molecular origin lung cancer

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T h e n e w e n g l a n d j o u r n a l o f medicine

n engl j med 359;13 www.nejm.org september 25, 2008 1367

review article

Molecular Origins of Cancer

Lung Cancer

Roy S. Herbst, M.D., Ph.D., John V. Heymach, M.D., Ph.D.,and Scott M. Lippman, M.D.

From the Departments of Thoracic/Headand Neck Medical Oncology (R.S.H.,J.V.H., S.M.L.), Cancer Biology (R.S.H.,J.V.H.), and Clinical Cancer Prevention(S.M.L.), University of Texas M.D. Ander-son Cancer Center, Houston. Address re-print requests to Dr. Lippman at the De-partment of Thoracic/Head and NeckMedical Oncology, Unit 432, M.D. Ander-

son Cancer Center, 1515 Holcombe Blvd.,Houston, TX 77030, or at [email protected].

Drs. Herbst and Heymach contributedequally to this article.

N Engl J Med 2008;359:1367-80.Copyright 2008 Massachusetts Medical Society.

Lung cancer is the leading cause of cancer deaths in the united

States1and worldwide. The two major forms of lung cancer are nonsmall-cell lung cancer (about 85% of all lung cancers) and small-cell lung cancer

(about 15%). Despite advances in early detection and standard treatment, nonsmall-cell lung cancer is often diagnosed at an advanced stage and has a poorprognosis. The treatment and prevention of lung cancer are major unmet needs thatcan probably be improved by a better understanding of the molecular origins and

evolution of the disease.Nonsmall-cell lung cancer can be divided into three major histologic subtypes:

squamous-cell carcinoma, adenocarcinoma, and large-cell lung cancer. Smokingcauses all types of lung cancer but is most strongly linked with small-cell lungcancer and squamous-cell carcinoma; adenocarcinoma is the most common typein patients who have never smoked (Fig. 12-8). This review will focus on majorrecent advances in the molecular study of the origins and biology of squamous-cellcarcinoma and adenocarcinoma, since they constitute the vast majority of diagnosedlung cancers (Table 15,8-14). These advances have been facilitated by the developmentof molecular techniques and biomarkers for defining cancer risk, prognosis, andoptimal therapy aimed at personalized prevention and treatment of lung cancer.

Molecular Origins

Host Susceptibility

Epidemiologic studies showing an association between family history and an in-creased risk of lung cancer provided the first evidence of host susceptibility (Fig. 1).Lung-cancer susceptibility and risk also are increased in inherited cancer syndromescaused by rare germ-line mutations in p53,15retinoblastoma,16and other genes,17as well as a germ-line mutation in the epidermal growth factor receptor (EGFR)gene.18More recently, three large genomewide association studies identified an as-sociation between single-nucleotide polymorphism (SNP) variation at 15q2415q25.1and susceptibility to lung cancer. The region of the SNP variation was recently

linked to lung carcinogenesis and includes two genes encoding subunits of thenicotinic acetylcholine receptor alpha, which is regulated by nicotine exposure.19-22

Lung-cancer susceptibility and risk also increase with reduced DNA repair ca-pacity (particularly when accompanied by exposure to tobacco smoke)23that re-sults, for example, from germ-line alterations in nucleotide excision repair genessuch as ERCC1.24Increased expression of DNA synthesis and repair genes, includ-ing RRM1(the regulatory subunit of ribonucleotide reductase) and ERCC1, in nonsmall-cell lung cancer correlates with a better prognosis overall but no benefitfrom platinum-based chemotherapy.25,26Table 1presents gene abnormalities in-volved in the development of different histologic types of lung cancer.

The New England Journal of Medicine

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Copyright 2008 Massachusetts Medical Society. All rights reserved.


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n engl j med 359;13 www.nejm.org september 25, 20081368

Normal Precancer Early-stage cancer Advanced cancer

Smoking-related

Not smoking-related

Molecularcellular

features

Treatmentapproaches

Commongerm-line

geneticvariations

Prevention

Cancer risk

Host susceptibility

Tobacco smoke

Unknown factors

Clonal patch

Tissue injury

Genetic,epigenetic

changes

Clonal patches

Dysregulatedpathways,

proliferation,apoptosis

Invasion, angiogenesis

Definitive local therapy with orwithout adjuvant therapy

Prognostic, predictive

Metastatic spread

Systemic therapy with orwithout radiation therapy

PredictiveRoles formolecularmarkers

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Figure 1.Molecular Evolution of Lung Cancer.

