Pathogenesis of lung adenocarcinoma

Several molecular changes frequently present in lung adenocarcinomas are also present in AAH lesions, and they are further evidence that AAH may represent true preneoplastic lesions (Figure 5.3).

The most important finding is the presence of KRAS(codon 12) mutations in up to 39% of AAHs,
which are also a relatively frequent alteration in lung adenocarcinomas [15,56]. Other molecular
alterations detected in AAH are overexpression of Cyclin D1 (∼70%), p53 (ranging from 10 to
58%), survivin (48%), and HER2/neu (7%) proteins overexpression [15,57,58]. Some AAH le-
sions have demonstrated LOH in chromosomes 3p (18%), 9p (p16INK4a, 13%), 9q (53%), 17q, and 17p (TP53, 6%), changes that are frequently detected in lung adenocarcinomas [59,60]. A study on lung adenocarcinoma with synchronous multiple AAHs showed frequent LOH of tuberous sclerosis complex (TSC)-associated regions (TSC1 at 9q,53%, and TSC2 at 16p, 6%), suggesting that theseare candidate loci for tumor suppressor gene in asubset of adenocarcinomas of the lung [60]. Activation of telomerase expressed by expression of
human telomerase RNA component (hTERC) and telomerase reverse transcriptase (hTERT) mRNA, has been detected in 27–78% of AAH lesions, depending in their atypia level [61]. Recently, it has been shown that loss of LKB1, a serine/threoninekinase that functions as a tumor suppressor gene, isfrequent in lung adenocarcinomas (25%) and AAH (21%) with severe cytological atypia, while it is rare in mild atypical AAH lesions (5%), suggesting that
LKB1 inactivation may play a role in the AAH progression to malignancy [62].
Several mouse models have been developed to better study various oncogenic molecular signaling pathways and the sequence of molecular events involved in the pathogenesis of peripheral lung tumors, and to test novel chemopreventive agents [63]. The KRAS oncogenic mouse model is characterized for the development of peripheral alveolar type of proliferations, including AAH, adenoma,and adenocarcinoma [63]. Using this mouse model,
several important findings that need to be further validated in human tissues have been reported.Kim et al. [64] identified the potential stem cell population (expressing Clara cells-specific protein and surfactant protein-C, termed bronchioalveolar stem cell, BASC) that maintains the bronchiolar Clara cells and alveolar cells of the distal respiratory epithelium and which could be considered the precursors of lung KRAS neoplastic lesions in
mice. Wislez et al. [50] provided evidence that the expansion of lung adenocarcinoma precursors
induced by oncogenic KRAS requires mammalian target of rapamycin (mTOR)-dependent signalingand, most importantly, that inflammation-related host factors, including factors derived from macrophages, play a critical role in mice adenocarcinoma progression. Recent findings reported by Collado et al. [65], suggest that KRAS oncogeneinduced senescence may help to restrict tumor progression of lung peripheral lesions in mice. They discovered that a substantial number of cells in mice premalignant alveolar type of lesions undergooncogene-induced senescence, but the cells that escape senescence by loss of oncogene-induced senescence effectors, such as p16INK4a or p53,progress to malignancy. Thus, senescence is a defining feature of premalignant lung lesions, but not invasive tumors.

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References9 Hanahan D, Weinberg RA. The hallmarks of cancer. Cell Jan 7, 2000; 100(1):57–70. 10 Weinstein IB. Cancer. addiction to oncogenes—the Achil

1 Mazin B, Qumsiyeh YY.Molecular methods in oncol-
ogy: cytogenetics. In: Vincent T, Devita SH, Rosen-
berg SA (eds). Cancer: Principles and Practice of Oncology,
7th edn. Philadelphia: Lippincott,Williams &Wilkins,
2005.
2 Nowell PC. The clonal evolution of tumor cell popu-
lations. Science Oct 1, 1976; 194(4260):23–8.
3 Varmus HE. Nobel lecture. Retroviruses and onco-
genes. I. Biosci Rep Oct 1990; 10(5):413–30.
4 Todaro GJ, Huebner RJ. The viral oncogene hypoth-
esis: new evidence. Proc Natl Acad Sci U S A 1972;
69:1009–15.
5 Knudson AG, Jr. Mutation and cancer: statistical
study of retinoblastoma. Proc Natl Acad Sci U S A Apr
1971; 68(4):820–3.
6 Friend SH, Bernards R, Rogelj S et al. A human DNA
segment with properties of the gene that predisposes
to retinoblastoma and osteosarcoma. Nature Oct 16–
22, 1986; 323(6089):643–6.
7 Futreal PA, Coin L, Marshall M et al. A census of hu-
man cancer genes. Nat Rev Mar 2004; 4(3):177–83.
8 SjoblomT, Jones S,Wood LD et al. The consensus cod-
ing sequences of human breast and colorectal cancers.
Science Oct 13, 2006; 314(5797):268–74.3p21.3: identification and evaluation of the resi-
dent candidate tumor suppressor genes. The Inter-
national Lung Cancer Chromosome 3p21.3 Tumor
Suppressor Gene Consortium. Cancer Res Nov 1, 2000;
60(21):6116–33.
25 Ji L, Nishizaki M, Gao B et al. Expression of several
genes in the human chromosome 3p21.3 homozy-
gous deletion region by an adenovirus vector results
in tumor suppressor activities in vitro and in vivo.
Cancer Res May 1, 2002; 62(9):2715–20.
26 Sekido Y, Fong KM, Minna JD. Molecular genetics of
lung cancer. Annu Rev Med 2003; 54:73–87.
27 Wistuba II, Gazdar AF. Lung cancer preneoplasia.
Annu Rev Pathol Mech Dis 2006; 1(1):331–48.
28 Wistuba II, Mao L, Gazdar AF. Smoking molecular
damage in bronchial epithelium. Oncogene Oct 21,
2002; 21(48):7298–306.
29 Franklin WA, Gazdar AF, Haney J et al. Widely dis-
persed p53 mutation in respiratory epithelium. A
novel mechanism for field carcinogenesis. J Clin In-
vest Oct 15, 1997; 100(8):2133–7.
30 Walsh CP, Chaillet JR, Bestor TH. Transcription of IAP
endogenous retroviruses is constrained by cytosine
methylation. Nat Genet Oct 1998; 20(2):116–7.
31 Yoder JA, Walsh CP, Bestor TH. Cytosine methyla-
tion and the ecology of intragenomic parasites. Trends
Genet Aug 1997; 13(8):335–40.
32 Gaudet F, Hodgson JG, Eden A et al. Induction of tu-
mors in mice by genomic hypomethylation. Science
Apr 18, 2003; 300(5618):489–92.
33 Eden A, Gaudet F,Waghmare A, Jaenisch R. Chromo-
somal instability and tumors promoted by DNA hy-
pomethylation. Science Apr 18, 2003; 300(5618):455.
34 Zochbauer-Muller S, LamS, Toyooka S et al. Aberrant
methylation ofmultiple genes in the upper aerodiges-
tive tract epithelium of heavy smokers. Int J Cancer
Nov 20, 2003; 107(4):612–6.
35 Zochbauer-Muller S, Fong KM, Virmani AK,
Geradts J, Gazdar AF, Minna JD. Aberrant promoter
methylation of multiple genes in non-small cell lung
cancers. Cancer Res Jan 1, 2001; 61(1):249–55.
36 Belinsky SA, Liechty KC, Gentry FD et al. Promoter
hypermethylation of multiple genes in sputum pre-
cedes lung cancer incidence in a high-risk cohort.
Cancer Res Mar 15, 2006; 66(6):3338–44.
37 Baylin SB, Ohm JE. Epigenetic gene silencing in
cancer—a mechanism for early oncogenic pathway
addiction? Nature Rev Feb 2006; 6(2):107–16.
38 Shames DS, Minna JD, Gazdar AF. DNA methylation
in health, disease, and cancer. CurrMolMed Feb 2007;
7(1):85–102.
39 Esteller M, Corn PG, Baylin SB, Herman JG. A gene
hypermethylation profile of human cancer. Cancer Res
Apr 15, 2001; 61(8):3225–9.
40 Baylin SB, Belinsky SA, Herman JG. Aberrantmethy-
lation of gene promoters in cancer—concepts, mis-
concepts, and promise. JNatl Cancer Inst Sep 20, 2000;
92(18):1460–1.
41 Bestor TH. Unanswered questions about the role of
promotermethylation in carcinogenesis. AnnNYAcad
Sci Mar 2003; 983:22–7.
42 Chan AO, Broaddus RR, Houlihan PS, Issa JP,
Hamilton SR, Rashid A. CpG island methylation in
aberrant crypt foci of the colorectum. AmJ PatholMay
2002; 160(5):1823–30.
43 Shivapurkar N, Stastny V, Suzuki M et al. Application
of a methylation gene panel by quantitative PCR for
lung cancers. Cancer Lett Apr 25, 2006; 247(1):56–71.
44 Zochbauer-Muller S, Minna JD, Gazdar AF. Aberrant
DNA methylation in lung cancer: biological and clin-
ical implications. Oncologist 2002; 7(5):451–7.
45 Issa JP. CpG island methylator phenotype in cancer.
Nat Rev Dec 2004; 4(12):988–93.
46 Sekido Y, Fong KM, Minna JD. Progress in un-
derstanding the molecular pathogenesis of human
lung cancer. Biochim Biophys Acta Aug 19, 1998;
1378(1):F21–59.
47 Roth JA, Nguyen D, Lawrence DD et al. Retrovirus-
mediatedwild-type p53 gene transfer to tumors of pa-
tients with lung cancer. Nat Med Sep 1996; 2(9):985–
91.
48 Gabrilovich DI. INGN 201 (Advexin): adenoviral p53
gene therapy for cancer. Expert Opin Biol Ther Aug
2006; 6(8):823–32.
49 Wikman H, Nymark P, Vayrynen A et al. CDK4 is a
probable target gene in a novel amplicon at 12q13.3-
q14.1 in lung cancer. Genes Chromosomes Cancer Feb
2005; 42(2):193–9.
50 Ratschiller D, Heighway J, Gugger M et al. Cyclin D1
overexpression in bronchial epithelia of patients with
lung cancer is associated with smoking and predicts
survival. J Clin Oncol Jun 1, 2003; 21(11):2085–93.
51 Burbee DG, Forgacs E, Zochbauer-Muller S et al. Epi-
genetic inactivation of RASSF1A in lung and breast
cancers and malignant phenotype suppression. J Natl
Cancer Inst May 2, 2001; 93(9):691–9.
52 Dammann R, Li C, Yoon JH, Chin PL, Bates S, Pfeifer
GP. Epigenetic inactivation of a RAS association do-
main family protein fromthe lung tumour suppressor
locus 3p21.3. Nat Genet Jul 2000; 25(3):315–9.
53 Kondo M, Ji L, Kamibayashi C et al. Overexpression
of candidate tumor suppressor gene FUS1 isolated

