Future Research Directionsin the Use of BiomarkersKari Hemminki,l Rajiv Kumar,' Vladimir J. Bykov,Jari Louhelainen,1 and Pavel Vodicka21Center for Nutrition and Toxicology, Karolinska Institute, Huddinge,Sweden; 2lnstitute of Experimental Medicine, Czech Academy ofSciences, Prague, Czech Republic

Many DNA adduct studies have been carried out in occupational groups that have been at a riskof cancer based on epidemiological results relating to exposures decades ago. Even new

epidemiological publications on cancer cannot accurately address the effective exposures afterabout 1970. This is one justification for biomarker studies. Another justification is exposures forwhich epidemiological studies have not been conducted or have provided inadequate results, inspite of suspicions raised by short-term or animal experiments. The modulation of environmentalcarcinogenesis by host polymorphism in genes for xenobiotic metabolizing and DNA repairenzymes is currently under extensive investigation. The studies relating phenotype/genotype tocancer are presently extended to various end points that may be related to cancer such as DNAadducts and cytogenetic damage. Adjustment for a metabolic phenotype or genotype may alsoincrease the precision in the measurement. Mutations in oncogenes and tumor suppressor genes

may give clues to the etiology of cancer. Environ Health Perspect 1 04(Suppl 3):459-464 (1996)

Key words: DNA adducts, UV-photoproducts, UV-induced mutations, mutation screening,genetic epidemiology, mutational epidemiology

IntroductionThe use of biomarkers (defined as indicatorsof exposure, effect, and individual suscepti-bility) is relatively recent. Many of themethods used have not been extensivelyvalidated and it is not known, in mostinstances, to what extent the biomarkerspredict the risk of mutation or cancer.

This paper was presented at the 2nd InternationalConference on Environmental Mutagens in HumanPopulations held 20-25 August 1995 in Prague,Czech Republic. Manuscript received 22 November1995; manuscript accepted 28 November 1995.

This study was supported by the Swedish CancerFund, the EC Environment Program, partially throughthe National Environmental Protection Board and theMedical Research Council, the Swedish RadiationProtection Board, and the Work-Environment Fund.

Address correspondence to Dr. Kari Hemminki,Center for Nutrition and Toxicology, KarolinskaInstitute, Novum 141 57 Huddinge, Sweden.Telephone: 46-8-6089243. Fax: 46-8-6081501. E-mail:kari.hemminki@cnt.ki.se

Abbreviations used: PAHs, polycyclic aromatichydrocarbons; IAC, immunoaffinity chromatography;BPDE, benzolalpyrene diol epoxide; dGMP, deoxy-guanosine 3'-monophosphate; HPLC, high-perfor-mance liquid chromatography; SSCP, single strandconformation polymorphism; DGGE, denaturant gra-dient gel electrophoresis; CDCE, constant denaturantcapillary electrophoresis; PDGF, platelet-derivedgrowth factor; EGF, epidermal growth factor; TGFa,transforming growth factor-a; ATBC, a-tocopherol,fcarotene; EGFR, EGF receptor.

In this paper we will review, subjec-tively, some areas of biomarker researchthat are familiar to us. The areas coveredinclude DNA adducts, adducts and muta-tions, and mutations in oncogenes andtumor suppressor genes, including theirproducts, oncoproteins. Mutation epi-demiology, whether carried out through atwin or a population registry or linked to acancer registry, may be a powerful sourceof new patients for genetic analysis.Because genes involved in familial cancermay also operate in common cancers, alarge interest in these genes has developed.

DNA AdductsIn the future, the emphasis in DNA adductresearch should be on quantification ofspecific adducts. For some 10 years, aro-matic adduct profiles have been presentedin the literature. While serving a purposeas a general adduct level, there is now aneed to improve the specificity of adductstudies, aiming at criteria of generalanalytical chemistry; however, because ofmultiple steps of analysis, a great deal ofstandardization is required. The moststraightforward approach is to use bothexternal and internal standards for the

adducts to be identified. This is notfeasible in exposures to complex mixturessuch as polycyclic aromatic hydrocarbons(PAHs). The approach is more feasible toadducts, which make only one main typeof adduct.

Another kind of general problem withadducts is the use of surrogate tissuesinstead of target tissues. Furthermore, half-lives of adducts are largely unknown evenin surrogate tissues. A few studies haveaddressed these problems.

Smoking is a known risk factor oflaryngeal cancer. Aromatic adducts oflaryngeal tissue obtained from surgery wereanalyzed; there was a relationship to smok-ing, most clearly in the tumor tissue. Bothtumor and normal laryngeal tissues showeda correlation of about 0.9 to the total whiteblood cells (1).

