American Journal of Medical Genetics (Semin. Med. Genet.) 115:183–188 (2002)

A R T I C L E

Cytogenetics and Molecular Genetics ofLung CancerYASUHIRO MITSUUCHI AND JOSEPH R. TESTA*

The development and progression of lung cancer is a multistep process characterized by the accumulation ofnumerous genetic and epigenetic alterations, some of which occur early in the course of disease. In this review, wesummarize cytogenetic imbalances and molecular genetic/epigenetic changes seen in human small-cell and non-small-cell lung cancer. Alterations of tumor suppressor genes and oncogenes leading to perturbations of key cell-regulatoryand growth-control pathways are highlighted. The translational implications of molecular biomarkers for riskassessment, early detection, and monitoring of chemoprevention trials are discussed. � 2002 Wiley-Liss, Inc.

KEYWORDS: lung cancer; cytogenetic imbalances; genetic/epigenetic changes; tumor suppressor genes, oncogenes; molecular biomarkers

INTRODUCTION

Lung cancer is a leading cause of cancer

death among men and women in in-

dustrialized countries, currently acco-

unting for approximately 160,000 deaths

annually in the United States. Cigarette

smoking constitutes approximately 85%

of attributable risk, with asbestos, radon,

other environmental exposures, and

genetic factors contributing to the re-

mainder of cases [Ginsberg et al., 1993].

Lung cancer is divided into two main

histologic groups—80% are non-small-

cell lung carcinomas (NSCLCs) and 20%

are small-cell lung carcinomas (SCLCs).

SCLCs express certain features of neuro-

endocrine cells, whereas most NSCLCs

lack these properties (Table I).

Both NSCLCs and SCLCs typically

display numerous numerical and struc-

tural chromosome alterations [Testa

et al., 1997]. Lung cancer cells accumu-

late many molecular genetic and epige-

netic changes, which appear necessary

for conversion of normal bronchial

epithelium to malignant lung cancer

[Sekido et al., 2001]. The molecular

changes often involve tumor suppressor

genes and oncogenes, and these altera-

tions lead to perturbations of key cell-

regulatory and growth-control path-

ways. Many of these changes are present

in both major histologic groups of lung

cancer, though abnormalities of certain

tumor suppressor genes tend to be more

common in SCLC, whereas dominant

oncogene expression is more frequent

in NSCLC. In this review, we summa-

rize cytogenetic and molecular genetic

characteristics of lung cancer and the

implications of these alterations in the

pathogenesis of this disease.

Yasuhiro Mitsuuchi, Ph.D., is a ResearchAssociate at Fox Chase Cancer Center inPhiladelphia, Pennsylvania.

Joseph R. Testa, Ph.D., is a Senior Memberat Fox Chase Cancer Center in Philadelphia,Pennsylvania. He is director of the Center’sHuman Genetics Program and holds theCarol and Ken Weg Chair in HumanGenetics.

Grant sponsor: NCI; Grant number: CA-58184 and CA-06927; Grant sponsor: AnnRicci Memorial Fund; Grant sponsor: Com-monwealth of Pennsylvania.

*Correspondence to: Dr. Joseph R. Testa,Fox Chase Cancer Center, 7701 BurholmeAvenue, Philadelphia, PA 19111.E-mail: JR_Testa@fccc.edu

DOI 10.1002/ajmg.10692

TABLE I. Frequent Molecular Genetic Changes in Lung Cancer*

SCLC NSCLC

Frequent allelic loss 3p, 4p, 4q, 5q, 8p, 10q,

13q, 17p, 22q

3p, 6q, 8p, 9p, 13q,

17p,19q

RAS mutations <1% 15–20%

BCL2 expression 75–95% 10–35%

MYC family overexpression 15–30% 5–10%

RB1 inactivation �90% 15–30%

p53 inactivation 80–90% �50%

p16INK4a inactivation 0–10% 30–70%

RARb 70% 40%

FHIT inactivation �75% 50–75%

*Adapted from Sekido et al. [2001].

Lung cancer cells accumulate

many molecular genetic and

epigenetic changes, which

appear necessary for conversion

of normal bronchial epithelium

to malignant lung cancer

� 2002 Wiley-Liss, Inc.