Environmental factors, such as tobacco smoke, and genetic susceptibility interact to influence carcinogenesis. Factors that are unrelated

to smoking including genetic, hormonal, and viral (e.g., human papillomavirus) factors have been suggested.2

Tissue injury (e.g.,from tobacco smoke, reflected in the discolored smoking-related lungs) initially occurs in the form of genetic and epigenetic changes (e.g.,mutations, loss of heterozygosity, and promoter methylation) and global transcriptome changes (e.g., inflammation and apoptosis path-ways). These changes can persist long term3,4and eventually lead to aberrant pathway activation and cellular function (e.g., dysregulatedproliferation and apoptosis) to produce premalignant changes, including dysplasia and clonal patches. Additional changes can result inangiogenesis, invasion and early-stage cancer, and advanced cancer and metastasis.5Many molecular changes in earliest-stage canceralso occur in advanced disease.6,7Premalignant patches contain clones and subclones (inset), which can involve loss of heterozygosity,microsatellite instability, and mutations (e.g., in p53and epidermal growth factor receptor [EGFR]). Lung cancers unrelated and related tosmoking have strikingly different molecular profiles, including those of mutations in p53, KRAS, EGFR,and HER2. Smoking-related patchesand primary cancers (usually squamous-cell carcinoma and small-cell lung cancer) most of ten develop in the central airway.4,8Most tumorsthat are not related to smoking are adenocarcinomas and develop in the peripheral airways. Molecular markers can signify risk (in people with-out cancer), prognosis (outcome independent of treatment), and sensitivity to treatment through predictive markers. Such stage-specific mark-ers can span the course of disease from its early stages through its late stages. They also can help define mechanisms of resistance to therapy.





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Clonal Evolution

Changes in certain genes (e.g., proinflammatoryinterleukin-8 [IL8] and some DNA-repair genes)occur in nonmalignant lung tissue of smokersand patients with lung cancer, a finding consis-tent with diffuse tissue injury.3,27-29These chang-es probably precede epithelial clonal evolution, animportant element of the molecular origins oflung and other cancers (Fig. 1). Patches of clon-

ally related cells, or clonal patches containing40,000 to 360,000 cells, have been mapped in thelung.30The size and number of subclones in aclonal patch may contribute to the cancer risk.31Early events in the development of nonsmall-cell lung cancer include loss of heterozygosity atchromosomal region 3p21.3 (site of RASSF1A, amember of the Ras association domain family,and FUS1), 3p14.2 (FHIT,a fragile histidine triad

Table 1.Genetic Abnormalities Specific in the Lung to NonSmall-Cell Lung Cancer and Small-Cell Lung Cancer.*

Abnormality NonSmall-Cell Lung Cancer Small-Cell Lung Cancer

Squamous-CellCarcinoma Adenocarcinoma

Precursor

Lesion Known (dysplasia) Probable (atypical adenomatous hyperplasia) Possible (neuroendocrine field)

Genetic change p53mutation KRASmutation (atypical adenomatous hyperplasiain smokers), EGFRkinase domain mutation(in nonsmokers)

Overexpression of c-MET

Cancer

KRASmutation Very rare 10 to 30% Very rare

BRAFmutation 3% 2% Very rare

EGFR

Kinase domain mutation Very rare 10 to 40% Very rare

Amplification 30% 15% Very rare

Variant III mutation 5% Very rare Very rare

HER2

Kinase domain mutation Very rare 4% Very rare

Amplification 2% 6% Not known

ALKfusion Very rare 7% Not known

MET

Mutation 12% 14% 13%

Amplification 21% 20% Not known

TITF-1amplification 15% 15% Very rare

p53mutation 60 to 70% 50 to 70% 75%

LKB1mutation 19% 34% Very rare

PIK3CA

Mutation 2% 2% Very rareAmplification 33% 6% 4%

* Nonsmall-cell lung cancer includes squamous-cell carcinoma and adenocarcinoma. Neuroendocrine fields have been detected only in tissue surrounding tumors and have been characterized by extremely high rates of allelic

lossand by c-MET overexpression (Salgia R: personal communication). Variations are based in part on smoking profiles. The percentages include increased gene copy numbers from amplification or polysomy and represent percentages from resected cancers.

The percentages are higher in primary tumors from patients with metastatic disease. Increased copy numbers have been reported insquamous dysplastic lesions but not in adenocarcinoma precursors.

