Epidemiological Studies Using Biological Markers: Issues ...Transitional Epidemiological Studies Transitional epidemiological studies are observational studies in humans, employing

Vol. 1, 13-19, November/December 1991 Cancer Epidemiology, Biomarkers & Prevention 13

ASPO Distinguished Achievement Award Lecture

Epidemiological Studies Using Biological Markers: Issues for

Epidemiologists1Barbara S. Hulka2

Kenan Professor and Chair, Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina 27599

On April 1 1, 199 1, Barbara Sorenson Hulka, M.D., MPH., received the Distinguished Achievement Award ofthe American Society of Preventive Oncology (ASPO) at the annual meeting of the Society in Seattle,Washington. The award is given by the Executive Committee of ASPO in recognition of contributions to theprevention of cancer over many years, not for a single accomplishment. Any member of ASPO may nominatea candidate for the award. The 15-member Executive Committee votes by secret ballot after considering asummary of the contributions to cancer prevention of the several candidates. Dr. Hulka won in a close electionover several other outstanding scientists.

Dr. Hulka has chaired the Department of Epidemiology at the University of North Carolina School ofPublic Health since 1983. During her tenure she has developed the Epidemiology Department into one of thebest in the country. The basis for her award, however, was her 25 years of work in the prevention of cancersof female reproductive organs. Dr. Hulka began her career with a series of critical studies of cervical cancer inunderserved populations. These were followed by studies of the role of hormones in endometrial and breastcancers, a field in which she continues to make important contributions. She has brought to these studies abalanced evaluation of the risks and benefits of hormones rarely found in epidemiological research.

Dr. Hulka has been incredibly generous in her service on state and national advisory committees. Since1973 she has served on 26 national advisory committees, been a member of the editorial boards of sevenjournals, and been a consultant to numerous public and private organizations.

Dr. Hulka is a graduate of Radcliffe College, the Julliard School of Music, and Columbia University’sCollege ofPhysicians and Surgeons and received her MPH. from Columbia University SchoolofPublic Health.It was an honor for ASPO to bestow its Distinguished Achievement Award on Barbara Hulka.

w. Thomas LondonPresident of ASPO

(1969- 199 1)

Biological markers in epidemiological research have along history. They were used initially in the study ofinfectious diseases from which came the genesis of epi-demiology as an approach to studying health and disease.Over the years, however, the issues in epidemiologyhave changed; quantitative statistical and epidemiologi-cal methods have blossomed and assumed preeminence.During the 1980s when laboratory advancements in mo-lecular and biochemical techniques were rapid, biolog-ical elements in epidemiological research again becameprominent. There are biological markers applicable toalmost all diseases and conditions that we study in epi-demiology. How these markers are used and the influ-ence they have on our study designs and methods,

Received 6/18/91.

1 This work was funded in part through Cooperative Agreement

CR813485 between the Health Effects Research Laboratory of the U.S.Environmental Protection Agency, and the University of North Carolina,Department of Epidemiology. Presented at the 15th Annual Meeting of

the American Society of Preventive Oncology in Seattle, Washington onApril 11, 1991.2 To whom requests for reprints should be addressed.

particularly in the area of cancer epidemiology, are thefocus of this paper.

I will review several topics, starting with the concep-tual framework for characterizing biomarkers and therationale for their use. Next I will mention several meth-odological issues pertinent to studies using biomarkersand show how these lead to modifications in traditionalepidemiological study designs. A series of studies that fitwithin the conceptual framework for biomarkers willserve as examples. Finally, I will offer some suggestionsabout future directions for studies using biomarkers.

Conceptual Framework

Fig. 1 is a modification of a diagram that has appeared in

many publications and presentations (1). It represents asequence of events from a toxic environmental exposureto the outcome of disease. Markers for these events arelabeled “internal dose,” “biologically effective dose,” “bi-ological response,” “altered structure/function,” “dis-ease,” and “prognosis.” I have added the concepts of�susceptibility” and “environmental/life style” factors,noting their potential importance as modifiers ofthe stepsalong the spectrum from exposure to disease.

on April 15, 2020. © 1991 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

http://cebp.aacrjournals.org/

14 ASPO Distinguished Achievement Award [ecture

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Fig. 1. The relationship of biological markers to exposure and disease.

