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Rationale _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _• Early detection of cancer provides physi-cians with optimal treatment strategies;screening practices for breast and prostatecancers have contributed to significantlyimproved survival. Early-stage lung canceris generally asymptomatic, and the tumorsare often radiologically invisible until theyare quite advanced. That lung cancer is theleading cause of cancer death in the worldcan be attributed in part to the advancedstage of the disease at diagnosis. When lungcancer is diagnosed at stage I, the survival issignificantly better.1 It is expected that di-agnosis of preneoplastic lesions before an-giogenesis, invasion, and micrometastasiswill not only contribute to improved sur-vival for patients with lung cancer but ulti-mately have an effect on overall mortalityfrom this disease.

• Histologic diagnoses of early-stage lungcancers are complicated by a lack of agree-ment among histolopathologists on whatconstitutes a preneoplastic lesion in thelung. Additionally, genetic mutations canbe detected in the histologically normalbronchial epithelium of patients with a his-tory of smoking but without cancer,2 sug-gesting that a purely histologic examina-tion would miss important indicators ofpotentially malignant tissue.

• As a result of the large number of individ-uals with a history of smoking, a practical,inexpensive, and accurate screening pro-gram is needed. Genomic and proteomicscreening techniques can be applied to tissuesamples obtained through nonsurgicalmeans such as bronchoalveolar lavage andbronchial brushings, or from the exfoliatedcells present in sputum. Because samples ob-

tained by these techniques are more likely tohave originated from the central airway thanfrom peripheral bronchioles and alveoli,fine-needle aspiration biopsies of pulmonarynodules could also be considered.

• Multiple abnormal molecular processeshave been identified in lung cancer, andthus development of any single test for thedisease is problematic. The key molecularevents in tumor ontogeny must be definedso clinicians can distinguish potentiallymalignant preneoplastic lesions from thosethat will remain benign. It is hoped thatone or more selective molecular identifierswill be found to accurately distinguish be-tween malignant and nonmalignant le-sions. However, given the heterogeneity ofcancers and the multiple mechanisms ofmolecular transformation, development ofan accurate screen will probably requiretesting panels of genes and/or performingmultiple tests.

Natural History of Lung CancerCurrently, researchers are studying the

natural history of lung cancers in thecontext of the genetic and epigeneticchanges that accompany the transformationfrom preneoplastic lesion to carcinoma insitu (CIS). No single preneoplastic lesionhas been clearly identified for small-celllung cancer (SCLC), but preneoplasticlesions of the bronchial epithelium havebeen classified by the World Health

Organization and are listed in Table 1.3 Theclassification assumes that the lesionsprogress in an orderly manner fromhyperplasia to metaplasia and then toincreasing degrees of dysplasia. Notablechanges in DNA ploidy, methylation, andchromosomal integrity as well as changes ingene expression have been noted in lungcancer cells and form the basis formolecular testing.

Gene Expression Changes in Lung Tumors

Changes in gene expression associatedwith malignant transformation in lungtissue can be identified in squamousmetaplasia, dysplasia, and CIS, and inatypical adenomatous hyperplasia (AAH).The transformation from dysplasia tometaplasia and then to hyperplasia is sup-ported by the appearance of proliferationmarkers such as Ki-67 that appear to in-crease with increasing atypia in squamouscell dysplasia and AAH.4 Other expres-sion markers that seem to progressivelychange as transformation proceeds in-clude p53,5 epidermal growth factor re-ceptor (EGFR), k-ras,6 and telomerase.7

p53 mutations are common in lungcancer and can also be identified in squa-mous metaplasia developing into dysplasiaand in AAH.8 Studies have found nostaining in normal epithelium, and stain-ing is minimal in metaplasia or adenoma-tous hyperplasia that is not atypical. The

Prepared by: Nancy Price, PhDReview by: Vinay K. Jain, MD,Chandra P. Belani, MD

Emerging Molecular Biomarkers for Early Detection of Lung Cancer in Patients at High Risk

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Table 1: Classification of Morphologically Recognizable Preneoplastic Lesions of the Bronchial Epithelium in NSCLC3

