR and AR are uncertain. ' Certain characteristics of sexual maturation and reproduction are associated with increased risk (age at menarche, age at menopause, age at first pregnancy, number of pregnancies) but are not amenable to practical preventive measures. ' Significant protection conferred by the use of oral contraceptives. 8 Compared to a situation in the 1960s and 1970s. c

785

35% of all cancers were published by Doll and Peto [5] and since then, this figure has been widely quoted as if it were a final conclusion based on strong scientific data. The authors, however, clearly stated that it was no more than a guesstimate to which they attached a confidence interval as wide as 10% to 70%. Shortly afterwards, Peto [6] published a thoughtful commentary making a distinction between what is suspected to be a cause, what is really known precisely, and finally what could be used to provide cancer prevention guidelines. Under this critical review, certainty as to the proportion of cancers preventable by dietary changes vanished from 35% to 0%. We should, therefore, resist the temptation to substitute diet for our ignorance as to the origin of most of the cancers occurring in developed countries. The reasoning by exclusion which says that, given that colon or stomach cancers do not seem to be related in any meaningful, quantitative way to tobacco, occupational exposure, environmental chemicals and so on, then they must be related to diet - the only possible exogenous factor left - is certainly attractive, but still much too vague to be included in the scientific knowledge on cancer etiology and, even less, to be used as the basis for public health intervention. A definition of which cancers are etiologically related to diet and what proportion of them are actually due to specific components of diet must be based on strong scientific evidence, particularly in view of the social, ethical and economic consequences that substantial changes in food consumption would have on society. Based on epidemiological data gathered in the last 10 years, there are only a few associations between dietary factors and cancer in developed countries for which there is consistent evidence, namely that: 1. Frequent, daily consumption of fruits and vegetables seems to be associated consistently with lower risk of cancers of the stomach, colon, rectum and lung [7, 8|. 2. High consumption of fat of animal origin and/or of meat is associated with an increased risk of cancer of the colon and rectum [9]. 3. High consumption of salt and salted foods is associated with increased risk of stomach cancer [3]. 4. Sustained, heavy alcohol consumption is causally linked to cancer of the upper aerodigestive tract (oropharynx, larynx, oesophagus) and liver [3]. For many other postulated associations between diet and cancer, results from different studies have thus far been contradictory. This applies particularly to the hypothesis on fat and breast cancer; several case-control studies and two large prospective studies have not provided a clear answer [3, 9-11]. It can tentatively be concluded that for the moment either there is no association or that, if there is one, it is so weak that the chances of detecting an increased risk consistently in different studies are low, especially if one considers the possibility of random misclassification of subjects caused by imprecise measurement of their fat intake.

However, the fact remains that if high intake of fat by adults or teenagers is even weakly associated with an increase in breast cancer risk, the proportion of cases attributable to dietary fat could be quite large since most of the women in our society are 'exposed' to excessive fat intake. If a threshold of 20%-25% of energy from fat is taken as the cut-off point between 'desirable' and 'excessive' fat intake, as international correlation studies have indicated, then up to 90% of women could be at increased risk of breast cancer because of their diet. The putative association between fat and breast cancer is related to another burning issue in the interpretation of dietary data from epidemiological studies, namely, the question of the possible effect of energy compared to that of each energy-providing nutrient: protein, carbohydrates, fat and alcohol. Besides some important progress in the conceptualization of separating the respective effects of energy and of the energycontributing nutrients in statistical analysis [12, 13], it should not be forgotten that statistics are based on data, and sophisticated statistical models cannot compensate for poor data. If there is inherent confounding because of inexact measurements and insufficient diversity in exposure, sophisticated statistical modelling is futile [14]. Similar reasoning can be applied to the analyses carried out on other nutrients such as vitamins and dietary fibre which provide no energy. Much has been written about the protective effects of, for example, vitamin C against stomach cancer, (3-carotene against lung cancer, fibre against colorectal cancer. Too often it is forgotten that in epidemiological studies the measurement of dietary intake is actually based on what people report, and they can only answer questions about their food consumption. Nutrients are estimated by means of food composition tables which, at best, reflect only approximately the actual composition of the foods. In addition, food composition tables do not necessarily contain information on food components relevant to cancer research but only a few (perhaps 30 or 40) nutrients of interest for 'nutrition'. For example, there is generally a lack of data on various carotenoids (several hundred are known) which have no vitamin A activity (i.e., cannot be transformed into retinol by human enzymes) and which until now have therefore not been included in the tables. Some of these carotenoids may be the substances of interest for lung cancer, though in many papers only vitamin A or at best p*-carotene are reported. In conclusion, we can apply to our present knowledge on diet and cancer the somewhat pessimistic conclusion of Moore et al. [15), who wrote that after three decades of research on breast cancer and hormones '... so much work has already been done .. that it can be stated with some certainty that there is little point in further studies in the usual case-control mode... The analytical work over the last 30 years clears the ground for new efforts to understand the relationship between