Tumor suppressor genes 4

RAS/RAF/MEK/ERK pathway
The RAS family of proto-oncogenes (HRAS, KRAS,
and NRAS) are 21-kD plasma membrane-associated
G-proteins that regulate key signal transduction
pathways involved in normal cellular differenti-
ation, proliferation, and survival [104]. Multiple
studies have shown that oncogenic KRAS (e.g.,
KRASV12 mutant) activates cell signaling pathways
important to cellular transformation [105]. As a
result, KRAS abnormalities represent an impor-
tant therapeutic target. RAS mutations (nearly al-
ways KRAS mutations in lung cancer) are found in
15–20% of NSCLCs, especially in adenocarcinomas
(20–30%), but never in SCLCs [26]. The mutations
occur in codons 12, 13, and 61, all of which in-
fluence intrinsic GTPase activity [104]. A number
of drugs that target different aspects of RAS func-
tion and metabolism have been developed and are
currently under clinical investigation [104]. These
include the farnesyl transferase inhibitors tipifarnib
and lonafarnib, which are now being tested in thecombination with cytotoxic drugs in phase III clini-
cal trials [106].
BRAF protein serine/threonine kinase is a down-
stream effecter of the Ras pathway and mutations
of BRAF occur in ∼70%melanoma, but in only 3%
of lung cancers [107–109]. However, for those rare
lung cancers, mutated BRAF protein is a potentially
important and specific therapeutic target. An orally
administered Raf kinase inhibitor, BAY 43-9006 (so-
rafenib), is currently being tested in phase I and
phase II trials in lung cancer [110–111].
Activated BRAF phosphorylates and activates
MEK1 and MEK2, which in turn phosphorylate
and activate ERK1 and ERK2. However, MEK or
ERK gene amplification ormutations have not been
found in lung cancers. Nevertheless, ERK1/ERK2
are constitutively activated in a subset of lung can-
cers and MEK and ERK remain therapeutic tar-
gets for lung cancer treatment using an oral MEK
inhibitor CI-1040 and its derivative PD03255901
[112].

Tumor suppressor genes 3

Receptor tyrosine kinases
The EGFR family
The EGFR family of receptors are transmembrane
TK receptors and are composed of EGFR, HER2,
HER3, and HER4 and each has unique proper-
ties. For example, HER2 lacks a functional ligand-
binding domain and HER3 lacks kinase activity [63].
Upon ligand binding, these EGFR family members
form active homo- and hetero-dimers, leading to
autophosphorylation and activation of intracellular
signaling cascades. EGFR is overexpressed in ∼70%
ofNSCLCs but rarely expressed in SCLCs [64]. There
are several drugs targeting EGFR or HER2 currently
available including the small molecule TKIs, gefi-
tinib, erlotinib, and the monoclonal antibodies, ce-
tuximab (targeting EGFR), and trastuzumab (target-
ing HER2).
Recently, several mutations in the TK domain of
EGFR have been described, and are not infrequent in
NSCLC (10–20%), but never occur in SCLC [65,66].
Of interest is that TK domain mutations are almost
exclusive to lung cancer, whereas intracellular re-
gion mutations are found in glioblastomas. In lung
cancer, these mutations are limited to the first four
exons of the TK domain and are categorized into
three different types (deletions, insertions, and mis-
sense point mutations). Inframe deletions in exon19 (44% of all mutations) and missense mutations
in exon 21 (41% of all mutations) are the most fre-
quent, accounting for more than 80% of all muta-
tions [67]. Importantly, the presence of mutations
in TK domain correlates with the drug sensitivity to
TKIs [65,66]. An intriguing characteristic of EGFR
mutations is that they occur in a highly selected
subpopulation: female East-Asian never smokers
with adenocarcinoma histology [68]. Notably, be-
fore EGFR mutations were discovered, all of same
clinicopathological factors were found to be associ-
ated with tumor responses to TKIs [69,70].
Although several studies have confirmed the re-
lationship between the presence of mutant EGFR
and the response to TKIs [65,66,71], a subset of
NSCLC patients with mutant EGFRs do not respond
to TKIs. These tumors often (>50%) have a “sec-
ond” TK domain mutation (T790M) usually found
in patientswho relapse after TKI treatment, suggest-
ing its contribution to acquired resistance to TKIs
[72,73]. However, several examples of the T790M
mutations occur in lung tumors not treated with
EGFR TKIs, and often themutation is only in a small
subset of the tumor cells. This contrasts with the
other EGFR TK domain mutations, which are in all
tumor cells.Also, a germline EGFR T790Mmutation
was reported to be associated with familial NSCLC,
suggesting that this mutation could predispose peo-
ple to lung cancer [74]. Fortunately, there are EGFR
TKIs that inhibit EGFR with the T790M mutation,
and these drugs are currently under clinical evalu-
ation [75].
Some patients without EGFR mutation also re-
spond to TKIs, and several predictive markers other
than EGFR mutation have been reported to corre-
late with TKI response, including EGFR amplifica-
tion, elevated EGFR protein, HER2 amplification,
HER3 amplification, and activation of AKT [76–
80]. In fact, KRAS mutations and EGFR mutations
are mutually exclusive. KRAS mutations are asso-
ciated with cigarette smoking, while EGFR muta-
tions generally occur in never smokers [81]. These
studies suggest that other biological features be-
sides EGFRmutation status determine TKI response.
Among biologic predictors, EGFR mutation and
amplification by fluorescence in situ hybridization
are highly correlated with TKI response whileEGFR protein expression gives conflicting results
[65,66,71,76,82]. There is also the possibility that
tumors with EGFR mutations are associated with
better survival independent of TKI treatment. Thus,
all survival studies after TKI treatment need to have
molecular analyses for comparison [80,83,84]. Two
well-controlled phase III studies were conducted for
these drugs. The results of these studies showed that
erlotinib prolonged survival of previously treated
NSCLC patients by 2 months (BR21 trial), while
gefitinib failed to show survival benefit (Iressa Sur-
vival Evalulation in Lung Cancer (ISEL)) [86,87].
Despite positive preclinical studies of the combi-
nation of TKI and chemotherapy, several phase
III studies have failed to show a survival bene-
fit of adding erlotinib or gefitinib to conventional
chemotherapy [88,89]. Finally, lung cancers with
EGFR mutations are more sensitive to ionizing ra-
diation than those without EGFR mutations, which
potentially provides a molecular basis for combined
modality treatment involving TKIs and radiother-
apy [90].
While standard criteria for selecting patients with
NSCLC for TKI therapy are being developed, in prac-
tice, East-Asian female patients with tumors that
have EGFR mutations or EGFR amplification and
that are never smokers often receive TKI therapy. To
address this issue, prospective clinical trials designed
to incorporate the patient’s clinicopathological data
as well as molecular biological features (EGFR mu-
tation and/or amplification) of the tumors are cur-
rently underway.
HER2 mutations occur in 2% of NSCLCs. All re-
ported HER2 mutations are in-frame insertions in
exon 20 and target the corresponding TK domain
region as in EGFR insertion mutations and occur in
the same subpopulation as those with EGFR muta-
tions (adenocarcinoma, never smoker, East Asian,
and woman) [68,91,92]. So far no small molecule
inhibitors show similar potency against HER2muta-
tions as seenwith EGFR TKIs and studies are needed
to see if mutant HER2 lung cancers respond to the
anti-HER2 antibody trastuzumab. HER4 mutations
were found in (2.3%) NSCLC tumor samples from
Asian patients including male smokers [93].
EGFR mutations occur as preneoplastic lesions
occurring in histologically normal bronchial epithe-lial cells adjacent to tumors with EGFR mutations.
The discovery of EGFR mutations could be used
as an early detection marker and chemoprevention
target [94]. Transgenic mice with either EGFR point
mutations or deletion mutations develop lung ade-
nocarcinomas with similar histology to those seen
in patients [95,96]. When the mutant gene was
“turned-off” in the mice through controlled gene
expression the lung tumors all regressed indicating
thatmutant EGFR is required for both initiation and
maintenance of the tumors.
c-KIT
SCLC but not NSCLC frequently express (40–70%)
both the receptor c-KIT and its ligand, stem cell fac-
tor (SCF) suggesting an autocrine loopmay promote
the growth of the SCLC cells [97]. However, unlike
gastrointestinal stromal tumors which frequently
contain c-KITmutations, activating c-KITmutations
are very rare in lung cancer [98,99].While imatinib,
an inhibitor of c-KIT kinase, inhibits cell growth in
some c-KIT expressing SCLC cell lines in vitro, two
phase II clinical studies and amouse xenograft study
failed to showtumor regression in SCLC by imatinib
monotherapy [100–103].

Tumor suppressor genes 2

3p tumor suppressor genes
Allele loss in 3p, including LOH and homozygous
deletion, occurs in nearly 100%of SCLCs and more
than 90% of NSCLCs and is one of the earliest
events in lung cancer development. Because of the
early changes in chromosome region 3p21.3 (oc-
curring in histologically normal lung epithelium)
the presence of 3p allele loss and inactivation of
expression of these 3p TSGs can be of use in de-
termining smoking related field effects. Three dis-
creet regions of 3p loss have been identified by
allelotyping in lung cancers, including, a 600-kb
segment in 3p21.3, the 3p14.2 (FHIT/FRAB3), and
the 3p12 (ROBO1/DUTT1) regions. The 3p21.3 re-
gion has been analyzed most extensively and 25
genes were identified from this region.
One of the best studied genes in this region is
RASSF1A, which is rarely mutated in lung cancer
but whose expression is frequently lost by tumor
acquired promoter methylation [51,52]. RASSF1A
is involved in multiple pathways critical to can-
cer pathogenesis, including cell cycle, apoptosis,
and microtubule stability. RASSF1A is methylated
in ∼90% of SCLCs and ∼40% of NSCLCs and has
the ability to suppress the growth of lung cancer
cell lines in tissue culture and as xenografts in nude
mice [51,52].
FUS1 is located next to RASSF1A and one of the
two alleles of the gene is often lost in lung cancers.
FUS1 is rarelymutated in lung cancers, does not un-
dergo promoter hypermethylation, yet the protein
product of this gene is frequently lost in lung can-
cer compared to normal lung tissues [53].Wild-type
FUS1 but not tumor-acquiredmutant FUS1 induces
G1 growth arrest and apoptosis [53].Administration
of FUS1 with in DOTAP:cholesterol (DOTAP:Chol)
nanoparticles (FUS1-nanoperticles) inhibits cancercell growth in vitro and in vivo. These preclinical
studies provide a basis for FUS1 gene therapy clin-
ical trials for the treatment of lung tumors using
FUS1-nanoparticles [54,55].
Two other 3p21.3 candidate tumor suppressor
genes, Semaphorin 3B (SEMA3B) and a family
member SEMA3F, are extracellular secreted mem-
bers of the semaphorin family, and are impor-
tant in axonal guidance. Wild-type SEMA3B, but
not missense mutant SEMA3B, induces apopto-
sis when re-expressed in lung cancers or added
as a soluble molecule [56,57]. Overexpression of
SEMA3F in tissue culture results in inhibition of
tumor cell growth and tumor cell invasion. Both
SEMA3B and SEMA3F are soluble, secreted pro-
teins, and therefore are promising candidates for
drug development.
Two other 3p genes with evidence to support
their candidacy as tumor suppressors are FHIT and
retinoic acid receptor beta (RARβ). FHIT is located
in 3p14.2, one of the most common fragile sites of
the human genome. FHIT is either homozygously
deleted or expresses aberrant transcripts in more
than 50% of lung cancers [58]. In addition, FHIT
overexpression induces apoptosis in lung cancer
cells. RARβ is located at 3p24 and functions as a
receptor for retinoic acid (RA). Although the RARβ
gene is not mutated in lung cancer, it undergoes
methylation in 72% of SCLCs and 41% of NSCLCs,
leading to loss of its expression [59]. Re-expression
of RARβ in lung cancer cell lines suppresses their
growth in the culture and nude mice [60].
Oncogenes and the pathways
they regulate
While there are multiple components to each of the
growth signaling pathways involved in lung can-
cer, we will focus the discussion on those proteins
that are frequently affected by genetic abnormalities
in cancer. It has become clear that these mutated
proteins, while driving cells toward transformation,
also “addict” the cells to their abnormal function.
This concept is referred to as “oncogene addiction”
and represents a cellular physiologic statewhere thecontinued presence of the abnormal function,while
oncogenic, also becomes required for the tumor to
survive [61]. This means that if the function is re-
moved or inhibited, for example, by a targeted drug,
the tumor cells die. By contrast, bystander normal
cells, which are not “addicted” to the mutant pro-
tein, are much less sensitive to the drug; thus, the
targeted drugs have great tumor cell specificity. The
most important example of this concept for lung
cancer is EGFR. Tumors withmutations in EGFR are
dependent on survival signals transduced by mu-
tant EGFR, and thus are particularly sensitive to ty-
rosine kinase inhibitors (TKIs) [62]. These findings
have led to massive genome-wide sequencing ef-
forts (discussed above) targeting thousands of genes
to find additional mutated oncogene targets for ra-
tional therapeutics design.