Smokers had elevated levels of 7-methylguanine, particularly in theirlymphocyte DNA as compared to thegranulocyte DNA (2). The adduct levelswere highest in the bronchial DNA ofsmokers, almost four times the level innonsmokers (3). In a small number ofsmokers, both target (bronchial) and surro-gate (lymphocyte) DNA were available,showing a correlation of 0.8.

Larynx tissue samples obtained fromsurgery have also been assayed for7-methylguanine-DNA adducts. Therewas a relationship to smoking, and larynxadduct levels were two times the level inwhite blood cells. There was a modest cor-relation only between 7-alkylguanines andaromatic adducts.

In the latter part of this section, we willdiscuss some examples of how the problemsof specificity and quantification can betackled on very different kinds of exposures.

PAH AdductsWe have attempted to study the nature ofthe aromatic adducts detected in the post-labeled samples from Silesia, an industrial-ized area of Poland, which has been a focusof our studies for years. The methodsapplied with samples before postlabelingincluded nuclease P1 treatment, butanolextraction, and immunoaffinity chroma-tography (IAC) using an antibody raisedagainst benzo[a]pyrene-modified DNA(4). The results on IAC are shown inFigure 1. The antibody binds benzo[a]py-rene diol epoxide (BPDE)-modified deoxy-guanosine 3'-monophosphate (dGMP),a microsomally produced mixture of 10

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HEMMINKI ETAL

30 -

4c0)

coE

cu

4-

BPDE- PAH Summer Winter Summer WinterdGMP mixture Environmental Occupational

Figure 1. Binding of aromatic adducts by benzo[a]-pyrene diol epoxide (BPDE)-DNA antibody. The boundmaterial was eluted with methanol. The ratiobound:unbound is shown for BPDE-dGMP standards,DNA reacted with 10 different PAHs in a microsomalsystem, and human white blood cell DNA from Silesia,showing coke workers with occupational exposure andSilesians with environmental exposure, sampled insummer and winter.

different PAH-DNA adducts, and DNAobtained from lymphocytes of coke workers(highly occupationally exposed to PAHs)and Silesian residents (environmentallyexposed to PAHs). However, samples ofboth coke workers and other Silesiansshowed more binding by the IAC columnin the winter, a time of heavy exposureto PAHs.

In high-performance liquid chromatog-raphy (HPLC) analysis using a flow-through radioactivity detector, typicalseasonal adduct peaks were noted; theywere particularly prominent in lymphocyteDNA collected in the winter. These peakseluted in the area of PAH-DNA adducts,giving additional support that the adductsare PAH-like (5).

Another problem with complex mix-tures is the difficulty in quantitating theresults. In an illustrative experiment, DNAadducts of a number of 3H-labeled PAHswere prepared in a microsomal system andused for optimization and measurements ofrecoveries in the postlabeling assay. Theoptimal labeling conditions for all testedcompounds were very similar. The recover-ies varied from 3 to 60% among differentPAHs, indicating that the levels of theseadducts could be considerably underesti-mated when analyzing human samplesfrom PAH-exposed populations (6).Because we have found similar results withan entirely different group of compounds,we concluded that different adducts requiredifferent conditions for optimal labeling(7). Thus, the absence of proper standards,or unknown adducts, makes quantitativeinterpretation of the postlabeling resultsdifficult, if not impossible.

Further methods and efforts are neededto characterize the levels of adducts ofindividual PAHs or other aromatic com-pounds, which is a large task.

AlkenesGasoline is one of the most commonsolvent vapors to which workers and thegeneral public are exposed. The exposure isparticularly to the volatile alkanes andalkenes. Additionally, many of the compo-nents of gasoline are found in vehicleexhaust either because of incomplete com-bustion or because of the chemical reac-tions taking place in engines. Incompletecombustion also creates new types of com-pounds that are not present, or only inminor quantities, in gasoline. Typicallythese are various PAHs, but aliphatic com-pounds such as alkenes ( e.g., ethene,propene, butadiene, isoprene) and variousaldehydes are also being formed. Fuel andengine development has been focused onthe reduction of the polycyclic aromaticcompounds. Catalytic converters, on theother hand, are effective in removing themain part of volatile hydrocarbons fromengine exhaust; however, even in optimalconditions, a fraction remains. In coldstarts and in malfunctioning converters,a high proportion of hydrocarbons isreleased unburned.