786

hormones and cancer of the breast'. And, I would add, between diet and all the most frequent cancer sites. Historical background of prospective cohort studies

The idea of setting up very large multi-centre prospective studies on nutrition and cancer in Europe was conceived during the 1980s. It was felt that further extension of the epidemiological approach followed during the preceding twenty years, mainly based on retrospective case-control studies, was unlikely to provide substantially new or more specific scientific evidence to clarify the role of diet in the etiology of cancer. The scientific validity of well designed, carefully conducted case-control studies as a basic principle of modern epidemiology is not questioned. However, case-control studies rely on three basic principles: 1) cases and controls are chosen in such a way as to avoid selection bias and confounding; 2) measurement of exposure is not distorted by disease status; and 3) measurement of exposure has 'some' validity and precision (if not on an absolute scale, at least on a relative scale in terms of correlation with reality). While it is no harder to satisfy the first condition in studies on diet than in studies on other risk factor, there is good reason to believe that there is generally a certain degree of inaccuracy in measurements of 'usual past diet', which are obtained retrospectively in case-control studies. Memory of past diet may be distorted by current dietary habits and even, in some instances, by disease status which may lead to systematic differences between cases and controls [16, 17]. One of the main advantages of prospective studies is that data on current diet can be collected well before the occurrence of the disease of interest at the time of the subjects' enrolment in the study. In addition, repeat measurements of diet can be made at any time during the follow-up period in order to investigate the effect on cancer risk of any dietary changes which may occur. This is not possible in case-control studies where diet can be evaluated only once, retrospectively after cancer has been diagnosed. The first large, modern prospective study in cancer epidemiology was the world-famous study on smoking among British doctors started by Doll and Hill in 1951 [18, 19]. In the 1950s the results of several case-control studies had already found a strong positive association between smoking and lung cancer, but questions were raised as to whether this was in fact a causal association. The prospective study by Doll and Hill provided the final demonstration that tobacco smoking was the main factor responsible for the dramatic epidemic of lung cancer which started in the 1920s and 1930s in the USA and the UK and spread all over the world in later decades. Doll's and Hill's prospective study indicated not only that mortality from lung cancer was much higher in smokers than in non-smokers but also that the risk increased with the amount of tobacco

smoked and that subjects who gave up smoking during the follow-up period had lower mortality that those who continued smoking. The reduction in risk after removal of the exposure (in ex-smokers) was, and still is, a very strong argument in favour of the causal nature of the association. The tobacco industry may still claim that the causal relationship between tobacco and cancer is unproven, but this is merely because they want to protect their market and they fear law suits; their argument carries no scientific weight among independent researchers. Today, mutatis mutandis, the scientific evidence on diet and cancer is to a certain extent in the same position as that on tobacco during the 1950s. A large number of case-control studies indicate that some components of diet are related to cancer, either by increasing or decreasing the risk of specific cancers, but the strong scientific evidence which could be provided by large, well designed prospective studies based on combined questionnaire data and biological markers is still limited. The first very large prospective cohort study which included questions on diet was started by Hirayama in Japan in 1965. A simple questionnaire covering tobacco, alcohol and diet was filled in by 265,118 Japanese who were subsequently followed up by means of death certificates [20]. However, the information collected on diet was very limited, as the questionnaire included only six questions on rice or wheat, meat, fish, milk, green-yellow vegetables, pickles and soya soup, and the questionnaire was administered only once at the start of the study (in a country where food habits changed dramatically between the 1960s and the 1980s). This study produced some interesting results in relation to diet, mainly in the sense of supporting the hypothesis that a diet rich in fresh vegetables is associated with reduced mortality from cancer, particularly those of the colon, stomach and lung. Contrasting results, however, have been observed in relation to other dietary variables. For example, frequent meat consumption was associated with higher mortality from breast cancer but lower mortality from colon cancer (and lower mortality from all causes). In Japan, per capita daily intake of meat increased during the study period from 18.7 g in 1960 to 67.9 g in 1989, and the subjects in the cohort study presumably followed this trend, but the lack of data on how individuals changed their meat intake during the 17 years follow-up period could have biased the results (based on consumption in 1961) in an unpredictable way. A few other prospective studies were begun during the 1960s, but they were too small to be really informative, as they were primarily designed to investigate risk factors for cardiovascular disease and were often based on dietary assessment methods (such as single 24-hour diet recall) which are inadequate for ranking subjects according to their usual diet [21]. Not until the end of the 1970s did a group of Har-

