clinicas de norteamerica

1. LUNG CANCER 0272–5231/02 $15.00 .00
MOLECULAR BIOLOGY OF LUNG
CANCER: CLINICAL IMPLICATIONS
Kwun M. Fong, MBBS, PhD, and John D. Minna, MD
Lung cancer accounts for the most cancer-related MOLECULAR EPIDEMIOLOGY:
deaths in men and women in the United States, INHERITED LUNG CANCER
causing about 29% of all cancer deaths, more than SUSCEPTIBILITY
prostate, colorectal, and breast cancers combined.59
There is intense effort now looking at the screening The major classes of carcinogens in tobacco
and early detection of lung cancer, with the in- smoke are the polycyclic hydrocarbons (such as
creasingly used low-dose spiral CT scanning tech- benzo (a) pyrene), the nitrosamines, and the aro-
nology. In addition, there has been a flurry of new, matic amines. Tobacco smoke carcinogens may be
biologically based therapy designed from knowl- activated enzymatically to chemically reactive elec-
edge of molecular and biologic changes in lung trophiles that form carcinogen DNA adducts. Al-
cancer cells. This review discusses the relevance of though most lung cancer cases are linked to smok-
recent molecular data on lung cancer pathogenesis ing, only a minority of heavy smokers develop
to clinical practice. The challenge is to translate lung cancer, leading to the notion that there may
discoveries regarding how lung cancers achieve be genetic factors that affect individual susceptibil-
uncontrolled growth, proliferation, and metastatic ity to develop lung cancer. Familial aggregation
behavior by disruption of key cell-cycle regulators (clustering of cases) was described some time ago,
and signal transduction cascades into improved with the observation of more lung cancer in rela-
clinical outcomes. The molecular basis of lung car- tives of lung cancer cases.129, 191 Segregation analy-
cinogenesis, essentially by genetically or epigenet- ses have suggested a mendelian codominant pat-
ically altering oncogenes and tumor suppressor tern of inheritance 161 perhaps most relevant to
genes, must be understood more fully and ex- early onset, never-smoking lung cancer cases.158 In
ploited to enhance survival in the presence of this addition, there has been much interest in identi-
highly lethal cancer. The molecular epidemiology fying the more common genetic variants or poly-
of individual susceptibility to tobacco smoke car- morphisms that are hypothesized to affect lung
cinogens may help in focusing on the highest risk cancer risk, particularly focusing on molecules as-
group for screening technologies, which are now sociated with carcinogen handling and DNA re-
capable of detecting very small lung nodules.63 pair. An individual’s susceptibility to cancer may
Moreover, clinicians need new clinical strategies to be affected partially by the balance between the
complement surgery, radiotherapy, and chemo- capacity to activate inhaled procarcinogens (phase
therapy, and to assist in primary and secondary I enzymes) and the capacity to detoxify carcino-
prevention efforts. gens (phase II enzymes), for example.178 It is recog-
nized increasingly that genetic polymorphisms
common in the population can affect each of these
We are grateful to Dr Maree Colosimo for reviewing processes, leading to the notion that an individu-
this manuscript. Supported by Lung Cancer SPORE al’s lung cancer susceptibility could be affected
Grant P50 CA70907 by genetic polymorphisms, modified by tobacco
From the Prince Charles Hospital, Chermside, Brisbane, Australia (KMF); and the Hamon Center for Therapeutic
Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas (JDM)
CLINICS IN CHEST MEDICINE
VOLUME 23 • NUMBER 1 • MARCH 2002 83

2. 84 FONG & MINNA
smoking. The genetic variations thought to be im- MOLECULAR CHANGES IN LUNG
portant in lung cancer include polymorphisms at CARCINOGENESIS: THERAPEUTIC
the P-450 gene loci, such as CYP1A1, CYP2D6 and IMPLICATIONS
the glutathione S-transferases gene cluster.77, 148, 209
Molecular changes in lung cancer and associated
Some studies have yielded conflicting results,
preneoplastic cells increasingly are being identi-
however, perhaps limited by the infrequency of
fied, particularly with the advent of tools (such
certain polymorphisms, ethnic confounding, and
as the fluorescent bronchoscope) that improve the
differences in smoking consumption—that is, risk
detection and sampling of bronchial intraepithelial
and tumor heterogeneity. Different metabolic en-
lesions, and tissue microdissection techniques (in-
zymes may be associated with susceptibility of
cluding the rapid laser capture microdissection
tumor subtypes to various tobacco smoke carcino-
system) that allow for careful analysis of specific
gens, for instance.96 Furthermore, the possibility lung epithelial cell populations. There is an intense
of gene–gene interactions means that large, well- effort to translate these data into surrogate bio-
designed studies are needed to understand fully markers for lung cancer risk assessment, for
the role of these genetic variations. achieving early diagnosis, for monitoring chemo-
Another area of study is the individual variabil- prevention studies, and for developing new thera-
ity in DNA repair, which may be hypothesized to pies, making an understanding of the major molec-
be linked to different rates of DNA repair and ular abnormalities acquired by bronchial cells
therefore, accumulation of DNA mutations. ‘‘Mu- during their transformation to lung cancer an es-
tagen sensitivity,’’ based on the number of chromo- sential resource for clinicians dealing with lung
some breaks in peripheral blood lymphocytes after cancer.
they were exposed in vitro to benzo (a) pyrene Lung cancer is the end stage of multistep carci-
diol-epoxide (BPDE), a carcinogenic derivative of nogenesis, in most cases driven by genetic and
benzopyrene, has been linked to increased lung epigenetic damage caused by chronic exposure to
cancer risk.8 In fact, more chromosomal 3p21.3 de- tobacco smoke carcinogens and tumor promoters.