Genomic EGFRvariant III mutations have been detected only in lung squamous-cell carcinoma, and these tumors are sensitive preclinicallyto irreversible EGFR tyrosine kinase inhibitors. The incidence of 5% is substantially lower than that of 30 to 40% for the detection insquamous-cell carcinoma or adenocarcinoma by immunohistochemical analysis or other techniques.

The anaplastic lymphoma kinase (ALK) fusion gene (involving chromosome 2p), consisting of parts of EML4andALK,is transforming infibroblasts and occurs in adenocarcinoma but not in other types of nonsmall-cell lung cancer or other nonlung cancers.





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gene), 9p21 (p16), and 17p13 (p53).32 All thesegenes are tumor-suppressor genes. Loss-of-hetero-zygosity patterns in squamous-cell carcinomaand adenocarcinoma differ (e.g., chromosome 3pdeletions are much more extensive in squamous-cell carcinoma). Mutations in the EGFR kinase

domain occur early in the development of adeno-carcinoma that is generally unrelated to smok-ing, and KRASmutations occur early in the devel-opment of smoking-related adenocarcinoma.33,34Clonal patches with methylation of promoter re-gions of genes (epigenetic changes),p53mutation,EGFRmutation, c-Mycamplification, loss of hetero-zygosity, and microsatellite instability can occurin normal tissue surrounding nonsmall-cell lungtumors5,30,32,34,35and may be associated with a

greater risk of recurrence and second primarytumors. These findings suggest that in the futuremolecular analyses of surgical margins may helpidentify patients most likely to benefit from adju-vant therapy.

Methylated genes in premalignant squamous-

cell lung lesions (e.g., metaplasia and dysplasia) arep16and FHIT(frequently and very early) and O-6-methylguanine-DNA methyltransferase (MGMT),death-associated protein kinase (DAPK), andRASSF1A(less frequently or rarely and in advancedprecancers).35-41The early methylation of p16inthe development of squamous-cell lung cancer(e.g., in normal lung tissue in approximately50% of smokers)36exemplifies differences withthat in the development of adenocarcinoma, in

Hypoxia

Ligand bindingand dimerization

EGFR

LKB1

AMPK

TSC2

HIF-1 mTOR

Akt

PI3K

Mek

Raf

Ras

Gene transcription, cellular effects

Proliferation Invasion Metastasis Resistance to apoptosis Angiogenesis

Other receptor tyrosinekinases (e.g., IGF-1R, c-Met)

VEGF

Figure 2.Epidermal Growth Factor Receptor (EGFR) Cell-Signaling Pathways.

EGFR activates several major downstream signaling pathways, including RasRafMek and the pathway consistingof phosphoinositide 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), which in turn may have aneffect on proliferation, survival, invasiveness, metastatic spread, and tumor angiogenesis through pathways that areeither dependent on or independent of the hypoxia inducible factor (HIF). These pathways also may be modulatedby other receptor tyrosine kinases, such as insulin-like growth factor 1 receptor (IGF-1R) and cMET, and by the

LKB1amp-activated protein kinase (AMPK) pathway, which is involved in energy sensing and cellular stress. Mostof these functions depend on signaling through the kinase domain. However, kinase-independent functions, suchas maintaining glucose transport, have been reported.46TSC2 denotes tuberous sclerosis complex 2, and VEGF vas-cular endothelial growth factor.





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which p16 methylation occurs very rarely andonly late in precursors (e.g., high-grade atypicaladenomatous hyperplasia).37Methylation markersin sputum are associated with the risk of lungcancer (e.g., methylated p16)38and the recurrenceof lung cancer (methylated ASC-TMS1,39 alsocalled PYCARD). Recent data show that promoter

methylation of various genes, including p16 instage I nonsmall-cell lung cancer, is associatedwith recurrence after resection.40,41Agents thatreverse epigenetic changes have shown promisein a mouse model of lung carcinogenesis and arebeing tested in humans with lung cancer.42

Bronchoalveolar stem cells, which may beprecursors of lung adenocarcinoma, were identi-fied recently in studies in mice.43The KRAS, Pten,phosphoinositide 3-kinase (PI3K), and cyclin-dependent kinase pathways have been implicatedin the proliferation of these stem cells.44,45The

potential role of bronchoalveolar stem cells andother tumorigenic stem-cell populations in thedevelopment and prognosis of human lung can-cer and its resistance to drugs is an importantarea of future investigation.