787

vard researchers initiate the first really large, well designed prospective study in which diet was measured by means of a semi-quantitative food frequency questionnaire, appropriately tested for validity and reproducibility [22]. The Nurses' Health Cohort Study, based on 121,700 women, is the largest existing prospective study in which diet has been measured repeatedly (every two years), and additional data have been collected prospectively on a number of diet-related factors such as physical activity and anthropometry [9]. In 1986 the same group of researchers started a similar study on male health professionals which included 51,529 subjects [23]. In parallel with these two Harvard cohorts, a prospective study was started in Canada in 1982 based on women enrolled in the Canadian National Breast Screening Study. A total of 56,837 women filled in a self-administered dietary questionnaire and have been followed up to determine cancer incidence and mortality [24]. Finally, a large prospective study was started in 1986 in The Netherlands, which included 120,852 men and women [25].

Biochemical markers Most of the studies mentioned above were originally conceived as purely questionnaire-based investigations in which assessment of exposure to dietary factors or indeed to other environmental and lifestyle factors was based on information provided by the study subjects, usually by means of a self-administered questionnaire. However, during the 1980s new horizons for cancer research were opened by progress made in the field of the molecular biology of cancer and by the development of research on biochemical markers for a variety of environmental exposures and host factors. New possibilities arose to use laboratory methods to measure exposure and to identify individual susceptibility, genetic predisposition and genetic damage from environmental factors. The idea that this could complement the traditional questionnaire-based epidemiological approach soon attracted a great deal of interest, and led to the definition of a new area of research. Biochemical and molecular epidemiology, however, is in its infancy and, in my view, still has a long way to go before moving from the limited dimension of 'laboratory studies in human subjects' to the more promising and sound perspective of laboratory investigations nested in large, well-designed epidemiological studies. One of the reasons, probably the most important, is that case-control studies (by far the most common type of epidemiological investigation) are inherently unsuitable for most of the molecular and biochemical investigations which could theoretically help to shed light on the role of nutrition and of other environmental factors in the etiology of cancer. In case-control studies, biological samples can obviously be collected only from patients who already have clinically diagnosed cancer,

with all the complex metabolic alterations, known and unknown, which can often accompany cancer from the very beginning of its clinical phase. This can best be achieved by a prospective approach in which biological samples are collected from healthy subjects and stored at a very low temperature so that comparisons can be made years later between results of laboratory measurements on samples from subjects who develop cancer and those who do not during the same follow-up period. The collection and storage at low temperature of blood and other biological samples constitutes a major characteristic of what I would call the 'new generation' of prospective studies on cancer and diet-related hormonal and nutritional factors. Among the first to use this novel approach in cancer epidemiology were researchers in New York and Milan who started two projects at about the same time, both focusing mainly on investigation of the etiology of breast cancer in relation to diet, endogenous and exogenous hormones, reproductive life and other lifestyle and individual physical characteristics (anthropometry, physical activity, drug use, etc.). The New York University cohort was started in 1985 and has included 17,000 women [26]. The ORDET study was started in 1988 in the Lombardy region and has enrolled 10,000 middle-aged women [27]. More recently, collection and storage at low temperature of blood samples has been started also in one of the two Harvard cohorts (the Nurses Health Cohorts) (Willett, personal communication). The European Prospective Study into Cancer and Nutrition (EPIC) The EPIC project was started in 1989-1990 in seven European countries, listed in order from north to south: UK, The Netherlands, Germany, France, Italy, Spain and Greece. The design of the study has evolved over the last two years in light of the results of a series of pilot and methodological studies which were conducted to test the validity and feasibility of collecting data through different types of questionnaires, of taking anthropometric measurements and of drawing, aliquoting and storing blood samples. Populations included in the study Table 2 summarizes the main characteristics of the cohorts in each of the seven countries. Overall, it is planned to include in the study about 400,000 subjects, with about equal numbers of men and women. Men will mainly be recruited in the age range of 40 to 63, while for women the age range will be 35 up to 69. The lower age limit for women was selected to ensure a sufficient number of subjects for investigating risk factors for premenopausal breast cancer. Geographical distributions and target populations from which to draw the subjects where chosen with