letions were found by fluorescent in situ hybridiza- The genetic instability in human cancers seems
tion (FISH) in peripheral blood lymphocytes of to exist at two levels—at the chromosomal level,
lung cancer cases than controls after BPDE expo- including large-scale losses and gains, and at the
sure, and increased rates of 3p21.3 deletions after nucleotide level, including single or several base
BPDE exposure were associated with approxi- changes and DNA promoter-region methylation.97
mately a 14-fold increased risk for having lung Lung cancers are characterized by genomic imbal-
cancer.210 This finding is of interest because the ances, consisting of many numerical chromosome
exact same 3p21.3 region also is lost in respiratory abnormalities (aneuploidy) and structural cytoge-
epithelial cells in smokers.204, 205 Studies of other netic abnormalities, including deletions, amplifi-
DNA repair genes (ERCC1, XPD, XPF, XRCC3, and cations, and nonreciprocal translocations. Chromo-
XRCC1) are reported increasingly and are promis- somal instability leading to aneuploidy can occur
ing for at least some of these molecules.24, 142 because of the loss of function of a mitotic check-
The major clinical implication lies in the poten- point, but the precise mechanism in lung cancer is
tial for these studies to provide new genetic epide- not known. In addition to karyotyping, chromo-
miology tools to help identify the highest risk pop- somal copy number aberrations can be mapped
ulation for developing lung cancer. These more finely using newer molecular cytogenetic
individuals then can be targeted for education, techniques.188 These techniques include compara-
novel screening methods, such as low-dose helical tive genomic hybridization, which has demon-
CT scan, and medical surveillance (Table 1). strated multiple abnormalities in lung cancer,99, 137
Table 1. MOLECULAR BIOLOGY OF LUNG CANCER: CLINICAL IMPLICATIONS
Issue Possible Approaches (in Conjunction with Traditional Methods)
Molecular Risk stratify according to individual susceptibility for prevention and screening
epidemiology
Early detection Detection of lung cancer–specific mutations, other genetic alterations, and methylation in sputum,
blood, bronchoscopy specimens
Develop intermediate biomarkers to gauge chemoprevention response
Diagnosis Adjunct to morphologic and histologic diagnosis
Assist in subtype differentiation
Therapy Biologically based approaches, such as monoclonal antibodies and inhibitors against oncogenes,
growth factors and receptors, tyrosine kinase inhibitors, replacement gene therapy (e.g., p53),
matrix metalloproteinase inhibitors, cyclin-dependent kinase inhibitors, farnesyltransferase
inhibitors, cyclooxygenase inhibitors, protein phosphatase and kinase modulators, antisense
molecules, apoptosis modulators, angiogenesis inhibitors, immunotherapy, vaccination,
antibody– or ligand–toxin conjugates
Prognosis Predict metastatic disease, prognosis, response, and side effects of chemotherapy and radiotherapy

3. MOLECULAR BIOLOGY OF LUNG CANCER: CLINICAL IMPLICATIONS 85
Table 2. MAJOR MOLECULAR ALTERATIONS IN LUNG CANCER
SCLC (%) NSCLC (%)
Tumor suppressor genes
p53 abnormalities
mutation with 17p13 LOH 75–100 50
abnormal p53 expression (IHC) 40–70 40–60
p16-cyclin D1-CDK4-RB pathway lesions
p16 mutation or DNA methylation
with 9p21 LOH 1 10–40
absent p16 expression (IHC2) 0–10 30–70
absent RB expression with 13q14 LOH 90 15–30
APC (5q21 LOH) DNA methylation 26 46
Chromosome 3p LOH (several sites) 100 90
RAR- 3p24 (DNA methylation) 76 40
RASSF1A 3p21.3 (DNA methylation) 90 30–40
FHIT 3p14.2 (DNA methylation and del) 64 50
CDH13 (HCAD) (DNA methylation) 20 45
Proto-oncogenes and growth stimulation
Putative autocrine loops GRP/GRP receptor HGF/MET
SCF/KIT NDF/ERBB
RAS mutation 1 15–20
MYC amplification* 15–30 5–10
Other molecular changes
BCL-2 expression 75–95 10–35
Telomerase activity 100 80–85
Microsatellite instability 35 22
Promoter hypermethylation marker dependent marker dependent
SCLC Small cell lung cancer; NSCLC non–small cell lung cancer; IHC immunohistochemistry; GRP gastrin releasing
peptide; SCF stem cell factor; HGF hepatocyte growth factor; MET metatasis oncogene; NGF new differentiation factor;
ERBB epidermal growth factor receptor.
*Overexpression without amplification is observed in other cases. SCLC amplifications include MYC, MYCN, and MYCL.
and FISH, which allows the examination of in- type. Microsatellite alterations have been reported
terphase cells from tissue sections or cell suspen- in approximately 35% of small cell lung cancers
sion, and therefore is potentially applicable for the (SCLC) and 22% of non–small cell lung cancers
analysis of preneoplasia.184 Chromosomal losses of- (NSCLC).159 Microsatellite alterations seem to be
ten are observed at sites that house known or associated with younger age,153 reduced survival,147
suspected tumor suppressor genes (TSGs; e.g., 3p, and advanced tumor stage.2 Finally, other DNA
5q, 17p), which have a fundamental tumorigenic repair pathways may be implicated in lung cancer,
role. Tumor suppressor genes classically are inacti- given that there are reports of occasional mutations
vated by structural deletion (loss) of one parental in the OGG1 gene involved in repairing oxidative
allele combined with a point or small mutation, or DNA damage31 and inactivation of the DNA repair
epigenetic methylation inactivation of the re- gene O6-methylguanine-DNA methyltransferase
maining allele (Table 2). expression by the epigenetic mechanism of pro-
At least two classes of cellular genes other than moter hypermethylation.43 These changes can be
TSGs are involved in tumorigenesis—proto-onco- detected by sensitive PCR-based tests in bronchial
genes and DNA repair genes. Oncogene activation washings, biopsies, and sputum and therefore are
often occurs through point mutation, gene ampli- being tested as molecular markers for risk assess-
fication, or rearrangement. Additionally, dysregu- ment, early detection, and monitoring prevention
lated (increased or decreased) gene expression can studies.
occur by other, as yet unknown, mechanisms.
Studies in lung cancer have not confirmed a prom- Tumor Suppressor Genes
inent role for somatically acquired abnormalities
of DNA repair genes, but there are alterations in The p53 TSG maintains genomic integrity in the
DNA repeat sequences that are reminiscent of the context of DNA damage from or ultraviolet irra-
replication error repair (RER ) phenotype seen diation and carcinogens. DNA damage or hypoxia
in colorectal and other tumors characterized by up-regulates p53, which acts as a sequence-specific
mutations in DNA mismatch repair genes. None- transcription factor regulating downstream genes,
theless, the phenotype in lung cancers seems dis- including p21, MDM2, GADD45, and BAX,
tinct, affecting fewer markers and causing a single thereby helping to regulate the G1/S cell cycle
‘‘shift’’ of individual allelic bands, in contrast to transition, G2/M DNA damage checkpoint, and
‘‘RER laddering’’ prompting the term microsatel- apoptosis or programmed cell death. Dysfunction
lite alteration to distinguish this lung cancer pheno- of p53 allows the inappropriate survival of geneti-

4. 86 FONG & MINNA
Figure 1. The p53 tumor suppressor gene pathway. p53 is situated on the short
arm of chromosome 17 (17p). It helps maintain genomic stability, inhibits the cell
cycle at G1, and causes apoptosis if DNA is damaged beyond repair. These functions
are lost because of mutation in p53 in 90% of small cell lung cancer (SCLCs) and
more than 50% of non–small cell lung cancer (NSCLCs). p53 activity is antagonized
by MDM2 (which thus functions as an oncogene), with the two forming a negative
feedback loop. MDM2 is not frequently abnormal in lung cancers. In addition, p19
alternate reading frame (ARF) is an alternatively spliced transcript from the same
chromosome 9p21 locus that gives rise to p16. It antagonizes MDM2, thus function-
ing as a tumor suppressor. p19ARF mutations are apparently not common in lung
cancer. p53 is inactivated by damage to both alleles, often by allelic loss and somatic
missense mutations. Tumor suppressor genes also can be inactivated by promoter
hypermethylation (see later) but this is apparently not common for p53. Gene therapy
with wild-type p53 replacement is clinically successful in some NSCLCs.