CYTOGENETIC ANALYSIS

Cytogenetic changes seen in lung cancer

have been reviewed in detail elsewhere

[Testa et al., 1997] and are summarized

briefly here. Karyotypic analyses pro-

vided the first evidence for recurrent

deletions of 3p in SCLCs [Whang-Peng

et al., 1982]. Loss of heterozygosity ana-

lysis of 3p markers confirmed an almost

universal loss of genetic material from

this region, suggesting that one or more

tumor suppressor gene important in the

pathogenesis of SCLC resides in this

chromosomal region. Other recurrent

karyotypic changes reported in SCLC

include losses of segments in chromo-

some arms 5q, 13q, and 17p. Compara-

tive genomic hybridization analysis of

primary SCLC specimens has found

frequent losses of 3p, 4p, 4q, 5q, 8p,

10q, 13q, and 17p; gains of 3q, 5p, 8q,

19q, and Xq are also common [Ried

et al., 1994; Levin et al., 1995].

Double minutes and homogene-

ously staining regions, two cytogenetic

manifestations of gene amplification,

have been reported in many SCLCs,

particularly in derived cell lines. These

alterations are associated with amplifi-

cation of various members of the MYC

oncogene family [Testa et al., 1997;

Sekido et al., 2001]. Karyotypes of

NSCLCs tend to be more complex than

those of SCLCs. Chromosome arms fre-

quently contributing to losses inNSCLC

include 3p, 6q, 8p, 9p, 9q, 13q, 17p, 18q,

19p, 21q, and 22q. Gains of 1p, 1q, 3q,

5p, 7p, 7q, 11q, and 12q are common.

Our comparative genomic hybridiza-

tion analyses of NSCLCs found frequent

gains of 1q, 3q, 5p, and 8q, whereas the

most frequently underrepresented arms

were 3p, 8p, 9q, and 17p [Pei et al.,

2001]. Many imbalances, such as gains

of 1q, 5p, and 8q, occurred at a high

frequency in both major histologic sub-

groups of NSCLC, that is, adenocarci-

noma and squamous cell carcinoma.

Several differences were noted, how-

ever, the most prominent being gain of

3q24-qter, which was seen in �80% of

squamous cell carcinomas but in only

30% of adenocarcinomas. Other com-

parative genomic hybridization analyses

of NSCLCs have shown that over-

representation of 1q23 and loss of 9q22

are associated with adenoid differentia-

tion, whereas loss of 2q36-37 and gains

of 3q21-22 and 3q24-qter are significant

markers for the squamous type [Petersen

et al., 1997]. In our investigation, gains

of 7q and 8q were associated with

higher-stage tumors and with either

positive nodal status or higher tumor

grade, suggesting that these changes are

indicative of tumor aggressiveness [Pei

et al., 2001].

MOLECULAR GENETICANALYSIS

Chromosome Arm 3p Deletions

Loss of heterozygosity at chromosome

arm 3p is thought to be an early genetic

change in the development of both

SCLCs (>90%) and NSCLCs (>50%)

[Sekido et al., 2001]. Several distinct

regions of 3p loss have been identifi-

ed by high-density allelotyping, that is,

3p25-26, 3p24, 3p21.3, 3p14.2, and

3p12, suggesting that several different

tumor suppressor genes reside in 3p. The

3p21.3 region has been examined ex-

tensively for putative tumor suppressor

genes, because homozygous deletions

have been found in several lung cancer

cell lines. Todd et al. [1997] identified

homozygous deletions in three squ-

amous cell lung tumors within a region

of 3p21 that had been described pre-

viously only in cell lines. They also

uncovered a homozygous deletion at

3p12 in an SCLC tumor specimen and

a 3p14.2 homozygous deletion in an

NSCLC cell line. Altogether, there is

evidence for at least four separate regions

of 3p, which undergo homozygous dele-

tions in either uncultured lung tumors or

cell lines [Todd et al., 1997].

A number of candidate genes in

deleted regions of 3p have been identi-

fied, including the b-retinoic acid re-

ceptor gene [Kok et al., 1997], the

protein-tyrosine phosphatase-g gene

[LaForgia et al., 1991], the semaphorin

IV and A(V) genes [Roche et al., 1996;

Sekido et al., 1996], the von Hippel–

Lindau tumor suppressor gene (VHL)

[Latif et al., 1993], and the fragile histi-

dine triad gene (FHIT) [Sozzi et al.,

1996]. As noted earlier, there is compel-

ling evidence for a lung cancer gene at

3p21.3. Although several putative tumor

suppressor genes located at 3p21 have

been identified, none of these genes

appeared to be consistently mutated in

lung cancer. Recent work, however, has

revealed frequent epigenetic inactiva-

tion of a RAS association domain family

protein from the lung tumor suppressor

locus 3p21.3 [Dammann et al., 2000].