One drawback of the diagram is the impression thatevery marker moves inexorably on to the next. For manyof the early stages in the marker spectrum, progressionis rare. Almost all biological alterations, as measured bya marker, are repaired, stabilized, or destroyed. Repaircapability is greatest and progression least frequent onthe left side of the diagram.

Rationale

The rationale for the use of biological and biochemicalmarkers in epidemiological research include thefollowing.

(a) Improve accuracy and reduce misclassificationin exposure variables. Misclassification of exposure is acommon problem in studies of occupational and envi-ronmental exposures. Internal dose or biologically effec-tive dose markers should be more accurate reflections ofthe true dose absorbed by the body than ambient mon-itors of the environment.

(b) Allow for study of preclinical disease and pro-vide opportunity for prevention. A classical example iscervical cytology, used in screening for precancerouslesions of the cervix. The Pap smear, having been acomponent of clinical practice for several decades, hasresulted in the diagnosis and treatment of preneoplasticdisease with a subsequent reduction in invasive cervicalcancer incidence and mortality.

(c) Provide more homogeneous classification of dis-ease. Subsets of acute nonlymphocytic leukemias, forexample, have been identified with distinctive chromo-somal abnormalities. These cases are more frcquentlyrelated to chemical exposures than cases without thesealterations. More accurate risk estimates can be obtainedfor the effect of chemicals in producing acute nonlym-phocytic leukemia by including chromosomal abnormal-ities in the case definition rather than diluting the asso-ciation with a heterogeneous case group (2).

(d) Identify interindividual differences in suscepti-bility to disease and utilize this information in classifyingrisk groups. Susceptibility markers such as genetic poly-morphisms or differences in metabolic activity are likelyto act as effect modifiers, or possibly confounders, instudies that evaluate relationships between exposure anddisease.

(e) Improve the methodology of clinical trials. Bio-logical markers may take the form of intermediate out-comes, proximate to the occurrence of actual disease,which is the ultimate outcome. Their employment mayreduce the sample size or length of follow-up required

in trials. Biological markers may also be used to classifytrial participants for prognostic purposes or as part of theeligibility criteria. Biological markers of compliance withintervention strategies used in clinical trials are oftenessential.

(f) Increase our understanding of disease mecha-nisms, allowing for stronger biologically based researchdesigns and interpretation of epidemiological data.

(g) Enhance the credibility of traditional epidemio-logical research, especially for studies of weak associa-tions. Very little current epidemiological research iden-tifies risk estimates of 3- to 4-fold or greater. Much ofour research is centered on studies providing risk esti-mates of 1.5 or less. Innovative epidemiological studiesemploying biomarkers, relevant to exposure or disease,can elucidate mechanisms for disease occurrence thatsupportthe epidemiological studies ofweak associations.Relevant findings from biomarker studies can increasethe likelihood that the weak associations are real and notjust artifacts due to inadequacies of study design, meth-ods, or analyses.

(h) Improve methods in risk assessment or estima-tion. Biological markers can be used to identify hazards,clarify dose-response relationships, describe the extentof exposure in human populations, and estimate risks(especially for low dose exposure).

(i) Increase scientific knowledge about diseaseprocesses and adverse health effects through multidisci-plinary research. The strategy of addressing health prob-lems through multiple disciplines, rather than a singularfocus, is gaining ascendency in scientific inquiry. Theability to integrate knowledge from a variety of disciplinesabout a defined health problem may be our most fruitfulavenue for developing a better understanding of diseasecausation and prevention.

Validation of Biological Markers

The ultimate validation of any marker is its ability topredict disease occurrence. However, preliminary stud-es with more limited goals for validation are usually

necessary before a marker is proposed for a study todemonstrate its predictive validity.

In epidemiology, sensitivity and specificity are theusual measures of validity for a screening test or otherprocedure that requires evaluation. But the definition ofthese terms varies across disciplines. Laboratory scientistsconsider sensitivity to be the minimum level of an analytethat an assay can detect. (More technically, sensitivity isthe smallest single value that can be distinguished fromzero with 95% confidence limits.) For analyte levelsabove this minimum, the assay is positive; for lowerlevels, the assay is negative. Specificity in the laboratoryindicates the ability to detect a unique analyte from agroup of closely related structures. This issue frequentlyarises with immunological assays, such as the enzyme-linked immunosorbent assay, in which antibodies arecreated in animal systems to interact with specific chem-ical structures in humans. Often these antibodies detectseveral closely related structures and are not uniquelyspecific.