cally damaged cells, setting the stage for the accu- p53, which inhibits cyclin and cyclin-dependent
mulation of multiple mutations and the subse- kinase (CDK) complexes at the G1 phase. Al-
quent evolution of a cancer cell (Fig. 1). p53 plays though not somatically mutated in lung cancer,
a critical role in lung cancer; its chromosome 17p13 p21 was overexpressed in 65% to 75% of NSCLCs,
locus frequently is deleted hemizygously and mu- especially in well-differentiated tumors. 106 One
tational inactivation of the remaining allele occurs NSCLC study reported that p21 expression was
in 75% or more of SCLCs and approximately 50% linked to a favorable outcome27; another suggested
of NSCLCs.17, 58, 185 Mutations of p53 in lung tumors that concordant expression of p21 and tumor
correlate with cigarette smoking and are primarily growth fact or- 1 (TGF- 1) predicts better chance of
the guanine to thymine (G-T) transversions ex- survival than discordant expression.16 The MDM2
pected of tobacco smoke carcinogens.58 Further- oncogene product inhibits p53 function by
more, benzo (a) pyrene selectively forms adducts blocking its regulation of target genes and en-
at the major p53 mutational hot spots in bronchial hances proteasome-dependent degradation of p53.
epithelial cells.39 Missense p53 mutations can pro- Conversely, p53 regulates (increases) the expres-
long the protein half-life, mutant p53 protein lead- sion of MDM2 by directly binding and activating
ing to easily detected by immunohistochemistry the MDM2 promoter. Because the investigator
(IHC).28 Other types of p53 mutations do not corre- would expect p21 expression to be lost with p53
late with IHC staining. Studies have shown abnor- loss of function, the overexpression indicates there
mal p53 expression by IHC in 40% to 70% of must be other mechanisms stimulating p21. In ad-
SCLCs and 40% to 60% of NSCLCs (squamous cell dition, the fact that a lung cancer develops in the
carcinomas higher than adenocarcinomas).21, 56, 120 face of p21 overexpression indicates there are other
Mutations of p53 have been linked to response to cellular defects that can bypass this negative
cis-platinum–based chemotherapy or radiotherapy growth regulator. The MDM2 protein is overex-
in NSCLC.79, 107 pressed in 25% of NSCLCs65 and MDM2 expres-
Two proteins homologous to p53 include p51 sion without abnormal p53 expression has been
and p73, both of which can induce growth sup- reported to be a favorable prognostic factor. Differ-
pression and apoptosis. Mutations of p51 and p73 ent ways of inactivating the p53 pathway therefore
appear infrequently in lung cancer, however.122, 217 may have different clinical outcomes.
The expression of the gene p21 is up-regulated by Therapeutic implications: The in vitro reintro-

5. MOLECULAR BIOLOGY OF LUNG CANCER: CLINICAL IMPLICATIONS 87
duction of wild-type p53 into lung cancer cells that in lung cancers, with protein abnormalities de-
have various other genetic abnormalities besides tected in approximately 90% of SCLCs and 15% to
p53 blocks tumor cell growth by inducing 30% of NSCLCs.25, 40, 56, 143 Functional RB loss can
apoptosis.1 Direct injection of a retroviral vector include deletion, nonsense mutations, or splicing
containing wild-type p53 into tumors in nine pa- abnormalities, frequently leading to a truncated
tients with NSCLC who had failed conventional RB protein. Whether absent RB expression is asso-
treatment led to tumor regression in one third of ciated with poor prognosis in NSCLCs is contro-
the patients.149, 150 Other promising strategies for versial.40, 56, 92, 212 Functionally, in vitro reintroduc-
delivering p53 gene therapy include liposome–p53 tion into tumor cells of a wild-type RB suppresses
complexes delivered endobronchially and adeno- SCLC growth.130 Although the inherited germline
virus-mediated p53 transfer given locally, endo- form of mutant RB is associated with the develop-
bronchially, or even systemically.222 Intratumoral ment of the childhood disease retinoblastoma, rela-
injection of adenoviral p53 gene therapy, however, tives of patients with retinoblastoma carrying
seemed to provide no additional benefit in patients germline RB mutation are about 15 times more
receiving first-line chemotherapy for advanced likely to die from lung cancer than the general
NSCLC.157 In addition, vaccine trials with mutant population.154 As a result, in this rare inherited
p53 peptides are ongoing. Finally, because most disorder, there seems to be an example of genetic
p53 mutations are missense, small molecules are predisposition to lung cancer. Two RB-related
being developed to try to reactivate p53 wild-type genes also have been implicated in lung cancer,
function by binding to and changing the conforma- including p107 and pRB2/p130; decreased expres-
tion of the mutant p53 protein. sion of these proteins is associated with more ag-
The p16–cyclin D1–cyclin-dependent kinase 4 gressive histologic behavior.110
(CDK4)–RB pathway is central to controlling the Therapeutic implications: Experiments in cell
G1 to S transition of the cell cycle, and its compo- culture and transgenic lung cancer models demon-
nents are altered functionally or mutated in many strate inhibition of tumor-cell growth in vivo after
cancers (Fig. 2). Inactivation of both alleles of the transfecting in wild-type pRB2/p130, but human
RB TSG at chromosome region 13q14 is common studies have not yet been reported.33, 119, 182
Figure 2. The p16-cyclin D1-CDK4-RB pathway. The product of the retinoblastome
(RB) tumor suppressor gene binds to the E2F transcription factor during the resting
(G0/G1) phase of the cell cycle. When complexed to RB, E2F cannot activate the
genes needed to initiate the S phase. Moreover, the RB-E2F complex also represses
the transcription of other target genes. RB is phosphorylated at the end of G1 by
cyclin/cyclin dependent kinase (CDK) complexes, for example cyclin D/CDK4-6, and
dephosphorylated at the end of mitosis (M). Phosphorylation of RB releases E2F,
which initiates the S phase, overcoming the block to the cell cycle. In quiescent
cells, RB is unphosphorylated, cyclin D levels are low, and CDK inhibitors, for
example p16, p21 and p27, inhibit the cyclin/CDK complexes. In lung cancers,
acquired loss of RB function allows continued cell cycling. This pathway can be
turned on by mutations inactivating RB, inactivating p16, overexpression of cyclin
D1, or overexpression of CDK4. Mutations inactivating p53 function also can impinge
on this pathway. p21 abnormalities have not been reported. Drugs that replace RB
or p16 function or that would inhibit cyclin D1 or CDK4 would represent new
therapeutics. In SCLC the abnormality is usually in RB and in NSCLC the abnormality
is usually in p16.