TheRASeffector homologue,RASSF1,

is located in a 120-kb region of minimal

homozygous deletion in 3p21.3, and

three transcripts have been identified,

one of which (transcript A) was mis-

sing in all SCLC cell lines analyzed

[Dammann et al., 2000]. Loss of expres-

sion was correlated with methylation of

the CpG-island promoter sequence of

RASSF1A. The promoter was highly

methylated in 40% of primary lung

tumors, �10% of which had missense

mutations. Reexpression of RASSF1A

in lung cancer cells inhibited in vitro

growth and tumor formation in nude

mice, supporting the potential role of

RASSF1A as a lung tumor suppres-

sor gene. Subsequent work has found

RASSF1A promoter hypermethylation

in 100% of SCLC cell lines, in 63% of

NSCLC cell lines, and in 30% of primary

NSCLC tumors [Burbee et al., 2001].

TheFHIT gene is located at 3p14.2,

one of the sites where rare homozygous

deletions have been reported in lung

cancer cell lines. FHIT spans the com-

mon fragile site FRA3B and encodes

a diadenosine triphosphate hydrolase

[Ohta et al., 1996]. Sozzi et al. [1996]

detected abnormal FHIT messenger

RNA transcripts in 80% of SCLCs and

40% of NSCLCs, and most of the tumors

Loss of heterozygosity at

chromosome arm 3p is thought

to be an early genetic change

in the development of both

SCLCs (>90%) and

NSCLCs (>50%)

184 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE

exhibited loss of FHIT alleles. Loss of

heterozygosity of the FHIT gene in lung

cancers has been observed in a much

higher percentage of smokers than

nonsmokers, suggesting that FHIT is a

molecular target of tobacco smoke

carcinogens [Sozzi et al., 1997]. Al-

though most lung cancers express aber-

rantFHIT transcripts, they almost always

also express wild-type FHIT transcripts

[Sozzi et al., 1996; Fong et al., 1997].

Moreover, unlike classic tumor suppres-

sor gene inactivation, point mutations in

the FHIT gene are rare [Sozzi et al.,

1996; Fong et al., 1997]. Despite these

apparent paradoxes, FHIT protein is

absent in many lung carcinomas and in

some preneoplastic lesions [Sekido et al.,

2001]. Functionally, FHIT appears to

play a role in the regulation of apoptosis

and in cell-cycle control [Sard et al.,

1999]. Transfection of wild-type FHIT

in lung cancer cells induces apoptosis

and inhibits tumorigenicity in nude

mice, supporting the contention that

FHIT functions as a tumor suppressor

gene [Ji et al., 1999].

The FHIT gene is located at

3p14.2, one of the sites

where rare homozygous

deletions have been reported

in lung cancer cell lines.

RAS Activation

RAS mutations are a common occur-

rence in lung cancer. The RAS family of

proto-oncogenes (HRAS, KRAS, and

NRAS) encodes 21-kd proteins loca-

lized to the inner plasma membrane.

RAS signaling amplifies stimuli from

divergent receptors to the nucleus to

regulate the cell cycle. RAS signals are

transmitted via a cascade of kinases,

resulting in the activation of mitogen-

activated protein kinases (MAPK),

ERK1 and ERK2, which translocate to

the nucleus and activate transcription

factors. KRAS mutations are found

in 15–20% of NSCLCs but are rare

in SCLCs [Richardson and Johnson,

1993]. RAS mutations usually occur by

point mutations at codons 12, 13, or 61

and have been reported to be late events

in the development of lung cancer,

particularly in squamous cell lung carci-

nomas [Sugio et al., 1994; Zhang et al.,

1996]. Li et al. [1994] reported, how-

ever, that RAS mutations may occur

very early in lung adenocarcinomas.

KRAS accounts for approximately 90%

of all RAS mutations in lung adenco-

carcinomas, with about 85% of the

mutations affecting codon 12 [Sekido

et al., 2001].

MYC Overexpression

The MYC family of genes (MYC,

MYCN, and MYCL) encode nuclear

phosphoproteins belonging to the

basic helix-loop-helix leucine-zipper

(bHLHZ) class of transcription factors.