Molecular Evolution

EGFR Family

EGFR regulates important tumorigenic process-es, including proliferation, apoptosis, angiogene-sis, and invasion (Fig. 2), and, along with itsligands, is frequently overexpressed in the devel-opment and progression of nonsmall-cell lungcancer.2,5,34,46Clinical trials of the EGFR tyrosinekinase inhibitor erlotinib for second-line or third-line treatment of such tumors and of the mono-clonal antibody against EGFR, cetuximab (com-bined with chemotherapy), for treatment ofpreviously untreated, advanced disease47,48vali-dated EGFR as a molecular target for therapy.EGFRmutations that were discovered during clini-cal trials led to extensive studies of the roles of

these mutations and EGFRamplification in thepathogenesis of the disease and its prognosisand sensitivity to treatment.

Several groups of investigators independentlyidentified somatic mutations in the kinase do-main of EGFRin lung adenocarcinoma in approxi-mately 10% of specimens from patients in theUnited States and in 30 to 50% of specimensfrom patients in Asia49(Fig. 3). The mutationsoccur with increased frequency in women and

nonsmokers and are tightly associated with sensi-tivity to the EGFR tyrosine kinase inhibitors gefi-

tinib and erlotinib and so appear to explain mostof the dramatic responses to these agents.50-52More than 80% of these mutations in lung can-cer involve in-frame deletions within exon 19 orthe L858R mutant within exon 21. EGFRmuta-tions are associated with an improved prognosisin nonsmall-cell lung cancer, even when treatedwith cytotoxic chemotherapy.53,54EGFRamplifi-cation is detected in dysplasia (especially of a highgrade), which is associated with lung-cancer risk

Extracellulardomain

Transmembranedomain

Ligand-bindingpocket

Intracellulardomain

EGFRvariant III del(del exons 27)Squamous-cell

carcinoma

Catalyticdomain

Exon 19 delAdenocarcinoma

L858R(exon 21 del)

Adenocarcinoma

ATP-binding pocket

T790MAdenocarcinoma

PhosphorylationP

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Figure 3.Effect of Deletions and Mutations in the Epidermal Growth FactorReceptor Gene (EGFR) on Disease Development and Drug Targeting.

Ligand binding to the EGFR extracellular domain results in receptor ho-modimerization and tyrosine phosphorylation, with the phosphate derivedfrom ATP bound within the kinase catalytic domain. EGFRmutations havetransforming potential in preclinical lung models and can occur early in hu-

man lung carcinogenesis. EGFRpoint mutations (e.g., L858R) and exon 19deletions, which occur predominantly in adenocarcinoma of the lung, arelocated within the catalytic domain and result in constitutive EGFR activa-tion. These mutations are associated with increased sensitivity to EGFR ty-rosine kinase inhibitors, such as erlotinib and gefitinib. In contrast, muta-tions in T790M (an amino acid located within the ATP binding site of theEGFRkinase domain) are associated with acquired resistance to these drugs.EGFRvariant III mutant deletions occur in the extracellular domain and areassociated with squamous-cell cancer.





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when detected in the sputum of smokers, and isassociated with a poor prognosis but also withsensitivity to EGFR inhibitors.55,56The epidemio-logic links highlight three factors whether thepatient is a nonsmoker, Asian, and female that are associated independently and collective-ly with an improved response to EGFR tyrosine

kinase inhibitors. However, erlotinib appearsto prolong survival in virtually all subgroups ofpatients with nonsmall-cell lung cancer.48Thereare major differences in clinical, pathological,sex-related, and molecular factors between smok-ers and lifelong nonsmokers in whom lung can-cer develops (Fig. 1).

The vast majority of patients who have aninitial response to erlotinib and gefitinib even-tually have a relapse.49,57,58Recent studies haveidentified EGFRT790M mutations (in exon 20) intumors before drug treatment59 and in tumors

of patients who had a relapse after therapy withstandard reversible EGFR tyrosine kinase inhibi-tors.57The binding kinetics of the mutant EGFRappear to be altered by the T790M mutation(Fig. 3). Irreversible EGFR inhibitors suppressT790M-mutant tumor cells in vitro and are prom-ising treatments for T790M-mutant tumors.60Amplification of the met proto-oncogene (MET),another major mechanism of acquired resistanceto EGFR tyrosine kinase inhibitors, marks a poorprognosis.61-63Other proposed resistance mech-anisms include activation of other receptor tyro-sine kinases, such as insulin-like growth factor 1receptor (which can bypass EGFR to activatecritical downstream signaling pathways [Fig. 2]),KRASmutations, and the epithelial-to-mesenchy-mal transition.58,64,65 The epithelial-to-mesen-chymal transition is a program of cell develop-ment involving loss of cell adhesion, repressedE-cadherin expression, and increased cell mobility.