6. 88 FONG & MINNA
Cyclin D1 inhibits the activity of RB by stimulat- somal regions in lung cancers. In the classical TSG
ing its phosphorylation by CDK4. Cyclin D1 over- paradigm, the presence of underlying TSGs is sug-
expression therefore is an alternative mechanism gested as the target of these genetic losses. The
for disrupting the p16–cyclin D1–CDK4–RB path- chromosomal regions showing hemizygous dele-
way. Cyclin D1 was overexpressed in 25% to 47% tions include 1p, 1q, 2q, 3p, 4p, 4q, 5q, 6p, 6q, 8p,
of primary NSCLCs and may be associated with 8q, 11p, 11q, 14q, 17q, 18q, and 22q.98, 123, 125, 131, 155,
poor prognosis. 18, 26 Transfection of a cyclin-D1 163, 194, 206
Although several of these chromosomal
antisense construct into lung cancer cell lines in- arms contain known or candidate TSGs (such as
duces destabilization of RB and retards growth.41 adenomatous polyposis coli [APC] at 5q21, Wilm’s
It is not clear whether amplification of CDK4, tumor (WT1) at 11p13, neurofibromatosis (NF2) at
which can occur in other malignancies, plays a 22q12), these genes are not known to be mutated
role in lung cancer. in lung cancer. Some, however, such as APC, can
Therapeutic implications: A CDK inhibitor, fla- be inactivated by tumor-acquired promoter hyper-
vopiridol, is in clinical trials against lung and methylation (discussed subsequently). Hemizy-
other cancers. gous loss of chromosome 3p regions, including
p16 regulates RB function by inhibiting CDK4 3p25-26, 3p21.3-22, and 3p14-cen, often occurs in
and CDK6 kinase activity. p16 (p16INK4/CDKN2) lung cancer, consistent with the presence of TSGs.64
is situated at chromosome 9p21 and frequently The notion of critical 3p TSGs also is supported
undergoes allele loss and mutation in lung can- by the observation of homozygously deleted re-
cer.159 Homozygous deletion or point mutations gions at 3p12-13, 3p14.2, and 3p21 in lung cancer
occur in 10% to 40% of NSCLCs.105, 152 An alterna- cell lines.159 One candidate is fragile histidine triad
tive abnormality down-regulating p16 expression (FHIT) gene at 3p14.2, which undergoes hemizy-
in 30% to 40% of cases is promoter hypermethyl- gous and, occasionally, homozygous deletion in
ation.109, 133 Thus, p16 inactivation represents a fur- lung cancer cells and encodes a dinucleoside hy-
ther mechanism for disrupting the p16-cyclin D1– drolase. Lung cancer cells frequently (40%–80%)
CDK4–RB pathway, particularly in NSCLC, func- express abnormal mRNA transcripts of FHIT but
tionally analogous to the preferential RB nearly always also express wild-type FHIT tran-
inactivation in SCLC.82, 86, 92, 117, 127, 162 The end result scripts.48, 177 Unlike classical TSG inactivation, FHIT
from all of these mechanisms is that 30% to 50% point mutations are rare, and abnormal transcripts
of early stage primary NSCLCs do not express can be found in normal lung tissue.190 Notwith-
p16. Coinactivation of RB and p16 in any one standing, FHIT is expressed in normal lung but
tumor is rare but cyclin D1 overexpression can FHIT protein expression loss occurs in primary
coexist with these abnormalities in the same tu- lung tumors. This loss frequently is caused by
mor.162 Notably, 10% to 30% of NSCLCs seem nor- promoter hypermethylation.221 Moreover, FHIT al-
mal for RB and p16, indicating involvement of lele loss is more common in smokers than non-
cyclin D1, CDK4, or other pathway members in smokers176 and may be associated with a poorer
these cases. p16 alteration and loss of function in likelihood of survival in NSCLC.23 Furthermore,
lung cancer may be associated with tumor progres- reintroduction of exogenous wild-type FHIT sup-
sion, clinical stage, and survival, although not all pressed tumorigenicity of a lung cancer cell line in
studies concur.56, 86, 92, 183 nude mice,74, 170 whereas others have reported that
The p16 locus also encodes a second alternative FHIT transfection does not suppress tumor growth
reading frame (ARF) protein, p19ARF, which over- of human cancer cell lines.132
laps with p16, and that also may be important in The 3p21.3 region has been examined exten-
growth regulation. p19ARF sometimes called p14ARF sively for putative TSGs, particularly at homozy-
binds to the MDM2-p53 complex and prevents gously deleted regions.89, 90, 201, 213 The RASSF1A
p53 degradation, resulting in p53 activation. IHC isoform of RASSF1 undergoes promoter hyper-
analysis suggests more frequent loss of p19ARF pro- methylation in approximately 30% of primary
tein expression in tumors with neuroendocrine fea- NSCLCs and more than 90% of SCLCs, causing
tures.54 One genetic locus at 9p21 therefore pro- loss of RASSF1 A expression. This promoter hyper-
duces two products, p16 and p19ARF, both of which methylation is correlated with poorer survival in
play a critical role in growth regulation; p16 with resected patients with NSCLC. Transfection and
the RB pathway, and p19ARF with the p53 pathway. re-expression of wild-type RASSF1 A result in sup-
Reduced expression of another CDK-inhibitor pression of lung cancer tumorigenicity. 22, 37 The
gene, p27KIP1, correlates with poor prognosis in RARB2 isoform also undergoes promoter hyper-
NSCLC.42, 216 In contrast, most SCLCs exhibit in- methylation in approximately 60% to 70% of
creased p27KIP1 staining, suggesting a possible link NSCLCs and SCLCs. This occurrence leads to loss
with neuronal differentiation.216 p 57KIP2 at chromo- of RARB2 expression, which occurs in most lung
some region 11p15 is imprinted with maternal ex- tumors and in some preneoplastic lesions. This
pression and p57KIP2 expression is down-regulated situation could account for the retinoid resistance
by selective loss of the maternal alleles in some seen in lung cancers.55, 195
lung cancers.91 Other candidate TSGs include the PTEN gene at
Other candidate tumor suppressor genes are im- chromosome 10q23, which encodes a phospha-
plied by the finding of many hemizygous and tase.100 Protein tyrosine phosphatases are able to
some homozygous deletions at multiple chromo- antagonize the growth-promoting protein kinases,

7. MOLECULAR BIOLOGY OF LUNG CANCER: CLINICAL IMPLICATIONS 89
and the PTEN gene is mutated in a subset of lung vated earlier in women in response to tobacco
cancers and homozygously deleted in several lung exposure.164
cancer cell lines.52 Another candidate at 10q, region Therapeutic implications: The in vitro formation
25.3-26.1, is DBMT1, which frequently is down- of soft agar clones and the in vitro growth of nude
regulated and occasionally homozygously deleted mouse xenografts of SCLC cell lines are inhibited
in lung cancer.210 There also seem to be important by a neutralizing monoclonal antibody directed
chromosomal deletions at 11q23-24, including the against GRP/BN and by antagonists of BN.35, 60 A
locus for PPP2RIB (beta isoform of the A subunit clinical trial of the anti-BN monoclonal antibody
of the human protein phosphatase-2A) gene, in has shown some antitumor activity in previously
which mutations have been described in lung and treated patients with SCLC, with phase I results
colon cancers, suggesting a TSG function.198, 199 published recently.29, 81 The development of peptide
Clinical implications: The increasing discovery antagonists by new small-molecule therapeutics is
of TSGs important in lung cancer pathogenesis, another attractive strategy.