Epidemiologists actually have two approaches toconsidering validity and, therefore, sensitivity and speci-ficity. These approaches relate to the goals of the studiesand to whether they focus on disease or exposure. Those



Cancer Epidemiology, Biomarkers & Prevention 15

of us who have learned the concept of validity from thestandpoint of screening for disease evaluate marker sen-sitivity and specificity relative to diseased and non-diseased persons. Environmental epidemiologists mayrefer to the sensitivity and specificity of a biologicalmarker relative to exposed and nonexposed persons.They may validate biological markers of exposure againstambient levels of the chemical or pollutant. However,the justification for this approach is not immediatelyobvious, since the biological marker should be a moreaccurate indicator of the impact of the chemical on thehuman organism than the external measurement; other-wise, why bother with the marker? Validating the biolog-ical marker against the external measurement appears to

lack conceptual coherence.What are the strategies for developing and validating

markers prior to the ultimate validation of their ability topredict disease occurrence? Preliminary work in animalmodel systems is necessary for assay development, togenerate dose-response curves, and to gain an under-standing of whether the marker is truly part of the path-ogenesis of the disease or merely an adaptive responseon the part of the organism. In the conceptual model ofbiological markers shown in Fig. 1, the focus of interestis on understanding disease causation and markers that

fit into the biological pathway between exposure anddisease. In those instances where surrogate markers areused (e.g., hemoglobin adducts versus DNA adducts intarget tissues), it is important to determine that the sur-rogate is an accurate, quantitative reflection ofthe markerof interest that lies in the disease pathogenesis pathway.

Preliminary studies must also be done on humantissues. In comparison to animal studies, humans willhave had very low exposures to the chemical or complexmixture of interest. Therefore, the sensitivity of the assay(laboratory definition of sensitivity) will be stressed. Assaytechniques that were successful in animal systems mayrequire modification for human studies. Human tissuesneed to be obtained from highly selected, heavily ex-posed persons. With these tissues, the feasibility, relia-bility, and laboratory sensitivity and specificity of theassay can be evaluated.

Transitional Epidemiological Studies

Transitional epidemiological studies are observationalstudies in humans, employing biological markers andbridging the gap between laboratory experimentationand population-based epidemiology. These studies maybe designed to evaluate exposures, health effects, orsusceptibility.

What are their goals? We expect them to addresspreliminary or developmental questions in anticipationof large-scale epidemiological studies, or they may an-swer substantive questions. For example, many of thestudies required to validate biomarkers would fall intothe category of transitional epidemiological studies.Some might be designed to assess intra- and interindivid-ual variability. Sources and extent of variability assumeimportance in transitional studies, since expansion ofsample size to reduce variability is often not a feasibleoption. Within-person variability is illustrated by varyinglevels of DNA adducts across organ sites or sister chro-matid exchange frequency among WBC. The variabilitywithin a person for these markers may be as great or

greater than the variability among persons. Another goalof these studies might be to evaluate the feasibility ofusing a marker in field conditions. Information must beobtained about the marker’s stability and the conditionsrequired for collection, transport, and storage. In addi-tion, there are situations in which transitional studies aredesigned to identify exposure/effect relationships.

What are the characteristics of these studies? Theymay use laboratory assays as exposure factors, healtheffects, confounders, or effect modifiers. These studieswill usually not be population based. Subjects will fre-quently be selected to meet particular disease or expo-sure criteria. For example, subjects may be selected tobe extreme, such as people who have been very highlyexposed or totally unexposed.

Commonly applied design criteria will include sub-ject restriction and matching. We may expect large be-tween-group differences in the outcome variable so thatsmall sample sizes (50 to 100 subjects or even less) maybe adequate. There will be more use of continuousvariables and transformed variables, new scoring meth-ods, and statistical techniques. Thoughtful use of analyticmodeling techniques will be necessary to minimize thenumber of independent variables in the models.

Background levels of markers may cause misclassi-fication. If, for example, one is interested in DNA adductsformed from polycyclic aromatic hydrocarbons and nitro-samines in tobacco smoke, the ubiquitous presence ofthese chemicals in the general environment and theresulting adduct formation in the tissues of nonsmokerswill make the association between adducts and smoking

difficult to detect. Recognition of background levels ofthe exposure should be part of the study design whenconsidering sample size and dose-response relationshipsfor the biomarker.