Preclinical and clinical data suggest that EGFRmutations are early events in the development ofnonsmall-cell lung cancer.66-70EGFRmutations,

including those involving exons 18, 19, and 20and L858R, can transform fibroblasts and lungepithelial cells.67Furthermore, in transgenic micewith lung-specific expression of exon 19 deletionor the L858R mutation, atypical adenomatoushyperplasia, which is considered to be a precur-sor lesion of peripheral adenocarcinoma, wasfollowed by lesions resembling bronchioalveolarcarcinoma at 5 to 6 weeks of age and invasiveadenocarcinomas at 8 to 10 weeks.68Deinduction

of mutant EGFRexpression led to regression oftumors, suggesting the need for persistent mu-tant EGFRactivity for continued tumor survival.Lung tumors also developed in transgenic micewith lung-specific expression of EGFRvariant IIImutation (in-frame deletion of exons 27 fromthe extracellular domain) (Fig. 3).13Mutations of

the region that encodes the tyrosine kinase do-main of EGFRhave been detected in specimensof atypical adenomatous hyperplasia from Asianpatients with no history of smoking.71These mu-tations also occur in normal epithelium withinand adjacent to tumors with EGFRtyrosine kinasemutations (a localized-field effect, possibly reflect-ing stem-cell expansion) and before EGFRampli-fication, a change associated with tumor progres-sion and metastasis.72

HER2mutations and amplification have beenidentified in patients with lung adenocarcinoma.

The frequency of such mutations is less than 5%,and the frequency of such amplification is 5 to10%. HER2kinase domain mutations (in-frameinsertions in exon 20) and EGFRkinase domainmutations have similar associations with femalesex, nonsmoking status, and Asian backgroundin patients with adenocarcinoma.73HER2amplifi-cation is associated with sensitivity to inhibitorsof the EGFR tyrosine kinase74; HER2mutations areassociated with resistance to such inhibitors butalso with sensitivity to HER2-targeted therapy.75HER3kinase domain mutations have not beendetected in patients with nonsmall-cell lungcancer.76Mutations in the HER4kinase domainwere found in 2 to 3% of Asian patients withthis disease, with a possible association withmale sex and smoking.77

RasRafMek

The RasRafMek pathway is involved in signal-ing downstream from EGFR and in other path-ways leading to the growth of cancer cells andtumor progression (Fig. 2). Activating KRASmu-

tations are limited to nonsmall-cell lung cancer(predominantly adenocarcinomas), virtually mu-tually exclusive of mutations in the EGFR andHER2kinase domains, and associated with resis-tance to EGFR inhibitors (tyrosine kinase inhibi-tors and cetuximab) and chemotherapy.58,78,79Most KRAS mutations in lung adenocarcinomaare smoking-related GT transversions (substitu-tions of a purine for a pyrimidine) and affectexon 12 (in 90% of patients) or exon 13.2,79A dis-





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tinct KRASmutational profile consisting of GAtransition mutations was recently detected inpatients with adenocarcinoma who had neversmoked; its functional significance is unclear.79Transversions (smokers) and transitions (non-smokers) also have been reported for p53muta-tions in lung adenocarcinoma.2KRASmutations

appear to be an early event (e.g., detectable in thepreinvasive lesions of atypical adenomatous hy-perplasia and bronchoalveolar carcinoma33) thatprecedes smoking-related lung adenocarcinoma.They generally mark a poor prognosis. Furtherevidence supporting this genes role in the patho-genesis of lung cancer comes from transgenicmice bearing a mutated KRASand in which multi-focal atypical adenomatous hyperplasia and ade-nocarcinoma develop.80 MET activation occursearly in KRAS-induced carcinogenesis in this mod-el.81BRAFmutations have also been detected in

nonsmall-cell lung cancer9and may be an earlyevent in lung tumorigenesis.82

PI3KAktmTOR

The pathway consisting of PI3K, Akt, and mam-malian target of rapamycin (mTOR), which isdownstream of EGFR, is activated early in lungcarcinogenesis.83 Akt is also overexpressed inbronchial dysplasia. Inhibition of Akt can induceapoptosis of human premalignant and malignantlung cells and prevent lung carcinogenesis in ananimal model. An mTOR inhibitor can block ma-lignant progression of atypical adenomatous hyper-plasia lesions in the KRasmouse model.84SincemTOR drives tumorigenesis in part through mac-rophages, a prominent component of the tumormicroenvironment, the antitumor effect of mTORinhibition requires the tumor microenvironment.There is mutation or amplification of PIK3CA,which encodes the PI3K catalytic subunit, in a sub-group of nonsmall-cell lung tumors, especiallysquamous-cell carcinoma, in association withincreased PI3K activity and Akt expression.85