The concerns about the harmful effectsof engine exhausts have traditionally beenfocused on polycyclic aromatic compounds(8,9). There has been increasing concernabout the effects of alkenes such as ethene,propene, butadiene, and isoprene becausetheir epoxides (metabolites in humans) arecarcinogenic (10). The present risk esti-mates of environmental cancer ascribecancers approximately equally to butadienealone as to polycyclic aromatic matter,including PAHs (11). It is projected thatbutadiene is increasing overwhelminglyover polycyclic compounds; in 2010, it isestimated that butadiene alone will causeapproximately five times more cancer thanpolycyclic material, based on a comparisonof motor vehicle exhausts (11).

The extraordinary carcinogenicity ofbutadiene in rodents influences such pro-jections (8); however, for this compoundhuman occupational data are also becom-ing available (12). New carcinogenicalkenes are still being detected. Isoprene,an analogue of butadiene that possessestwo double bonds capable of cross-linkingDNA has recently been found to be apotent carcinogen in rodents (10). It canbe assumed that other dialkenes will be

found in vehicle exhaust that will alter therisk estimates for particular compounds.Effort should be focused on DNA bindingproducts of alkenes, including butadieneand isoprene. The occupational groupsthat are most heavily exposed include tanktruck drivers, tank ship unloaders, butadi-ene manufacturing workers, and garageworkers. The method of adduct detectioncan rely on the newly developed postlabel-ing technique for monoadducts (7), whichhas been successfully used in studies ofexperimental animals exposed to 1-alkenes(Figure 2) (13).

UV-AdductsFor cross-links, the technique used forcisplatinum and UV cross-links can beapplied (14,15). This modification of thepostlabeling technique (Figure 3) is neces-sary because cross-linked dinucleotideslabel vary poorly (14,15). In the modifi-cation, a normal nucleotide is left on the5'-side of the cross-linked dinucleotide,

8

c,5

6

LiverE~ E* Lymphocytes

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O *-2

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Ethene Propsne Butene Pentene Hexene Heptene Octene

Alkenes

Figure 2. Liver and lymphocyte alkene adduct levels inrats exposed to 300 ppm of alkenes for 12 hr on 3 con-secutive days. Data from Eide et al. (13).

NpNpNp P p P pNpNDNase ISVPPAP

NpP p P +N+p

T4 kinase + ATP

p Np P p P

Figure 3. The scheme of postlabeling for cross-linkedadducts. o, radioactive phospate. Data from Forsti etal. (14) and Bykov et al. (15).

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resulting in a number of labeled trinu-cleotides. This kind of modification is sofar the only way to label cross-linked prod-ucts. The results on excised human skin,irradiated at approximately 310 nm, areshown in Figure 4, analyzed by HPLCradioactivity detection.

UV-Adducts and MutationsThe incidence of malignant melanoma andother skin cancers has increased markedlyin many countries with primarily fair-skinned populations (16). In Sweden theincidence of malignant melanoma and non-melanomatous skin cancer has increasedmore than any type of cancer, representing4.5 and 3.2% annual increases during thelast 20-year time period, respectively.

Solar ultraviolet (UV)-irradiation isthought to be an important cause of thenonmelanomatous skin cancer, but it mayalso contribute to melanoma (16). UV lighthas complex action on biological organismsand is considered a complete carcinogen,with both initiation and promotion capaci-ties in model systems (16,17). UV irradia-tion causes specific dipyrimidine adducts inDNA that are likely to be related to themutagenicity and tumor-initiating potentialof UV light. Mammalian cells can repairthe adducts at various rates (18). The rela-tionship between DNA repair and cancer isillustrated by several skin diseases such asxeroderma pigmentosum, in which repairdefect predisposes to skin cancer. Decreasedrepair of UV damage also contributes to

30,000 -

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common skin tumors such as basal cellcarcinoma (18).

Specific UV-induced photoproductsmay be measured by the novel modificationof the 32P-postlabeling technique (15).Further methods development involvesadaptation of the method to human skin insitu. UV-induced adducts can be deter-mined in parallel with mutation measure-ments in the p53 gene. The assay ofUV-specific CC to TT mutations in thep53 gene in human skin has been published(19). CC to TT mutations in the p53 geneare rare in internal organs, which implicatesUV as the main causative factor. Thecodons conveying transforming propertiesshould be complemented with silent muta-tions that give no growth advantage, thusserving as measures of mutation frequency.