The MYC proteins regulate normal

cell growth through direct activation of

genes involved in DNA synthesis, RNA

metabolism, and cell-cycle progression

[Grandori et al., 2000]. The ability of

overexpressed MYC proteins to pro-

mote proliferation and inhibit terminal

differentiation befits the fact that tumors

of various types exhibit genetic altera-

tions of MYC family genes. The usual

mechanism of MYC activation in lung

cancer is gene amplification with result-

ing overexpression (reviewed in [Sekido

et al., 2001]). MYC amplification occurs

in 15–30% of SCLCs and 5–10% of

NSCLCs [Richardson and Johnson,

1993; Sekido et al., 2001]. Amplification

of MYC genes has been shown to affect

survival adversely in SCLC patients

[Brennan et al., 1991].

BCL2 Overexpression

The BCL2 proto-oncogene, located at

chromosome 18q21, is overexpressed in

many lung tumors. The product of the

BLC2 gene is a key anti-apoptotic

protein, whose expression is regulated

negatively by p53. Upregulated BCL2

expression has been noted in 75–95%

of SCLCs, compared with 10–35%

of NSCLCs [Sekido et al., 2001]. In

NSCLC, an inverse relationship has

been found between overexpression of

BCL2 and mutant p53, suggesting that

either alteration may have a fundamental

role in the pathogenesis of this disease

[Fontanini et al., 1995]. Because of the

important role of BCL2 in suppres-

sing apoptosis and, thus, in potentially

hindering a patient’s response to con-

ventional therapies, there has been con-

siderable interest in developing antisense

BCL2 therapeutics, which are entering

clinical trials at this time [Sekido et al.,

2001].

The BCL2 proto-oncogene,

located at chromosome 18q21,

is overexpressed in many lung

tumors. The product of the

BLC2 gene is a key

anti-apoptotic protein, whose

expression is regulated

negatively by p53.

p53 Inactivation

The p53 gene (TP53) is located at

17p13.1, a common site of chromo-

somal loss in lung cancer. p53 plays

a critical role in regulating cell-cycle

progression and in maintaining geno-

mic integrity when cells sustain DNA

damage. The p53 protein functions as a

nuclear transcription factor that regu-

lates expression of various genes encod-

ing proteins involved in cell-cycle

checkpoints (e.g., p21WAF1/CIP1), apop-

tosis, and DNA repair. TP53 is mutated

in 80–90% of NSCLCs and in �50%

of SCLCs [Sekido et al., 2001]. Most

missense mutations in the TP53 gene

occur in the DNA-binding domain,

abolishing its transactivation function.

Because mutant p53 cannot activate

p21WAF1/CIP1, the cell cycle can proceed

unabated.

Most TP53 mutations occur in

exons 5–8. Codon 157 is a mutational

hot spot in lung cancer, whereas codon

248 and 273 mutations are hot spots in

other cancers, for example, colon and

liver cancer, suggesting that different

ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) 185

mutagens cause different TP53 mu-

tations. These codons contain CpG

islands, and the presence of 5-methyl

cytosine greatly enhances binding of a

major cigarette smoke carcinogen, ben-

zo[a]-pyrene diol epoxide, to guanine,

such that benzo[a]-pyrene diol epoxide

selectively forms adducts at TP53 hot

spots [Denissenko et al., 1997]. In lung

tumors, most of the mutations are G-to-

T transversions expected from tobacco

smoke carcinogens [Hussain and Harris,

1998]. TP53 mutations also have been

found in premalignant lung lesions,

indicative of an early inactivation of

p53 function in lung carcinogenesis

[Vahakangas et al., 1992].

Retinoblastoma Gene

(RB1) Inactivation

The RB1 gene, located at 13q14.11, is

frequently inactivated in lung cancer,

particularly SCLC. Absent or mutant

RB1 protein has been reported in more

than 90% of SCLCs, compared with 15–

30% of NSCLCs [Sekido et al., 2001].

Ectopic expression of recombinant RB1

protein has been shown to reverse the

tumorigenicity of RB1-deficient SCLC

cells [Ookawa et al., 1993]. The RB1

gene encodes a nuclear phosphoprotein

that regulates entry into the cell cycle

from the quiescent state through its

interaction with G1 cyclins and cyclin-

dependent kinases (CDKs). When RB1

is dephosphorylated, it binds the E2F

transcription factor, resulting in suppres-

sion of G1-to-S-phase cell-cycle transi-

tion. Phosphorylation of RB1 by the

cyclin E/CDK2 complex results in the

release and activation of E2F, thereby

promoting progression to S phase.

p16INK4a Inactivation

The CDKN2A (INK4a)/ARF locus,

encoding the tumor suppressor gene

products p16INK4a and p14ARF, is located

at chromosome region 9p21 and frequ-

ently is altered in many types of cancer.