WHO Preneoplastic ClassificationsCancer Histology

Squamous dysplasia/CIS

AAH

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia

Squamous Cell

Adenoma

Carcinoid

Abbreviation: WHO = World Health Organization

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expression of p53 may be an indicator fora preinvasive to invasive lesion. p53 inter-acting proteins such as the p53-induciblecyclin-dependent kinase inhibitor p219

and MDM2,10 which inhibits p53-medi-ated transcriptional activity, appear to bedysregulated from p53 expression in somelesions. K-ras mutations are found in ap-proximately 30% of non–small-cell lungcancer (NSCLC)s and appear to be morecommon in adenocarcinoma than in squa-mous cell carcinoma, and correspondinglyappear to be more common in AAH thanin squamous dysplasia.11

Telomerase activity is observed in cancercells and is associated with cellular immor-talization. Yashima and colleagues looked attelomerase enzyme activity in normal, pre-neoplastic, and cancerous lung tissue andfound that dysregulation of telomerase oc-curs early in lung pathogenesis.7 Metapla-sia, dysplasia, and CIS are all associatedwith telomerase activity, and the expressionof the RNA subunit of telomerase(hTERC) correlates with telomerase activi-

ty. Additionally, telomerase activity wasoften present in CIS foci near invasive can-cers, suggesting that the presence ofhTERC expression in CIS may indicate im-minent invasion. The presence of hTERCalso correlated with telomerase activity, thetelomerase-associated protein, and thetelomerase catalytic subunit in 100%, 93%,and 89%, respectively, of 92 lung cancer tis-sue samples evaluated by Arinaga and col-leagues.12 In normal lung tissue adjacent toneoplasias, the telomerase-associated pro-tein and telomerase catalytic subunit wereexpressed in 94% and 100% of cases, re-spectively, but the RNA subunit was detect-ed in only 1 of 32 samples.

Serum MarkersProteins that are overexpressed in tu-

mors might enter the circulation and ap-pear in serum to act as surrogate markers oflung cancer, like prostate-specific antigenin prostate cancer. Common upregulatedproteins in lung and other cancers are sum-marized in Table 2. Some studies have in-

dicated that P53 and EGFR are elevated inserum samples from patients with lungcancer, as are autoantibodies against mu-tant p53. Schneider and colleagues com-pared P53 and EGFR protein levels andP53 autoantibodies from patients with dif-ferent histologic types of lung cancer withthose in healthy controls.13 No increase inP53 protein or antibodies or EGFR pro-tein was observed in patients with lungcancer compared with controls.

Pleiotropin is a heparin-binding growthfactor important in embryonic develop-ment of nervous tissue. It also exhibits mi-togenic and angiogenic properties in breastand melanoma carcinoma cells. Serumpleiotropin was determined in 63 patientswith SCLC and 22 patients with NSCLCand compared with 41 healthy controls ina study conducted by Jäger and colleagues.14

Pleiotropin levels were higher in the lungcancer population (81%; P < 0.001) andpresent in 87% of SCLCs and 63% ofNSCLCs. The mean serum levels ofpleiotropin were 10.8 times greater in thetumor group (P < 0.001). Although notspecific for lung cancer, serum pleiotropinlevels appear to be a good serum indicatorof the presence of a tumor. In combinationwith other methods, pleiotropin levelscould be a potential serum indicator forlung cancer screening.

Glutathione S-transferase (GST) is anenzyme responsible for the reduction ofglutathione (GSH), an important cellularreducing agent. Glutathione S-transferasewas found to be significantly greater in thelavage fluid taken from an area of the lungassociated with tumor compared with thattaken from a normal area of lung tissue.15

Ferruzi and colleagues assessed theGST/GSH system in NSCLC in the clin-ical setting.15 Matched blood and tissuesamples (normal and malignant) from 52patients with NSCLC or head and necksquamous cell carcinoma were assessed.The GSH levels and GST activity weregreater in NSCLC (P = 0.0004 for GSHand P = 0.0002 for GST). Importantly,the GSH level in whole blood and GSTactivity in cancer tissue was highly corre-lated with GST expression in NSCLC(P = 0.003), and GSH levels in NSCLC

Table 2: Potential Serum Markers for Lung Cancer

ProteinFunction

SurrogateMarker

P53

EGFR

P53Autoantibodies

Pleiotropin

Glutathione

GlutathioneS-Transferase

Abbreviation: NA = not applicable

Tumor suppressorinvolved in gene

regulationand apoptosis

Growth factorreceptor important

in mitogenesis

NA

Heparin-binding growth factor in the growth

and differentiation ofneuronal tissue

Intra- and extracellularreducing agent

NA

CancerHistology

NSCLC, SCLC

NSCLC, SCLC

NSCLC, SCLC

NSCLC, SCLC

NSCLC

NSCLC

Clinical Indications andRelevance as a Serum Biomarker

Initially reported as upregulated in lung cancer patients. P53

levels in the serum in lung cancer cases did not differ significantly

from healthy controls.