LKB1

LKB1(also called STK11) is frequently mutated innonsmall-cell lung tumors and is thought to actas a tumor-suppressor gene through interactionswith p53and CDC42,modulating the activity ofAMPK (a multifunctional protein kinase) andother possible mechanisms that are just begin-ning to be studied.86,87LKB1is thought to func-tion in early tumorigenesis, subsequent differen-

tiation, and the development of metastases.88Results in transgenic KRas-mutant mice in whichLKB1was inactivated suggest that the gene playsa role in the differentiation and invasive behaviorof such tumors.87The presence of LKB1mutationsalone (i.e., without KRasmutations) was not asso-ciated with the development of lung cancer in

mice. Low levels of LKB1 protein were associatedwith high grades of dysplasia in atypical adeno-matous hyperplasia lesions, suggesting that LKB1has an early role in the development of premalig-nant lesions in the lung.89LKB1mutations (includ-ing point mutations and deletions) were found in34% of adenocarcinomas and 19% of squamous-cell carcinomas from 144 human specimens ofnonsmall-cell lung cancer.87However, much low-er rates of LKB1mutation (


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of bronchial dysplasia from low grade to highgrade.95Bronchial hyperplasia, metaplasia, andcarcinoma in situ are associated with increasedmicrovessel density, and a distinctive patternknown as angiogenic squamous dysplasia canoccur.96Factors associated with increased tumorangiogenesis correlate with the development and

prognosis of lung cancer.97-99Circulating VEGFlevels may predict the clinical benefit of VEGFinhibitors in patients with this disease. Many an-giogenic factors are regulated at least in partthrough the hypoxia-regulated pathways, such ashypoxia-induced factor (HIF) 1 and 2.100,101Inaddition to hypoxia, VEGF and other angiogenicfactors are also regulated by EGFR through HIF-dependent and independent mechanisms102andby oncogenes such as KRASand p53. VEGF hasrecently been validated as a therapeutic target onthe basis of the results of a phase 3 trial, which

led the Food and Drug Administration (FDA) toapprove the VEGF monoclonal antibody bevaci-zumab in combination with standard chemother-apy for previously untreated, advanced nonsmall-cell lung cancer.103

Interactions between the VEGF and EGFRpathways and an association between acquiredresistance to EGFR blockade and increased VEGFexpression in preclinical models104 led to thehypothesis that dual blockade of VEGF and EGFRmight be more effective than either approachalone. Randomized phase 2 trials of dual inhibi-tion with bevacizumab plus erlotinib105 or theVEGF receptorEGFR tyrosine kinase inhibitorvandetanib (combined with chemotherapy)106,107had promising results. Phase 3 testing of bothapproaches in patients with platinum-resistantdisease is ongoing.

molecular profiling

technical advances

Molecular profiling, including the profiling of

genes and proteins, to guide treatment may im-prove the clinical outcome in patients with nonsmall-cell lung cancer (Fig. 1). Progress in theidentification of markers, mutations, and genomicsignatures far outstrips the modest improvementin treatments that are based on these molecularadvances. Formidable obstacles to developing ef-fective markers include tumor heterogeneity, thehighly complex interplay between the environ-ment and host and the complexity, multiplicity,

and redundancy of tumor-cell signaling networksinvolving genetic, epigenetic, and microenviron-mental effects. Emerging high-throughput tech-niques for assessing genomic DNA, messengerRNA (mRNA), microRNA, methylation, and pro-tein or phosphoprotein signaling networks shouldhelp address these obstacles (Fig. 4). The Cancer

Genome Atlas is a large-scale project designedto provide a comprehensive profile of humantumors according to their gene mutations, alter-ations in gene copy number, and epigeneticchanges. Squamous-cell carcinoma of the lungwill be one of the f irst tumors profi led by thisatlas.