The issue of confounding assumes some new dimen-sions in these studies. Often, little is known about riskfactors for the marker (other than the exposure of inter-est), and even less attention may be given to the distri-bution of risk factors among exposure groups. The po-tential for confounding is particularly great when nonspe-cific markers of genotoxicity are used. In this situation, avariety of exposures may produce the same biologicalresponse. For example, metabolic products of dietaryconstituents can produce adducts with the same chemi-cal structures as environmental toxins (3). If diet andenvironmental exposure are correlated, confoundingmay occur.

Specific nutrients may act as independent predictorsof a marker, confounders, or effect modifiers. Often thereis uncertainty about which dietary factors to study andhow to measure them, although both biomarkers ofdietary constituents and questionnaire information aregenerally useful. The need for greater attention to dietaryconstituents and methods for their measurement is evi-dent from the scientific literature both within and outsideof epidemiology.

Other factors may act as confounders or possibleeffect modifiers. Endogenous exposure to normal bodyconstituents, such as estrogens in women, can influencethe body’s response to xenobiotics. In the female ratmodel, estrogens act to induce the P450 isoenzymesystem in the liver, increasing metabolic production ofelectrophilic metabolites of genotoxic xenobiotics with aresultant increase in DNA adduct formation (4). In



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Everson, M. Schramm, I. Griffith, and M. MCann. Cotinine concentra-lions in semen, urine and serum of smokers and nonsmokers, submitted

for publication.

16 ASPO Distinguished Achievement Award Lecture

women, only the induction of liver enzymes by estrogenshas been demonstrated; the subsequent biochemicalpathways have not been studied.

An example of possible effect modification was re-ported by Perera et al. (5). They showed differences inDNA adduct levels in normal lung tissue from personswith and without lung cancer. Seasonal differences werealso observed; higher adduct levels were found in tissuesobtained between July and October as compared toother times of the year.

What do we do to minimize confounding? First,

maximize design options to reduce confounding, thusminimizing the need to control confounding in the analy-sis. With small numbers of subjects, analytic control isunlikely to be reliable or stable. Design options for mm-mizing confounding include restriction, matching, and

exclusion. Limit the distribution of potential confoundersacross subjects through restriction. Consider matchingon demographic characteristics, i.e. , age, gender, race,and social class, which will also help to control for un-known confounders. The ultimate form of restriction isexclusion. Application of exclusion criteria in epidemio-logical studies is standard procedure and perhaps is usedexcessively when the number of people excluded be-comes a significant proportion of the total study popu-lation. Overzealous application of exclusion criteria re-quires the recruitment of more study subjects and couldbias the study results.

In transitional epidemiology, the first rule of analysisis to keep it simple. Graphic display of the individualdata, followed by descriptive statistics and stratified anal-yses, is informative. Multivariate models should be usedsparingly, and the models should be sparse, with fewindependent variables. Transformation of continuousvariables is often appropriate. Log or square transforma-

tions are desirable if there is a need to equalize thevariance across a distribution of values and to normalizea skewed distribution (6). Transformed data are oftenmore appropriate for analysis.

Metaanalysis may be an important analytical toolwhen dealing with multiple small studies, none of whichhave adequate power to demonstrate statistically signifi-cant effects. Although we anticipate large differences inthe effect measure for exposed and unexposed personsor cases and controls, transitional epidemiological studiesare likely to be small, with modest power. A metaanalysisreported by Margolin and Shelby (7) brought togethermultiple small studies of sister chromatid exchange dis-tributions in different race and gender groups. The me-taanalysis demonstrated that women on average havehalf a sister chromatid exchange more per cell than men,a finding not previously recognized from the individualstudies.

Illustration of a Transitional Epidemiological Study

A transitional exposure study, cross-sectional in design,is currently in progress in the Epidemiology and Biosta-tistics Departments in the School of Public Health at theUniversity of North Carolina in conjunction with scien-tists in the Health Effects Research Laboratories of theEnvironmental Protection Agency (8). The study employscotinine as a marker for tobacco smoke in the semen,serum, and urine of smokers and nonsmokers. The ex-ample is relevant to cancer as well as reproductive epi-

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demiology since the mechanisms of genotoxicity and theagents involved are frequently the same in both fields,although the target cells and organs may differ. Cigarettesmoke contains mutagens and carcinogens, which areknown to cause adverse reproductive outcomes and

cancers of multiple sites. An adverse effect of cigarettesmoking on sperm count, motility, and morphology hasbeen suggested in some studies, although statisticallysignificant associations are rare. If cotinine, a metaboliteof nicotine, could be detected in the semen of smokers,it would support the concept that mutagenic substancesin tobacco smoke could also reach sperm cells andpotentially lead to heritable damage. Cigarette smokealso provides a model system for other environmentalpollutants that could lead to heritable mutations.