The protein encoded by p16INK4a is

capable of binding to CDK4 and thereby

inhibits the catalytic activity of the

CDK4/cyclin D enzymes. Therefore,

loss or inactivation of p16INK4a would

lead to loss of cell-cycle regulation. In

contrast to the frequent loss of the

RB1 gene in SCLC, recent studies have

shown that the p16INK4a gene is inacti-

vated in up to 70% of NSCLC tumor

specimens and cell lines but rarely in

SCLCs. RB1-positive lung cancer cell

lines have been shown to have reduced

or absent expression of p16INK4a, which

normally functions to prevent RB1

phosphorylation. Therefore, aberrant

expression of either one of these tumor

suppressor proteins can disrupt down-

stream signals and lead to deregulation of

the cell cycle. This reciprocal relation-

ship, involving either RB1 or p16INK4a

inactivation in a given tumor, suggests

that dysfunction within the RB pathway

could be a major target in the genesis of

lung cancer [Shapiro et al., 1995].

The critical alteration of p16INK4a

results from homozygous deletion, mu-

tation, or hypermethylation of CpG

islands in the promoter region of the

gene [Sanchez-Cespedes et al., 1999].

Interestingly, homozygous deletion or

mutation of p16INK4a is seen only in

tumors from smokers, whereas the

p16INK4a gene is inactivated in tumors

from nonsmokers only through pro-

moter hypermethylation [Sanchez-

Cespedes et al., 2001]. Belinsky and

colleagues have shown that aberrant

methylation of p16INK4a is an early event

in lung cancer and a potential biomarker

for early diagnosis [Belinsky et al., 1998].

In lung squamous cell carcinomas, the

frequency of p16INK4a silencing increas-

ed during disease progression. Subse-

quent studies showed that aberrant

methylation of the p16INK4a or O6-

methyl-guanine-DNA methyltransfer-

ase promoters or both can be detected

in DNA from sputum in 100% of

patients with squamous cell lung carci-

noma up to 3 years before clinical diag-

nosis [Palmisano et al., 2000]. Thus,

these studies indicate that aberrant

methylation of p16INK4a may represent

a valuable biomarker for early detection

and monitoring in chemoprevention

trials.

CONCLUSION

Lung carcinomas arise via a multistep

process involving many genetic and epi-

genetic changes, including perturbations

of key cell-cycle genes. These alterations

accumulate in the bronchial epithelium,

eventually leading to clonal cell expan-

sion. In lung cancer patients, there is

evidence that numerous small clonal

patches exist not only in malignant

lesions but also in histologically nor-

mal-appearing areas adjacent to tumors

[Hittelman et al., 1996]. Several groups

have utilized microsatellite analysis to

investigate the presence of clonal genetic

alterations in samples of histologically

normal and abnormal epithelium from

lung cancer patients or in samples ob-

tained from smoking and nonsmoking

patients [Mao et al., 1997; Wistuba et al.,

1999]. Such studies have shown that pre-

neoplastic genetic changes are extensive

and that these alterations begin to accu-

mulate early in the pathogenesis of lung

cancer. The detection of genetic and

epigenetic (e.g., p16INK4a) changes in

histologically normal-appearing epithe-

lium may provide markers of increased

cancer susceptibility in at-risk popula-

tions. Furthermore, the identification of

such alterations also may be efficacious

in guiding chemopreventive measures,

The critical alteration

of p16INK4a results from

homozygous deletion,

mutation, or hypermethylation

of CpG islands in the

promoter region of the gene.

Interestingly,

homozygous deletion or

mutation of p16INK4a is seen

only in tumors from smokers,

whereas the p16INK4a gene is

inactivated in tumors from

nonsmokers only through

promoter hypermethylation

186 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE

by applying pharmaceutical agents that

target these pivotal genetic changes in

cancer progression.