Gene Profiling

Tumor molecular heterogeneity is a major reasonthat patients with nonsmall-cell lung cancer witha similar clinical stage and tumor histology can

have dramatically different clinical outcomes andresponses to treatment. Microarray techniquesthat profile the expressions of tens of thousandsof genes simultaneously can measure this tumorheterogeneity at a global level. Gene-expressionprofiles that are associated with subtypes of nonsmall-cell lung cancer108,109 and with reducedrecurrence-free or overall survival of patientshave been identified.110-113Combined clinical andmolecular information provides better indicationsof cancer risk114and prognosis.115

In a recent analysis of 672 invasion-associatedgenes from 125 frozen specimens of early-stagetumors,111microarray and reverse-transcriptasepolymerase-chain-reaction (RT-PCR) analyses iden-tified a molecular signature of five genes as anindependent predictor of relapse-free and overallsurvival. In two validation cohorts, another re-cently developed gene-expression profile (meta-gene) predicted clinical outcome with an accuracyof 72% and 79% (greater than that for tumorstage, tumor diameter, nodal status, or otherclinical measures) and predicted the outcome in

patients with stage IA tumors.112

Randomized,controlled trials will need to validate these sig-natures and establish whether the patients withstage IA tumors who were identified as being athigh risk will benefit from adjuvant therapy. Onesuch phase 3 trial, coordinated by the Cancerand Leukemia Group B, is approved and underfinal review. It will evaluate a large predictive setof metagenes (or subgroups of gene-expressionprofiles consisting of 25 to 200 genes) in patients





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with stage IA tumors who are undergoing adju-vant chemotherapy.

For the majority of patients with advanced ormetastatic nonsmall-cell lung cancer, the most

important potential effect of molecular markersis likely to be in predicting the response to spe-cific therapies with the goal of personalizingtreatment (Fig. 4). Many exciting potential pre-dictive markers have been developed in vitro andneed validation in tumor samples and clinicaltrials.113For example, gene-expression signatureshave been developed for cisplatin and pemetrexedon the basis of in vitro sensitivity; the cisplatinin vitro signature predicted the likelihood of

response. Recently developed in vitro profilespredicting the sensitivity of tumors to EGFR in-hibitors and other therapies have yet to be as-sessed clinically.113

MicroRNA has recently emerged as an impor-tant regulator of gene expression. High-through-put analyses have shown that microRNA expres-sion is commonly deregulated in lung and othercancers.116,117Using real-time RT-PCR, investi-gators recently identified a five-microRNA sig-nature that is associated with treatment out-come.116 Loss of microRNA-128b, a putativeregulator of EGFR that is located on chromo-some 3p, has been shown to correlate with the

Host

Tumor, local environment

Germ-line genetic profiling(e.g., SNP arrays)

Blood-based profiling

ProteomicsCytokine, angiogenic factorsCirculating DNA or RNACirculating cells (e.g., tumor,endothelial, lymphocytes)

Genetic or epigenetic

MutationDeletionAmplificationTranslocationMethylation

Gene expression

Expression arraysMicroRNA profilingPCR-based approaches

Proteins

IHCProteomics

Personalizedmedicine

Interactions involving the host, tumor,and therapy

Tumor sensitivity to therapy

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Figure 4.Molecular-Profiling Approaches to the Development of Personalized Therapy.

Host profiling involves innate characteristics of the cancer patient. All markers that are involved in profiling lung cancer can apply to thetumor or its local environment. Predictive markers identify groups of patients who are likely to have increased sensitivity or resistance toa given therapy, a critical step in personalizing treatment. It has been traditional to assess individual genetic or protein prognostic orpredictive markers (e.g., HER2 for breast cancer), but emerging techniques permit global analyses of the genomic, gene-expression, epi-

genetic, and protein profiles of the host (innate), including markers in blood and in tumor or nonmalignant lung tissue. These methodsinclude single-nucleotide polymorphism (SNP) arrays to assess genomic alterations, bisulfite sequencing, and methylation-specific poly-merase chain reaction (PCR) to assess epigenetic changes, microarrays for assessing gene expression or microRNA levels, and proteom-ic methods (such as mass spectroscopy, reverse-phase protein arrays, and multiplex beads) to assess intracellular signaling in tumor tis-sue and cytokines and angiogenic factors in blood. Blood-based profiling includes markers derived from the host (e.g., lymphocytes)and the tumor and local environment (e.g., circulating tumor cells and tumor-derived cytokines) (red arrows). IHC denotes immunohis-tochemical analysis.