particularly those at 3p, which occur early in lung
cancer pathogenesis, is likely to lead to the oppor-
tunity to develop new diagnostic and therapeutic ERBB Family
strategies. These strategies would include monitor-
ing target tissue for 3p allele loss and promoter Non–small cell lung cancers, rather than SCLCs,
hypermethylation, treatment with demethylation often demonstrate abnormalities of the neuregulin
agents, and replacement gene therapy for 3p TSGs. receptors, ERBB2 and ERBB1, which are part of a
family of transmembrane receptor tyrosine ki-
nases. On ligand binding, ERBB receptors homo-
Proto-oncogenes and Growth Stimulation or heterodimerize, thereby inducing intrinsic ki-
nase activities that initiate intracellular signal
Autocrine and paracrine growth stimulatory transduction cascades, including the MAP kinase
loops exist in lung cancers as a consequence of the pathway. ERBB2 (also called HER2/neu) is highly
expression of growth factors, regulatory peptides, expressed in approximately 30% of NSCLCs, espe-
and their receptors by the cancerous or adjacent cially adenocarcinomas.141, 202 Transfection experi-
normal cells. Several but not all components of ments suggest that ERBB2 overexpression contri-
these stimulatory pathways are proto-oncogene butes to tumorigenicity in immortalized human
products. bronchial epithelial cells.121 High ERBB2 levels are
The gastrin-releasing peptide/bombesin (GRP/ associated with the multiple-drug-resistance phe-
BN) growth stimulatory loop has a role in lung notype 192 and increased metastatic potential in
development and repair.179 Immunohistochemical NSCLC,219 which may help explain the poor clini-
studies show that approximately 20% to 60% of cal outcome linked to ERBB2 overexpression re-
SCLCs express GRP/BN, whereas NSCLCs express ported by some investigators.83, 139
GRP/BN less frequently.144 The human GRP/BN Therapeutic implications: In preclinical studies
receptor subtypes belong to the G-protein–coupled with tumor cell lines, trastuzumab (Herceptin), a
receptor superfamily and include receptors for monoclonal antibody against the ERBB2 receptor,
GRP, neuromedin B, and bombesin subtype-3; all was found to have additive and synergistic effects
of these can be expressed in SCLC, NSCLC, and with some chemotherapeutic agents. Clinical trials
in some bronchial epithelial biopsies from smok- investigating combination chemotherapy with
ers.45, 166 Why these embryonic regulatory loops trastuzumab and a variety of chemotherapeutic
become ‘‘reactivated’’ in lung cancers is uncertain agents are in progress in lung cancer,4 and a com-
because mutations of GRP/BN or its receptor are mercial US Food and Drug Administration (FDA)-
seemingly absent. Nonetheless, the GRP/BN auto- approved diagnostic kit is now available for detec-
crine loop is an important growth-stimulatory loop tion of ERBB2 (HER-2/neu) expression.
in lung cancer, particularly SCLC. It may play an ERBB1 (also called EGF receptor) regulates epi-
early pathogenic role because there is an increased thelial proliferation and differentiation and usually
likelihood of expression of the GRP receptor is activated in lung cancer cells by overexpression
mRNA in the respiratory epithelium of some indi- by an unknown mechanism. The production of
viduals with a history of prolonged tobacco expo- ERBB1 ligands such as EGF and TGF- by lung
sure, and expression of the GRP receptor mRNA cancer cells expressing cognate receptors indicates
is accompanied by in vitro responsiveness of these an autocrine loop.141, 151 More common in NSCLC,
respiratory epithelial cells to the mitogenic effects ERBB1 activation may be related to tumor stage
of bombesin-like peptides. 166 In addition, there and differentiation.38, 187
may be gender-specific GRP abnormalities, inter- Clinical implications: Monoclonal antibodies
esting because of possible differences in lung can- against the ERBB1 receptor (C225, ImClone) are
cer risk between the genders. A recent study, for entering clinical trials combined with chemother-
instance, found that the GRP receptor gene situ- apy, with early phase I results available.14 In addi-
ated on the X chromosome is expressed more fre- tion, several tyrosine kinase inhibitors that have
quently in women than in men in the absence of some selectivity as EGF receptor (ERBB1) blockers
smoking and that expression of this gene is acti- (CP358774, ZD1839-Iressa, OS1774) also are being

8. 90 FONG & MINNA
tested in clinical trials, with a major advantage PDGF tyrosine kinases) and has promising activity
that most are orally active and able to be given in SCLC cell lines.200
with conventional chemotherapy (Fig. 3). Members of the RAS proto-oncogene family
Other membrane tyrosine kinases include the (KRAS, HRAS, and NRAS) encode plasma mem-
hepatocyte growth factor (HGF), which normally brane proteins and are activated in some lung can-
stimulates epithelial cell proliferation, mobility, cers by point mutations, resulting in inappropriate
and differentiation programs. During fetal lung signaling for continued cell division (Fig. 4). KRAS
development, HGF acts as a mesenchyme-derived is the most frequently activated RAS gene in lung
morphogenic factor. The constitutively low levels cancer, usually by mutations at codon 12 but, occa-
of HGF increase in response to lung or distant sionally, codons 13 and 61. KRAS mutations affect
injury.214 Hepatocyte growth factor stimulates mi- approximately 20% to 30% of lung adenocarcino-
togenesis or motogenesis of human bronchial epi- mas and 15% to 20% of all NSCLCs, but rarely
thelial, alveolar type II, and SCLC cells in vitro. SCLCs.144 There is subtype heterogeneity—for ex-
The receptor for HGF is the product of the metasta- ample, KRAS mutations are present in parenchy-
sis (MET) proto-oncogene. It generally is expressed mal but not bronchial adenocarcinomas34 and gob-
in normal lung and SCLC and NSCLC; HGF, how- let-cell subtypes of adenocarcinoma have the
highest frequency of KRAS mutations. 193 KRAS
ever, is expressed mainly in NSCLCs, suggesting
mutations correlate with smoking,171 often being
an autocrine loop specific for NSCLC.62, 128 Clini-
the guanine to thymine (G-T) transversions associ-
cally, high HGF levels are associated with a poor
ated with polycyclic hydrocarbons and nitrosa-
outcome in resectable patients with NSCLC.168 The mines.58 KRAS mutations may imply a poor prog-
KIT proto-oncogene encodes the tyrosine kinase nosis in NSCLC, although this observation is
receptor, CD117. It is coexpressed together with debated.57, 113, 146, 167, 172 Neither chemotherapy sensi-
its ligand, stem cell factor, in many SCLCs,93, 160 tivity nor survival is correlated with KRAS muta-
representing another autocrine loop that may pro- tions in a prospective study of advanced lung ade-
vide a growth advantage or mediate chemoattrac- nocarcinoma.145 Moreover, KRAS mutations were
tion. Other putative loops involve insulin growth not associated with in vitro resistance against a
factor (IGF)-I, IGF-2, and type I IGF receptors, range of chemotherapeutic agents in NSCLC cell
which frequently are coexpressed in SCLC and lines.192
NSCLC140 as are PDGF and its receptor.11 Therapeutic implications: To be active in the cell,
Therapeutic implications: These growth loops Ras has to undergo lipid modification (farnesyla-
provide additional avenues for the development tion), a process regulated by the farnesyltransfer-
of novel, biologically based therapies. The tyrosine ase enzyme. This process potentially can be
kinase inhibitor, STI 571, for instance, can inhibit blocked by specific farnesyltransferase inhibitors
the KIT tyrosine kinase (in addition to bcr-Abl and (FTIs), several of which are in clinical trials against
Figure 3. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibition.