In our study, male volunteers, smokers and non-smokers, were recruited by newspaper solicitation toprovide blood, urine, and semen specimens and to corn-plete a self-administered questionnaire. All specimenswere analyzed for cotinine by radioimmunoassay, and astandard clinical semen analysis was performed to deter-mine sperm count, density, morphology, and motility.Exclusion criteria were applied to obtain a group ofhealthy men, aged 18 to 35, who had not had otherexposures that might alter sperm form or function. Eighty-eight men participated in the study.

Fig. 2 shows that cotinine concentrations in semenare similar to those in serum; higher concentrations arefound in urine. A dose-response relationship for non-smokers and light and heavy smokers is also evident. Innonsmokers (Fig. 3) cotinine appears in all three bodyfluids, presumably due to environmental tobacco smoke

exposure. There is a suggestion of a dose-response rela-tionship between h/day of environmental tobacco smokeexposure and cotinine concentration in urine.

We found high correlations between log cotinineconcentrations in each of the three body fluids, rangingfrom 0.87 to 0.94. Fig. 4 shows the linear associationbetween log cotinine concentrations in serum and se-men.1 The correlations between log cotinine in each



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Cancer Epidemiology, Biomarkers & Prevention 17

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body fluid and number of cigarettes smoked was moremodest, ranging from 0.40 to 0.53. There were smallnegative correlations, -0.20 to -0.22, between cotinineconcentration and sperm density, measured in numberof cells/mi.

Cotinine provides an excellent internal dose markerfor nicotine in cigarette smoke. But the presence of

additional biological markers that appear at later stagesin the exposure/disease spectrum would strengthen theargument for biological effects of smoke constituents onsperm cells. In order to provide this evidence, additionalstudies are in progress. Specifically, we are attempting todetect smoking-related DNA adducts, using 32P postla-

beling assays, in the sperm cells of smokers. If adductsare found, these would provide evidence of mutagenicinsult to DNA. Although assay results are not yet avail-able, the design of the adduct study is relevant to anadditional aspect of our work.

In the adduct study, subjects were recruited toachieve triplets, composed of a nonsmoker, a lightsmoker, and a heavy smoker. The heavy smoker andnonsmoker provided the exposure extremes, whereasthe light-smoking member provided the opportunity fordose-response analysis. Members of each triplet werematched on age and education; exclusion criteria wereapplied such that only healthy Caucasian men withoutexposure to other suspect mutagenic agents were in-cluded. Furthermore, only men with a sperm count of atleast 40 million were included in order to ensure anadequate amount of DNA for the 32P postlabeling assay.

As the study progressed, it became evident thatheavy smokers were being excluded disproportionately

from the triplets because of the sperm count criterion.In fact, it was necessary to supplement the advertising torecruit more heavy smokers. This experience caused usto return to the literature on studies of smoking andsperm count or sperm density. The literature yieldedseveral observations. There were 1 7 interpretable studiesin the English language, which fell into two groups, thosethat were based on healthy volunteers and those thatwere based on infertility clinic patients. Summary datafrom these studies are plotted in Fig. 5 (9). Each study is

represented by a data point (triangles for infertility clinicpatients and squares for healthy volunteers). The datapoints plot the difference in mean sperm density be-

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18 ASPO Distinguished Achievement Award Lecture

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for all subjects in each study. In all of the studies, thedifference is positive; nonsmokers have higher averagesperm density than smokers, although in the originalreports, only one study had indicated that the differencewas statistically significant. The regression lines for thetwo sets of studies are similar in their slopes, and, as ithappens, our study lies near the midpoints of these lines.This graphic display is convincing; smokers have a loweraverage sperm density than nonsmokers. In our studythe average sperm density was 93 x 106/ml for non-smokers and 72 x 106/mI for smokers of 20+ cigarettes/day. A structured metaanalysis is in progress.