788 Table 2. Summary of study subject recruitment for the European prospective investigation into cancer and nutrition. Country

Geographical area

Target population

No. of subjects

Sex

Age

France

Nationwide

Teachers

70,000"

F

40-65

h

Italy

North (Turin, Varese) Centre (Florence) South (Ragusa)

Blood donors and participants in breast cancer screening

30,00() 20,000

M F

40-65 35-65

Spain

North (Asturias, Basque Country, Navarra) South-east (Murcia, Granada)

Blood donors

30,000 20,000

M F

40-65 35-65

UK

Nationwide and Cambridge region

General population

32,000 43,000

M F

40-74 40-74

Greece

Nationwide

Teachers

25,000 25,000

M F

40-65 35-65

Germany

Nationwide

Member of national health insurance

30,000 30,000

M F

40-65 35-65

Netherlands

Regional

General population and participants in breast cancer screening

12,000c 32,000

M F

20-60 20-69

a

100,000 subjects have already answered the first two mailed questionnaires and will receive a dietary questionnaire in 1992. Compliance of 70% is expected. b 10,000 of the 35,000 women represent an extension of an on-going cohort study on hormones and diet (ORDET), conducted in Varese province. c Extension of an on-going cohort study on monitoring of risk factors in which about 20,000 men and women have already been enrolled.

different, and sometimes contrasting, requirements in mind to ensure scientific value, efficiency and feasibility. The wide geographical distribution is one of the most attractive features of the study, as it provides populations with very heterogeneous dietary habits, ranging from the Mediterranean diets of Greece and southern Italy and Spain to the 'middle European' food patterns of Germany and The Netherlands. Within each country the choice of a particular population or a particular region was oriented to combine wide geographical distributions with the best available possibilities for recruitment and follow-up. For example, in Italy, Spain and The Netherlands, it was decided to carry out the study in regions where the entire population is covered by existing cancer registries. In France, Germany and Greece, where there are legal and technical limitations on following up subjects through cancer registries or death certificates, the solution adopted was to seek the collaboration of nationwide health insurance programs for school employees (France, Greece) or blue- and white-collar workers (Germany). Finally, in Great Britain, where cancer registries cover the whole of England, Scotland and Wales, it was decided to recruit subjects in collaboration with general practitioners and to combine a cohort of 25,000 subjects in a limited geographical area (Cambridgeshire and Norfolk) with another cohort of 50,000 scattered over the entire country. Follow-up methods Follow-up of the entire cohort will be based on cancer incidence data obtained either through population can-

cer registries or health insurance programs. For each case of cancer occurring in the cohort, every effort will be made to obtain clinical data on anatomical localization of the tumour, histology and other diagnostic procedures and stage at diagnosis. Mortality data will be obtained for the entire cohort, either by record linkage with death certificate files or through health insurance files. Additional sources of information for identifying subjects who move to different areas will be provided by population files, blood donor association computerized files, health insurance schemes and national health service files. Dietary assessment methods The methods of dietary assessment to be used in large epidemiological studies must at once be of good technical quality, feasible within the logistical constraints of the project, and culturally adapted to the population recruited for the study. Extensive reviews and discussions on the merits and drawbacks of dietary methods for use in epidemiological studies have been published [28, 29] as well as a substantial number of methodological studies which tested different dietary methods under varying conditions. From this previous work, it appears that there is no single 'best method' on usual current diet which could be applied as a standard in all epidemiological studies. It is therefore necessary when planning a large international study on diet, to test different possible methods in order to estimate their validity and reproducibility under real, local conditions. In the context of EPIC, various methods were tested