After ligand binding, the EGFR receptor dimerizes and signals downstream mole-
cules through the activity of a specific tyrosine kinase, which can be inhibited with
specificity by drugs with specificity for the EGFR.

9. MOLECULAR BIOLOGY OF LUNG CANCER: CLINICAL IMPLICATIONS 91
Figure 4. Mutant KRAS pathway. RAS is active when bound to guanosine triphos-
phate (GTP) and inactive when bound to guanosine diphosphate (GDP). The intrinsic
GTPase activity of RAS is stimulated by GAP resulting in inactive RAS. Oncogenic
RAS mutations inhibit GTPase activity causing it to remain permanently activated
with ensuing positive signaling to downstream molecules. Drugs that could interfere
with the localization of mutant KRAS in the membrane (and thus inactivate it) or
interfere with its expression such as antisense compounds, or with components of
the downstream signaling cascade such as mitogen stimulated extracellular regu-
lated kinase (MEK) inhibitors all have therapeutic potential.
lung cancer (e.g., BMS214662, RII5777, SCH 66336). growth inhibition of an SCLC cell line by all-trans-
Trials of vaccination with mutant KRAS peptides retinoic acid is associated with increased neuroen-
also are underway. Although tumor responses docrine differentiation, increased MYCL, and de-
have been seen, KRAS the commonly mutated creased MYC expression.134 Gene therapy in the
form in lung cancer, probably is not blocked by form of antisense technology, although encourag-
FTIs. Other experimental approaches to block ing in cell culture systems, has not yet been re-
KRAS have included antisense treatment, ribo- ported in clinical trials.
zymes to block protein expression, and signal Many new therapeutic strategies therefore are
transduction interference further down the KRAS- being tested to exploit the molecular abnormalities
activated pathway. observed in lung cancer cells. Developments in
antiangiogenic and antimetastatic agents are de-
scribed later, and these new therapeutic ap-
MYC Proto-oncogenes proaches based on specific tumor biology offer
much potential promise.
RAS signaling ultimately activates nuclear
proto-oncogene products such as MYC, which, on
heterodimerization, transcriptionally activates MOLECULAR CHANGES IN LUNG
downstream genes that drive cells to grow. The CARCINOGENESIS: EARLY DETECTION
myc family comprises MYC, MYCN, and MYCL AND DIAGNOSTIC IMPLICATIONS
(originally isolated from a lung cancer118). MYC is
activated most frequently and affects SCLC and Rapid advances in molecular biology, radiology,
NSCLC, whereas the others usually only affect bronchoscopy, and other biopsy methods have oc-
SCLC. Activation occurs by gene amplification curred since the classic tests of chest radiography
( 20–100 copies per cell) or by transcriptional dys- and sputum cytology, neither of which seemed to
regulation, both of which lead to protein overex- alter outcome when used as a screening modality.
pression. Richardson et al144 concluded from 17 Knowledge of the molecular evolution is crucial if
studies that 18% to 31% of SCLCs had amplifica- tumor characteristics are to be exploited as a tool
tion of one MYC family member. Conversely, only for early detection and diagnosis.
8% to 20% of NSCLCs were affected.144 Cancers arise from precursor lesions in a
Therapeutic implications: MYC amplification multistep fashion, with preneoplastic cells somati-
seems to occur more frequently in chemotherapy- cally acquiring genetic alterations, ultimately
treated patients, and the ‘‘variant’’ SCLC subtype,76 evolving into invasive cancer by clonal expansion.
and may correlate with adverse survival. In vitro Morphologically distinct preneoplastic changes

10. 92 FONG & MINNA
(hyperplasia, metaplasia, dysplasia, and carcinoma cently.69 There is the increasing ability to obtain
in situ) can be observed in bronchial epithelium cells and cellular products such as DNA and RNA
before overt, invasive cancer develops. The se- from clinical specimens such as sputum, bronchial
quential morphologic changes that occur in pre- biopsies, brushings, lavage fluids, and blood to
neoplastic lesions are best described for squamous use as substrates for these new techniques. A
cell carcinomas. Other potential premalignant le- promising monoclonal antibody that recognizes
sions now have been recognized, however, includ- hnRNP A2/B1 seems to have good sensitivity for
ing atypical adenomatous hyperplasia as a precur- detecting lung cancers, with prospective study re-
sor for peripheral adenocarcinomas; angiogenic sults awaited.47, 189 Microsatellite alterations, detect-
squamous dysplasia, in which dysplastic lesions able by PCR, have been suggested as useful mark-
occur with an angiogenic response; and diffuse ers of lung cancer and can be found in tumors and
idiopathic neuroendocrine cell hyperplasia for car- corresponding sputum samples.111 The finding of
cinoids. Some reversal of morphology can occur microsatellite alterations in bodily fluids from pa-
after smoking cessation even though the elevated tients with chronic obstructive pulmonary disease
risk for lung cancer does not return completely and individuals without clinical lung cancer, how-
to baseline, raising the possibility of irreversibly ever, raises an important issue.46, 165 Any potential
genetically damaged areas of at-risk bronchial epi- biomarker needs to be detectable easily in bodily
thelium. These preneoplastic cells and even macro- fluids (i.e., is sensitive) but must be specific for
scopically normal bronchial epithelium adjacent to neoplastic transformation and not just reflect
cancers can contain genetic abnormalities identical smoking-related lung damage. The ease of ob-
to those in invasive cancer cells. These abnormali- taining peripheral blood has led to attempts to
ties include allele loss at several loci (3p, 9p, 8p, identify blood-based biomarkers, especially given
17p), MYC and RAS up-regulation, cyclin D1 ex- that tumor DNA is released into plasma. In this
pression, p53 immunoreactivity, BCL2 overexpres- regard, microsatellite alterations have been re-
sion, and DNA aneuploidy.19, 20, 67, 101, 156 Examina- ported in plasma DNA,30 but recent efforts have
tion of microdissected, preneoplastic lesions focused on detecting methylation changes in circu-
suggests that the earliest change is allele loss at lating DNA in blood samples.