Alterations of various genes, such as

p16INK4a, TP53, and FHIT, may occur

early in lung tumorigenesis and may

represent viable targets for intervention

in lung cancer and their precursor

lesions. Restoration of the expression

of such tumor suppressors as RB1,

p16INK4a, p53, or FHIT by transfer of

wild-type gene sequences or abrogation

of cyclin D1 overexpression by antisense

techniques has been shown to restore

cell-cycle regulation, resulting in growth

inhibition of lung cancer cells [Schrump

et al., 1996; Ji et al., 1999]. With ad-

vances in gene-transfer techniques and

the development of less toxic pharma-

ceutical agents that target specific mole-

cular defects, it should be possible to

develop improved therapeutic options

for lung cancer patients.

REFERENCES

Belinsky SA, Nikula KJ, Palmisano WA, MichelsR, Saccomanno G, Gabrielson E, BaylinSB, Herman JG. 1998. Aberrant methyla-tion of p16(INK4a) is an early event in lungcancer and a potential biomarker for earlydiagnosis. Proc Natl Acad Sci USA 95:11891–11896.

Brennan J, O’Connor T, Makuch RW, SimmonsAM, Russell E, Linnoila RI, Phelps RM,Gazdar AF, Ihde DC, Johnson BE. 1991.myc family DNA amplification in 107tumors and tumor cell lines from patientswith small cell lung cancer treated withdifferent combination chemotherapy regi-mens. Cancer Res 51:1708–1712.

Burbee DG, Forgacs E, Zochbauer-Muller S,Shivakumar L, Fong K, Gao B, Randle D,Kondo M, Virmani A, Bader S, Sekido Y,Latif F, Milchgrub S, Toyooka S, GazdarAF, Lerman MI, Zabarovsky E, White M,Minna JD. 2001. Epigenetic inactivation ofRASSF1A in lung and breast cancers andmalignant phenotype suppression. J NatlCancer Inst 93:691–699.

Dammann R, Li C, Yoon JH, Chin PL, Bates S,Pfeifer GP. 2000. Epigenetic inactivation ofa RAS association domain family proteinfrom the lung tumour suppressor locus3p21.3. Nature Genet 25:315–319.

Denissenko MF, Chen JX, Tang MS, Pfeifer GP.1997. Cytosine methylation determines hotspots of DNA damage in the human P53gene. Proc Natl Acad Sci USA 94:3893–3898.

Fong KM, Biesterveld EJ, Virmani A, Wistuba I,Sekido Y, Bader SA, Ahmadian M, Ong ST,Rassool FV, Zimmerman PV, Giaccone G,Gazdar AF, Minna JD. 1997. FHIT andFRA3B 3p14.2 allele loss are common in

lung cancer and preneoplastic bronchiallesions and are associated with cancer-related FHIT cDNA splicing aberrations.Cancer Res 57:2256–2267.

Fontanini G, Vignati S, Bigini D, Mussi A, LucchiM, Angeletti CA, Basolo F, Bevilacqua G.1995. Bcl-2 protein: a prognostic factorinversely correlated to p53 in non-small-celllung cancer. Br J Cancer 71:1003–1007.

Ginsberg R, Kris M, Armstrong J. 1993. Cancerof the lung. In: De Vita VT, Hellman S,Rosenberg SA, editors. Principles and pra-ctice of oncology. Philadelphia: Lippincott-Raven Publishers. p 673–758.

Grandori C, Cowley SM, James LP, EisenmanRN. 2000. The Myc/Max/Mad networkand the transcriptional control of cell be-havior. Annu Rev Cell Dev Biol 16:653–699.

Hittelman WN, Kim HJ, Lee JS, Shin DM,Lippman SM, Kim J, Ro JY, Hong WK.1996. Detection of chromosome instabilityof tissue fields at risk: in situ hybridization.J Cell Biochem Suppl 25:57–62.

Hussain SP, Harris CC. 1998. Molecular epide-miology of human cancer: contribution ofmutation spectra studies of tumor suppressorgenes. Cancer Res 58:4023–4037.

Ji L, Fang B, Yen N, Fong K, Minna JD, Roth JA.1999. Induction of apoptosis and inhibitionof tumorigenicity and tumor growth byadenovirus vector-mediated fragile histidinetriad (FHIT) gene overexpression. CancerRes 59:3333–3339.

Kok K, Naylor SL, Buys CH. 1997. Deletionsof the short arm of chromosome 3 in solidtumors and the search for suppressor genes.Adv Cancer Res 71:27–92.