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response to EGFR inhibition in patients withlung cancer.117

Studies suggest that information about tumor-specific genetic and epigenetic changes also maybe obtained from the blood of patients with lungcancer. Circulating DNA can be detected in theplasma and serum of such patients, and levels of

this DNA are associated with a poor progno-sis.118,119Tumor-specific DNA alterations (suchas loss of heterozygosity), promoter methylation,and KRASand EGFRmutations have also beendetected in the blood of patients with lung can-cer.120-122New techniques for capturing circulat-ing tumor cells allow the detection of EGFR-activating mutations and the drug-resistanceallele T790M. Such techniques appear to be moresensitive than those for capturing circulatingDNA. Furthermore, a decline in the number ofcirculating tumor cells was associated with tu-

mor response on radiography.123These studiessuggest that blood profiling may provide usefulinformation about genetic changes in tumorsthat could ultimately help detect lung cancer andguide therapy.

Protein Profiling

Profiling of genomic and mRNA expression pro-vides an incomplete picture of the heterogeneityof nonsmall-cell lung cancer. Levels of mRNAdo not always correlate with protein levels and donot provide information on proteinprotein inter-actions or post-translational modifications suchas phosphorylation that may be critical for regu-lating protein activity.124Furthermore, most tar-geted therapeutic agents are designed to inhibitthe activity of proteins such as tyrosine kinases.Therefore, protein-based profiling is likely to beessential in understanding the complexity of pro-tein signaling networks and developing molecu-lar signatures that predict a response to therapy.

Immunohistochemical analysis remains themost widely applied method for assessing indi-

vidual proteins and may be useful for estimatingprognosis and predicting the response to ther-apy.25,26 Emerging high-throughput proteomictechniques, such as mass spectrometry and pro-tein microarrays, have the potential to view sig-nal transduction networks more globally than ispossible with immunohistochemical analysis.Such techniques are feasible in small amounts oftumor tissue.125Proteomic signatures for prog-

nosis and predicting the response to chemother-apy or EGFR inhibitors have been developed intumors and cell lines.126,127

Proteomic profiling from blood is also understudy, allowing repeated measurements duringtreatment without the need for tumor tissue.Serum mass spectrometry profiles can distin-

guish patients with nonsmall-cell lung cancerfrom normal controls124,128and patients with bet-ter outcomes from those with worse outcomesafter treatment with EGFR tyrosine kinase inhib-itors.129New techniques also permit the multi-plex analysis of dozens of cytokines and angio-genic factors in small amounts of serum orplasma. This approach is being used in develop-ing predictive markers in nonsmall-cell lungcancer. Although promising, blood- and tissue-based proteomic approaches remain investiga-tional and await prospective testing and valida-

tion in large, randomized trials before they canbe applied clinically.

Conclusions

The molecular origins of lung cancer lie in com-plex interactions between the environment andhost genetic susceptibility. Lung cancer thenevolves through genetic and epigenetic changes,including deregulated signaling pathways, whichare potential targets for chemoprevention andtherapy. Emerging techniques for genomic, gene-expression, epigenetic, and proteomic profil-ing92,114,125,130,131could revolutionize clinical ap-proaches across the spectrum of lung-cancer typesand subtypes by identifying practical molecularmarkers of risk (in precancer), early detectionand prognosis (in early-stage cancer), and treat-ment sensitivity (in early-stage and advanced-stage cancer). Genomewide and other molecularassessments are helping elucidate germ-line vari-ations that may contribute to lung cancer risk,19-21prognosis,132and treatment sensitivity133,134and

somatic genetic alterations that occur in lungadenocarcinomas14,50-52 and in high-risk lungtissue associated with tumors or in smokers.3,28,29Molecular targeted research has produced the re-cently FDA-approved EGFR and VEGF inhibitorserlotinib and bevacizumab, which have modestlyimproved the outcome in patients with nonsmall-cell lung cancer.48,103Molecular profiling of thetype described in this review has begun in clini-





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cal trials112,135-137and promises to select patientswho are most likely to benefit from therapy andto guide the development of more effective agentsthat will personalize standard medicine for lungcancer.138

Dr. Herbst reports receiving consulting fees and researchgrants from Bristol-Myers Squibb, ImClone, Genentech, Amgen,

AstraZeneca, and OSI and lecture fees from Genentech; Dr.

Heymach, serving on advisory boards for and receiving researchgrants from AstraZeneca, Pfizer, and GlaxoSmithKline and serv-ing on an advisory board for Genentech; and Dr. Lippman, servingon advisory boards for OSI and Genentech. No other potentialconflict of interest relevant to this article was reported.

We thank Lauren A. Byers, Balvindar S. Johal, and Ignacio I.Wistuba for their careful comments and other contributions re-garding aspects of this work; and Bich N. Tran, Suzanne E. Davis,and Kendall Morse for their contributions to the preparation of

the manuscript.

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