If we return at this point to the conceptual frame-work for biological markers, ranging from exposurethrough pathogenesis to disease, it becomes clear howthe studies I have described relate to the sequence ofevents in that spectrum. Questionnaire information onactive and passive smoking was obtained to measureexogenous exposure. We obtained information on inter-nal dose with cotinine assays in body fluids and onbiologically effective dose using 32P postlabeling assaysfor DNA adducts in sperm cells. Sperm density fits in theconceptual framework at the level of functional andstructural alterations. These interrelated studies, usingthe methods of epidemiology, biochemistry, molecularbiology, and biostatistics, have enabled us to identifymultiple markers that lie at different points in the bio-marker spectrum. This accumulation of information oncigarette smoke, semen, and sperm strengthens the sci-entific basis for possible adverse reproductive effects ofsmoking on men.

Constraints on Biological Markers inEpidemiological Research

The application of biological markers to epidemiologycreates opportunities but also presents difficulties. Firstis the issue of collaborative research among scientistsfrom different disciplines. Although the application ofknowledge and skills from multiple disciplines to a singlehealth problem is a significant reason for doing researchwith biological markers, it is also a constraint. Scientistsfrom different disciplines have different goals. Laboratoryscientists want to develop new assays, to be on thecutting edge of new laboratory technology. Epidemiolo-gists want standardized, reproducible, inexpensive assays

suitable for high-volume work. We work in differentlaboratories; the epidemiologist’s is population basedand the laboratory scientist’s is bench based, using smallnumbers of subjects (specimens) under highly controlledconditions. Laboratory scientists tend to have a deter-ministic orientation toward science, whereas in epide-miology we think in a probabilistic mode. Years of edu-cation, training, and experience are reflected in our atti-tudes toward and values in science. This indoctrinationmakes it difficult to appreciate fully the importance ofscientific disciplines other than one’s own. Differencesin orientation and assumptions are major reasons for thedifficulties that arise in multidisciplinary research.

The greatest constraint in our current environment,however, is research funding. There are few fundingsources that cross disciplinary lines. If funding sourcesare found, the review process may be an obstacle, sinceboth laboratory scientists and epidemiologists must beenthusiastic about a proposal, which may be viewed ashybrid research and may not be attractive to either expertgroup. To the extent that the individual components ofa proposal can be viewed in the context of the whole,the significance of the research will receive higher prior-

ity and more proposals will be funded.The search for environmental exposures that have

significant health effects is the task of environmentalepidemiology, which is devoted to identifying biologicaleffects in humans from low-level exposures. One prob-em is that frequently these exposures are neither suffi-

ciently potent nor sufficiently high to produce a meas-urable effect. In order to counteract these constraints,more sensitive assays are developed, such as the �2Ppostlabeling assay, for which the sensitivity approaches1 adduct/10’1 nucleotides. But one must ask, at whatpoint does enhancement of sensitivity result in the de-tection of alterations that are insignificant with respect tohuman health and disease?

Replication of findings is necessary in order to gaincredibility. In epidemiology, we are accustomed to seek-ing replication in multiple populations, geographic set-tings, and time periods. This strategy is not satisfying toall disciplines, however, because of the priority that isgiven to making new discoveries. Replication of assaysin multiple laboratories is necessary in order to be as-sured of assay reliability, and results should be verifiedin multiple study groups and settings.

More information is needed on a variety of topicsrelevant to biomarkers. The background level of specificmarkers in different populations is one of these. Identi-fication of risk factors and potential confounders fordifferent markers is another. Health effects in relation to

various markers are virtually unexplored. Sources andextent of variability assume more importance. Theseinclude both biological variability (intra- and interindivid-ual), which can have both genetic and environmentalsources, and laboratory variability, which has both ran-dom and systematic components. Understanding thenatural history of biomarkers, analogous to the naturalhistory of disease, will help us to use biomarkers effec-tively in epidemiological studies.

Future Directions

Biological markers will play a prominent role in epide-miological research during the 1990s. Multiple markers,



cancer Epidemiology, Biomarkers & Prevention 19

including different points on the spectrum of exposureto disease, will be used. Nonspecific markers and chem-ical-specific markers may be used concurrently, depend-ing on the goals of the study.

The concept of “completing the parallelogram” willreceive more empirical validation (10). The parallelogram

relates findings from epidemiological studies, both tran-sitional and traditional, to those from animal models andin vitro systems. Knowledge about findings from theseother systems allows epidemiologists to design studiesthat can build on existing laboratory data.