LaForgia S, Morse B, Levy J, Barnea G,Cannizzaro LA, Li F, Nowell PC,Boghosian-Sell L, Glick J, Weston A, HarrisCC, Drabkin H, Patterson D, Croce CM,Schlessinger J, Huebner K. 1991. Receptorprotein-tyrosine phosphatase gamma is acandidate tumor suppressor gene at humanchromosome region 3p21. Proc Natl AcadSci USA 88:5036–5040.

Latif F, Tory K, Gnarra J, Yao M, Duh FM, OrcuttML, Stackhouse T, Kuzmin I, Modi W, GeilL, Schmidt L, Zhou F, Li H, Wei MH, ChenF, Glenn G, Choyke P, Walther MM, WengY, Duan DS, Dean M, Glavac D, RichardsFM, Crossey PA, Ferguson-Smith MA, LePaslier D, Chumakov I, Cohen D, ChinaultAC, Maher ER, Linehan WM, Zbar B,Lerman MI. 1993. Identification of the vonHippel–Lindau disease tumor suppressorgene. Science 260:1317–1320.

Levin NA, Brzoska PM, Warnock ML, Gray JW,Christman MF. 1995. Identification of novelregions of altered DNA copy number insmall cell lung tumors. Genes ChromosomCancer 13:175–185.

Li ZH, Zheng J, Weiss LM, Shibata D. 1994. c-k-ras and p53 mutations occur very early inadenocarcinoma of the lung. Am J Pathol144:303–309.

Mao L, Lee JS, Kurie JM, Fan YH, Lippman SM,Lee JJ, Ro JY, Broxson A, Yu R, MoriceRC, Kemp BL, Khuri FR, Walsh GL,Hittelman WN, Hong WK. 1997. Clonalgenetic alterations in the lungs of currentand former smokers. J Natl Cancer Inst89:857–862.

Ohta M, Inoue H, Cotticelli MG, Kastury K,Baffa R, Palazzo J, Siprashvili Z, Mori M,McCue P, Druck T, Croce CM, HuebnerK. 1996. The FHIT gene, spanning thechromosome 3p14.2 fragile site and renalcarcinoma–associated t(3;8) breakpoint, isabnormal in digestive tract cancers. Cell 84:587–597.

Ookawa K, Shiseki M, Takahashi R, Yoshida Y,Terada M, Yokota J. 1993. Reconstitutionof the RB gene suppresses the growth ofsmall-cell lung carcinoma cells carryingmultiple genetic alterations. Oncogene 8:2175–2181.

Palmisano WA, Divine KK, Saccomanno G,Gilliland FD, Baylin SB, Herman JG,Belinsky SA. 2000. Predicting lung cancerby detecting aberrant promoter methylationin sputum. Cancer Res 60:5954–5958.

Pei J, Balsara BR, Li W, Litwin S, Gabrielson E,Feder M, Jen J, Testa JR. 2001. Genomicimbalances in human lung adenocarcino-mas and squamous cell carcinomas. GenesChromosom Cancer 31:282–287.

Petersen I, Bujard M, Petersen S, Wolf G, GoezeA, Schwendel A, Langreck H, Gellert K,Reichel M, Just K, du Manoir S, CremerT, Dietel M, Ried T. 1997. Patterns ofchromosomal imbalances in adenocarci-noma and squamous cell carcinoma of thelung. Cancer Res 57:2331–2335.

Richardson GE, Johnson BE. 1993. The biologyof lung cancer. Semin Oncol 20:105–127.

Ried T, Petersen I, Holtgreve-Grez H, SpeicherMR, Schrock E, du Manoir S, Cremer T.1994. Mapping of multiple DNA gains andlosses in primary small cell lung carcino-mas by comparative genomic hybridization.Cancer Res 54:1801–1806.

Roche J, Boldog F, Robinson M, Robinson L,Varella-Garcia M, Swanton M, Waggoner B,Fishel R, Franklin W, Gemmill R, DrabkinH. 1996. Distinct 3p21.3 deletions in lungcancer and identification of a new humansemaphorin. Oncogene 12:1289–1297.

Sanchez-Cespedes M, Reed AL, Buta M, Wu L,Westra WH, Herman JG, Yang SC, JenJ, Sidransky D. 1999. Inactivation of theINK4A/ARF locus frequently coexists withTP53 mutations in non-small cell lungcancer. Oncogene 18:5843–5849.