789

and evaluated in pilot studies carried out by collaborat- This may cause some systematic over- or underestimaing centres in each of the seven countries. These meth- tion of the average intake of a given nutrient or food in odological studies were designed to test the validity of some of the cohorts. In order to adjust for these potenthe questionnaires prepared for use in the prospective tial distortions in dietary measurements, EPIC will instudy with a reference method of proven validity but clude a built-in calibration study which will involve colmuch too complex to be used in the entire cohort. The lecting precise measurements of single-day food intake reference methods selected were 24-hour weighed diet in a subsample of about 2% of the total cohort. record repeated for 16 days (4 days x 4) over one year Several thousand 24-hour diet recalls will be collect(UK) and 24-hour diet recall repeated for 12 days (1 ed with highly standardized procedures and used to day x 12) over one year in the other six countries. calibrate (adjust) between countries at the group level, In addition, the questionnaires and the reference the measurements provided by the questionnaires. In methods were compared with biochemical markers of other words, the concept of 'calibration to a common diet measured from urine and blood samples collected standard' which has been known for many years in during the study period. In particular, the average good analytical laboratory work will be applied to excretion of urinary nitrogen was measured in four to questionnaire measurements of diet from different eight 24-hour urine collections for each study subject, countries. and used to evaluate the protein intake estimated by the reference method and by the questionnaires. Plas- Biological markers matic levels of vitamin C, vitamin E and five specific carotenoids were measured to validate vitamin intake The plan for collecting, aliquoting and storing at very estimated by the different dietary assessment methods. low temperature blood samples from about 400,000 At each centre, the pilot studies included 100 to 150 subjects was initially considered to be too complex and subjects for a total of 1150 subjects in the seven coun- costly to be realized. However, a number of relatively tries. These studies were completed at the end of 1991 innovative solutions have been found to standardize in France, Italy, Spain and the UK, and will be com- and simplify the procedures, particularly regarding the pleted towards the end of 1992 in Germany, Greece aliquoting of blood samples. and The Netherlands. Although the simplest solution would appear to be While detailed reports on these methodological to store serum or plasma in large tubes for each subject, studies have not yet been completed, it can be antici- this would mean it would be necessary to thaw, depated that three parallel methods will be used: freeze and refreeze the samples each time an analysis a) A detailed self-administered dietary history ques- was needed on a sample. tionnaire with quantification of usual portion size It was therefore decided to store plasma, serum, based on food photographs has provided very buffy coat and red blood cells in small plastic straws good compliance in France, Italy, The Nether- with a capacity of only 0.5 ml in order to have available lands and Greece. Results available for France for the future small aliquots more adapted to new laand Italy indicate that the concordance with the boratory 'micromethods'. The straws will be stored in reference method and with biochemical markers very large liquid nitrogen containers at -196°C to enwas reasonably good. A version of this method, sure maximum stability of the biological samples. improved on the basis of the results of the pilot The main advantage of this approach is to invest study, will be used in Italy and France and, most now in the best available methods for aliquoting and probably, in Greece, Germany and The Nether- storing biological samples and to leave the door open lands once complete results are available from the for as many different types of laboratory analyses as pilot studies still on-going in these three coun- may be scientifically justified and technically possible tries. in the next 10-15 years. b) A dietary history method obtained by personal In fact, there are several types of laboratory analyses interview provided good compliance and good (on biological samples collected during prospective validity in Spain and southern Italy where it will studies) which could in theory provide useful informabe used in the main cohort study. tion for investigating the relationship between diet c) A combined method based on a one-week food and cancer. These laboratory measurements can be diary and a short food frequency questionnaire grouped into the following main categories: will be used in the UK where the combination of these two self-administered methods has provid- 1) Markers of diet which can help to measure, either ed the best results compared to the reference in absolute terms or more often in relative terms, the method. actual intake of a given nutrient. The best examples are: urinary excretion of nitrogen as a marker of total proBetween-country calibration of dietary measurements tein intake; levels in blood of vitamin C, different carotenes and vitamin E; fatty acid composition in plasma In multi-centre studies it is reasonable to expect a lipids, red blood cell membranes or exfoliated cells degree of systematic bias in the measurement of diet. (e.g., oral mucosa cells); levels of minerals in blood,