Laboratory capability is essential for biomarker stud-ies, and how epidemiologists relate to the laboratory isalso important. A number of options for functional rela-tionships between epidemiologists and laboratory sci-entists exist. Perhaps most desirable for the epidemiolo-gist is to have laboratory capability within one’s owndomain. This will require the acquisition of laboratoryskills by epidemiologists or epidemiological skills by lab-oratory scientists. Individuals who can cross disciplinesand run laboratories themselves will become valuedmembers of epidemiology departments.

Epidemiologists should establish research collabo-rations with recognized laboratory experts who have themost advanced laboratory technology and capability.This collaboration provides the strongest scientific linksand paves the way for eventual in-house laboratorycapabilities.

As assays are replicated, and their use for clinical orregulatory purposes becomes evident, technology trans-fer from research to clinical and commercial laboratorieswill take place. To the extent that assays can be done inlarge numbers and hopefully at modest cost, productionlaboratories, including commercial ones, will be usefulfor epidemiology.

A resource for biomarker research is the availabilityof repositories of biological specimens. Tissue and bodyfluid repositories have the potential to make studiesfeasible and efficient from the standpoint of cost versusinformation obtained. Repositories of biological mediathat have been collected and stored in the context ofprior epidemiological studies are obviously valuable, butarchived fixed tissue specimens, which exist in pathologydepartments, should not be ignored. Although sub-jects may not be well characterized on epidemiologicalparameters, innovative means to overcome this defi-ciency may be possible. Use of archived specimens hasimportant implications for assay technology in that assaysusing fixed tissues have different technical problems fromthose using fresh or frozen material.

My last point pertains to those of us in the AmericanSociety of Preventive Oncology who are concernedabout cancer control and prevention. Biological markersare likely to become a more prominent part of our

research and implementation agendas. Even though pastefforts to develop markers for cancer screening havebeen disappointing, it is hard to envision a significantalternative to the continued search for sensitive andspecific biomarkers. The domain of clinical trials is also afertile ground for marker application. Markers can beused to detect precursor lesions (the response variablein some clinical trials) and intermediate outcomes. Theymay also serve as part of the inclusion criteria for admis-sion into trials and as indicators of compliance with theintervention.

Acknowledgments

The author gratefully acknowledges the many scientific and technicalcollaborators on the research reported in this paper. Primary participantsfrom the Health Effects Research Laboratory, U.S. Environmental Protec-

lion Agency, include Drs. Richard Everson and Jack Griffith. From theUniversity of North Carolina at Chapel Hill the investigators include Drs.

Marilyn Vine, Barry Margolin, Young Truong, and Ping-chuan Hu.

References

1 . Subcommittee on Reproductive and Neurodevelopmental Toxicology,

Committee on Biological Markers, Board of Environmental Studies andToxicology, Commission on Life Sciences, National Research Council.Biological Markers in Reproductive Toxicology, p. 1 7. Washington, D.C.:National Academy Press, 1989.

2. SandIer, D. P., and Collman, G. W. Cytogenetic and environmental

factors in the etiology of the acute leukemias in adults. Am. J. Epidemiol.,126: 1017-1032, 1987.

3. Poirier, M. C. DNA adduct determination in human tissues and bloodcell DNA samples. Presented at the American Cancer Society Mary Lasker

Conference, Sarasota, FL, April 3, 1991.

4. Lucier, G. W. Receptor-mediated carcinogenesis. Presented at theworkshop on “Approaches to Classifying Carcinogens According to Mech-anisms of Action,” for the International Agency for Research on Cancer,

Lyon, France, June 11-18, 1991.

5. Perera, F., Mayer, J., Jaretzki, A., Hearne, S., Brenner, D., Young, T.L., Grimes, M., Grantham, S., Tang, M. x., Tsai, W-Y., and Santella, R. M.Comparison of DNA adducts and sister chromalid exchange in lung

cancer cases and controls. Cancer Res., 49: 4446-4451, 1989.

6. Kleinbaum, D. G., Kupper, L. L., and Muller, K. E. Applied RegressionAnalysis and Other Mullivariable Methods. Boston: PWS-Kent PublishingCompany, 1988.

7. Margolin, B. H., and Shelby, M. D. Sister chromalid exchanges: areexamination of the evidence for sex and race differences in humans.

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1991;1:13-19. Cancer Epidemiol Biomarkers Prev B S Hulka epidemiologists.Epidemiological studies using biological markers: issues for ASPO Distinguished Achievement Award Lecture.

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