Initially reported as upregulated in lung cancer; EGFR mutations

have been reported in some NSCLCs.

Some studies have correlated P53 antibodies with lung cancer occurrence and others have not.

Pleiotropin was elevated in 81% of the lung cancer population, was significantly different from

healthy controls (P < 0.001), and correlated with disease stage.

High serum levels positively correlated with several cancers,

including NSCLC.

High serum levels positively correlated with several cancers,

including NSCLC.

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were significantly correlated with whole-blood GSH levels (P = 0.006), suggestingthat circulating GSH may behave as aclinically relevant surrogate marker.

Chromosomal Aberrations in Lung Tissue

DNA deletions and translocations arecommon in lung cancer cells. Commonearly changes in squamous cell carcinomaand adenocarcinoma involve allele-specif-ic loss at 3p, 9p, and possibly 17q. Loss of3p almost always occurs in CIS and inva-sive squamous cell carcinoma. However,chromosomal loss at these locations canalso be seen in histologically normal ep-ithelia, suggesting that allele-specific lossof heterozygosity plays an important rolein the development of invasiveness.

DNA MethylationDNA methylation is an epigenetic al-

teration that occurs at CpG sequences.CpG sequence islands are often present inthe 5´ untranslated regions of genes, sug-gesting that methylation affects transcrip-tional activation.15 DNA methylationpatterns in tumor cells are significantly al-tered compared with those of normal

cells.16,17 Genome-wide hypomethylationis often observed concurrently with localregions of hypermethylation. Deletionsand translocations often occur in hy-pomethylated areas and may reflect thepermissive activation of parasitic elementssuch as retroviruses or retrotransposonsthat are otherwise restricted by DNAmethylation.16,17 Hypermethylated genestend to be silenced,18 and several observedhypermethylated genes are involved in cellcycle regulation, adhesion, apoptosis, andsignal transduction (Table 3).

As a screening mechanism, polymerasechain reaction amplification of methylatedgenomic DNA could be highly promisingif a cancer-specific DNA methylation sig-nature can be identified. Such a signaturecould be used for diagnosis, to monitor re-sponse to therapy, or as a prognostic tool.The methylation status of > 40 genes inlung cancer has been assessed (reviewed inTsou et al19). Methylation patterns differbetween carcinomas originating in differ-ent organs and between different cancerhistologies from the same organ. Becauseof the heterogeneity of methylation pat-terns, panels of genes will have to be as-sessed to assure accuracy.

Clinical RelevanceMolecular surrogate markers to screen

for lung cancer potentially represent apractical and possibly inexpensive meansto identify lung cancer in its early stages.Several assayable tumor-specific geneticmodifications are known and could betested through serum markers, tumor-specific gene expression, DNA methyla-tion, and chromosomal aberrations. Thedevelopment of molecular screening de-pends on a more comprehensive under-standing of the natural history of lung tu-mors in the context of genetic change.Genetic mutations can be found in can-cerous lung tissue and in the histological-ly normal bronchial epithelium of formersmokers who do not have cancer. Specificidentification of molecular markers thatindicate premalignant transformation isessential for early detection of lung cancerand the possible application of these tech-niques as screening tools.

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Table 3: Hypermethylated Genes in Silenced Lung Cancer

FunctionGene

Adenomatouspolyposis coli

H-cadherin

Cyclin-dependentkinase inhibitor 2A(p16/INK4A)

Fragile histidine triad

O6-methylguanine-DNAmethyltransferase

Retinoic acidreceptor–�

RAS effectorhomologue

*Data from cell line rather than tumor tissue.

Protein associated with Wnt signaling. Function

unknown but may be an adaptor or scaffold protein

Adhesion

Cell cycle control

Hydrolase involved in purine metabolism

DNA repair

Nuclear receptor

Signaling modulator

Methylatedin NSCLC

46%-96%

21%-75%

37%-64%

17%-81%

40%-43%

30%-63%

Methylatedin SCLC

26%-58%*

0-17%

64%*

23%-91%

62%-76%

72%-100%

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