790

hair or nails; other markers found in urine such as 3-methylhistidine (from animal protein intake), etherocyclic amines (from cooked animal protein), sodium, potassium. 2) Markers of diet-related factors which do not directly indicate intake of a given nutrient but which may be related to dietary patterns and be relevant to the process of carcinogenesis: Examples of new and promising markers are lipid peroxidation products (e.g., malondialdehyde) and indicators of antioxidant status (e.g., plasma levels of ceruloplasmin, transferrin, glutathione, total anti-oxidant capacity of serum). 3) Markers of endogenous hormones which can either be influenced by diet or which can be related to other individual characteristics which are known or suspected risk factors for cancer. In this broad area of research, attention has been focused on sex hormones and their transport globulins, particularly in relation to reproductive factors and hormone-dependent cancer; insulin and growth hormones. 4) Markers of exposure to xenobiotics present in foods as natural or chemical contaminants or in the general environment. This is a very large, heterogeneous group of compounds for which at least three different approaches are available today or are being developed: a) The traditional analytical approach involving direct chemical measurement of the xenobiotic concentration in biological samples. b) The method based on measurement of the 'fingerprint' (adducts) left by the xenobiotic on DNA or proteins. Methods for adducts of a variety of compounds interacting with DNA or albumin or haemoglobin have been developed for nitrosamines, aflatoxins, ochratoxins and other mycotoxins, nitroarenes, 4-amino-biphenyl and many others. c) The most promising recent methods which identify specific patterns of DNA point mutations which could be related to specific chemicals or at least to specific families of chemicals.

drugs, history of previous illnesses and current physical activity. In addition, height, weight and waist and hip circumferences will be measured with standardized procedures. Conclusions The European Investigation into Cancer and Nutrition is being started as this is written and is expected to produce scientific results within the next 10-15 years. For the moment the study represents a major example in Europe of collaboration in cancer research not only between countries but also, which is sometimes even more difficult, between disciplines, as it brings together epidemiologists, nutritionists, statisticians, clinicians, molecular biologists, pathologists, biochemists and public health managers. The enthusiasm and dedication of all these people have made it possible to undertake this mammoth project (as it was recently defined by A. Coghlan in the New Scientist [30]), and we hope that these qualities will also guarantee the quality of its scientific outcome. Acknowledgement

The work described in this paper is being carried out with the support of the 'Europe Against Cancer' Programme of The Commission of the European Communities. * On behalf of the EPIC collaborative group composed of: F. Clavel, F. Lucas, V. Ezratty (France); J. Wahrendorf, H. Boeing (Germany); A. Trichopoulou, K. Katsouyanni, C. Gnardelis, G. Kiriazi, P. Laviou, P. Mavrika, E. Polychronopoulos, A. Valaora (Greece); F. Berrino, E. Buiatti, L. Gafa, V. Krogh, D. Palli, P. Pisani, R. Tumino, P. Vineis (Italy); H. B. Bueno de Mesquita, H. J. A. Collette, D. Kromhout, G. L. Obermann-de Boer, M. C. Ocke, P. H. M. Peeters, M. A. Pols, J. Seidell, H. A. Smit (The Netherlands); C. Gonzalez, A. Del Moral, M. Dorronsoro, C. Martinez, C. Navarro, J. R. Quiroz, M. Torrent (Spain); S. Bingham, N. Day, D. Forman, P. Hannaford, T. Key, K..-T. Khaw (UK); M. Wilpart (EC); E. Riboli, R. Saracci, R. Kaaks, Other epidemiological data of interest in cancer research N. Slimani, C. Casagrande, B. Hemon (IARC).

Even though the focus of EPIC is on diet, it is clear that there is a large number of other factors which can be relevant in cancer epidemiology, either because they are related to nutritional status (e.g., anthropometry) or because they are such strong causes of certain cancers and must be taken into account in the statistical analysis (e.g., tobacco, some occupational exposures). In summary, quite detailed data will be collected using a standardized questionnaire on history of tobacco smoking, history of alcohol consumption, occupation, socioeconomic status, age at puberty and reproductive history, use of contraception and of hormonal

References 1. Tannenbaum A. The genesis and growth of tumours: I. Effects of caloric restriction perse. Cancer Res 1942; 2: 460-7. 2. Tannenbaum A. The genesis and growth of tumours: II. Effects of a high-fat diet. Cancer Res 1942; 2: 468-74. 3. Tomatis L (ed.), Aitio A, Day NE et al. Cancer: Causes, Occurrence and Control (IARC Sci Publ 100). Lyon, International Agency for Research on Cancer, 1990. 4. Pollack S. Breast cancer: Piecing together the puzzle. Harvard Public Health Review 1992; 3: 10. 5. Doll R, Peto R. The causes of human cancer: Quantitative esti-

791

6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16.