Tumor suppressor genes

Several key tumor-suppressor pathways are fre-
quently inactivated in lung cancer. These include
the p53 and the p16INK4a
—CyclinD1-CDK4-RB
pathways.
The p53 pathway
The tumor suppressor gene p53 is the most fre-
quently mutated gene in human cancer, and p53
is inactivated by mutation in ∼90% of SCLCs and
∼50% of NSCLCs, respectively [26,46]. Most in-
activating mutations in p53 are caused by point
mutations in the DNA-binding domain (missense
mutation, 70–80%) of one parental allele and LOH
(deletion) of the other. Occasionally homozygous
deletions are observed. p53 is located at chromo-
some 17p13.1, and codes for a protein that functions
as a key transcription factor. The transcriptional tar-
gets of p53 include a number of cell cycle regula-
tory proteins such as p21 andMYC, as well as many
proteins involved in apoptosis such as BAX, 14-3-
3σ, and GADD45. p53 regulation occurs primarily atthe level of protein stability. p53 controls transcrip-
tion of MDM2, an E3 ubiquitin ligase, which in turn
regulates p53 stability in a feedback loop. This par-
ticular connection in the p53 pathway is a frequent
target of dysregulation in tumor cells.
The p53 pathway is activated in response cel-
lular stress and DNA damage induced by gamma-
irradiation, ultraviolet light, DNA damaging drugs,
and carcinogens. p53 stabilization results in the
expression of downstream genes, which induces
either cell cycle arrest to permit DNA repair, or
programmed cell death when there is too much
damage. Loss of p53 function allows cells to di-
vide in spite of genetic damage, which can result
the clonal expansion of premalignant cells. In most
cases, only mutant, missense p53 is present because
of LOH involving thewild-type p53 allele. However,
in some cases, mutant p53 proteins can form het-
erodimers with wild-type p53 inactivating its tumor
suppressive function even before LOH. These “gain-
of-function” mutations contribute to increased tu-
morigenicity and invasiveness of several types of
cancers [26,46]. However, despite large-scale stud-
ies, it is not clear whether NSCLCs with p53 muta-
tions have impaired survival compared to lung can-
cers with only wild-type p53.
There are two important upstream regulators
in the p53 pathway: MDM2 and p14ARF. MDM2
functions as an oncogene by reducing p53 levels
through enhancing proteasome-dependent degra-
dation. Amplifications of MDM2 were reported in
∼7% (2/30) of NSCLCs, resulting in loss of p53
function [46]. p14ARF
derives from the p16 locus
with an alternatively spliced 5-exon that results in
an alternative reading frame for translation. p14 en-
codes a protein that binds toMDM2 thereby inhibit-
ing its ubiquitination activity,which leads to the sta-
bilization of p53. Immunohistochemistry analyses
of p14ARF on lung cancers have shown that p14ARF
protein expression was lost in ∼65% of SCLCs and
∼40% of NSCLCs. Thus, through p53 mutation or
changes in MDM2 or p14, the p53 pathway is inac-
tivated in the majority of all lung cancers.
Lung cancer cells are addicted to loss of p53 func-
tion. When wild-type p53 is re-expressed in lung
cancer cells with mutant or deleted p53, the tumor
cells undergo apoptosis. These findings have led to
clinical trials of p53 gene replacement therapy. The
results frompreclinical and early-stage clinical trials
of p53 gene replacement therapy using a replication
incompetent retrovirus p53 expression vector in pa-
tients with NSCLCs, show evidence of antitumor
activity and the feasibility and safety of gene ther-
apy [47]. INGN 201 (Ad5CMV-p53, AdvexinTM),
a replication-impaired p53 adenoviral vector has
been evaluated in clinical trials, and is both safe and
effective for the treatment of several different types
of cancer [48]. This treatment has been approved in
China for the treatment of primary head and neck
cancers in combination with radiation therapy and
is currently undergoing phase III trials in head and
neck cancer in the United States.
The RB pathway
The RB pathway plays a central role in G1/S
cell transition. Hypophosphorylated RB exerts its
growth suppressive effect by binding to and inhibit-
ing the E2F transcription factor, which promotes
cells through the G1/S transition. RB is phosphory-
lated by the CyclinD1/CDK4 complex. Once these
kinases phosphorylate RB, it releases E2F, resulting
in transition from G1 to S. Thus, loss of RB function
though deletion ormutation leads to loss of theG1/S
checkpoint, and is a common event in lung cancer,
particularly SCLCs (>90%), while inactivation of
RB is found in 15–30% of NSCLCs [26].
The activity of the CDK4/Cyclin D1 complex is
regulated by p16. p16 keeps RB hypophosphory-
lated (and growth suppressingmode) by preventing
CDK4 from phosphorylating RB. Thus, loss of p16
function results in loss of function of the RB path-
way. By contrast to RB, p16 is more frequently in-
activated in NSCLCs (∼70%) than in SCLCs (10%)
[26]. Inactivation of p16 is caused by LOH coupled
with deletion, intragenic mutations or promoter
hypermethylation of the remaining allele. In lung
cancer, promoter methylation is the most frequent
method of inactivation of p16.
Overexpression of either CDK4 or Cyclin D1
inhibits RB pathway function by saturating the
growth suppressive activity of p16. CDK4 is am-
plified in some cases of NSCLCs, but cyclin D1 is
overexpressed in more than 40% of NSCLCs as as-
sessed by immunohistochemistry [26,49]. Recently,overexpression of Cyclin D1 in normal-appearing
bronchial epithelial of patients with NSCLCs has
been reported to be associated with smoking and to
predict shorter survival, suggesting the possible util-
ity of Cyclin D1 as a molecular marker to identify
high-risk individuals [50]. Thus through changes in
either RB, p16, CDK4, or cyclin D1, this important
growth regulatory pathway is inactivated and dis-
rupted in the large majority of lung cancers.

Epigenetic basis of lung cancer—DNA methylation and tumor suppressor gene inactivation

Lung cancers turn out to have at least as many
epigenetic alterations as genetic changes. Epige-
netic phenomena are heritable characteristics (phe-
notypes) that cannot be explained by differences
in the primary structure of DNA. In normal cells,
genomic DNA is packaged into chromatin. Chro-
matin regulates the spatial arrangement and acces-
sibility of DNA to transcription factors in the nu-
cleus. DNAmethylation is an important component
of epigenetic gene regulation in normal cells and its
dysregulation is crucial to cellular transformation
on at least two levels: genome-wide hypomethyla-
tion and gene-specific promoter hypermethylation.
Genome-wide hypomethylation affects heterochro-
matic regions of the genome, which do not ordinar-
ily code for protein. These regions were believed to
be transcriptionally inert, or “junk” DNA, but recent
evidence suggests that the transcriptional capacity
genome has been underestimated, and thus could
encode sequences important for cancer [20].
Genome-wide hypomethylation has several im-
plications in preneoplastic cells, affecting both
transcription and genetic integrity. Transcriptional
effects include loss of imprinting, re-expression
of genes involved in fetal development, and
transcriptional activation of repetitive elements
[19,30,31]. The genetic effects are indirect andinvolve larger-scale processes such as overall chro-
matin architecture, aneuploidy, and DNA replica-
tion [20,32,33].
There is overwhelming evidence that tumor-
acquired promoter hypermethylation, leading to
loss of expression of the associated gene, is a com-
mon event during themultistep pathogenesis of hu-
man lung cancer [26,34–37]. Over the past decade,
nearly 150 genes have been identified that show
tumor-specific methylation in primary tumor sam-
ples, includingmany in lung cancer (Table 4.2) [38].
Certain loci are preferentially methylated in certain
cancer types [39,40].Gene-specific promoter hyper-
methylation is an early event in tumorigenesis and
occurs in conjunction with transcriptional silencing
of the associated gene. In addition, aberrant pro-
moter hypermethylation often coincides with loss
of heterozygosity resulting in complete loss of ex-
pression and thus function of the affected locus
[16,37]. However, the molecular mechanisms that
drive tumor-acquired promoter hypermethylation
in cancer progression are not yet known [41].
DNA methylation-dependent transcriptional si-
lencing frequently affects genes that are involved
in transcriptional regulation, DNA repair, negative
regulation of the cell cycle, as well as growth reg-
ulatory signaling pathways (Table 4.2). Similar to
genetic changes, promoter hypermethylation in-
creases during tumor progression. However, in-
creasing promoter hypermethylation also occurs
with increasing age and with carcinogen exposure-
related cancers such as that of the colon and lung
[42]. In the lung, a continuumof increasingmethy-
lation fromhyperplasia through invasive carcinoma
is evident [27,28,34,43,44]. Aberrant promoter hy-
permethylation has been found in a variety of pre-
neoplastic lesions, which supports the hypothesis
that this epigenetic alteration is an early event in
carcinogenesis. This observation has resulted in sub-
stantial interest from the medical community in
that detection of methylation in sputum, blood, or
bronchial washingsmay have utility in the early de-
tection of cancer.
Some genes, such as the important TSG p53, are
never inactivated by promoter hypermethylation
because they do not have a promoter region CpG
islands. Other genes, such as the tumor suppressorgene RASSF1A, which has a prominent CpG island
are nearly always inactivated by LOH and promoter
hypermethylation in both SCLC andNSCLC. Thus, a
curious feature of aberrant promoter hypermethy-
lation is that it does not appear to affect all genes
with equal probability. An even more conspicuous
example of this phenomenon is evidenced by the
difference between p16 and RB; the protein prod-
ucts of these two genes interact directly and inac-
tivation of one or the genes (and thus this regu-
latory pathway) is nearly universal in tumors. In-
terestingly in SCLC, RB (13q14) is nearly univer-
sally inactivated, whereas in NSCLC, it is usually
p16 (9p21) that is lost. Both genes have large CpG
islands in their promoter regions, but only p16 is
methylated with significant frequency, whereas in-
activation of RB almost always occurs through DNA
mutations. This suggests tumor-acquired promoter
hypermethylation is nonrandom, and that there is
something about certain loci that makes them par-
ticularly susceptible to aberrant methylation or to
mutation [37,45].

Preneoplasia and the early detection of lung cancer

As discussed above, lung cancer results from the
accumulated effects of genetic and epigenetic al-
terations over time. Strong evidence for this posi-
tion derives from molecular genetic studies whichshow that some genetic alterations found in frank
tumors can also be identified in preneoplastic lung
cells. Using a series of microsatellite markers and
precise microdissection of cancer and lung preneo-
plastic lesions in smoking-damaged lung epithelium
as well as primary lung cancers, several groups have
shown that as cells progress histologically from hy-
perplastic epithelium through dysplasia, carcinoma
in situ, to invasive carcinomas, they acquire more
frequent and extensive genetic alterations [27,28].
The earliest genetic change that has been identified
in preneoplastic bronchial epithelial cells often in-
volves the short arm of chromosome 3. The specific
region is a 630-kb minimal homozygously deleted
portion of cytoband 3p21.3 [24]. This locus encom-
passes approximately 20 genes, including RASSF1A,
FUS1, and SEMA3B, which are discussed in the next
section.
Themost common genetic alterations and the rel-
ative timing of their appearance during lung tumori-
genesis are of particular interest because knowledge
of their occurrence can be potentially used for risk
assessment of who is themost likely to develop lung
cancer. However, these changes primarily represent
a “full defect” induced by cigarette smoking and
only rarely do sites of these changes progress to full-
fledged cancer.
In exposure-related cancers such as lung cancer,
progenitor epithelial cell clones frequently undergo
epigenetic and genetic alterations that expand into
“fields” of cells, exacerbating the problem of clonal
instances of genetic damage. The presence of spe-
cific genetic changes such as a definedmutation can
be used to track clonally-related cells. In one such
study, a group of pathologists examined 10 widely
dispersed sites in the tracheobronchial tree of a pa-
tient who died of severe atherosclerosis and found
patches of cells with the identical p53 point muta-
tion in seven of these sites [29]. While there was
no evidence of cancer in any organ at autopsy, the
presence of this mutation indicated that a lung cell
with the stem-like properties existed and migrated
throughout the lung.
The combination of chronic exposure to cigarette
smoke and chromosomal instability lead to LOH
in 3p21.3 (several genes), 9p21 (p16), and 17p.13
(p53) and frequent amplifications in eight (c-Myc),which contained defined tumor suppressor genes
or oncogenes. Loss of tumor suppressor gene func-
tion and activation of oncogenes contribute to the
initiation, development, and maintenance of lung
cancer by conferring six distinct properties, called
the “hallmarks of cancer” [9]. The hallmarks in-
clude self-sufficiency in growth signals (activation
of oncogenes), insensitivity to growth-inhibitory
signals (inactivation of TSGs), evading apoptosis,
immortalization, sustained angiogenesis, and tissue
invasion and metastases. In the following section,
we will discuss the genes involved in conferring
these “hallmarks” on lung cancer cells.

Chromosomal instability, aneuploidy, and loss of heterozygosity

There are several types of genetic damage that con-
tribute to lung cancer pathogenesis: (i) changes in
chromosome number; (ii) changes in chromosome
structure; (iii) allelic alterations and loss of het-
erozygosity (LOH); and (iv) sequence alterations in
the form of point mutations or small amplifications
or deletions [17]. The first three types of genetic
damage fall under the rubric of genomic instability
and can occur anywhere in the genome, whereas
the final type involves mutations in protein coding
sequences. In this section, we will discuss genomic
instability in the context of chromosomal instabil-
ity, aneuploidy, and loss of heterozygosity. In the
next section, we will discuss the genes frequently
affected by mutational events in human lung can-
cer, and howknowledge of their functionwill trans-
late into novel, effective therapeutics.While we dis-
cuss genomic instability and loss or gain of gene
function in different sections, it is important to re-
alize that these factors are not mutually exclusive
and both contribute to cellular transformation in
complex and cooperative ways. The consequences
of specific alterations in DNA sequence, be they
large-scale translocations or single-pointmutations,
are rarely binary events; rather, it is the accrued ef-
fects of multiple, sequential genetic alterations over
time that gives each tumor its idiosyncratic clinical
course and outcome.
It has been argued that the term genetic insta-
bility properly refers to the rate at which genetic al-
terations occur [17]. Vogelstein and others correctly
argue that the rate of genetic change cannot be in-
ferred from the extant alterations in a given sam-
ple, but rather should be determined experimen-
tally. As a result, here we will distinguish between
the terms genomic instability and genetic instability,
and use the termgenomic instability to refer only to
the fact of alterations in chromosome number (ane-
uploidy) or gross alterations in chromosome struc-
ture through translocation, amplification, and dele-
tion (chromosomal instability). Genomic instability
can involve LOH, particularly in the context of tu-
mor suppressor genes. In this case, one allele has a
mutation or epigenetic change inactivating one al-
lelewhile the otherwild-type allele is lost alongwith
many other genes leaving the cell with a completely
inactive tumor suppressor gene. This commonly oc-
curs in the case of the well-known TSGs p53, p16,
and RB.
LOH refers to the loss of one allele of a given lo-
cus, but says nothing about the number of copies
of that locus. This distinction is important be-
cause tumor cells frequently duplicate their chro-
mosome complement on a background of LOH
such that one parental allele of a chromosome
is lost, but the other is duplicated. The net ef-
fect is that daughter cells are hemizygous for
a given allele, but retain a normal karyotype
for that particular chromosome. The mechanisms
that cause genomic instability include exposure
to carcinogens, hypoxia, hypomethylation of het-
erochromatic DNA, loss of mitotic checkpoint con-
trols, defective DNA repair, and telomere shortening
[18–20].
Karyotypic studies were the first to shed light on
the genetic complexity of cancer pathogenesis, and
one of the first observations was that cancer cells of-
ten exhibit significant aneuploidy. Solid tumors fre-
quently undergo genome duplication early in their
evolution, and many malignancies exhibit a hy-
potetraploid genotype. Genome duplication occurs
during mitosis, and may involve centrosome am-
plification and the formation of multipolar spindles
prior to cytokinesis [21]. Genome duplication prob-
ably occurs in normal cells, but functional mitotic
checkpoints and sentinel DNA damage response
proteins such as p53 and ATM detect aberrant spin-
dle formation and either induce apoptosis or repair
the damage. In preneoplastic cells with mutations
in p53 or other crucial genes, this type of damage
can go undetected.
Karyotypic studies also yielded the first informa-
tion about large genetic alterations in lung cancer.
A major step to achieve lung cancer chromosome
analysis occurred with the ability to grow lung
cancer cells in tissue culture, which allowed prepa-
ration of cancer cell metaphases for analysis [22].
Indeed, karyotypic studieswhere the first to demon-
strate genetic similarities and differences between
NSCLC and SCLC [23]. Frequent sites of chromoso-
mal losses in SCLC include 3p, 5q, 13q, and 17p.
These occur together with double minutes asso-
ciated with amplification of the myelocytomatosis
viral oncogene homolog (MYC), particularly the c-
Myc, family of genes. In NSCLCs, deletions of 3p,
9q, and 17p; +7, i(5)(p10), and i(8)(q10) are com-
mon [23]. Molecular cytogenetic methods includ-
ing array-based comparative genomic hybridization
(CGH), microsatellite marker analysis, and single-
nucleotide polymorphism (SNP) studies have con-
firmed and extended earlier work. CGH analysis
incorporates whole genome-scale analyses with
relatively high-resolution quantitative information
and revealed gains in 5p, 1q24, and Xq26, and dele-
tions in 22q12.1–13.1, 10q26, and 16p11.2 [23].
Comparative genomic studies led to the finding
that nearly all SCLCs and many NSCLCs suffer LOHon chromosome 3p, suggesting the presence of one
or more tumor suppressor genes in this chromo-
some region. Although LOH by itself is not sufficient
to indicate the presence of a tumor suppressor lo-
cus, subsequent, high-resolution analyses showed
that in some SCLCs, several genes in the minimally
deleted 3p21.3 and 3p14.2were deleted on both the
maternal and paternal alleles and thus completely
gone from the cancer cell genome, a so-called ho-
mozygous deletion [24]. Homozygous deletions are
rare in cancer cell genomes, and are taken as a
strong indication that a tumor suppressor gene ex-
ists in the affected region. Other homozygous dele-
tions common in lung cancer occur on 9p21 and
17p13. These loci turned out to include the tumor
suppressor genes p16 and p53, respectively. Subse-
quentwork showed the 3p14.2 region to include the
TSG fragile histidine triad, FHIT, while the 3p21.3
region encodes several closely linked TSGs includ-
ing RASSF1A, FUS1, NPRG2, 101F6, SEMA3B, and
SEMA3F [24,25].
Another common type of genomic instability in
lung cancer primarily affects short repetitive se-
quences of DNA, which are called microsatellites.
These microsatellites, while polymorphic can un-
dergo tumor-specific (compared to normal DNA
from the same patient) alterations in length as a
result of insertion or deletion of the repeating units.
Tumors vary significantly in the rate of microsatel-
lite instability (MSI), which may be due to differ-
ences in extant DNA repair pathways as is the case
in colon cancer [17]. MSI can be measured by us-
ing a series of microsatellite markers in polymerase
chain reaction (PCR)-based assays. The overall fre-
quencies of MSI from 13 studies are 35%for SCLCs
and 22% for NSCLCs [26]. However, it remains to
be determined whether MSI is a cause or corollary
of lung tumorigenesis.

Molecular genetics of lung cancer

Tobacco smoke and lung cancer
It is well known that tobacco smoke is the major
cause of lung cancer. Smokers are 14-fold more
likely to develop lung cancer than nonsmokers
[14]. There are more than 60 carcinogens in to-
bacco smoke, many of which are activated by the
p450 enzymes in the cytosol and then interact co-
valently with DNA, forming DNA adducts [15].
Human cells have evolved specialized mechanisms
that repair different types of DNA adducts, as well
as a specific DNA polymerase (DNA polymerase)
that can bypass the most common types of DNA
mutations. Benzopyrene and 5-methylchrysene (as
well as other components of tobacco smoke)
form large adducts that cannot be bypassed by
DNA polymerase eta, and need to be removedby nucleotide excision repair (NER). Enzymes in
this DNA repair pathway remove large adducts
by cleaving the DNA helix where adducts have
formed, replacing the affected base, and then ligat
the broken DNA chain. This pathway also repairs
inter- and intra-strand DNA cross-links. Another
important family of tobacco smoke carcinogens, the
N-nitrosamines, frequently induce miscoding mu-
tations. The most common type of miscoding mu-
tation involves alkylation of guanine at the 6O
position, and 6O-methylguanine methyltransferase
repairs this particular change.
Several studies have explored the nature of to-
bacco smoke-induced mutations in lung cancer pa-
tients and have found that themost common type of
mutation is a G-T transversion. When the profile of
tumor-acquired point mutations in the p53 tumor
suppressor gene is compared between smokers and
nonsmokers with lung cancer, there are clear dis-
tinctions in the position and type of mutation that
occur. In smokers, the most frequent type of muta-
tion is G:C>T:A transversion, whereas in lung can-
cer patients with no smoking history, themost com-
mon type of mutation is G:C>A:T transition at CpG
sites (the cytosine in the CpG dinucleotides is partic-
ularly susceptible to spontaneous deamination re-
sulting in a conversion to thymine) [15,16]. Fur-
ther distinctions are apparent when the positions of
G-T transversions are compared between smoking-
related lung cancer and other common types of can-
cer [15].

Overview of lung cancer etiology, incidence, and treatment

Pathologists have described various different his-
tologies of lung cancer. There are two major sub-
types: small cell lung cancer (SCLC), and nonsmall
cell lung cancer (NSCLC). SCLC accounts for 25%of
lung cancer cases in the United States, and NSCLC
accounts for the remaining 75%. NSCLCs can be
further subdivided into several subtypes: adenocar-
cinoma (Ad), squamous cell carcinoma (SCC), large
cell carcinoma (LCC), bronchioalveolar carcinoma
(BAC), and variousmixed subtypes.While this clas-
sification system is based on histology, there are sig-
nificant molecular differences between SCLC and
NSCLC. Thus, there is an ongoing effort to describe
these differences in terms of mRNA expression pro-
files as well as the acquired genetic and epigenetic
changes between the different subtypes. There are
significant clinical differences in terms of prognosis
and treatment strategies for the different subtypes.
Therefore, another effort is directed at determin-
ing if specific molecular abnormalities predict stage
and prognosis as well as the different responses to
chemotherapy and radiation therapy well described
in patients.
We need to understand the molecular differences
between tumors arising in current smokers, for-
mer smokers, and lifetime never smokers. Are there
different acquired molecular abnormalities in lung
cancers arising in women and men or in persons of
different ethnicity or age? Can we use the molec-
ular abnormalities found in lung tissue, sputum,or those shed into the blood as aids for very early
diagnosis or learning who is at the highest risk for
developing cancer? These patients would be can-
didates for extensive screening and early detection
efforts. Could some of the changes be targets for de-
veloping tumor-specific vaccines or targeting drugs
to specific molecular abnormalities for therapeutic
purposes? A molecular diagnostic platform was re-
cently approved for use in breast cancer, and similar
designsmust be developed for use in suspected lung
cancer cases. Possible targets for these platforms in-
clude altered gene expression patterns, serum pro-
tein profiles, and aberrantly methylated DNA.
Although it seems intuitive that the large mass
of tumor cells that make up the bulk of the tu-
mor should be the target of cancer drugs, recent
evidence suggests that this bulk tumor cell popu-
lation may be less important to tumor progression
than a rare cancer stem cell that can self-renew,
initiate invasion, and propagate metastases. These
cancer stem cells are often less sensitive to cytotoxic
chemotherapy than the bulk primary tumor, and
evade first-line therapy as a result. The key to tar-
geting these rare cells is the development of molec-
ularly targeted therapies based on the profile of the
individual tumors.
Individual tumors exhibit significant phenotypic
and epigenetic variation, yet they are normally
clonal with respect to crucial genetic alterations.
This means that the evolution of a particular tumor
is driven, at least in part, by the oncogenic changes it
has acquired, and suggests that the continued prop-
agation of the tumor also depends on the activ-
ity of the oncogenes it contains. Bernard Weinstein
likened this effect of oncogene dependence to an
Achilles “heal” for the tumor: he proposed that be-
cause tumors are “addicted” to the presence of a par-
ticular oncogene, they might be uniquely sensitive
to compounds or natural products (antibodies) that
specifically target the function of the activated onco-
gene [10]. Similarly, a given tumor with a mutation
in a “gatekeeper” tumor suppressor gene whose loss
of function is absolutely required for a particular tu-
mor to develop may be hypersensitive to replacing
the activity of that tumor suppressor gene (TSG).
These concepts are the basis for rational, or molec-
ularly targeted, therapeutic approaches. Severalexamples of this new therapeutic approach are now
available and are having a significant impact in the
clinic (Table 4.1).
To fully exploit the potential targets in human
cancer cells for rational drug design, an understand-
ing of the mutational repertoire of human cancer
is necessary. Recently there have been several re-
ports on large-scale sequencing of candidate genes
in cancer cells (such as all tyrosine kinases) that
have identifiedmutations in several genes that drug
targets such as PI3 kinases [12]. Following this, the
NIH, in collaborationwith the Broad Institute atMIT
and Johns Hopkins University among others, has
begun to collect data for The Cancer Genome At-
las (TCGA). Over the next decade, this project will
produce a wealth of information that will need to
be analyzed and put into biological context to be
exploited for pharmaceutical development [13].

CHAPTER 4 The Molecular Genetics of Lung Cancer

A brief history of cancer genetics
That cancer is a genetic disease was first understood
near the turn of the last century [1]. After the dis-
covery that DNA was the genetic material, cytoge-
netic studies showed that neoplasms were nearly
always clonal with respect to karyotype and chro-
mosomal pattern. These early genetic studies led to
the concept of the clonal inheritance of somatically
acquired genetic abnormalities in cancer pathogen-
esis [2].
By the late 1960s, there were two competing hy-
potheses both of which derived from the observa-
tion that retroviral-like sequences of DNA and RNA
were frequently found in tumor cells: the idea popu-
larized by Temin involved retrotranscription of viral
genes into host cell DNA (this hypothesis eventu-
ally led to a Nobel Prize for Temin and his postdoc-
toral researcherDavid Baltimore); the other idea de-
veloped by Huebner and Todaro led to the concept
of the oncogene—genes that promote the develop-
ment of cancer [3,4]. The distinction between these
two ideas was the source of the oncogenic element:
in Temin’s view, the carcinogen derived from an in-
fectious agent,whereas for Todaro and Huebner, the
source was an endogenous, vertically transmitted,
retroviral-like gene. Both proposals turned out to
partially correct. By directly testing these compet-
ing hypotheses,Nobel laureates, Varmus and Bishop
were able to show that normal cells contain gene
sequences that are homologous to viral oncogenes;
these sequences are “proto-oncogenes” ready to be
activated during cancer pathogenesis.
Around the same time Alfred Knudson used
statistical inference to devise the complementary
concept of recessive anti-oncogenes. In a classic pa-
per, Knudson postulated that if the overall muta-
tion rate between patients with the inherited form
of retinoblastoma versus the sporadic version were
similar, then the frequent incidence of multifocal
or bilateral retinoblastomas in familial cases must
occur on the background of a germline mutation
in a critical gene [5]. The implication of this study
was that, at least in retinoblastoma, two mutations
were sufficient for the onset of disease: the so-called
“two-hit hypothesis.” Several years later, the gene
that is responsible for familial retinoblastoma was
cloned [6]. In the two decades since retinoblastoma
was first cloned and characterized, several hundred
genes—either oncogenes or tumor suppressors or
their accomplices—have been implicated in cancer
pathogenesis [7,8].
The brief overview presented above suggests that
there are at least two distinct genetic components
to cellular transformation: there are large, clonal
chromosome aberrations (aneuploidy) including
translocations, amplifications, and deletions, and
there are alterations that occur at the level of
the gene, which often include point mutations,
small amplifications, and deletions. By studying the
genetic lesions that frequently occur in primary
tumor material, cancer geneticists have made sig-
nificant inroads into a general understanding of the
mechanisms of cancer pathogenesis [9]. Modern
molecular biology techniques including cloning, the
polymerase chain reaction, and genome-wide DNA
Lung Cancer, 3rd edition. Edited by Jack A. Roth, James D. Cox,
and Waun Ki Hong. c  2008 Blackwell Publishing,
ISBN: 978-1-4051-5112-2.microarrays have increased the rate with which
new genes are discovered and disease associations
determined. The next step in the biotechnology
revolution will be to translate our growing under-
standing of cancer genetics into rational diagnos-
tic and drug development platforms, and ultimately
into better treatment strategies. This chapter dis-
cusses the genetic basis of lung cancer in light of the
ongoing translational and clinical challenges these
diseases present to physicians, and describes new
approaches to developingmolecularly targeted ther-
apies to treating lung cancer.

References

1 American Cancer Society: Cancer Facts & Figures.
Atlanta, GA: American Cancer Society, 2007.
2 Hsu TC, Spitz MR, Schantz SP. Mutagen sensitivity: a
biological marker of cancer susceptibility. Cancer Epi-
demiol Biomarkers Prev 1991; 1(1):83–9.
3 Spitz MR Hsu TC, Wu X, Fueger JJ, Amos CI, Roth
JA.Mutagen sensitivity as a biologicalmarker of lung
cancer risk in African Americans. Cancer Epidemiol
Biomarkers Prev 1995; 4(2):99–103.4 Wu X, Delclos GL, Annegers JF et al. A case–control
study of wood dust exposure, mutagen sensitivity,
and lung cancer risk. Cancer Epidemiol Biomarkers Prev
1995; 4(6):583–8.
5 Zheng YL, Loffredo CA, Yu Z et al. Bleomycin-induced
chromosome breaks as a riskmarker for lung cancer: a
case–control study with population and hospital con-
trols. Carcinogenesis 2003; 24(2):269–74.
6 Smith-Warner SA, Spiegelman D, Yaun SS et al.
Fruits, vegetables and lung cancer: a pooled analysis
of cohort studies. Int J Cancer 2003; 107(6):1001–11.
7 Peto R, Darby S, Deo H, Silcocks P,Whitley E, Doll R.
Smoking, smoking cessation, and lung cancer in the
UK since 1950: combination of national statisticswith
two case–control studies. BMJ 2000; 321(7257):323–
9.
8 Bach PB, KattanMW, ThornquistMD et al. Variations
in lung cancer risk among smokers. J Natl Cancer Inst
2003; 95(6):470–8.
9 Darby S,Whitley E, Silcocks P et al. Risk of lung cancer
associated with residential radon exposure in south-
west England: a case–control study. Br J Cancer 1998;
78(3):394–408.
10 Taylor R, Cumming R, Woodward A, Black M. Pas-
sive smoking and lung cancer: a cumulative meta-
analysis. Aust N Z J Public Health 2001; 25(3):203–
11.
11 Gorlova OY, Zhang Y, SchabathMB et al. Never smok-
ers and lung cancer risk: a case–control study of epi-
demiological factors. Int J Cancer 2006; 118(7):1798–
804.
12 Tokuhata GK, Lilienfeld AM. Familial aggregation
of lung cancer in humans. J Natl Cancer Inst 1963;
30:289–312.
13 Ooi WL, Elston RC, Chen VW, Bailey-Wilson JE,
Rothschild H. Increased familial risk for lung cancer.
J Natl Cancer Inst 1986; 76(2):217–22.
14 Samet JM,Humble CG, PathakDR. Personal and fam-
ily history of respiratory disease and lung cancer risk.
Am Rev Respir Dis 1986; 134(3):466–70.
15 Shaw GL, Falk RT, Pickle LW, Mason TJ, Buffler PA.
Lung cancer risk associated with cancer in relatives.
J Clin Epidemiol 1991; 44(4–5):429–37.
16 Osann KE. Lung cancer in women: the importance of
smoking, family history of cancer, andmedical history
of respiratory disease. Cancer Res 1991; 51(18):4893–
7.
17 Schwartz AG, Yang P, Swanson GM. Familial risk of
lung cancer among nonsmokers and their relatives.
Am J Epidemiol 1996; 144(6):554–62.
18 Mayne ST, Buenconsejo J, Janerich DT. Previous
lung disease and risk of lung cancer among men
and women nonsmokers. Am J Epidemiol 1999;
149(1):13–20.
19 Bromen K, Pohlabeln H, Jahn I, AhrensW, Jockel KH.
Aggregation of lung cancer in families: results from a
population-based case–control study in Germany. Am
J Epidemiol 2000; 152(6):497–505.
20 Etzel CJ, Amos CI, SpitzMR. Risk for smoking-related
cancer among relatives of lung cancer patients. Cancer
Res 2003; 63(23):8531–5.
21 KreuzerM,Kreienbrock L,GerkenMet al. Risk factors
for lung cancer in young adults. Am J Epidemiol 1998;
147(11):1028–37.
22 SchabathMB, Delclos GL,MartynowiczMMet al.Op-
posing effects of emphysema, hay fever, and select
genetic variants on lung cancer risk. Am J Epidemiol
2005; 161(5):412–22.
23 Cockcroft DW, Klein GJ, Donevan RE, Copland GM.
Is there a negative correlation between malignancy
and respiratory atopy? Ann Allergy 1979; 43(6):345–
7.
24 Talbot-Smith A, Fritschi L, Divitini ML, Mallon DF,
Knuiman MW. Allergy, atopy, and cancer: a prospec-
tive study of the 1981 Busselton cohort. Am J Epi-
demiol 2003; 157(7):606–12.
25 Vena JE, Bona JR, Byers TE,Middleton E, Jr, Swanson
MK, Graham S. Allergy-related diseases and can-
cer: an inverse association. Am J Epidemiol 1985;
122(1):66–74.
26 Gabriel R, Dudley BM, Alexander WD. Lung cancer
and allergy. Br J Clin Pract 1972; 26(5):202–4.
27 McDuffie HH. Atopy and primary lung cancer. Histol-
ogy and sex distribution. Chest 1991; 99(2):404–7.
28 Castaing M, Youngson J, Zaridze D et al. Is the risk
of lung cancer reduced among eczema patients? Am
J Epidemiol 2005; 162(6):542–7.
29 Santillan AA, Camargo CA, Jr, Colditz GA. A meta-
analysis of asthma and risk of lung cancer (United
States). Cancer Causes Control 2003; 14(4):327–34.
30 Feskanich D, Ziegler RG, Michaud DS et al. Prospec-
tive study of fruit and vegetable consumption and risk
of lung cancer among men and women. J Natl Cancer
Inst 2000; 92(22):1812–23.
31 Voorrips LE, Goldbohm RA, van Poppel G, Sturmans
F, Hermus RJ, van den Brandt PA. Vegetable and fruit
consumption and risks of colon and rectal cancer
in a prospective cohort study: The Netherlands Co-
hort Study on Diet and Cancer. Am J Epidemiol 2000;
152(11):1081–92.
32 Brennan P, Fortes C, Butler J et al. A multicenter
case–control study of diet and lung cancer among
non-smokers. Cancer Causes Control 2000; 11(1):49–
58.

LC risk assessment models

Statistical models relating multiple risk factors to
cancer risk can identify high-risk subsets of smokers.
There are three criteria to evaluate the performance
of risk assessment models: calibration (reliability),
discrimination, and accuracy [280]. Calibration as-
sesses the ability of a model to predict the num-
ber of endpoint events in subgroups of the popula-
tion and is evaluated by using the goodness-of-fit
statistic. Discrimination is a measure of a model’s
ability to distinguish between those who will and
will not develop disease, and is quantified by cal-
culating the concordance statistic, or area under a
receiver operating characteristic (ROC) curve. Ac-
curacy including positive and negative predictive
values refers to themodel’s ability to categorize spe-
cific individuals. The best-known cancer prediction
model is the Gail model for breast cancer [281]. It
has been validated in several populations [282–285]
and appears to give accurate predictions for women
undergoing routine mammographic screening but
probably overestimates the risk for young women
not undergoing routine mammography [286]. The
modest discrimination ability of the Gail model calls
for the incorporation of promising biological fac-
tors [287–290]. Prediction models for other cancers
(melanoma [291,292], colorectal cancer [293], and
LC [7,8]) have also emerged.
The few published LC risk assessment models
mainly focus on smoking behavior and demo-
graphic characteristics. Bach et al. [8] used data col-
lected from CARET, a large, randomized trial of LC
prevention, to derive a LC risk prediction model.
The model used the subject’s age, sex, asbestos ex-
posure history, and smoking history to predict LCrisk andwas derived by use of data fromfive CARET
study sites and then validated by assessing the ex-
tent it could predict events in the sixth study site.
The model was then applied to evaluate the risk of
LC among smokers enrolled in a study of LC screen-
ing with computed tomography (CT). The model
identified smoking variables (duration of smoking,
average number of cigarettes smoked per day, dura-
tion of abstinence), age, asbestos exposure and the
study drug, β-carotene and retinyl palmitate as sig-
nificant predictors of LC. Themodel provided strong
evidence that LC risk varies greatly among smok-
ers and was internally validated and well calibrated
with a cross-validated concordance index of 0.72.
Bach’s model is most applicable to heavy smokers
aged between 50 and 75 years. Recently, Spitz et al.
[294] developed lung cancer risk models for never,
former, and current smokers, respectively. In their
models, factorswith strong etiological roles, e.g., en-
vironmental tobacco smoke, family history of can-
cer, dust exposure, prior respiratory disease, and
smoking history variables were all identified as sig-
nificant predictors of lung cancer risk. The models
were internally validated with cross-validated con-
cordance statistics for the never, former, and current
smoker models of 0.57, 0.63, and 0.58, respectively.
The computed 1-year absolute risk of lung cancer
for a hypotheticalmale current smokerwith an esti-
mated relative risk close to 9was 8.68%. The ordinal
risk index performed well in that true-positive rates
in the designated high-risk categories were 69%
and 70% for current and former smokers, respec-
tively. When externally validated, this risk assess-
ment procedure could use easily obtained clinical
information to identify individualswhomay benefit
from increased screening surveillance for lung can-
cer. In summary, current LC risk prediction models
have been focused on smoking variables and there
is potential to developmore accuratemodels by col-
lecting more data and incorporating additional risk
factors. Moreover, external validation of existing
models to independent populations is important.Concluding remarks
The results of many reported associations of single
polymorphism analyses are incongruent and couldnot be replicated even with key study parameters
similar to the original ones. Beyond a possible effect
frompopulation heterogeneity, shortcomings in ex-
perimental design and statistical methodology such
as small sample size, lack of control for confound-
ing, selection bias, and multiple comparisons may
account for a large part of these discrepancies. Since
cancer is a multistep and multifactorial disease, the
influence of individual variants identified frommost
candidate gene approach studies on overall cancer
riskmight beminimal.Moreover,many cancer risk-
associated genetic variants lack functional valida-
tion. To circumvent these caveats, pathway-based
approaches have been exploited that simultane-
ously analyze the impact of multiple variants in
the same carcinogenesis-related signaling or func-
tion pathway on cancer predisposition. This strategy
might amplify the effect from single variants; how-
ever, the pathway-based approach also depends on
a priori knowledge from basic investigations sug-
gesting the involvement of the pathway in tumori-
genesis. A haplotype-based genome scan approach
has also been proposed to identify causal variants
in the whole-genome scale without any presump-
tion based on prior knowledge, as has been success-
fully applied to isolate causal polymorphisms in a
variety of common human diseases. This approach
mandates stringent study designs, adequate sample
size, and statistical power. In addition, high-power
computational methodologies of data analysis and
error shooting should be developed to probe the vast
amount of interactions amongst genetic and envi-
ronmental factors, and molecular function assays
should be carried out to determine the genotype–
phenotype correlations and validate the biological
significance of the identified high risk alleles.

Growth factor

IGFs
Both insulin growth factors (IFG1 and IGF2) play
a major role in fostering cell proliferation, survival,
migration and inhibiting apoptosis [264]. The inter-
actions between IGFs and IGFRs are regulated by
IGF binding proteins (IGFBPs) functioning in both
IGF-dependent and IGF-independent manners to
regulate cellular growth [265]. High plasma levels
of IGF1 were associated with increased risk of LC in
a dose-dependent manner [266]. To date, only two
SNPs were reported to predispose to LC. The ho-
mozygous variant at the −202 nucleotide position
of IGFBP3 promoter region was reported to be neg-
atively correlated to LC susceptibility in a Korean
population [267]. This was supported by the ob-
servation that IGFBP3 may have a dual role in the
biosynthesis of IGFs [268], and serum-circulating
IGFBP3 protein might prolong the half-life of IGF
through influencing its interaction with the mem-
brane receptors [269]. A recent study evaluating
1476 nsSNPs of cancer-related genes identified a sig-
nificantly altered LC risk associated with Trp138Arg
of IGFBP5 [270]. Using the Pathway Assist software,
11 out of 1476 SNPs exhibiting significant LC risk
association were mapped to the GH–IGF axis [270],
indicating the importance of this pathway in LC
development.
EGF
Epidermal growth factor (EGF), a small molecule
ligand that activates receptor tyrosine kinase (RTK),
mediates signal transduction pathways. An A to G
transition in the 5
UTR of EGF gene has been asso-
ciated with reduced LC risk in a Korean population
[271].
VEGF
Vascular endothelial growth factor (VEGF) is a
proangiogenesis protein implicated in carcinogene-
sis and metastasis of many cancers. Three common
polymorphisms in the promoter region (−634G>C,
−1154G>A, and −2578C>A) regulate VEGF pro-
tein level, vascular density, aswell as vascularization
status of tumor tissues from NSCLC patients [272].
However, no study has assessed their implications
in LC risk.Methylation-related genes
Aberrant methylation of pivotal cell growth-related
genesmay lead to carcinogenesis through regulating
their protein expression and common genetic vari-
ants in methylation maintenance genes may also
impact cancer susceptibility.DNMT 3B
DNMT3B is responsible for the generation of ge-
nomicmethylation patterns. To date, three DNMT3B
polymorphisms have been evaluated in LC suscep-
tibility. Wang et al. reported that a C to T single
base substitution in the promoter region was as-
sociated with enhanced promoter activity [273].
Genotypes encompassing the variant allele were as-
sociated with a 1.88-fold excess of LC risk com-
pared to the common homozygotes in Caucasians
[274]. In a Korean population, Lee et al. noted that
the variant alleles of another two promoter poly-
morphisms (−283C>T and −579G>T) were both
associated with reduced risk for LC [275]. The re-
sults of the these studies were in concordance as
both reported that the allele leading to enhanced
DNMT3B expression was associated with increased
cancer risk.
MBD1
MBD1 is a mediator of the DNA methylation-
induced gene silencing. Jang et al. reported that
the wild-type allele of a −634G>A SNP in the pro-
moter region was associated with LC risk with OR
of 3.10 (1.24–7.75) in a Korean population [276].
For another two SNPs (−501delT and Pro401Ala),
the wild-type alleles were correlated with increased
risk of adenocarcinoma but not with other LC
subtypes. Luciferase assays demonstrated that the
haplotype containing the risk-conferring alleles ex-
hibited higher promoter activity, indicating the
presence of a negative correlation between MBD1
expression and LC development.
MTHFR
MTHFR gene encodes an essential enzyme involved
in the production of the S-adenosylmethionine in-
termediate for DNA methylation [277]. Besides a
role in DNA methylation, MTHFR is also important
in maintaining normal cellular folate levels. So far,
two nsSNPs (677C>T and 1298A>C) have been as-
sessed in studies with inconsistent results [278].
SUV39H2
Suppressor of variegation 3–9 homolog 2
(SUV39H2) is a site-specific histone methyl-
transferase responsible for the methylation oflysine 9 in histone 3. A1624G>C SNP in the 3
UTR region was associated with a 2.63-fold (1.10–
6.29) increased risk of LC in ever smokers when
variant-containing genotypes were compared to
homozygous wild-types [279]. In vitro assays
showed a more than 2-fold higher transcript level
for the variant allele, indicating that this SNP
might be the causal agent functioning through
influencing protein expression.

Tumor microenvironment

Microenvironmental factors
Matrix metalloproteins (MMPs) degrade a range of
extracellular matrix and nonmatrix proteins. Since
MMP expression level has been implicated in can-
cer development, several polymorphisms in the pro-
moter regions of MMPs that might affect gene ex-
pression have been evaluated.
MMP1
MMP1 is a highly expressed interstitial collagenase,
which degrades fibrillar collagens. MMP1 is upregu-
lated by tobacco exposure [226] and overexpression
of MMP1 in tumors has been linked to tumor inva-
sion and metastasis [227,228]. A 1G/2G polymor-
phism in the MMP1 promoter was associated with
altered gene expression [229]. Promoters contain-
ing the 2G allele displayed higher transcriptional ac-
tivity than those with the 1G allele. An increased LC
risk was identified with the 2G/2G genotype by Zhu
et al. [230]. Two other studies, Su et al. [231] and
Fang et al. [232] both noted an increase in LC risk
with the 2G allele, but this did not reach statistical
significance.
MMP2
MMP2 is a gelatinase whose substrates include
gelatins, collagens, and fibronectin. The expression
levels of MMP2 have been commonly used to pre-
dict cancer prognosis. A promoter SNP (−1306C>T)
has been associated with reduced activity due to
a possible interference with the SP1-binding site
[233]. Interestingly, compared to the variant allele
associated with lower gene expression, the wild-
type allele exhibited an association with LC risk
with an OR of 2.18 (1.70–2.79) in a Chinese pop-
ulation [234]. Another promoter SNP (−735C>T),
which was linked with −1306C>T, was also associ-
ated with risk for the wild-type allele with an OR of
1.57 (1.27–1.95) [235] which retains the SP1 bind-
ing site as well as a higher transcriptional activa-
tion efficiency [236].Moreover, itwas noted that an
even higher risk was associated with the haplotype
containing the wild-type alleles at both loci and this
risk showed a multiplicative interaction effect with
smoking [235].
MMP3
MMP3 is a stromelysin whose substrates include
collagens, gelatin, aggrecan, fibronectin, laminin,
and casein. The most commonly studied MMP3
polymorphism is a promoter variant located at
−1171 nucleotide, containing either five or six
adenosines that may affect promoter transcription
activity [237]. In a Caucasian population, a hap-
lotype containing the 6A allele exhibited a higher
LC risk in never smokers [238]. However, in a Chi-
nese study, Fang et al. reported that smokers with
the MMP3 5A allele had a 1.68-fold (1.04–2.70) in-
creased risk to develop NSCLC [232].
MMP7
MMP7 is a matrilysin whose substrates include col-
lagens, aggrecan, decorin, fibronectin, elastin, and
casein.MMP7 is highly expressed in lungs of patients
with pulmonary fibrosis and other conditions asso-
ciated with airway and alveolar injury. The variant
allele of a promoter SNP, –181A>G, might lead to
higher promoter activity and increased mRNA lev-
els [239]. Consistently, the variant-harboring geno-
types, when compared to the common homozy-
gotes, have been proven to predispose to risk of
NSCLC [240].
MMP9
MMP9 is a gelatinase and the major structural com-
ponent of the basement membrane. Hu et al. re-
ported that two common nsSNPs, Arg279Gln and
Pro574Arg,might confer LC susceptibility in a dose-
dependent fashion [241].
MMP12
MMP12 is a metalloelastase required for
macrophage-mediated extracellular matrix pro-
teolysis and tissue invasion. A promoter SNP
(−82A>G) might regulate MMP12 expression
through modulating the binding affinity of tran-
scription activation protein 1 [242]. Another
nsSNP (1082A>G) leads to the substitution of
serine for asparagine. Although no significant LC
risk associations were identified for either SNP, a
haplotype containing the −82A and 1082G alleles
was associated with higher LC risk among never
smokers in comparison to haplotypes containing
−82G and 1082A [238].
Inflammation
Airway inflammation may promote tumorigenesis
through multiple mechanisms such as inducing ox-
idative stress and lipid peroxidation [243]. To date,
only a few polymorphisms in inflammation genes
have been evaluated for their roles in lung tumori-
genesis and the results have beenmostly discrepant.
Anti-inflammatory genes
The major functions of anti-inflammation genes
such as IL4, IL10, IL13, and PPARs are to resolve the
acute inflammatory reactions. Among these, IL10 is
produced by monocytes and lymphocytes and ex-
hibits multiple functions in the regulation of cell-
mediated immunity, inflammation, and angiogene-
sis [244]. Three SNPs in the promoter region of IL10
have been identified (−1082A>G, −819C>T, and
−592C>A). In a Chinese study, the variant allele
of the −1082A>G SNP was associated with a 5.26-
fold (2.65–10.4) increased LC risk [245], which was
in agreement with another study in small cell LC
[246]. The variant allele has been shown to affect
IL10 protein level through regulating gene tran-
scription [247–249].
Proinflammatory genes
Engels et al. [250] systematically evaluated a panel
of 59 single nucleotide polymorphisms (SNP) in 37
inflammation-related genes among non-Hispanic
Caucasian lung cancer cases (N = 1,553) and con-
trols (N = 1,730). They found that Interleukin 1
beta (IL1B) C3954T was associated with increased
risk of lung cancer and that one IL1A-IL1B hap-
lotype, containing only the IL1B 3954T allele, was
associated with elevated lung cancer risk. These as-
sociations were stronger in heavy smokers, partic-
ularly for IL 1B C3954T. IL1B activates a mixture
of inflammatory signaling mediators including NF
Kappa B, leading to an amplified proinflammatory
effect. A variable number of tandemrepeats (VNTR)
polymorphism in intron 2 of the IL1RN gene [251]
influences the expression of both IL1B and IL1RN
[252,253]. Lind et al. [251] observed an increased LC
risk in individuals with both the IL1RN∗
1 and the

Apoptosis

Genetic polymorphisms in
apoptotic pathways
Apoptosis (programmed cell death) is an essential
cellular defense mechanism. Two principal signal-
ing pathways, the intrinsic pathway and the ex-
trinsic pathway, are implicated in the coordination
of the apoptotic process. In the extrinsic apopto-
sis pathway, polymorphisms influencing the FASL–
FAS interaction might affect LC predisposition. In
a Chinese case–control study, two promoter SNPs
of FAS (−1377G >A) and FASL (−844T >C) [219]
were associated with increased LC risks with ORs
of 1.59 (1.21–2.10) and 1.79 (1.26–2.52), respec-
tively, when the rare homozygotes were compared
to the common homozygotes. A multiplicative in-
teractive effect was noted. In the intrinsic apop-
tosis pathway, CASP9 is the only gene that has
been assessed for a role in LC development. In a
Korean study, Park et al. found that two CASP9
promoter SNPs (−1263A >G and −712C >T) were
associated with significantly altered LC risk with
OR’s of 0.64 (0.42–0.98) and 2.32 (1.09–4.94),
respectively, when their homozygous variant geno-
types were compared to the homozygous wild-type
reference group [220]. Furthermore, the haplo-
type composed of the G allele of −1263A >G and
the C allele of −712C>T was associated with a
significantly decreased risk. This was consistent
with the results from single SNP analysis and
was functionally validated by a promoter-luciferase
assay.
Phenotypic assays in apoptotic pathways
Two groups have reported that impaired mutagen-
induced apoptotic capacity was associated with in-
creased risk of LC [217] using the TUNEL (Terminal
transferase dUTP nick end labeling) method [221].
Biros et al. [192] reported that in LC patients, indi-
viduals with the variant allele of p53 Pro72Arg poly-
morphismexhibited a lower level of apoptoticwhite
blood cells. This finding was consistent with the re-
sults fromWu et al. [187] who showed that the hap-
lotype containing the wild-type alleles of the three
p53 polymorphisms (intron 3, exon 4, and intron
6) was associated with higher apoptotic index than
those with at least one variant allele.
Telomere and telomerase
Telomeres are TTAGGG repeat complexes bound by
specialized nucleoproteins at the ends of chromo-
somes in eukaryotic cells. By capping the ends of
chromosomes, telomeres prevent nucleolytic degra-
dation, end-to-end fusion, irregular recombination
and other events lethal to cells [222].Wu et al. [223]
measured telomere length in the peripheral blood
lymphocytes of LC patients and age-matched con-
trols and found significantly shorter telomere length
in LC cases.
To date, only two studies have indicated that com-
mon sequence variants in the TERT genomic region
might predispose to LC. TERT is the protein moi-
ety of telomerase, the key enzyme in the mainte-
nance of telomere length by synthesizing TTAGGG
nucleotide repeats. In the majority of human can-
cers, telomerase is activated and cells overcome
senescence and become immortalized. Wang et al.
[224] showed that a polymorphic tandem repeat
minisatellite (MNS16A) in the promoter region of
an antisense transcript of the TERT gene regulates
the antisense transcript expression. Cells with short
tandem repeats displayed higher promoter activity
compared to those with longer tandem repeats. A
subsequent case–control study showed that the long
tandem repeat variant was associated with a more
than 2-fold increase in LC risk in a recessive pat-
tern, supporting the conjecture that the antisense
transcript might serve as a tumor suppressor gene
inhibiting the expression of TERT [224]. Another
polymorphism was recently identified in the Ets2
binding site of the TERT promoter region [225].
Compared to the common homozygotes, the rare
homozygotes exhibited reduced telomerase activity.