UV-induced photoproducts causedexperimentally and through suntanningcan be studied in an early biologicallyeffective target dose in human skin ofhealthy individuals and patients with skindiseases. The in situ transformation of theadducts, including those on the p53 gene,to p53 mutations could be measured inseveral codons as an early indication ofpotential hazard for skin cancer in individ-uals. This would tie different photoprod-ucts to mutations that appear relevant toskin cancers in healthy and predisposedhumans. Adducts indicate the target doseat a level of a nucleotide in the p53 gene,DNA repair indicates the efficiencyof damage removal, and p53 mutations

20 40Retention time, min

Figure 4. Human epidermis exposed to UVB and analyzed by the method used for cross-links. Somecyclobutane dimers (such as TT=C) and 6-4 photoproducts (such as TT-T) are shown in HPLC radioactivity

indicate fixation of damage as mutationsrelating to cancer risk.

Mutation Studies inTumor Suppressor GenesMany cancer-related genes are excessivelylarge (20). This applies to both tumor sup-pressor genes (Table 1) and to many DNArepair genes. Thus, the screening of theretinoblastoma (Rb) gene with 27 exonsrequires a huge number of polymerasechain reactions (PCR) because only a fewhundred nucleotides can be accuratelyassayed at one time. There are many up-to-date methods used in the detection ofunknown mutations at the gene, mRNA,and protein levels (21-28):* direct sequencing* denaturing/constant gradient gel elec-

trophoresis (DGGE/CDGE), < 600 bp* capillary electrophoresis (CDCE)* ligation-mediated assay* single-strand conformation polymor-

phism (SSCP), -300 bp* chemical deavage, < 2000 bp* application of mismatch repair enzymes* protein truncation assays, large* mRNA level* functional tests.Although protein truncation assays (24)are powerful in detecting deletion andframeshift mutations, they fail to detectmissense mutations.

It is of utmost importance to be ableto screen mutant species in a reliable andfast fashion. The results would be helpfulin the analysis of all mutations, irrespectiveof whether mutations are inherited orsomatic and to the extent that the muta-tions are scattered in different parts of thegenome. This would result in simplifiedanalysis of disease carriers in genetic dis-eases if parental DNA samples are notavailable and of samples from somaticmutations in cancer patients, even in largegenes. The aim is to widen the mainbottleneck in the analysis of mutations.

Capillary electrophoresis has been usedextensively in protein sequencing andto some extent in DNA sequencing. The

Table 1. Sizes of some tumor supressor genes (in basepairs) and of the encoded proteins.

\Jy] Gene Size, bp Exons Amino acids

p16 -2,000 3 14860 p53 20,000 1 1 393

APC >100,000 15 2,843Rb >1 00,000 27 928

>standard BRCA 1 100,000 22 1,863detection. Data from Ponder (20) and Hogervorst et al. (24).

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primary feature of capillary electrophoresisis its high separation power, giving abaseline separation of long nucleotidesequences that differ in size by onenucleotide only. For mutational analysisthe applications are new but essentiallyanalogous to single-strand conformationpolymorphism (SSCP) (25) or denaturantgradient gel electrophoresis (DGGE) (26).In the first application, genomic DNAsequences are amplified, denatured, andanalyzed on capillary electrophoresis assingle-stranded molecules. In the secondapplication, dubbed constant denaturantcapillary electrophoresis (CDCE) (26),melting profiles of the nucleotide sequencesare analyzed; it is important that thesequence to be analyzed contain domainsmelting both at high and low tempera-tures. This ensures separation of heterodu-plexes containing mismatches identical toDGGE but with a constant denaturantconcentration in the capillary. This is dif-ferent from the SSCP type of analysis inthat the samples are analyzed as partiallymelted heteroduplexes and not as singlestrands. In both methods, the DNA is avail-able for sequence analysis; however, experi-ence with analysis of certain sequences withcapillary electrophoresis will lead to someunderstanding of the types and locations ofthe mutations. An extensive sequenceanalysis is required before the ground rulescan be established.We used the 19 commonly found ras

mutants cloned in a plasmid, (Figure 5)(27). We have devised primers that allowus to use SSCP- and DGGE-type capillaryelectrophoresis and sequences of differentlengths in order to compare the separationpower of the two methods. Most of themutations could be detected as homo-duplexes and the rest as heteroduplexes(Figure 5) (28).

Oncoproteins andGrowth FactorsCellular growth signaling can be dividedinto four separate stages: a) extracellulargrowth factors (e.g., platelet-derivedgrowth factor [PDGF], epidermal growthfactor [EGF], and transforming growthfactor-a [TGFa]); b) growth factor recep-tors at cell membranes (e.g., PDGF recep-tor and a common receptor for EGF andTGFla); c) intracellular signaling proteins,G-proteins that interact between the mem-brane receptors and nuclear processes.(these forms involve many multistep path-ways and include proteins such as ras [p21]and raf); d) nuclear factors of many

functions such as transcription factors, cell-cycle control proteins, and DNA repairproteins, which have many interactions.p53 protein appears to take part in each ofthese functions.