17. 18. 19. 20.

mates of avoidable risks of cancer in the United States. J Natl Cancer Inst 1981; 66: 1192-308. Peto R. Why cancer? The Times Health Supplement, London (6 Nov. 1991). Steinmetz KA, Potter JD. Vegetables, fruit, and cancer. I. Epidemiology. Cancer Causes & Control 1991a; 2: 325-57. Steinmetz KA, Potter JD. Vegetables, fruit, and cancer. II. Mechanisms. Cancer Causes & Control 1991b; 2:427-42. Willett WC, Stampfer MJ, Colditz GA et al. Relation of meat, fat, and fiber intake to the risk of colon cancer in a prospective study among women. N Engl J Med 1990; 323: 1164-72. Howe GR, Hirohata T, Hislop G et al. Dietary factors and risk of breast cancer: Combined analysis of 12 case-control studies. J Natl Cancer Inst 1990; 82: 561-9. Willett WC, Stampfer MJ, Colditz GA et al. Dietary fat and the risk of breast cancer. New Engl J Med 1987; 316: 22-8. Willett WC. Implications of total energy intake for epidemiologic studies of breast and large-bowel cancer. Am J Clin Nutr 1987; 45: 354-60. Howe GR, Miller AB, Jain M. Total energy intake: Implications for epidemiologic analyses. Am J Epidemiol 1986; 124: 157-9. Riboli E. Methodological issues in the investigation of diet and cancer in humans. In Miller AB (ed): Diet and the Aetiology of Cancer (ESO Monograph Series). Heidelberg: Springer 1989; pp. 55-63. Moore JW, Thomas BS, Wang DY. Endocrine status and the epidemiology and clinical course of breast cancer. Cancer Surveys 1986; 5: 537-59. Giovannucci E, Stampfer MJ, Colditz GA et al. A comparison of prospective and retrospective assessments of diet in the study of breast cancer. Am J Epidemiol 1991; 134: 714 (Abstract). Friedenreich CM, Howe CR, Miller AB. The effect of recall bias on the association of calorie-providing nutrients and breast cancer. Epidemiology 1991; 2: 424-9. Doll R, Hill AB. The mortality of doctors in relation to their smoking habits. A preliminary report. Br Med J 1954; i: 14515. Doll R, Peto R. Mortality in relation to smoking: 20 years' observations on male British doctors. Br Med J 1976; 2: 1525-6. Hirayama T. Life-style and mortality: A large-scale census-

21. 22. 23. 24. 25. 26. 27.

28. 29. 30.

based cohort study in Japan (Contributions to Epidemiology and Biostatistics, Vol. 6). Basel, Karger, 1990. Stemmermann GN, Nomura AMY, Heilbrun LK. Dietary fat and the riskof colorectal cancer. Cancer Res 1984; 44:4633-7. Willett WC, Stampfer MJ, Underwood BA et al. Validation of a dietary questionnaire with plasma carotenoid and alphatocopherol levels. Am J Clin Nutr 1983; 38: 631-9. Giovannucci E, Stampfer MJ, Colditz G et al. Relationship of diet to risk of colorectal adenoma in men. J Natl Cancer Inst 1992; 84: 91-8. Howe GR, Friedenreich CM, Jain M et al. A cohort study of fat intake and risk of breast cancer. J Natl Cancer Inst 1991; 83: 336-40. Brandt van den PA, Goldbohm RA, Veer van 't P et al. A largescale prospective cohort study on diet and cancer in the Netherlands. J Clin Epidemiol 1990; 43: 285-95. Toniolo PG, Pasternack BS, Shore RE et al. Endogenous hormones and breast cancer: A prospective cohort study. Breast Cancer Res Treat 1992; 18:s23-s26. Berrino F, Pisani P, Muti P et al. Prospective study of hormones and diet in the etiology of breast cancer. In Riboli E, Saracci R (eds): Diet, Hormones and Cancer: Methodological Issues for Prospective Studies. I ARC Technical Report No. 4, 1988, pp. 34-8. Block J. A review of validation of dietary assessment methods. Am J Epidemiol 1982; 115:492-505. Willett W. Nutritional epidemiology: Issues and challenges. Int J Epidemiol 1987; 16:312-7. Coghlan A. Europe's search for the winning diet. New Scientist 1992; 1797: 29-33.

Received 21 July 1992; accepted 28 July 1992. Correspondence to: Elio Riboli, M.D., Sc.M. Head, Programme of Nutrition and Cancer Unit of Analytical Epidemiology International Agency for Research on Cancer 150 Cours Albert-Thomas 69372 Lyon, Cedex 08, France