chromosome region 3p, then 9p, 8p, 17p (with p53
mutation), and 5q, followed by RAS mutations.72,
87, 180, 207
There are data suggesting that KRAS acti- Tumor-acquired Promoter
vation also can occur at early stages, and muta- Hypermethylation as a Common Method
tions can be found in atypical alveolar hyperpla- of Inactivating Gene Expression in
sia.34, 101, 203 The genetic changes found in invasive Tumors
cancers and preneoplasia also can be identified in Methylation of CpG islands in the promoter re-
morphologically normal-appearing bronchial epi- gions of genes is a mechanism for controlling gene
thelium from current or former smokers.104, 208 The expression. Abnormalities of DNA methylation oc-
earliest change appearing in histologically normal cur consistently in human neoplasia and promoter-
epithelium seems to be allele loss at 3p, in about region hypermethylation may inactivate genes
50% of specimens from current or former smokers. transcriptionally, including TSGs (Fig. 5). In
There is also a progressive increase in the fre- NSCLCs, hypermethylation contributes to down-
quency and size of the areas undergoing allele loss regulation of p16 expression, occurs at an early
as they progress from normal to hyperplasia or stage in lung carcinogenesis,15, 109, 133 and correlates
metaplasia (representing mildly abnormal lesions) with smoking.85 An increasing number of genes
to dysplasia, to carcinoma in situ.207, 208 These ob- have been observed to undergo promoter methyla-
servations are consistent with ‘‘field canceriza- tion in lung cancer but not in associated normal
tion,’’ whereby the whole tissue region is exposed
lung, including p16, DAPK, GSTPI, MGMT, FHIT,
repeatedly to tobacco smoke and is at risk for
RARB, APC, CDHI3(HCAD), and RASSFIA22, 43, 195,
developing multiple, separate, clonally unrelated 220, 222
. Other regional sites of hypermethylation
foci of neoplasia, a notion supported by the wide-
have been found in lung cancer, including sites at
spread presence of aneuploidy in the respiratory
3p, 4q34, 10q26, and 17p13, although the precise
tree of smokers.173 A case report of a smoker with-
gene targets at these sites are uncertain and the
out invasive lung cancer with an identical somatic
significance not yet apparent.88, 103
p53 gene mutation at geographically dispersed
preneoplastic bronchial lesions is consistent with
the clonal expansion of a single progenitor clone Clinical Implications
throughout the respiratory tree.53
Hypermethylation of certain gene promoters
also may have relevance in predicting clinical out-
Diagnostic Implications
come for patients with lung cancer.22, 85 Because
The considerable inroads made in developing methylated DNA sequences can be found even in
new tools for the early detection of lung cancer, the setting of a high background of constitution-
including antibodies for immunostaining, poly- ally unmethylated normal DNA, they are attractive
merase chain reaction (PCR) techniques, and com- candidates for early molecular detection tools and
puterized image analysis, have been reviewed re- for following chemoprevention studies. A small

11. MOLECULAR BIOLOGY OF LUNG CANCER: CLINICAL IMPLICATIONS 93
Figure 5. Tumor acquired promoter hypermethylation leads to silencing of
gene expression. Schematic diagram of how hypermethylation of cytosine (C)
in CpG islands in the promoter region of a gene in a tumor cell may down-regulate
gene expression. Active gene expression often is associated with unmethylated
promoter CpG islands. In lung cancers, hypermethylation of promoter CpG is-
lands is associated with down-regulation of gene expression and thus is a
mechanism for inactivating tumor suppressor genes. These methylated DNA
products can be detected by methylation specific polymerase chain reaction
(PCR) in tissue or body fluid specimens, and the expression of these genes can
be restored by treatment with methylation inhibitors such as 5 aza-cytidine, which
is augmented by treatment with histone deacetylase inhibitors.
study reported the detection of aberrant methyla- have telomerase activity that is able to compensate
tion of p16 or O6-MGMT in DNA from sputum in for telomere shortening by replacing the hexameric
patients with squamous cell lung cancer up to 3 repeats, providing cellular ‘‘immortality.’’ Three
years before clinical diagnosis.135 In addition, ab- major components of human telomerase—RNA
normal identically methylated DNA can be de- component, telomerase-associated protein, and ca-
tected in serum from cases in which the tumor talytic subunit—frequently are overexpressed in
was methylated.44 Apart from potential diagnostic lung cancers, and activity can be determined by
use, the ability for methylation to be reversed with measuring telomerase enzyme activity (TRAP
drugs such as 5-aza-2 deoxycytidine leads to po- assay). 13 Nearly all SCLCs and 80% to 85% of
tential therapeutic developments. Retinoic acid, for NSCLCs have high levels of telomerase activity.6, 70
instance, plays an important role in lung develop- High telomerase activity is associated with in-
ment and differentiation, acting primarily through creased cell proliferation rates and advanced stage
nuclear receptors encoded by the retinoic acid re- in NSCLCs. Additionally, telomerase activity and
ceptor- (RAR- ) gene. RAR- often is hypermeth- dysregulated RNA expression frequently are
ylated in lung cancers, particularly SCLC,195 and found in carcinoma in situ lesions, implicating
chemical demethylation may provide an avenue involvement early in lung cancer development.215
for the re-expression of RAR- .
Clinical Implications
Telomerase Activation
The widespread presence of dysregulated te-
Another potential biomarker in lung cancer is lomerase in lung cancers has prompted examina-
telomerase activation. Human telomeres are struc- tion of its role as a molecular marker. Telomerase
tures located at the ends of chromosomes compris- positivity can be found in bronchoalveolar lavage
ing TTAGGG tandem DNA repeats bound to spe- fluid and may provide a useful adjunct to tradi-
cific proteins. During normal cell division, the tional cytology.211 On the other hand, it seems to
absence of telomerase activity is associated with have limited value in the molecular staging of
progressive telomere shortening, perhaps repre- established NSCLC. 5 The development of te-
senting an intrinsic ‘‘cellular clock,’’ leading to nor- lomerase inhibitors is being pursued in new thera-
mal cell ‘‘senescence’’ and ‘‘mortality.’’ In contrast, peutic drug development.
germ cells, some stem cells, and most cancer cells Biomarkers based on other molecular mutations

12. 94 FONG & MINNA
also hold promise for early detection, although sion), BCL-xL antisense in NSCLC, and a bispecific
sensitivity remains an issue. Some 15% to 25% BCL-2–BCLxL antisense to target SCLC and
of patients with lung cancer develop antibodies NSCLC.
against the p53 protein, for instance, indicating
that mutant p53 protein overexpression can lead
to a humoral immune response. Whether serum Angiogenesis
p53 antibodies can be used as a diagnostic marker
Tumors cannot exceed 1 to 2 mm3 volume with-
is presently unclear.95, 102, 114 Additionally, p53 muta-
out developing new blood vessels and therefore
tions can be detected in DNA from tumor cells in
require angiogenic factors early in their pathogene-
plasma.169
sis. Microvessel density may be regarded as a mor-
phologic measure of tumor angiogenesis and has
been linked variably to metastases and impaired
MOLECULAR CHANGES IN LUNG
survival in lung cancer.10, 49, 124, 136 Tumor angiogen-
CARCINOGENESIS: PROGNOSTIC
esis is complex and controlled by a diverse family
IMPLICATIONS
of inducers and inhibitors governing angiogenesis
and regulating endothelial cell proliferation and
Refining the staging of lung cancer with reliable
migration. 61 Vascular endothelial growth factor
and robust prognostic markers may allow the indi-
(VEGF) and basic fibroblast growth factor are im-
vidualization of therapy. Molecular abnormalities,
portant angiogenesis inducers. Vascular endothe-
being a fundamental step in tumorigenesis, may
lial growth factor expression is associated with
prove to be useful in this context.36, 94, 115 Before
intratumoral microvessel density and tumor-cell
adoption into routine clinical use, however, large,
proliferation in squamous lung cancer.7, 51, 108 Vascu-
high-quality, prospective studies of potential bio-
lar endothelial growth factor expression is higher
markers are required to validate their role because
in lung cancers with nodal metastasis than those
most studies have been retrospective, relatively
without126 and is associated with poor prognosis
small in size, possibly exposed to bias such as
in NSCLC.51, 196 In squamous cell carcinomas, the
selection bias, examined markers with different
VEGF receptor, Flt-1, is expressed frequently, sug-
techniques, and came to different conclusions.
gesting a possible autocrine role of VEGF.196 Mu-
Meta-analyses of p53 as a potential prognostic bio-
tant p53 acts synergistically with hypoxia to in-
marker have been reported and serve to highlight
duce VEGF expression,84 whereas wild-type p53
the heterogeneity of the studies to date.71, 112 Apart
up-regulates expression of the antiangiogenic pro-
from the prognostic relationships of the various
tein, TSP-1.116 Fibroblast growth factor is expressed
biomarkers discussed earlier, other potentially im-
in approximately 70% of NSCLCs, but its prognos-
portant biomarkers of lung cancer progression and
tic significance is controversial.186, 197
outcome are described herewith.