Sanchez-Cespedes M, Decker PA, Doffek KM,Esteller M, Westra WH, Alawi EA, HermanJG, Demeure MJ, Sidransky D, Ahrendt SA.2001. Increased loss of chromosome 9p21but not p16 inactivation in primary non-small cell lung cancer from smokers. CancerRes 61:2092–2096.

Sard L, Accornero P, Tornielli S, Delia D, BunoneG, Campiglio M, Colombo MP, GramegnaM, Croce CM, Pierotti MA, Sozzi G. 1999.The tumor-suppressor gene FHIT is involv-ed in the regulation of apoptosis and in cellcycle control. Proc Natl Acad Sci USA 96:8489–8492.

Schrump DS, Chen A, Consoli U. 1996. Inhibi-tion of lung cancer proliferation by antisensecyclin D. Cancer Gene Ther 3:131–135.

Sekido Y, Bader S, Latif F, Chen JY, Duh FM, WeiMH, Albanesi JP, Lee CC, Lerman MI,Minna JD. 1996. Human semaphorins A(V)and IV reside in the 3p21.3 small cell lungcancer deletion region and demonstrate

ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) 187

distinct expression patterns. Proc Natl AcadSci USA 93:4120–4125.

Sekido Y, Fong KM, Minna JD. 2001. Molecularbiology of lung cancer. In: De Vita VT Jr,Hellman S, Rosenberg SA, editors. Cancer:principles and practice of oncology. Phila-delphia: Lippincott Williams & Wilkins. p917–925.

Shapiro GI, Edwards CD, Kobzik L, GodleskiJ, Richards W, Sugarbaker DJ, Rollins BJ.1995. Reciprocal Rb inactivation andp16INK4 expression in primary lung can-cers and cell lines. Cancer Res 55:505–509.

Sozzi G, Veronese ML, Negrini M, Baffa R,Cotticelli MG, Inoue H, Tornielli S, PilottiS, De Gregorio L, Pastorino U, Pierotti MA,Ohta M, Huebner K, Croce CM. 1996. TheFHIT gene 3p14.2 is abnormal in lungcancer. Cell 85:17–26.

Sozzi G, Sard L, De Gregorio L, Marchetti A,Musso K, Buttitta F, Tornielli S, PellegriniS, Veronese ML, Manenti G, Incarbone M,

Chella A, Angeletti CA, Pastorino U,Huebner K, Bevilaqua G, Pilotti S, CroceCM, Pierotti MA. 1997. Association be-tween cigarette smoking and FHIT genealterations in lung cancer. Cancer Res 57:2121–2123.

Sugio K, Kishimoto Y, Virmani AK, Hung JY,Gazdar AF. 1994. K-ras mutations are arelatively late event in the pathogenesis oflung carcinomas. Cancer Res 54:5811–5815.

Testa JR, Liu Z, Feder M, Bell DW, Balsara B,Cheng JQ, Taguchi T. 1997. Advances inthe analysis of chromosome alterationsin human lung carcinomas. Cancer GenetCytogenet 95:20–32.

Todd S, Franklin WA, Varella-Garcia M, KennedyT, Hilliker CEJ, Hahner L, Anderson M,Wiest JS, Drabkin HA, Gemmill RM. 1997.Homozygous deletions of human chromo-some 3p in lung tumors. Cancer Res57:1344–1352.

Vahakangas KH, Samet JM, Metcalf RA, WelshJA, Bennett WP, Lane DP, Harris CC. 1992.Mutations of p53 and ras genes in radon-associated lung cancer from uranium miners.Lancet 339:576–580.

Whang-Peng J, Kao-Shan CS, Lee EC, Bunn PA,Carney DN, Gazdar AF, Minna JD. 1982.Specific chromosome defect associated withhuman small-cell lung cancer; deletion3p(14-23). Science 215:181–182.

Wistuba I, Behrens C, Milchgrub S, Bryant D,Hung J, Minna JD, Gazdar AF. 1999. Se-quential molecular abnormalities are in-volved in the multistage development ofsquamous cell lung carcinoma. Oncogene18:643–650.

Zhang Z, Nakamura M, Taniguchi E, Shan L,Yokoi T, Kakudo K. 1996. Late occurrenceof K-ras gene mutations in the pathogenesisof squamous cell carcinoma of the lung:analysis in sputum. Anal Quant Cytol Histol18:501–502.

188 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE