Review: Molecular Pathogenesis of Premalignancy Series Cancer

Prevention
Research
Early Events in the Molecular Pathogenesis of
Lung Cancer
Humam Kadara1,2, Paul Scheet2,3, Ignacio I. Wistuba1, and Avrum E. Spira4

Abstract
The majority of cancer-related deaths in the United States and that underlie lung cancer development and that would constitute
worldwide are attributed to lung cancer. There are more than markers and targets for early detection and prevention. This review
90 million smokers in the United States who represent a signif- summarizes the state of knowledge of human lung cancer path-
icant population at elevated risk for lung malignancy. In other ogenesis and the molecular pathology of premalignant lung
epithelial tumors, it has been shown that if neoplastic lesions lesions, with a focus on the molecular premalignant field that

Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/9/7/518/1937107/518.pdf by guest on 21 May 2023

can be detected and treated at their intraepithelial stage, patient associates with lung cancer development. Lastly, we highlight new
prognosis is significantly improved. Thus, new strategies to detect approaches and models to study genome-wide alterations in
and treat lung preinvasive lesions are urgently needed in order to human lung premalignancy in order to facilitate the discovery of
decrease the overwhelming public health burden of lung cancer. new markers for early detection and prevention of this fatal
Limiting these advances is a poor knowledge of the earliest events disease. Cancer Prev Res; 9(7); 518–27. Ó2016 AACR.

Introduction carcinomas (LUAD), lung squamous cell carcinomas (LUSC),

and large-cell lung carcinomas (LCLC; refs. 3, 4, 10). The
Lung cancer is the leading cause of cancer-related deaths in
different lung cancer subtypes are thought to develop through
the United States and worldwide (1, 2). The 5-year survival
diverse and unique molecular pathways. This supposition is
rates for advanced late-stage lung cancer remain poor (
5%;
supported by the divergent anatomical locations and suggested
refs. 3, 4), leading to the supposition that early diagnosis of
cells of origin of the different lung cancer subtypes. For exam-
lung cancer or its treatment in premalignant stages will reduce
ple, compared with LUSCs and SCLCs that typically arise
the public health burden of this fatal malignancy (5–8). The
from the major bronchi and are centrally located, LUADs are
vast majority (
85%) of lung cancers are causally linked to
thought to develop from small bronchi, bronchioles or alveolar
tobacco carcinogen exposure (3, 4). Heavy smokers as well as
epithelial cells and are typically peripherally located as
patients who have survived a prior aerodigestive cancer com-
reviewed elsewhere (10, 11).
prise a high-risk population that may be targeted for early
Accumulating evidence suggests that malignant lung tumors
detection and chemoprevention efforts (7). Also, while the risk
develop through progressive pathologic changes known as
of developing lung cancer decreases after smoking cessation,
preneoplastic or premalignant lesions (12, 13). Limited molec-
the risk never returns to baseline (9). Therefore, novel
ular aberrations (discussed below) have been described in lung
approaches for early detection and treatment of lung cancer
premalignant lesions. Studies have shown that cytologically
among former smokers are urgently needed. Curtailing these
normal airway epithelial cells lining the entire respiratory tract
key advances is a limited understanding of the earliest events in
and that have been exposed to smoking carry molecular altera-
lung cancer pathogenesis.
tions that may signify the onset of lung cancers (14, 15), a
Lung cancer consists of several histologic types, including
paradigm commonly referred to as the "airway field of injury"
small-cell lung carcinoma (SCLC) and non–small cell lung
(refs. 14, 15; Fig. 1A). Additional studies have also pointed to
carcinoma (NSCLC), with the latter comprising the majority
molecular alterations (e.g., tumor-associated gene expression
of diagnosed (
85%) lung tumors (4, 10). NSCLC is composed
patterns) that are shared between NSCLCs and adjacent cyto-
of the following three major histologic subtypes: lung adeno-
logically normal airway epithelia (referred to as molecular and
adjacent airway field of cancerization; ref. 14; Fig. 1B). These
premalignant fields can provide biologic insights into the
1
Department of Translational Molecular Pathology, The University of development of lung tumors (refs. 14, 15; Fig. 1). Particularly,
Texas MD Anderson Cancer Center, Houston, Texas. 2The University of the airway field of injury, because it is widespread in the lung
Texas Graduate School of Biomedical Sciences, Houston, Texas.
3
Department of Epidemiology, The University of Texas MD Anderson and precedes the development of tumors and premalignant
Cancer Center, Houston, Texas. 4Section of Computational Biomedi- lesions, also provides potential clinical opportunities for early
cine, Boston University School of Medicine, Boston University, Boston, detection and chemoprevention in high-risk smokers and in
Massachusetts.
patients who have survived a cancer of the upper aerodigestive
Corresponding Author: Humam Kadara, The University of Texas MD Anderson tract (14, 16).
Cancer Center, 1515 Holcombe Boulevard, Unit 2951, Houston, TX 77030. Phone:
It is clear that early detection of lung cancer or intraepithelial
713-745-3186; Fax: 713-834-6082; E-mail: hkadara@mdanderson.org
lesions and effective prevention are crucial to reducing lung
doi: 10.1158/1940-6207.CAPR-15-0400 cancer mortality. However, and to date, large-scale lung cancer
Ó2016 American Association for Cancer Research. chemoprevention trials have been negative with neutral, or

518 Cancer Prev Res; 9(7) July 2016

 Early Molecular Events in Lung Cancer Pathogenesis

A Airway field of injury

B Adjacent airway field of
cancerization

Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/9/7/518/1937107/518.pdf by guest on 21 May 2023

C Normal Preneoplastic Invasive
Epithelium “Field of injury ” lesion Carcinoma

Molecular changes Molecular changes in

involved in development of progression of the airway
the airway field of injury field of injury to preneoplasia

© 2016 American Association for Cancer Research

Figure 1.
Premalignant airway fields in the molecular pathogenesis of lung cancer. A, smoking induces widespread molecular alterations, such as gene expression
changes (small red circles), in exposed epithelia throughout the respiratory tract. This airway field of injury has been described in smokers with or
without lung cancer and is highly relevant for the identification of markers for minimally invasive and early detection of lung cancer. B, the adjacent airway
field of cancerization represents the field in normal-appearing airways adjacent to lung tumors. It has been suggested that in this adjacent field of
cancerization, there is closer molecular genealogy between lung cancers and airways that are in closest proximity to the tumors compared with
airways that are more distant from the tumors [illustrated in the figure by the decreased number of red circles (i.e., aberrations) in airways with
greater spatial distance from the tumor]. C, the progression of the molecular airway field of injury to preneoplasia and lung malignancy is still not clear.
Understanding molecular changes involved in the development of the airway field of injury and changes mediating progression of this field to lung
preneoplasia would enable the identification of early markers for lung cancer detection and chemoprevention.

even harmful, results (6, 17). Additionally, early discovery and genome-wide changes in lung premalignant phases as potential
diagnosis of lung cancers is still suboptimal (5). These limita- means to identify new markers that can guide strategies for early
tions are largely attributed to the scarcity of reliable molecular detection and prevention of lung cancer.
targets (e.g., genomic loci or genes) on which to assess the
efficacy of agents in clinical trials (6, 16, 17) and markers for
early detection of lesions (5, 18). Identification of these mar- Lung Cancer Pathogenesis
kers and targets is thought to heavily rest on comprehending Multistep tumorigenesis, in which tumors develop through a
early lung oncogenesis. In spite of this urgency, we still do not series of progressive pathologic changes (preneoplastic or pre-
know the molecular mechanisms by which the airway field of cursor lesions) with corresponding genetic and epigenetic aber-
injury evolves to a lung cancer (Fig. 1C) and we are unable to rations, has been demonstrated in various organs, including the
identify the individuals at higher risk (e.g., heavy smokers) who skin, lung, and colon (19–23). Hyperplasia, squamous meta-
are more likely to develop these lesions. Further and in par- plasia, squamous dysplasia, and carcinoma in situ (CIS) com-
ticular, we still have no effective means to predict which prise changes in the large airways that precede or accompany
premalignant lesions will go on to progress to malignant lung invasive LUSC (10, 12, 13). Dysplastic squamous lesions them-
cancer and, if so, how we can prevent or block this process. selves are typically categorized by different intensities—mild,
Here, we review the current state of knowledge of the molecular moderate and severe—based on a continuum of cytologic and
pathology of lung preneoplasia and oncogenesis. The review also histologic atypical changes (10, 12, 13). The sequential pre-
describes the airway field of injury phenomenon and how molec- neoplastic pathologic and molecular changes associated with
ular alterations in this epithelial field are relevant to the biology of LUSCs point to a multistage manner in the development of this
lung cancer and to early detection and prevention of the disease. lung cancer subtype from the respiratory mucosa (22). On the
We will also consider new approaches to better understand other hand, studies have suggested that the accumulation of

www.aacrjournals.org Cancer Prev Res; 9(7) July 2016 519

 Kadara et al.

molecular abnormalities beyond a certain threshold, rather There are shared molecular alterations between LUADs and
than the sequence of alterations, is the basis for development AAHs and they provide further evidence that AAHs represent
of the malignant phenotype (14). For example, sequential preneoplastic lesions in the course of LUAD pathogenesis (46).
premalignant changes have been poorly documented for Previous studies have reported that
30% to 40% of AAHs
LUADs, neuroendocrine tumors, and SCLCs. Atypical adeno- display KRAS (codon 12) mutations (25). In addition, EGFR
matous hyperplasia (AAH) is the only sequence of morpho- mutations are shared at similar frequencies between AAHs,
logic change identified so far for the development of invasive AIS lesions, and LUADs, particularly in sequence of lesions
LUADs (12, 13, 24, 25). Diffuse idiopathic pulmonary neuro- preceding development of the TRU subtype of LUADs (11). It is
endocrine cell hyperplasia (DIPNECH), a rare lesion worthy to note that EGFR mutations were detected in adjacent
that includes local extraluminal proliferation in the form of normal-appearing peripheral respiratory epithelium in 43% of
tumorlets, is considered to be the precursor lesion for neuro- EGFR-mutant LUAD but not in patients wild-type for the
endocrine carcinoids of the lung (26). Notably, there are no oncogene (44), suggesting that there may be different cells of
known preneoplastic lesions for the most common type of origin (bronchiolar epithelium and AT2 cells) for EGFR-mutant
neuroendocrine lung tumors, SCLCs. Increasing our under- LUADs. Other molecular aberrations that were identified
standing of early events in lung cancer pathogenesis would in AAHs include overexpression of Cyclin D1, survivin, and
allow us to better pinpoint the manner (multistep vs. linear) in ERBB2 oncoproteins (24, 47, 48), loss-of-heterozygosity (LOH)

Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/9/7/518/1937107/518.pdf by guest on 21 May 2023

which the malignant lung phenotype arises (27). Development in chromosomes 3p (18%), 9p (CDKN2A), 9q (tuberous scle-
of the lung malignant phenotype appears to be both due to rosis complex 1/TSC1), 17q, and 17p (TP53; refs. 10, 49) and
stepwise, sequence-specific and multistage molecular patho- reduced expression of the tumor suppressor serine threonine
genesis and due to accumulation and combination of genetic kinase 11 (STK11 also known as LKB1; ref. 50). Epigenetic
and epigenetic abnormalities. alterations including DNA methylation of CDKN2A and
PTPRN2 were also reported in AAHs (51). Studies aimed at
Molecular pathogenesis of LUAD interrogating differential gene expression and copy-number
A large body of evidence suggests that at least two molecular profiles between low-grade lesions (e.g., AAH and AIS) and
pathways are involved in the development of LUAD, the Kirsten LUADs found that amplification of the EGFR oncogene was
rat sarcoma viral oncogene (KRAS) and epidermal growth the predominant differential molecular feature between early
factor receptor (EGFR) pathways in smoker and non-smoker lesions and LUADs (27).
patients, respectively. Mutations in EGFR, particularly in-frame Studies have also shown that AAH and AIS lesions express
deletions of exon 19 and L858R and L861Q variants in exon 21, high levels of NKX2-1 (also known as TITF-1; ref. 43), a home-
are strongly associated with never-smoking status, female gen- odomain-containing transactivating factor predominantly
der, and East Asian ethnicity (28–31). On the other hand, expressed in the terminal lung bronchioles and lung periphery
mutations in KRAS are strongly associated with development (52, 53). In addition, NKX2-1 is crucial for branching morpho-
of LUADs linked to tobacco consumption (4, 28, 32–35). Other genesis during normal lung development (52–54) and transac-
commonly occurring point mutations and copy-number altera- tivates the expression of surfactant proteins important for the
tions (CNA) have been described in LUAD (32, 36, 37) and are differentiation of AT2 cells (55). NKX2-1 is commonly gained or
reviewed elsewhere (38). amplified (14q13.3 locus) in LUAD pointing to a cell-lineage
As mentioned before, AAH is the only sequence of morpholo- specific oncogenic function for this transcriptional factor in
gic change identified so far for the development of LUADs (12, 13, LUADs (32, 36, 56, 57). It is important to mention that while
24, 25) and there is consensus that the pathogenesis of many various reports have pointed to a growth-promoting role for
adenocarcinomas is largely unknown. Earlier studies have pro- NKX2-1 in human LUAD cells (56–58), studies in Kras(LSL-
posed that both Clara cells and alveolar type II (AT2) cells are the G12D/þ);p53(flox/flox) mice demonstrated that Nkx2-1 sup-
progenitor cells of LUADs (10, 39). However, accumulating presses murine lung tumor growth and metastatic potential
evidence now strongly suggests that surfactant protein C (SPC) in vivo (59).
expressing AT2 cells, and not Clara (also known as club) cells, are
the cell of origin of LUADs (40, 41). Due to the difficulty in Molecular pathogenesis of LUSC
distinction between AAHs and bronchioalveolar carcinoma It has been postulated that basal cells in the large airways
(BAC), new classification guidelines for LUAD have discontinued exhibit pluripotent capacity following cigarette smoke expo-
the BAC terminology, which is now replaced with adenocarcino- sure, giving rise to metaplastic and dysplastic squamous cells,
ma in situ (AIS; for LUADs with pure lepidic growth) or minimally which in turn function as precursors of squamous cell carci-
invasive adenocarcinoma (MIA; predominant lepidic growth with nomas (10, 12, 13). Earlier studies aimed at examining sequen-
less than 5-mm invasion; ref. 42). It is worthwhile to note that tial molecular abnormalities in the pathogenesis of LUSC
AAHs are thought to be involved in the linear progression of cells indicated that genetic abnormalities commence in histologi-
of the "terminal respiratory unit" (TRU) to AIS and subsequently cally normal epithelium and augment with progression of
invasive LUADs (11, 27, 43) due to the expression of common histopathologic changes (22, 60, 61). Allelic losses at multiple
genes between the TRU and AAH. These studies in East Asian 3p (3p21, 3p14, 3p22–24 and 3p12) chromosome sites and
LUAD patients have postulated that most, if not all, peripheral 9p21 (CDKN2A) in normal-appearing bronchial epithelia were
LUADs progress from alveoli through AAH as a preneoplastic described as the earliest detected changes in the sequence of
lesion. On the other hand, other studies suggested that LUADs pathogenesis of LUSCs (22, 61). Allelic imbalance in 8p21–23,
may arise from bronchiolar epithelium and small bronchi based 13q14 (RB1) and 17p13 (TP53) were detected in squamous
on the finding of driver EGFR mutations (e.g., EGFR p.L858R) in preinvasive lesions (22, 61, 62). DNA methylation of CDKN2A
bronchioles adjacent to EGFR-mutant LUADs (44, 45). is also found in early squamous preinvasive lesions with

520 Cancer Prev Res; 9(7) July 2016 Cancer Prevention Research
 Early Molecular Events in Lung Cancer Pathogenesis

frequencies that increase with histopathologic progression and colleagues provides important clues into early mecha-
(24% in squamous metaplasia and 50% in CIS; ref. 63). Vas- nisms of LUSC pathogenesis and potential biomarkers for
cular endothelial growth factor (VEGF) isoforms and VEGF chemoprevention. It is important to note that the findings were
receptors (VEGFR) have been found to be elevated in bronchial limited by a small number of LUSC patients (n ¼ 4; ref. 76),
squamous dysplastic lesions compared with normal bronchial thus warranting future studies with larger sample sizes. In
epithelia (64), supporting the notion that angiogenesis devel- addition, similar studies that comprise orthogonal (e.g., DNA
ops early in lung carcinogenesis and that these abnormalities sequencing) analyses will allow the identification of drivers in
provide a rationale for the development of targeted antiangio- the development of squamous preinvasive lesions and in their
genic chemoprevention strategies. Further, angiogenic squa- progression to malignant lung tumors.
mous dysplasias (ASD), a subset of squamous dysplasias,
exhibit vascular budding in the subepithelial tissues with SCLC pathogenesis
elevated microvessel density pointing to an architectural rear- To date, there is no phenotypically identifiable preneoplastic
rangement of the capillary microvasculature (65). Additionally, lesion for SCLC. Compared with NSCLC, very little is known
the presence of this lesion in high-risk smokers suggests that about early events in the molecular pathogenesis of SCLC. A
aberrant patterns of microvascularization may occur at an early study assessing LOH at several chromosomal sites and micro-
stage of bronchial carcinogenesis (65, 66). Besides altered satellite instability in histologically normal and hyperplastic

Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/9/7/518/1937107/518.pdf by guest on 21 May 2023

angiogenesis, other pathways, including fatty acids and retinoic bronchial epithelia adjacent to lung tumors demonstrated a
acid signaling, are also shared between squamous preinvasive significantly higher incidence of alterations in epithelia nearby
lesions and LUSCs and are reviewed elsewhere (67, 68). SCLCs than those adjacent to NSCLCs (77). These earlier find-
The SOX2 oncogene, demonstrated to be amplified (3q26.3) ings point to widespread and extensive molecular damage in
in LUSCs, promotes survival of tumors with amplification of normal tissues surrounding SCLCs and suggest that SCLC may
the gene (69, 70). Additionally, SOX2 was found to foster the develop directly from histologically normal or mildly abnormal
growth of lung tumor cells, particularly cells of squamous epithelium without passing through a more complex histologic
histology, and to mediate stemness of lung cancer stem cells sequence. It is noteworthy that studies using genetically engi-
(69, 71, 72). Alterations in SOX2 are also evident in premalig- neered mouse (GEM) models demonstrated that SCLC devel-
nant phases of LUSC development. Yuan and colleagues dem- opment is driven by inactivation of TP53 and RB1 (78) as well
onstrated that SOX2 protein expression was completely absent as by activation of the hedgehog pathway (79, 80). In a study
in lung adenocarcinoma pathogenesis and highly expressed in using integrative genomic analyses, human SCLCs were found
preinvasive squamous lesions and in LUSCs (73). McCaughan to exhibit known alterations in TP53 and RB1, recurrent muta-
and colleagues demonstrated, by analysis of the 3q chromo- tions in the histone modifiers CREBBP, EP300, and MLL as well
somal region, that high-grade bronchial dysplasias but not as amplifications in the FGFR1 oncogene (81). More recently,
low-grade lesions exhibited amplification of SOX2 (74). Nota- proteomic profiling studies have identified alterations in SCLC
bly, SOX2 gain was associated with clinical progression of high- that comprise therapeutically viable targets, including PARP1
grade preinvasive squamous lesions (74). In a manner analo- and DNA repair proteins (82, 83). Also, Mohammad and
gous to NKX2-1 in LUADs, it is plausible to surmise that SOX2 colleagues described the histone demethylase lysine demethy-
functions as a lineage-restricted oncogene in the early patho- lase 1 (LMD1) as a therapeutic target for SCLC (84). It is
genesis of LUSCs (73, 74). noteworthy that the roles of these aforementioned alterations
In contrast to studies in malignant tumors, genome-wide and targets in normal and premalignant phases of SCLC devel-
analyses of lung premalignancy have been extremely rare. It is opment are still poorly comprehended.
conceivable that molecular profiling of premalignant lung
lesions will substantially increase our understanding of early
events in lung cancer pathogenesis. An important step in this Field of Injury and Field Carcinogenesis
direction was the study by Nakachi and colleagues in which Earlier work by Danely Slaughter assessing oral premalig-
single-nucleotide polymorphism (SNP) arrays were used to nant and malignant lesions revealed widespread histologic
characterize novel chromosomal alterations in preinvasive dys- abnormalities in epithelial tissues that were distant to the oral
plastic lesions (75). The study revealed known and novel cavity (e.g., in esophagus and the lung) and at the margins of
recurrent chromosomal alterations in dysplastic lesions (75). invasive cancers (85). The studies by Slaughter and colleagues
These chromosomal regions included losses of putative tumor point to a "field cancerization," an effect in which the entire
suppressor genes RNF20 and SSBP2 and amplification of the carcinogen-exposed epithelial field is primed for cancerization
RASGRP3 oncogene (75). Notably, some of the chromosomal and tumor development (85). Field carcinogenesis has been
alterations were found in multiple premalignant lesions (75), described in various epithelial cell malignancies, including
indicative of widespread field effects (discussed further below). gastric, esophageal, hepatic, cervical, skin, and lung cancers
In another study by Ooi and colleagues, laser-capture micro- (14, 86–88). In 1961, Auerbach and colleagues suggested that
dissected and paired normal basal cells, premalignant lesions cigarette smoke induces extensive histologic changes in the
and LUSCs were analyzed by RNA-sequencing (RNA-seq; bronchial epithelia of smokers, leading to widespread prema-
ref. 76). This study pinpointed gene expression profiles mod- lignant lesions throughout the respiratory epithelium, sugges-
ulated early on between normal cells and premalignant lesions tive of a field effect (89). The advent of molecular high-
as well as profiles that changed between preneoplastic tissues throughput assays allowed the extension of Slaughter's concept
and the LUSCs (76). Using pathways and gene network anal- to molecular changes, a phenomenon termed airway field of
yses, these expression changes were found to be indicative of injury (15). It is now thought that the injured (e.g., by tobacco)
TP53 inhibition and MYC activation (76). The study by Ooi airway epithelium exhibits various molecular alterations (e.g.,

www.aacrjournals.org Cancer Prev Res; 9(7) July 2016 521

 Kadara et al.

aberrant gene expression, LOH) that precede and thus inform were associated with clinical response as measured by regres-
of the onset of primary lung cancer (15, 90–95). Because sion of the dysplastic lesion (104). More recently, Shaykhiev
airways of smokers invariably exhibit some degree of inflam- and colleagues demonstrated that airway basal cells in healthy
mation and inflammatory-related damage, the field of injury in smokers undergo reprogramming toward an embryonic stem
the lung may also be explained by both direct effect of tobacco cell–like phenotype and postulated that this process represents
carcinogens and initiation of the inflammatory response, and an early event in the development of lung carcinomas in
this concept has been extensively reviewed elsewhere (15). smokers (105). It is worthwhile to mention that expression
Various studies have suggested that adjacent normal-appear- profiling studies also demonstrated widespread extent of
ing airway epithelia exhibit molecular alterations that are spread of the molecular field of injury, revealing common
characteristic of the nearby lung tumor, an effect referred to alterations in bronchial, nasal, and buccal epithelia of smokers
by our group and others as the molecular and adjacent airway (106). Also, 119 genes were found to be modulated similarly by
field of cancerization (14, 96, 97). As mentioned before, smoking in both bronchial and nasal epithelium (107),
detailed analysis of histologically normal, premalignant, and although the bronchial and nasal compartments also exhibited
malignant epithelia from LUSC patients revealed that multiple, notable differences in gene expression.
sequentially occurring allelic imbalance events such as LOH Field carcinogenesis has also been shown to comprise aber-
commence in clonally independent foci early in the multistage rant modulation of microRNAs (miRNA; ref. 108). Global

Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/9/7/518/1937107/518.pdf by guest on 21 May 2023

pathogenesis of LUSC (22, 61). Notably, 31% of histologically alterations in miRNAs were found in the airways of smokers
normal epithelium adjacent to lung tumors exhibited LOH at relative to non-smokers (109). More recently, a study using
chromosomal regions 3p and 9p (22). LOH was also described microRNA-sequencing (miRNA-seq) strategy by Perdomo and
in normal bronchial epithelia of smokers without cancer and, colleagues identified a novel miRNA, miR-4423, that exhibited
importantly, appeared to have persisted for many years after lineage-specific properties because it was found to be exclu-
smoking cessation (92, 95). Activating mutations in the KRAS sively restricted in expression to the airway epithelium (110).
and EGFR oncogene have also been described in histologically This study also revealed that miR-4423 was decreased in lung
normal tissue adjacent to lung tumors (44, 98) and widespread tumors and inhibited features of the lung malignant pheno-
mutations in TP53 were reported in bronchial epithelia of type in vitro and in vivo (110), suggesting that miRNAs also lie
cancer-free smokers (99). Epigenetic alterations are also present at the intersection between field carcinogenesis and early
in the airway field of injury (63, 100–102). Belinsky and events in lung cancer pathogenesis.
colleagues demonstrated aberrant methylation of CDKN2A The spatiotemporal dynamics of the airway field of injury
and DAPK in the normal-appearing bronchial epithelium of and of the adjacent (to lung tumor) molecular field of cancer-
lung cancer cases and cancer-free controls (100). The same ization were analyzed in recent studies by assessment of mul-
study also revealed high level of concordance in CDKN2A tiple field samples per patient. A study analyzing airway gene
methylation status between lung tumors and adjacent normal expression at several time points (up to 3 years) following
bronchial epithelium (100). Other genes that were described surgery in definitively treated early-stage smoker NSCLC
as methylated in the airway field of injury in cancer-free patients, who were enrolled in a phase II surveillance trial,
smokers include RAR-b2, RASSFF1A, and GSTP1 (101, 102). provided novel insights into spatiotemporal characteristics of
Studies that used high-throughput approaches, such as gene the airway field of injury (96, 111). Although this study was
expression profiling, to interrogate the airway field of injury limited by lack of airway samples prior to surgery and when the
that is widespread throughout the lung have provided new tumor was still in situ, it identified candidate pathways and
insights into early events in lung cancer pathogenesis (103). markers that were differentially modulated with time in the field
Hackett and colleagues analyzed expression of 44 anti-oxidant– (96, 111). These included increased bronchial expression of
related and demonstrated significant upregulation of 16 anti- phosphorylated forms of the AKT1 and ERK1/2 oncogenes
oxidant genes in the airways of smokers relative to non-smokers (111). These findings suggest that the airway field of injury
(91). Normal-appearing bronchial epithelia of healthy cancer- exhibits temporal molecular changes that may be associated
free smokers were shown to comprise global changes in gene with lung cancer recurrence in surgically treated early stage
expression when compared with epithelium in non-smokers patients (96). In another study by the same group, paired
(93). Importantly, irreversible changes in airway gene expres- NSCLCs, uninvolved normal lung tissues and multiple nor-
sion following several years of smoking cessation were mal-appearing airways in the adjacent field of cancerization
described and were proposed to be associated with the persis- were analyzed by whole-transcriptome profiling (97). Impor-
tent risk former smokers exhibit for developing lung cancer tantly, the study demonstrated that gene expression patterns in
(90, 93). An important milestone in field of injury studies was the adjacent airway field of cancerization were enriched with a
the development of a gene-expression signature in cytologically signature that can distinguish smokers with lung cancer from
normal mainstem bronchus epithelium that can distinguish smokers with benign disease (97). This study also identified
smokers with lung cancer from those with benign disease (94). notable spatial properties in the airway field of cancerization in
Later, Gustafson and colleagues derived a PIK3CA pathway which gene profiles of adjacent but not distant airway cells
activation signature, which was found to be elevated in the closely embodied expression patterns of the nearby NSCLC
cytologically normal bronchial airway of smokers with lung (97). One of these genes, LAPTM4B, a lysosome-associated
cancer or with premalignant lesions relative to cancer-free oncogene, was later shown to be a novel activator of the NRF2
controls. Importantly, among smokers with premalignant stress response pathway in lung cancer cells (112). Like the
lesions, this PIK3CA signature decreased in the field of injury report by Gustafson and colleagues (104), these studies
following chemoprevention with the PIK3CA inhibitor myo- (97, 111, 112) suggest that airway field of injury and the
inositol, and changes in this airway gene-expression signature relatively more adjacent or local field of cancerization comprise

522 Cancer Prev Res; 9(7) July 2016 Cancer Prevention Research
 Early Molecular Events in Lung Cancer Pathogenesis

activation of therapeutically pliable signaling pathways, thus similar studies in premalignant lung tissues have been very
creating an opportunity for the development of early targeted limited.
chemoprevention strategies. Beane and colleagues interrogated the transcriptome of air-
The above studies in field carcinogenesis hold the potential way epithelia from a small number of smokers with and
to directly impact clinical care by providing novel strategies without lung cancer by RNA-seq (120). This study identified
for early detection and diagnosis (7). Although bronchoscopy markers and operative signaling cues, in the airway field of
is routinely used as an initial diagnostic tool in smokers with injury, that appear pertinent to lung oncogenesis (120). More
a suspicious lesion found on chest imaging, it is frequently recently, using deep-sequencing technology, Perdomo and col-
non-diagnostic, often resulting in additional invasive testing, leagues identified a novel lineage-specific miRNA, miR-4423,
including surgical lung biopsy even when the lesion is benign that is primarily expressed in normal airway epithelium (110).
(113, 114). In order to address this clinical unmet need, Following RNA-seq, the study then demonstrated that miR-
Whiteny and colleagues extended the initial observations of 4423 regulated airway epithelial cell differentiation in vitro and
a cancer-specific gene-expression signature in the mainstem suppressed features of the lung malignant phenotype in vitro
bronchus (94) by developing a 17-gene classifier, derived from and in vivo (110). Recently and as described above, RNA-seq
bronchial epithelial cells collected during bronchoscopy, able was used to interrogate genes that are differentially expressed
to discern current or former smokers with lung cancer from between paired normal epithelia, preinvasion squamous

Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/9/7/518/1937107/518.pdf by guest on 21 May 2023

those without the malignancy (115). In a recent translational lesions, and LUSCs (76). This study identified genes modulated
study by Silvestri and colleagues, the performance of the "early" between normal epithelia and preneoplasia as well
17-gene classifier as a bronchial biomarker for lung cancer as markers modulated later in the pathogenesis of the lesions
detection was prospectively validated in 639 suspect smokers to LUSCs, although findings were limited by the small cohort
undergoing bronchoscopy in two multicenter trials, Airway size (76). Adequately powered NGS studies of the airway field
Epithelial Gene Expression in the Diagnosis of Lung Cancer and lung preneoplasia will undoubtedly expand our under-
(AEGIS) 1 and 2 trials (116). It is worthwhile to note that the standing of the biology of lung cancer pathogenesis.
classifier and bronchoscopy exhibited a combined sensitivity of
97% for diagnosing lung cancer in both cohorts, with high
sensitivity for the classifier irrespective of size, location, cell Intra-lesional heterogeneity
type, or stage of the cancer (116). Notably, among smokers Concepts in evolutionarily biology have provided impor-
with a non-diagnostic bronchoscopy and an intermediate pre- tant insights into the evolution and progression of cancer
test probability of malignancy (where the physician is most (121, 122). Earlier work by Nowell suggested that tumor
uncertain about cancer status), the negative predictive value of cells acquire mutations some of which provide survival advan-
the classifier was 91%, potentially impacting clinical decision tages to selective clones of cells (123). Accumulating evi-
making by allowing these patients to be followed with CT dence points to a branched evolution pattern for tumor
surveillance as opposed to invasive biopsies in patients without pathogenesis in which subclones expand independently,
lung cancer (116). acquiring different mutations over time (121, 122). In addi-
tion, these multiple subclonal populations can coexist togeth-
er (121). Evidence of heterogeneity among tumor cells was
Emerging Concepts and Approaches to
first observed by molecular pathologists through cytogenetic
Study Lung Cancer Pathogenesis analysis and examination of karyotypes (121, 124, 125).
The application of standard genomic technologies (e.g., Heterogeneity was also previously genetically discerned by
arrays and sequencing) to survey genome-wide alterations has the identification of differential copy-number signals among
largely enhanced our understanding of the biology of malig- different cells in the same tumors (reviewed in ref. 126). With
nant lung tumors. In sharp contrast, our understanding of the the advent of NGS technologies, intratumoral heterogeneity
molecular pathology of the airway fields of injury and cancer- (ITH) can now be assessed genome wide at the single-nucle-
ization and of lung premalignant lesions is extremely limited. otide level (121). Genomic ITH has now been described in
Understanding the landscape of genomic changes in normal or various malignancies, including lung cancer (121, 127, 128).
premalignant phases in lung oncogenesis may provide valuable Zhang and colleagues used whole-exome sequencing to survey
insights into the earliest events that drive the pathogenesis of the spectrum of ITH in 11 early-stage LUADs (128). Notably,
lung cancer. this study alluded to an association between the extent of
ITH and post-surgical relapse, although findings were limited
Deep next-generation sequencing by the relatively small cohort and warrant validation in
Next-generation sequencing (NGS) technology, through additional studies (128). It is tempting to speculate that
whole-genome, whole-exome, and whole-transcriptome appro- paired analysis of molecular spectrum of ITH in the matched
aches, holds great promise for providing invaluable insights normal-appearing airway epithelia, premalignant lesions and
into lung cancer biology, diagnosis, prevention, and therapy primary NSCLCs will shed light on shared genealogy among
(117). NGS enables the sequencing of expressed genes, exons, the different stages of lung oncogenesis. It is conceivable to
and complete genomes, providing data on levels of expres- surmise that smoking-exposed airway epithelia in the fields of
sion, with a substantially larger dynamic range compared with injury/cancerization or premalignant lesions may exhibit cel-
array technology, sequence alterations, single-nucleotide var- lular subclones with different mutational patterns, and thus
iations as well as structural genomic aberrations (117). While variable capacity to progress to NSCLC. Resolving these fea-
various reports have used NGS to interrogate the molecular tures will allow us to better understand the spatiotemporal
pathology of malignant lung tumors (32, 36–38, 118, 119), dynamics of lung cancer evolution.

www.aacrjournals.org Cancer Prev Res; 9(7) July 2016 523

 Kadara et al.

Identification of subtle alterations in lung premalignancy subtle events of allelic imbalance in the adjacent normal-
Airway epithelia with field carcinogenesis or premalignant appearing airway that are also present in the nearby NSCLC
lesions present unique challenges when profiling their molecular as well as within different multiregion intratumoral biopsies
changes via standard genomic technologies. These technologies within the same early-stage NSCLC tumor. Applying these
were originally designed for scenarios of higher purity of the sensitive methods to infer the order of somatic events should
targeted tissue, or at least more balanced contributions of com- lend valuable insights into studies of field carcinogenesis and
ponents of interest, i.e., the cells of the premalignant lesion preneoplasia and offer windows into the earliest genomic
harboring somatic mutation (129, 130). When the proportion changes associated with lung cancer pathogenesis and, thus,
of aberrant cells in the targeted tissue is low, analogous to low targets for chemoprevention.
tumor purities ("cellularity," or proportion of DNA harboring an
aberration), the task of identifying alterations becomes exceed- Concluding Remarks
ingly difficult. For the normal-appearing airway fields of injury/
Despite numerous efforts that have centered on characterizing
cancerization and preinvasive lesions, genomic alterations will
molecular profiles in lung cancer, this malignancy still accounts
certainly be subtle, and in some cases rare (within samples and
for the biggest fraction of cancer-related deaths in the United
across individuals).
States and worldwide (1, 2, 7). Despite encouraging findings from
There are two main sources of information when assessing

Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/9/7/518/1937107/518.pdf by guest on 21 May 2023

the National Lung Screening Trial (142, 143), early detection of
evidence for CNAs: (i) total copy-number gains and losses from
lung cancer is challenging due to the lack of biomarkers for the
allelic intensities (from SNP microarrays) or read coverage
early diagnosis of the disease and due to the presence of multiple
(from NGS); and (ii) imbalance in allele-specific signals (B-
neoplastic molecular pathways that mediate lung carcinogenesis
allele frequencies; BAF) at germline heterozygous loci (131).
(5, 18, 103, 114). Moreover, little progress has been made in
Generally, standard computational methods may discern BAF
personalized and targeted chemoprevention strategies for this
dispersions when the aberration proportions are above 10% to
fatal malignancy (6, 17). Preneoplastic changes have been used
15% (132–135). However, methods that model the dispersion
as surrogate endpoints for chemopreventive studies. However, it
in BAFs induced by a CNA ignore critical information in subtle
was suggested that this "shooting in the dark" approach may
shifts of the BAF values (136). Because it is likely that a single
explain the reasons behind the general failures of clinical che-
chromosomal event, such as a CNA (i.e., a loss, gain, or cn-
moprevention studies (16). Therefore, novel approaches to iden-
LOH), induced the allelic imbalance in a particular contiguous
tify the best population to be targeted for early detection and
region on a chromosome, the direction the BAFs move from the
chemoprevention should be devised, and risk factors for lung
canonical value for a heterozygote (one-half) is not random
cancer development or relapse need to be better defined. Achiev-
(136). They, instead, move according to the allelic composition
ing this rests heavily on understanding early events (e.g., field
of an inherited chromosome, also known as a haplotype (136).
carcinogenesis) in the molecular pathogenesis of lung cancer. This
Thus, methods that model the distribution of BAF values jointly
notion is exemplified by the recent and encouraging findings of
can gain considerable power, particularly at low cell fractions,
the prospective AEGIS-1 and -2 clinical trials demonstrating
over standard algorithms that ignore this information and
sensitive and specific detection of lung cancer based on a molec-
instead consider only the magnitude of their shifts but not
ular gene classifier derived from relatively accessible normal
their direction (136). Precancerous lesions present greater
airway cells of suspect smokers (116). Comprehensive analysis
challenges than characterizing cancer genomes, and incorpo-
of early molecular events in lung cancer pathogenesis will
ration of haplotype information in this space is even more
undoubtedly unravel biomarkers that can aid early detection and
critical.
prevention efforts deliver their longstanding promise to oppose
Recently, computational methods have been developed that
lung cancer.
incorporate this information and deal directly with uncertainty
in haplotype estimation, hapLOH (136), and J-LOH (137).
These highly sensitive haplotype-based computational meth- Disclosure of Potential Conflicts of Interest
ods can deal with low-cellularity samples, or situations where A.E. Spira is a consultant/advisory board member for Veracyte Inc. No
the aberrant cell fraction is below 5% by assessing consistency potential conflicts of interest were disclosed by the other authors.
between BAF values and inherited chromosomes, estimated
statistically (138, 139), capturing the direction of the BAF
values (136, 137, 140, 141). This extra level of specificity is Grant Support
critical when attempting to characterize phenomena that are This study was supported in part by Departments of Defense (DoD)
rare (infrequent along the genome or in few samples) and/or grant W81XWH-10-1-1007 (H. Kadara, I.I. Wistuba, and A. Spira) and
W81XWH-11-2-0161 (A. Spira), NIH grant R01HG005859 (P. Scheet), and
subtle (occurring in a low proportion of the cells from which Cancer Prevention and Research Institute of Texas (CPRIT) award RP150079
the DNA was obtained), settings anticipated in normal-appear- (P. Scheet and H. Kadara).
ing the airway field of injury or in preneoplasia. When applied
to study chromosomal alterations in the airway field of cancer- Received November 23, 2015; revised February 29, 2016; accepted March 1,
ization, these haplotype-based methods may be able to discern 2016; published OnlineFirst March 22, 2016.

References
1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global
2015;65:5–29. cancer statistics, 2012. CA Cancer J Clin 2015;65:87–108.

524 Cancer Prev Res; 9(7) July 2016 Cancer Prevention Research
 Early Molecular Events in Lung Cancer Pathogenesis

3. Goldstraw P, Ball D, Jett JR, Le Chevalier T, Lim E, Nicholson AG, et al. factor receptor gene mutations in lung cancers. J Natl Cancer Inst
Non-small-cell lung cancer. Lancet 2011;378:1727–40. 2005;97:339–46.
4. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 2008; 31. Sun S, Schiller JH, Gazdar AF. Lung cancer in never smokers–a different
359:1367–80. disease. Nat Rev Cancer 2007;7:778–90.
5. Kanodra NM, Silvestri GA, Tanner NT. Screening and early detection 32. Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis
efforts in lung cancer. Cancer 2015;121:1347–56. E, et al. Mapping the hallmarks of lung adenocarcinoma with massively
6. Keith RL, Miller YE. Lung cancer chemoprevention: current status and parallel sequencing. Cell 2012;150:1107–20.
future prospects. Nat Rev Clin Oncol 2013;10:334–43. 33. Le Calvez F, Mukeria A, Hunt JD, Kelm O, Hung RJ, Taniere P, et al. TP53
7. Spira A, Halmos B, Powell CA. Update in Lung Cancer 2014. Am J Respir and KRAS mutation load and types in lung cancers in relation to tobacco
Crit Care Med 2015;192:283–94. smoke: distinct patterns in never, former, and current smokers. Cancer Res
8. Szabo E, Mao JT, Lam S, Reid ME, Keith RL. Chemoprevention of lung 2005;65:5076–83.
cancer: diagnosis and management of lung cancer, 3rd ed: American 34. Ridanpaa M, Karjalainen A, Anttila S, Vainio H, Husgafvelpursiainen K.
College of Chest Physicians evidence-based clinical practice guidelines. Genetic alterations in p53 and k-ras in lung-cancer in relation to histo-
Chest 2013;143:e40S–60S. pathology of the tumor and smoking history of the patient. Int J Oncol
9. Karam-Hage M, Cinciripini PM, Gritz ER. Tobacco use and cessation for 1994;5:1109–17.
cancer survivors: an overview for clinicians. CA Cancer J Clin 2014;64: 35. Rodenhuis S, Slebos RJ, Boot AJ, Evers SG, Mooi WJ, Wagenaar SS, et al.
272–90. Incidence and possible clinical significance of K-ras oncogene activation
10. Wistuba II, Gazdar AF. Lung cancer preneoplasia. Annu Rev Pathol in adenocarcinoma of the human lung. Cancer Res 1988;48:5738–41.
2006;1:331–48. 36. Cancer Genome Atlas Research N. Comprehensive molecular profiling of

Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/9/7/518/1937107/518.pdf by guest on 21 May 2023

11. Yatabe Y. EGFR mutations and the terminal respiratory unit. Cancer lung adenocarcinoma. Nature 2014;511:543–50.
Metastasis Rev 2010;29:23–36. 37. Govindan R, Ding L, Griffith M, Subramanian J, Dees ND, Kanchi KL, et al.
12. Colby TV, Wistuba II, Gazdar A. Precursors to pulmonary neoplasia. Genomic landscape of non-small cell lung cancer in smokers and never-
Adv Anat Pathol 1998;5:205–15. smokers. Cell 2012;150:1121–34.
13. Kerr KM. Pulmonary preinvasive neoplasia. J Clin Pathol 2001;54: 38. Devarakonda S, Morgensztern D, Govindan R. Genomic alterations in
257–71. lung adenocarcinoma. Lancet Oncol 2015;16:e342–51.
14. Kadara H, Wistuba II. Field cancerization in non-small cell lung cancer: 39. Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, et al.
implications in disease pathogenesis. Proc Am Thorac Soc 2012;9:38–42. Identification of bronchioalveolar stem cells in normal lung and lung
15. Steiling K, Ryan J, Brody JS, Spira A. The field of tissue injury in the lung cancer. Cell 2005;121:823–35.
and airway. Cancer Prev Res 2008;1:396–403. 40. Desai TJ, Brownfield DG, Krasnow MA. Alveolar progenitor and stem cells
16. Gold KA, Kim ES, Lee JJ, Wistuba II, Farhangfar CJ, Hong WK. The BATTLE in lung development, renewal and cancer. Nature 2014;507:190–4.
to personalize lung cancer prevention through reverse migration. 41. Mainardi S, Mijimolle N, Francoz S, Vicente-Duenas C, Sanchez-Garcia I,
Cancer Prev Res 2011;4:962–72. Barbacid M. Identification of cancer initiating cells in K-Ras driven lung
17. Greenberg AK, Tsay JC, Tchou-Wong KM, Jorgensen A, Rom WN. Che- adenocarcinoma. Proc Natl Acad Sci U S A 2014;111:255–60.
moprevention of lung cancer: prospects and disappointments in human 42. Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe
clinical trials. Cancers 2013;5:131–48. Y, et al. International association for the study of lung cancer/American
18. Brothers JF, Hijazi K, Mascaux C, El-Zein RA, Spitz MR, Spira A. Bridging thoracic society/European respiratory society international multidisci-
the clinical gaps: genetic, epigenetic and transcriptomic biomarkers for the plinary classification of lung adenocarcinoma. J Thorac Oncol 2011;6:
early detection of lung cancer in the post-National Lung Screening Trial 244–85.
era. BMC Med 2013;11:168. 43. Yatabe Y, Mitsudomi T, Takahashi T. TTF-1 expression in pulmonary
19. Cho KR, Vogelstein B. Suppressor gene alterations in the colorectal adenocarcinomas. Am J Surg Pathol 2002;26:767–73.
adenoma-carcinoma sequence. J Cell Biochem Suppl 1992;16G:137–41. 44. Tang X, Shigematsu H, Bekele BN, Roth JA, Minna JD, Hong WK, et al.
20. Cho KR, Vogelstein B. Genetic alterations in the adenoma–carcinoma EGFR tyrosine kinase domain mutations are detected in histologically
sequence. Cancer 1992;70:1727–31. normal respiratory epithelium in lung cancer patients. Cancer Res 2005;
21. Vogelstein B, Kinzler KW. The multistep nature of cancer. Trends Genet 65:7568–72.
1993;9:138–41. 45. Tang X, Varella-Garcia M, Xavier AC, Massarelli E, Ozburn N, Moran C,
22. Wistuba II, Behrens C, Milchgrub S, Bryant D, Hung J, Minna JD, et al. et al. Epidermal growth factor receptor abnormalities in the patho-
Sequential molecular abnormalities are involved in the multistage genesis and progression of lung adenocarcinomas. Cancer Prev Res
development of squamous cell lung carcinoma. Oncogene 1999;18: 2008;1:192–200.
643–50. 46. Kitamura H, Kameda Y, Ito T, Hayashi H. Atypical adenomatous hyper-
23. Yuspa SH. The pathogenesis of squamous cell cancer: lessons learned plasia of the lung. Implications for the pathogenesis of peripheral lung
from studies of skin carcinogenesis–thirty-third G. H. A. Clowes Memorial adenocarcinoma. Am J Clin Pathol 1999;111:610–22.
Award Lecture. Cancer Res 1994;54:1178–89. 47. Nakanishi K, Kawai T, Kumaki F, Hiroi S, Mukai M, Ikeda E. Survivin
24. Westra WH. Early glandular neoplasia of the lung. Respir Res 2000;1: expression in atypical adenomatous hyperplasia of the lung. Am J Clin
163–9. Pathol 2003;120:712–9.
25. Westra WH, Baas IO, Hruban RH, Askin FB, Wilson K, Offerhaus GJ, et al. 48. Tominaga M, Sueoka N, Irie K, Iwanaga K, Tokunaga O, Hayashi S, et al.
K-ras oncogene activation in atypical alveolar hyperplasias of the human Detection and discrimination of preneoplastic and early stages of lung
lung. Cancer Res 1996;56:2224–8. adenocarcinoma using hnRNP B1 combined with the cell cycle-related
26. Carr LL, Chung JH, Duarte Achcar R, Lesic Z, Rho JY, Yagihashi K, et al. The markers p16, cyclin D1, and Ki-67. Lung Cancer 2003;40:45–53.
clinical course of diffuse idiopathic pulmonary neuroendocrine cell 49. Takamochi K, Ogura T, Suzuki K, Kawasaki H, Kurashima Y, Yokose T,
hyperplasia. Chest 2015;147:415–22. et al. Loss of heterozygosity on chromosomes 9q and 16p in atypical
27. Yatabe Y, Borczuk AC, Powell CA. Do all lung adenocarcinomas follow a adenomatous hyperplasia concomitant with adenocarcinoma of the lung.
stepwise progression? Lung Cancer 2011;74:7–11. Am J Pathol 2001;159:1941–8.
28. Mounawar M, Mukeria A, Le Calvez F, Hung RJ, Renard H, Cortot A, et al. 50. Ghaffar H, Sahin F, Sanchez-Cepedes M, Su GH, Zahurak M, Sidransky D,
Patterns of EGFR, HER2, TP53, and KRAS mutations of p14arf expression et al. LKB1 protein expression in the evolution of glandular neoplasia of
in non-small cell lung cancers in relation to smoking history. Cancer Res the lung. Clin Cancer Res 2003;9:2998–3003.
2007;67:5667–72. 51. Selamat SA, Galler JS, Joshi AD, Fyfe MN, Campan M, Siegmund KD, et al.
29. Rudin CM, Avila-Tang E, Harris CC, Herman JG, Hirsch FR, Pao W, et al. DNA methylation changes in atypical adenomatous hyperplasia, adeno-
Lung cancer in never smokers: molecular profiles and therapeutic impli- carcinoma in situ, and lung adenocarcinoma. PLoS One 2011;6:e21443.
cations. Clin Cancer Res 2009;15:5646–61. 52. Minoo P, Su G, Drum H, Bringas P, Kimura S. Defects in tracheoesopha-
30. Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, geal and lung morphogenesis in Nkx2.1(-/-) mouse embryos. Dev Biol
et al. Clinical and biological features associated with epidermal growth 1999;209:60–71.

www.aacrjournals.org Cancer Prev Res; 9(7) July 2016 525

 Kadara et al.

53. Yuan B, Li C, Kimura S, Engelhardt RT, Smith BR, Minoo P. Inhibition of 75. Nakachi I, Rice JL, Coldren CD, Edwards MG, Stearman RS, Glidewell SC,
distal lung morphogenesis in Nkx2.1(-/-) embryos. Dev Dyn 2000;217: et al. Application of SNP microarrays to the genome-wide analysis of
180–90. chromosomal instability in premalignant airway lesions. Cancer Prev Res
54. Kimura S, Hara Y, Pineau T, Fernandez-Salguero P, Fox CH, Ward JM, et al. 2014;7:255–65.
The T/ebp null mouse: thyroid-specific enhancer-binding protein is 76. Ooi AT, Gower AC, Zhang KX, Vick JL, Hong L, Nagao B, et al. Molecular
essential for the organogenesis of the thyroid, lung, ventral forebrain, profiling of premalignant lesions in lung squamous cell carcinomas
and pituitary. Genes Dev 1996;10:60–9. identifies mechanisms involved in stepwise carcinogenesis. Cancer Prev
55. Ikeda K, Clark JC, Shaw-White JR, Stahlman MT, Boutell CJ, Whitsett JA. Res 2014;7:487–95.
Gene structure and expression of human thyroid transcription factor-1 in 77. Wistuba II, Berry J, Behrens C, Maitra A, Shivapurkar N, Milchgrub S, et al.
respiratory epithelial cells. J Biol Chem 1995;270:8108–14. Molecular changes in the bronchial epithelium of patients with small
56. Kwei KA, Kim YH, Girard L, Kao J, Pacyna-Gengelbach M, Salari K, et al. cell lung cancer. Clin Cancer Res 2000;6:2604–10.
Genomic profiling identifies TITF1 as a lineage-specific oncogene ampli- 78. Meuwissen R, Linn SC, Linnoila RI, Zevenhoven J, Mooi WJ, Berns A.
fied in lung cancer. Oncogene 2008;27:3635–40. Induction of small cell lung cancer by somatic inactivation of both
57. Weir BA, Woo MS, Getz G, Perner S, Ding L, Beroukhim R, et al. Trp53 and Rb1 in a conditional mouse model. Cancer Cell 2003;4:
Characterizing the cancer genome in lung adenocarcinoma. Nature 2007; 181–9.
450:893–8. 79. Park KS, Martelotto LG, Peifer M, Sos ML, Karnezis AN, Mahjoub MR, et al.
58. Tanaka H, Yanagisawa K, Shinjo K, Taguchi A, Maeno K, Tomida S, et al. A crucial requirement for Hedgehog signaling in small cell lung cancer.
Lineage-specific dependency of lung adenocarcinomas on the lung devel- Nat Med 2011;17:1504–8.
opment regulator TTF-1. Cancer Res 2007;67:6007–11. 80. Watkins DN, Berman DM, Burkholder SG, Wang B, Beachy PA, Baylin SB.

Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/9/7/518/1937107/518.pdf by guest on 21 May 2023

59. Winslow MM, Dayton TL, Verhaak RG, Kim-Kiselak C, Snyder EL, Feldser Hedgehog signalling within airway epithelial progenitors and in small-
DM, et al. Suppression of lung adenocarcinoma progression by Nkx2–1. cell lung cancer. Nature 2003;422:313–7.
Nature 2011;473:101–4. 81. Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, Kasper LH, et al.
60. Park IW, Wistuba II, Maitra A, Milchgrub S, Virmani AK, Minna JD, et al. Integrative genome analyses identify key somatic driver mutations of
Multiple clonal abnormalities in the bronchial epithelium of patients small-cell lung cancer. Nat Genet 2012;44:1104–10.
with lung cancer. J Natl Cancer Inst 1999;91:1863–8. 82. Byers LA, Wang J, Nilsson MB, Fujimoto J, Saintigny P, Yordy J, et al.
61. Wistuba II, Behrens C, Virmani AK, Mele G, Milchgrub S, Girard L, et al. Proteomic profiling identifies dysregulated pathways in small cell lung
High resolution chromosome 3p allelotyping of human lung cancer and cancer and novel therapeutic targets including PARP1. Cancer Discov
preneoplastic/preinvasive bronchial epithelium reveals multiple, discon- 2012;2:798–811.
tinuous sites of 3p allele loss and three regions of frequent breakpoints. 83. Cardnell RJ, Feng Y, Diao L, Fan YH, Masrorpour F, Wang J, et al.
Cancer Res 2000;60:1949–60. Proteomic markers of DNA repair and PI3K pathway activation predict
62. Wistuba II, Behrens C, Virmani AK, Milchgrub S, Syed S, Lam S, et al. response to the PARP inhibitor BMN 673 in small cell lung cancer.
Allelic losses at chromosome 8p21–23 are early and frequent events in the Clin Cancer Res 2013;19:6322–8.
pathogenesis of lung cancer. Cancer Res 1999;59:1973–9. 84. Mohammad HP, Smitheman KN, Kamat CD, Soong D, Federowicz KE,
63. Belinsky SA, Nikula KJ, Palmisano WA, Michels R, Saccomanno G, Van Aller GS, et al. A DNA hypomethylation signature predicts antitumor
Gabrielson E, et al. Aberrant methylation of p16(INK4a) is an early event activity of LSD1 inhibitors in SCLC. Cancer Cell 2015;28:57–69.
in lung cancer and a potential biomarker for early diagnosis. Proc Natl 85. Slaughter DP, Southwick HW, Smejkal W. Field cancerization in oral
Acad Sci U S A 1998;95:11891–6. stratified squamous epithelium; clinical implications of multicentric
64. Merrick DT, Haney J, Petrunich S, Sugita M, Miller YE, Keith RL, et al. origin. Cancer 1953;6:963–8.
Overexpression of vascular endothelial growth factor and its receptors in 86. Braakhuis BJ, Tabor MP, Kummer JA, Leemans CR, Brakenhoff RH. A
bronchial dypslasia demonstrated by quantitative RT-PCR analysis. Lung genetic explanation of Slaughter's concept of field cancerization: evidence
Cancer 2005;48:31–45. and clinical implications. Cancer Res 2003;63:1727–30.
65. Keith RL, Miller YE, Gemmill RM, Drabkin HA, Dempsey EC, Kennedy TC, 87. Gomperts BN, Spira A, Massion PP, Walser TC, Wistuba II, Minna JD, et al.
et al. Angiogenic squamous dysplasia in bronchi of individuals at high risk Evolving concepts in lung carcinogenesis. Sem Respir Crit Care Med
for lung cancer. Clin Cancer Res 2000;6:1616–25. 2011;32:32–43.
66. Hirsch FR, Prindiville SA, Miller YE, Franklin WA, Dempsey EC, Murphy 88. Graham TA, McDonald SA, Wright NA. Field cancerization in the GI tract.
JR, et al. Fluorescence versus white-light bronchoscopy for detection of Future Oncol 2011;7:981–93.
preneoplastic lesions: a randomized study. J Natl Cancer Inst 2001;93: 89. Auerbach O, Stout AP, Hammond EC, Garfinkel L. Changes in bronchial
1385–91. epithelium in relation to cigarette smoking and in relation to lung cancer.
67. Hirsch FR, Lippman SM. Advances in the biology of lung cancer chemo- N Engl J Med 1961;265:253–67.
prevention. J Clin Oncol 2005;23:3186–97. 90. Beane J, Sebastiani P, Liu G, Brody JS, Lenburg ME, Spira A. Reversible and
68. Khuri FR, Cohen V. Molecularly targeted approaches to the chemopre- permanent effects of tobacco smoke exposure on airway epithelial gene
vention of lung cancer. Clin Cancer Res 2004;10:4249s–53s. expression. Genome Biol 2007;8:R201.
69. Bass AJ, Watanabe H, Mermel CH, Yu S, Perner S, Verhaak RG, et al. SOX2 91. Hackett NR, Heguy A, Harvey BG, O'Connor TP, Luettich K, Flieder DB,
is an amplified lineage-survival oncogene in lung and esophageal squa- et al. Variability of antioxidant-related gene expression in the airway
mous cell carcinomas. Nat Genet 2009;41:1238–42. epithelium of cigarette smokers. Am J Respir Cell Mol Biol 2003;29:
70. Hussenet T, Dali S, Exinger J, Monga B, Jost B, Dembele D, et al. SOX2 is an 331–43.
oncogene activated by recurrent 3q26.3 amplifications in human lung 92. Mao L, Lee JS, Kurie JM, Fan YH, Lippman SM, Lee JJ, et al. Clonal genetic
squamous cell carcinomas. PLoS One 2010;5:e8960. alterations in the lungs of current and former smokers. J Natl Cancer Inst
71. Lu Y, Futtner C, Rock JR, Xu X, Whitworth W, Hogan BL, et al. Evidence 1997;89:857–62.
that SOX2 overexpression is oncogenic in the lung. PLoS One 2010;5: 93. Spira A, Beane J, Shah V, Liu G, Schembri F, Yang X, et al. Effects of cigarette
e11022. smoke on the human airway epithelial cell transcriptome. Proc Natl Acad
72. Xiang R, Liao D, Cheng T, Zhou H, Shi Q, Chuang TS, et al. Down- Sci U S A 2004;101:10143–8.
regulation of transcription factor SOX2 in cancer stem cells suppresses 94. Spira A, Beane JE, Shah V, Steiling K, Liu G, Schembri F, et al. Airway
growth and metastasis of lung cancer. Br J Cancer 2011;104:1410–7. epithelial gene expression in the diagnostic evaluation of smokers with
73. Yuan P, Kadara H, Behrens C, Tang X, Woods D, Solis LM, et al. Sex suspect lung cancer. Nat Med 2007;13:361–6.
determining region Y-Box 2 (SOX2) is a potential cell-lineage gene highly 95. Wistuba II, Lam S, Behrens C, Virmani AK, Fong KM, LeRiche J, et al.
expressed in the pathogenesis of squamous cell carcinomas of the lung. Molecular damage in the bronchial epithelium of current and former
PLoS One 2010;5:e9112. smokers. J Natl Cancer Inst 1997;89:1366–73.
74. McCaughan F, Pole JC, Bankier AT, Konfortov BA, Carroll B, Falzon M, 96. Gomperts BN, Walser TC, Spira A, Dubinett SM. Enriching the molecular
et al. Progressive 3q amplification consistently targets SOX2 in preinva- definition of the airway "field of cancerization:" establishing new para-
sive squamous lung cancer. Am J Respir Crit Care Med 2010;182:83–91. digms for the patient at risk for lung cancer. Cancer Prev Res 2013;6:4–7.

526 Cancer Prev Res; 9(7) July 2016 Cancer Prevention Research
 Early Molecular Events in Lung Cancer Pathogenesis

97. Kadara H, Fujimoto J, Yoo SY, Maki Y, Gower AC, Kabbout M, et al. 119. Kim Y, Hammerman PS, Kim J, Yoon JA, Lee Y, Sun JM, et al. Integrative
Transcriptomic architecture of the adjacent airway field cancerization in and comparative genomic analysis of lung squamous cell carcinomas in
non–small cell lung cancer. J Natl Cancer Inst 2014;106:dju004. East Asian patients. J Clin Oncol 2014;32:121–8.
98. Nelson MA, Wymer J, Clements N, Jr. Detection of K-ras gene mutations in 120. Beane J, Vick J, Schembri F, Anderlind C, Gower A, Campbell J, et al.
non-neoplastic lung tissue and lung cancers. Cancer Lett 1996;103:115–21. Characterizing the impact of smoking and lung cancer on the airway
99. Franklin WA, Gazdar AF, Haney J, Wistuba II, La Rosa FG, Kennedy T, et al. transcriptome using RNA-Seq. Cancer Prev Res 2011;4:803–17.
Widely dispersed p53 mutation in respiratory epithelium. A novel mech- 121. de Bruin EC, Taylor TB, Swanton C. Intra-tumor heterogeneity: lessons
anism for field carcinogenesis. J Clin Invest 1997;100:2133–7. from microbial evolution and clinical implications. Genome Med
100. Belinsky SA, Palmisano WA, Gilliland FD, Crooks LA, Divine KK, Winters 2013;5:101.
SA, et al. Aberrant promoter methylation in bronchial epithelium and 122. Yates LR, Campbell PJ. Evolution of the cancer genome. Nat Rev Genet
sputum from current and former smokers. Cancer Res 2002;62:2370–7. 2012;13:795–806.
101. Soria JC, Rodriguez M, Liu DD, Lee JJ, Hong WK, Mao L. Aberrant 123. Nowell PC. The clonal evolution of tumor cell populations. Science
promoter methylation of multiple genes in bronchial brush samples 1976;194:23–8.
from former cigarette smokers. Cancer Res 2002;62:351–5. 124. Gorunova L, Hoglund M, Andren-Sandberg A, Dawiskiba S, Jin Y, Mitel-
102. Zochbauer-Muller S, Lam S, Toyooka S, Virmani AK, Toyooka KO, Seidl S, man F, et al. Cytogenetic analysis of pancreatic carcinomas: intratumor
et al. Aberrant methylation of multiple genes in the upper aerodigestive heterogeneity and nonrandom pattern of chromosome aberrations.
tract epithelium of heavy smokers. Int J Cancer 2003;107:612–6. Genes Chromosomes Cancer 1998;23:81–99.
103. Gesthalter YB, Vick J, Steiling K, Spira A. Translating the transcriptome 125. Teixeira MR, Heim S. Cytogenetic analysis of tumor clonality. Adv Cancer
into tools for the early detection and prevention of lung cancer. Thorax Res 2011;112:127–49.

Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/9/7/518/1937107/518.pdf by guest on 21 May 2023

2015;70:476–81. 126. Navin NE, Hicks J. Tracing the tumor lineage. Mol Oncol 2010;4:267–83.
104. Gustafson AM, Soldi R, Anderlind C, Scholand MB, Qian J, Zhang X, et al. 127. Parker JS, Perou CM. Tumor heterogeneity: focus on the leaves, the trees,
Airway PI3K pathway activation is an early and reversible event in lung or the forest? Cancer Cell 2015;28:149–50.
cancer development. Sci Transl Med 2010;2:26ra5. 128. Zhang J, Fujimoto J, Zhang J, Wedge DC, Song X, Zhang J, et al. Intratumor
105. Shaykhiev R, Wang R, Zwick RK, Hackett NR, Leung R, Moore MA, et al. heterogeneity in localized lung adenocarcinomas delineated by multi-
Airway basal cells of healthy smokers express an embryonic stem cell region sequencing. Science 2014;346:256–9.
signature relevant to lung cancer. Stem cells 2013;31:1992–2002. 129. LaFramboise T. Single nucleotide polymorphism arrays: a decade of
106. Sridhar S, Schembri F, Zeskind J, Shah V, Gustafson AM, Steiling K, et al. biological, computational and technological advances. Nucleic Acids Res
Smoking-induced gene expression changes in the bronchial airway are 2009;37:4181–93.
reflected in nasal and buccal epithelium. BMC Genomics 2008;9:259. 130. Reuter JA, Spacek DV, Snyder MP. High-throughput sequencing technol-
107. Zhang X, Sebastiani P, Liu G, Schembri F, Dumas YM, Langer EM, et al. ogies. Molecular cell 2015;58:586–97.
Similarities and differences between smoking-related gene expression in 131. Biesecker LG, Spinner NB. A genomic view of mosaicism and human
nasal and bronchial epithelium. Physiol Genomics 2010;41:1–8. disease. Nat Rev Genet 2013;14:307–20.
108. Perdomo C, Spira A, Schembri F. MiRNAs as regulators of the response to 132. Assie G, LaFramboise T, Platzer P, Bertherat J, Stratakis CA, Eng C. SNP
inhaled environmental toxins and airway carcinogenesis. Mutat Res arrays in heterogeneous tissue: highly accurate collection of both germ-
2011;717:32–7. line and somatic genetic information from unpaired single tumor sam-
109. Schembri F, Sridhar S, Perdomo C, Gustafson AM, Zhang X, Ergun A, et al. ples. Am J Hum Genet 2008;82:903–15.
MicroRNAs as modulators of smoking-induced gene expression changes in 133. Beroukhim R, Lin M, Park Y, Hao K, Zhao X, Garraway LA, et al. Inferring
human airway epithelium. Proc Natl Acad Sci U S A 2009;106:2319–24. loss-of-heterozygosity from unpaired tumors using high-density oligo-
110. Perdomo C, Campbell JD, Gerrein J, Tellez CS, Garrison CB, Walser TC, nucleotide SNP arrays. PLoS Computat Biol 2006;2:e41.
et al. MicroRNA 4423 is a primate-specific regulator of airway epithelial 134. Li A, Liu Z, Lezon-Geyda K, Sarkar S, Lannin D, Schulz V, et al. GPHMM:
cell differentiation and lung carcinogenesis. Proc Natl Acad Sci U S A an integrated hidden Markov model for identification of copy number
2013;110:18946–51. alteration and loss of heterozygosity in complex tumor samples using
111. Kadara H, Shen L, Fujimoto J, Saintigny P, Chow CW, Lang W, et al. whole genome SNP arrays. Nucleic Acids Res 2011;39:4928–41.
Characterizing the molecular spatial and temporal field of injury in early- 135. Staaf J, Lindgren D, Vallon-Christersson J, Isaksson A, Goransson H,
stage smoker non-small cell lung cancer patients after definitive surgery by Juliusson G, et al. Segmentation-based detection of allelic imbalance
expression profiling. Cancer Prev Res 2013;6:8–17. and loss-of-heterozygosity in cancer cells using whole genome SNP arrays.
112. Maki Y, Fujimoto J, Lang W, Xu L, Behrens C, Wistuba II, et al. LAPTM4B is Genome Biol 2008;9:R136.
associated with poor prognosis in NSCLC and promotes the NRF2- 136. Vattathil S, Scheet P. Haplotype-based profiling of subtle allelic imbal-
mediated stress response pathway in lung cancer cells. Sci Rep 2015;5: ance with SNP arrays. Genome Res 2013;23:152–8.
13846. 137. Xia R, Vattathil S, Scheet P. Identification of allelic imbalance with a
113. Rivera MP, Mehta AC, Wahidi MM. Establishing the diagnosis of lung statistical model for subtle genomic mosaicism. PLoS Comput Biol
cancer: diagnosis and management of lung cancer, 3rd ed: American 2014;10:e1003765.
college of chest physicians evidence-based clinical practice guidelines. 138. Excoffier L, Slatkin M. Maximum-likelihood estimation of molecular
Chest 2013;143:e142S–65S. haplotype frequencies in a diploid population. Mol Biol Evol 1995;12:
114. Wang Memoli JS, Nietert PJ, Silvestri GA. Meta-analysis of guided bron- 921–7.
choscopy for the evaluation of the pulmonary nodule. Chest 2012;142: 139. Stephens M, Smith NJ, Donnelly P. A new statistical method for
385–93. haplotype reconstruction from population data. Am J Hum Genet
115. Whitney DH, Elashoff MR, Porta-Smith K, Gower AC, Vachani A, Fergu- 2001;68:978–89.
son JS, et al. Derivation of a bronchial genomic classifier for lung cancer in 140. Baugher JD, Baugher BD, Shirley MD, Pevsner J. Sensitive and specific
a prospective study of patients undergoing diagnostic bronchoscopy. detection of mosaic chromosomal abnormalities using the parent-of-
BMC Med Genomics 2015;8:18. origin-based detection (POD) method. BMC Genomics 2013;14:367.
116. Silvestri GA, Vachani A, Whitney D, Elashoff M, Porta Smith K, Ferguson 141. Nik-Zainal S, Van Loo P, Wedge DC, Alexandrov LB, Greenman CD, Lau
JS, et al. A bronchial genomic classifier for the diagnostic evaluation of KW, et al. The life history of 21 breast cancers. Cell 2012;149:994–1007.
lung cancer. N Engl J Med 2015;373:243–51. 142. Aberle DR, Abtin F, Brown K. Computed tomography screening for lung
117. Meyerson M, Gabriel S, Getz G. Advances in understanding cancer cancer: has it finally arrived? Implications of the national lung screening
genomes through second-generation sequencing. Nat Rev Genet 2010;11: trial. J Clin Oncol 2013;31:1002–8.
685–96. 143. Aberle DR, Adams AM, Berg CD, Black WC, Clapp JD, Fagerstrom RM,
118. Cancer Genome Atlas Research N. Comprehensive genomic characteri- et al. Reduced lung-cancer mortality with low-dose computed tomo-
zation of squamous cell lung cancers. Nature 2012;489:519–25. graphic screening. N Engl J Med 2011;365:395–409.

www.aacrjournals.org Cancer Prev Res; 9(7) July 2016 527