The a-tocopherol, ,-carotene (ATBC)cancer-prevention-trial serum bank includ-ing 30,000 middle-aged smoking men canbe used to identify the possible associationbetween the level of growth factors andoncoproteins (jointly called oncoproteins)in respect to lung and colorectal cancer.The cancer types were selected because oftheir high incidence and known increase inmutations or elevation of oncoproteins(29-31). The oncoproteins selectedincluded ras p21 protein, p53 protein, and,for squamous lung cancer only, epidermalgrowth factor receptor (EGFR). The spe-cial advantage of this study, as compared toothers carried out in this field, is the large,well-characterized study population, whichenables the assessment of the oncoproteinsyears before clinical diagnosis. Interviewdata and serological analysis enable thecontrol for confounding variables.

The objectives of this work are 4-fold.One objective is to analyze more thor-oughly the role of oncoproteins in earlystages of cancer. Evaluation of the lag timebetween detection of oncoproteins in serumand clinical diagnosis of cancer can beassessed in a large number of cancers devel-oped in this population. A second objectiveis to analyze the prognostic value becausemany oncoproteins are already being usedfor prognostic purposes in the treatment ofcancer (32,33). As a third objective, theappearance and possible fluctuation of thelevels of oncoproteins provide informationabout irreversibility, which is mechanisti-cally important. And fourth, the variablesaffecting the normal levels of oncoproteinswill also become available.

For predictive and preventive purposes,it is important to develop markers eitherfor general population screening or screen-ing of some risk groups. The criteria ofpredictivity are well established in generalpopulation screening; this generally impliesthat both false positive (low specificity) andnegative (low sensitivity) results undermine

A

Primer

GGT AGT

B

GGT AGT AC/GTHeteroduplexes

2 4 6 8 10 12 14 16

Minutes

Figure 5. Analysis of ras exon (codon 12) as wild type (GGT) and mutant (AGT) homoduplexes (A) and heterodu-plexes (B) by constant denaturant capillary electrophoresis at 72°C. Data from Kumar et al. (28).

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the marker. Based on the multiple pathwaysto oncogenesis, it would be overly opti-mistic to assume that one or a few onco-protein markers would fulfill the criteriafor population screening. However, onco-protein screening may serve a purpose inthe case of special groups such as thoseseeking medical advice about their symp-toms, families with a high incidence ofcancer, or tobacco smokers or other heavilyexposed populations. In such cases, the cri-teria of population screening do not hold,and the tolerance in predictivity can belower (34). The direction of false diagno-sis largely depends on the individual situa-tion. Yet, it is important that diagnosticefficiency is maintained, for example, thecost-benefit is reasonable.

Genetic EpidemiologyStudies have been initiated with theSwedish Cancer Registry (-1.2 millionpatients) and the Twin Registry (-50,000twin pairs) to analyze familial cancer in theCancer Registry, which covers all ofSweden since the 1950s (Figure 6) and tofind out to what extent mono- and het-erozygotic twins are presented in theRegistry. Because the Swedish Cancer

Generation registry Cancer registry3 to 4 Generations 1.2 Million cases

Cancer in families

Familial cancerSamples from relatives

Figure 6. A scheme for using a population registercovering 3 to 4 generations linked to a cancer registerto detect families with unusual frequencies of cancer.

Registry is one of the largest in the worldand the Twin Registry is the largest, thesedata sources will provide unique patientmaterial for analysis of common cancersusing sib pair analysis. However, as the col-lection of material involves several genera-tions, including a large number of deadpersons, logistics have to be worked out forthe identification of important genes incommon cancers.

ConclusionsThis work uses a number of parametersthat are currently available to predict thehealth outcome of the exposures ofconcern (Figure 7). As emphasized by the

Biomarker data (relevant lines)ExposureEffect

Susceptibility

Relevanlt exposures

~~~~~~~~~~~~~ \\

1960 1970 1980 1990

Figure 7. Use of biomarkers to estimate effects ofrecent exposures as contrasted to epidemiological datain which relevant exposures usually occurred decadesearlier. Three kinds of exposure patterns are shown.The relevant periods for biomarkers are indicatedon top.

proponents of molecular epidemiology, thepresent-day findings can be translated torisk estimates in the absence of epidemio-logical data (35). The relevant exposuredata from epidemiological studies datedecades back and are usually uncertain (seeFigure 7); this impedes direct extrapolationto the risks of the current exposure.Furthermore, individual metabolic factorscan be taken into consideration.

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