Genetic variations in LC risk assessment 4

ATM
In human cells, ATM is required for the early re-
sponse to ionizing radiation. ATM senses genomic
damage and initiates DNA repair through interact-
ing with the MRN (MRE11, RAD50, and NBS1)
complex and subsequently activating a series of
downstream signaling mediators [153]. In a Korean
study, an SNP in intron 62 (IVS62 +50G>A) ex-
hibited a significantly increased LC risk with an OR
of 1.6 (1.1–2.1) [154], and higher risks for haplo-
types and diplotypes containing the variant allele
with ORs of 7.6 (1.7–33.5) and 13.2 (3.1–56.1), re-
spectively. The close proximity of this SNP to ATM
PI3K and FAT domains suggests a potential func-
tional impact on ATM kinase activity.
NBS1
In the HR pathway, the first event is the resec-
tion of the DNA to yield single-strand overhangs
[118]. NBS1 is part of an exonuclease complex that
takes part in this step. Zienolddiny et al. and Mat-
ullo et al. showed no association between the NBS1
Glu185Gln polymorphism and LC risk [125,155],
but Lan et al. reported that homozygotes for this al-
lele had an increased risk of LC with an OR of 2.53
(1.05–6.08) [156].
XRCC3
XRCC3 is an RAD51-related protein involved in cat-
alyzing the DNA strand exchange reaction during
HR [118]. Five epidemiological studies found no
association between the thr241Met polymorphism
with LC risk [125,143,148,155,157].
LIG4
In the NHEJ pathway, LIG4 has an important role in
linking the ends of a double-strand break together
[118]. Matullo et al. found no association between
two LIG4 polymorphisms (Ala3Val and Thr9Ile) and
LC risk [155]. Sakiyama et al. reported that the vari-
ant allele of the Ile658Val polymorphism of LIG4
was associated with a reduced risk of squamous cell
carcinoma with an OR of 0.4 (0.1–0.8) [158].
MMR
The MMR system maintains the stability of the
genome during repeated duplication, by repairing
base–base mismatches, caused not only by errors
of DNA polymerases that escape their proofreading
function, but also by insertion/deletion loops that
result from slippage during replication of repetitive
sequences or during recombination [118]. So far,
only a few studies have investigated the connection
between MMR and LC.
MLH1 –93G>A polymorphism was studied for its
association with risk of LC and no overall associa-
tionwas identified [159]; Jung et al. investigated the
association of MSH2 –118T>C, IVS1 +9G>C, IVS10
+12A>G, and IVS12 –6T>C genotypes with LC risk
[160] and found that the presence of at least one
IVS10 +12G allele was associated with a decreased
risk of adenocarcinoma as compared with the IVS10
+12AA genotype with OR of 0.59 (0.40–0.88), and
the presence of at least one IVS12 –6C allele was as-
sociated with an increased risk of adenocarcinoma
as compared with the IVS12 –6TT genotype with an
OR of 1.52 (1.02–2.27) [160].
DNA damage and repair phenotypic assays
The phenotypic assays for DNA damage and repair
include measuring: (a) DNA damage/repair after a
chemical or physicalmutagen challenge (such as the
mutagen sensitivity, comet, and induced adduct as-
says); (b) unscheduled DNA synthesis; (c) cellular
ability to remove DNA lesions from plasmid trans-
fected into lymphocyte cultures in vitro by expres-
sion of damaged reporter genes (the host–cell reac-
tivation assay); (d) activity of DNA repair enzyme
(repair activity assay for 8-OH-Guanine) [161,162].
Mutagen sensitivity
The mutagen sensitivity assay quantifies chromatid
breaks induced by mutagens in cultured lympho-
cytes in vitro as an indirect measure of DRC
[163,164]. Bleomycin is a clastogenic agent that
mimics the effects of radiation by generating free
oxygen radicals capable of producing DNA single-
and double-strand breaks that initiate BER and DSB
repair [165].Wu et al. showed that higher BPDE and
bleomycin sensitivities were independently signifi-
cantly associated with increased risks of LC, a find-
ing that has been confirmed by other studies [3–
5,166,167].
Comet assay
The comet assay is a single-cell gel electrophoresis
method used to measure DNA damage in individ-
ual cells. It is a sensitive and versatile method with
high throughout potential [168,169]. The alkaline
version (pH > 13) of the comet assay can detect
DNA damage such as single-strand breaks, double-
strand breaks, and alkaline labile sites [170]. Com-
mon mutagens used in this assay include BPDE,
bleomycin, and γ-radiation. Wu et al. found that
higher γ-radiation- and BPDE-induced olive tail
moments, one of the parameters for measuring
DNA damage, were significantly associated with
2.32- and 4.49-fold risks of LC, respectively [171].
Rajaee-Behbahani et al. reported lower repair rate of
bleomycin-induced DNA damage using the alkaline
comet assay in LC patients compared with controls
[172].
DNA adducts
Using 32P postlabeling techniques, two studies by
the same group indicated a significant association
between the level of in vitro BPDE-induced DNA
adducts and risk for LC [173,174], suggesting sub-
optimal ability to remove the BPDE-DNA adduct re-
sulted in increased susceptibility to tobacco carcino-
gen exposure [174].
Host cell reactivation assay
The host cell reactivation assaymeasures globalNER
as a biomarker for LC susceptibility [175–177], by
quantifying the activity of a reporter gene (CAT
or LUC gene) in undamaged lymphocytes trans-
fectedwith BPDE-treated plasmids. Because a single
unrepaired BPDE-induced DNA adduct can block
reporter gene transcription [178], the measured
reporter gene activity reflects the ability of the trans-
fected cells to remove the adducts fromthe plasmid.
Reduced capacity to repair adducts is observed in
cases compared to controls and is associated with
an increased risk of LC with evidence of a signifi-
cant dose–response association between decreased
DRC and risk of LC [175–177].
8-OGG assay
The enzyme 8-oxoguanine DNA N-glycosylase is
encoded by the OGG1 gene and initiates the BER
pathway. The OGG activity assay monitors the abil-
ity ofOGG to remove an 8-oxoguanine residue from
a radiolabeled synthetic DNA oligonucleotide, gen-
erating two DNA products that can be distinguished
on the basis of size [179]. Paz-Elizur et al. showed
that OGG activity was significantly lower in periph-
eral blood mononuclear cells from LC patients than
in those fromcontrols. Individuals in the lowest ter-
tile of OGG activity exhibited an increased risk of
NSCLC compared with those in the highest tertile
(OR=4.8; 95%CI, 1.5–15.9) [179].Gackowski et al.
also reported that the repair activity of OGG was
significantly higher in blood leukocytes of healthy
volunteers than in LC patients [180].
Cell cycle control
The intricate cell cycle regulatory network is essen-
tial for cells to undergo replication, division, prolif-
eration, and differentiation. Anomalies of cell cycle
regulation genes are frequently observed in a vari-
ety of human malignancies including LC, and are
considered to be one of the most critical early-stage
events in carcinogenesis [181–184].
Genetic polymorphisms in cell
cycle-related genes
p53
p53 is the most important tumor suppressor gene of
the genome defense system regulating pivotal cel-
lular activities such as DNA damage response, DNA
repair, cell cycle control, and apoptosis. Three poly-
morphisms of the p53 gene have been commonly
studied in cancer susceptibility.Weston et al. first re-
ported the association between the Arg72Pro nsSNP
in exon 4 and increased LC risk [185], which was
confirmed by a number of subsequent studies in
various populations [186–191]. Functional assays
corroborated this finding by demonstrating the as-
sociation between the variant allele and increased
p53 mutations in tumor tissues, as well as a reduced
rate of apoptosis in white blood cells of LC patients
[189,192,193]. Wu et al. reported an association
with the variant genotype of both the intron 3 16-bp
deletion/insertion and the intron 6 polymorphisms
[187]. Analyses of haplotypes reconstructed using
these three polymorphisms demonstrated an in-
creased LC risk for the variant-harboring haplotypes
compared to the haplotype with wild-type alleles at
all three loci. This result was supported by func-
tional studies showing that the variant-harboring
haplotypes exhibited a reduced apoptotic index and
reduced DNA repair capacity [187]. Moreover, the
association with the intron 3 polymorphism was
confirmed in a recent large-scale European study,
which reported a 2.98-fold [194] increased LC risk
for the homozygous variant genotype. Although
most studies suggest a positive association between
these p53 polymorphisms and LC risk, disagree-
ments exist including a recent meta-analysis of
13 LC studies showing no LC risk association for
any of these polymorphisms [195].
p73
p73 may activate p53 down-stream transcriptional
effectors such as p21 to control cell cycle progression
and apoptosis [196]. A dinucleotide polymorphism
in the 5
UTR of p73 is associated with an increased
risk of LC in a Caucasian population but a protec-
tive effect in a Chinese population [197,198], sug-
gesting the possible existence of ethnic-specific risk
differentiation. Furthermore, a gene–dosage effect
by combining both p53 and p73 variant alleles to-
gether was demonstrated [199].
MDM2
MDM2, a ubiquitin ligase, negatively regulates p53
activity either by binding to the transactivation do-
main of p53 protein and inhibiting its transcrip-
tional activation of p21, or by targeting p53 pro-
tein to ubiquitin-mediated proteasome degradation
[200].AT toGtransversion in the intronic promoter
region of MDM2 was associated with increased LC
risk in Chinese [190], Koreans [201], and Euro-
peans [202]. Other studies exhibited similarly el-
evated risk, although not reaching statistical sig-
nificance [203,204]. The agreement amongst these
studies recapitulates the in vivo observation that
the variant allele upregulates MDM2 expression and
thus reduces p53 protein level [205].
HRAD9
HRAD9 is a phosphorylation target of ATM kinase
that plays a crucial role in DNA repair and cell cycle
arrest in response to DNA damage [206]. A nsSNP