Therapeutic implications: There are multiple
new therapeutic strategies in lung cancer directed
against angiogenesis in general and the VEGF sys-
Apoptosis
tem in particular. There currently are clinical trials
testing rhuMAbVEGF, an antiangiogenic human-
Tumor cells often escape the normal physiologic
ized monoclonal antibody. Other potential agents
response termed programmed cell death or
include a monoclonal antibody against VEGF re-
apoptosis when challenged by cellular and DNA
ceptor 2 (KDR/FLK1, ImCone), a tyrosine kinase
damage. A key player in apoptosis is the product
inhibitor (SU5416, Sugen), and a ribozyme directed
of the breakpoint cluster lymphoma (BCL2) antia-
against the VEGF receptor 1 (Flt-1) mRNA (Angio-
poptotic proto-oncogene. Its expression is higher
zyme).
in SCLCs (75%–95%) than in NSCLCs,75, 78 in which
expression is greater for squamous cell carcinoma
than adenocarcinoma.12, 66, 75, 138 The former finding Molecules Involved in Metastatic
is interesting because SCLCs are more responsive Behavior
to chemotherapy, a modality generally resulting in
tumor apoptosis. Clinically, there is a trend toward Tumor metastasis is a complex, multistep, host-
longer survival in SCLCs that express BCL-2,78 but dependent process. Cell adhesion molecules have
this relationship is controversial in NSCLC.12, 50, 66, 138 been implicated in epithelial differentiation, carci-
BAX counteracts BCL-2 and promotes apoptosis nogenesis, and metastasis. E-cadherin is responsi-
and is a downstream transcription target of p53. ble for the organization, maintenance, and mor-
Expression of BAX and BCL-2 is related inversely phogenesis of epithelial tissues, and reduced
in neuroendocrine cancers; most typical and atypi- expression has been associated with tumor dedif-
cal carcinoids have low BCL-2 and high BAX ex- ferentiation, increased lymphogenous metastasis,
pression, with the reverse in most SCLCs and large and poor survival rates in NSCLC.181 The integrins
cell neuroendocrine cancers.21 Therapeutic Implica- that bind to collagen and laminin in basement
tions: Among the anti-apoptosis strategies in pre- membranes are down-regulated in NSCLC.174 Re-
clinical trials are studies of antisense BCL-2 in duced integrin- 3 expression is linked with poor
SCLC (to down-regulate BCL-2 protein expres- prognosis in adenocarcinomas.3 Others have re-

13. MOLECULAR BIOLOGY OF LUNG CANCER: CLINICAL IMPLICATIONS 95
ported that the v6 isoform of the CD44 lymphocyte 3. Adachi M, Taki T, Huang C, et al: Reduced integrin
homing receptor correlates with adverse prognosis 3 expression as a factor of poor prognosis of pa-
in NSCLC.68 The degradation of the extracellular tients with adenocarcinoma of the lung. J Clin Oncol
matrix and basement membrane, particularly type 16:1060–1067, 1998
4. Agus DB, Bunn PA Jr, Franklin W, et al: HER-2/neu
IV collagen, by tumor cells is essential for invasion
as a therapeutic target in non–small-cell lung cancer,
and metastasis. Type IV collagen expression of ad- prostate cancer, and ovarian cancer. Semin Oncol
enocarcinomas is lost progressively with increas- 27:53–63; discussion 92–100, 2000
ing tumor dedifferentiation.32 5. Ahrendt SA, Yang SC, Wu L, et al: Comparison of
Therapeutic implications: The matrix metallo- oncogene mutation detection and telomerase activ-
proteinase enzymes that degrade stroma and ity for the molecular staging of non-small cell lung
therefore could lead to tumor invasion and metas- cancer. Clin Cancer Res 3:1207–1214, 1997
tasis include collagenases, gelatinases, stromely- 6. Albanell J, Lonardo F, Rusch V, et al: High te-
sins, membrane-type matrix metalloproteinases, lomerase activity in primary lung cancers: Associa-
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vanced pathologic stage. J Natl Cancer Inst 89:
instance, is expressed in SCLCs ( 50%) and
1609–1615, 1997
NSCLCs ( 65%).80 Stromelysin-3 has been shown 7. Ambs S, Bennett WP, Merriam WG, et al: Vascular
to be overexpressed by stromal elements in pri- endothelial growth factor and nitric oxide synthase
mary NSCLCs compared with adjacent normal expression in human lung cancer and the relation
lung specimens.9 Several matrix metalloproteinase to p53. Br J Cancer 78:233–239, 1998
inhibitors (Prinomastat, Marimastat, and BAY 8. Amos CI, Xu W, Spitz MR: Is there a genetic basis
129566) are in clinical trials. for lung cancer susceptibility? Recent Results Can-
cer Res 151:3–12, 1999
9. Anderson IC, Sugarbaker DJ, Ganju RK, et al: Stro-
SUMMARY melysin-3 is overexpressed by stromal elements in
primary non-small cell lung cancers and regulated
This review summarizes the rapidly expanding by retinoic acid in pulmonary fibroblasts. Cancer
Res 55:4120–4126, 1995
knowledge of the molecular pathogenesis of lung
10. Angeletti CA, Lucchi M, Fontanini G, et al: Prognos-
cancer. It is clear that respiratory epithelial cells tic significance of tumoral angiogenesis in com-
require many genetic alterations to become inva- pletely resected late stage lung carcinoma (stage
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plastic and malignant cells, therefore offering the 11. Antoniades HN, Galanopoulos T, Neville-Golden J,
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tored prevention and early detection efforts. Such lung carcinomas coexpress in vivo platelet-derived
molecular screening and detection efforts will growth factor (PDGF) and PDGF receptor mRNAs
and their protein products. Proc Natl Acad Sci U S
likely be coupled to advances in low-dose com-
A 89:3942–3946, 1992
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mography scans, and other imaging modalities.63, Prognostic value of the expression of p53, bcl-2, and
73, 175, 218
Although this molecular marker approach bax oncoproteins, and neovascularization in pa-
has great potential, there is not yet a molecular tients with radically resected non-small-cell lung
marker validated in large prospective trials that cancer. J Clin Oncol 15:2456–2466, 1997
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