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Lung Cancer Biomarkers

Present Status and Future Developments

Philip T. Cagle, MD; Timothy Craig Allen, MD, JD; Randall J. Olsen, MD, PhD

� The publication of the ‘‘Molecular Testing Guideline forSelection of Lung Cancer Patients for EGFR and ALKTyrosine Kinase Inhibitors: Guideline From the College ofAmerican Pathologists, International Association for theStudy of Lung Cancer, and Association for MolecularPathology’’ has now provided a guideline for biomarkertesting for first-generation lung cancer tyrosine kinaseinhibitors. Biomarker testing has forever altered the role ofpathologists in the management of patients with lungcancer. Current, unresolved issues in the precisionmedicine of lung cancer will be addressed by thedevelopment of new biomarker tests, new drugs, andnew test technologies and by improvement in the cost tobenefit ratio of biomarker testing.

(Arch Pathol Lab Med. 2013;137:1191–1198; doi:10.5858/arpa.2013-0319-CR)

In April 2013, the College of American Pathologists (CAP),the International Association for the Study of Lung

Cancer (IASLC), and the Association for Molecular Pathol-ogy (AMP) officially released the ‘‘Molecular TestingGuideline for Selection of Lung Cancer Patients for EGFR[epidermal growth factor receptor] and ALK [anaplasticlymphoma kinase] Tyrosine Kinase Inhibitors [TKIs].’’ 1 TheCAP/IASLC/AMP guideline provides the first standardizedevidence-based approach for performing biomarker testingto select patients with lung cancer for EGFR or ALK TKItherapy that is multidisciplinary and multicontinental inscope. Subsequent to the guideline release, the CAP is alsoreleasing a template protocol for the reporting of lungcancer–predictive biomarkers as an addendum to anatomicpathology reports, and the guidelines, themselves, will be

regularly updated. The number of predictive biomarkers tobe tested for lung cancer, the number of targeted therapies,and the types of test methodologies are all expected toincrease during upcoming months and years. The need forpredictive biomarker testing on pathology specimens hasforever altered the role of pathologists in the care of patientswith lung cancer. This review provides an overview ofcurrent developments, expected developments, and relatedissues for lung cancer–predictive biomarker testing.

HISTORICAL PERSPECTIVE

For many years, lung cancer has been the leading cause ofcancer deaths in the United States2 and worldwide.3 Mostpatients with lung cancer present with advanced disease,and conventional treatment options have been limited,resulting in an overall 5-year survival rate of only 10% to15%, for many decades.4,5

Approximately 85% of lung cancers are non–small celllung cancers (NSCLCs), traditionally divided into 3 majorcell types: adenocarcinoma (approximately 50%), squa-mous cell carcinoma (approximately 35%), and large cellcarcinoma (approximately 15%, although this is a dimin-ishing cell type category).6,7 About 70% of NSCLCs presentwith advanced disease not considered curable by surgicalresection, either locally advanced (stage IIIB) or often withmetastatic disease (stage IV). Clinical stage IIIB NSCLCsare associated with a 5-year survival rate of 7% and stageIV NSCLCs, with a 5-year survival rate of 2%.8 Conven-tional therapy for stage IV NSCLC is doublet chemother-apy that includes cisplatin or carboplatin. Affected patientsmay additionally receive radiation therapy. Of the patientswith lung cancer who initially respond to first-line therapy,nearly all subsequently experience disease progression.These patients may receive second-line therapy, or possiblymore lines of therapy, in an attempt to control theirdisease. Eventually, virtually all of these patients die fromlung cancer as reflected in the statistics mentionedpreviously.9–16

Given the dismal prognosis and limited treatment optionsfor patients with advanced-stage lung cancer, it is notsurprising that the US Food and Drug Administration (FDA)approval of targeted molecular therapies for advanced-stagelung cancer has been a game changer for these patients.

The first generation of EGFR TKIs approved for treatmentof advanced-stage lung cancer includes gefitinib (Iressa;AstraZeneca, London, United Kingdom) and erlotinib(Tarceva; Genentech, South San Francisco, California, andOSI Pharmaceuticals, Long Island, New York). Already

Accepted for publication May 28, 2013.From the Department of Pathology and Genomic Medicine, The

Methodist Hospital, Houston, Texas, and the Department ofPathology and Laboratory Medicine, Weill Medical College ofCornell University, New York, New York (Drs Cagle and Olsen);and the Department of Pathology, The University of Texas HealthScience Center at Tyler (Dr Allen).

The authors have no relevant financial interest in the products orcompanies described in this article.

Presented at the New Frontiers in Pathology: An Update forPracticing Pathologists meeting; Homestead Resort; August 3–5,2012; Glen Arbor, Michigan.

Reprints: Philip T. Cagle, MD, Department of Pathology andGenomic Medicine, The Methodist Hospital, 6565 Fannin St, MainBldg, Room 227, Houston, Texas 77030 (e-mail: [email protected]).

Arch Pathol Lab Med—Vol 137, September 2013 Lung Cancer Biomarkers: Present and Future—Cagle et al 1191

since the 1980s it was known that most NSCLCsoverexpress EGFR,17 but early clinical trials of EGFR TKIsaffected only a small percentage of patients with lungcancer. In 2004, several separate investigators reported thatthe presence of somatic mutations of EGFR gene in a lungcancer predicted the likelihood of its response to EGFRTKIs.18–20 Beginning in 2009, a number of clinical trials havereported better response rates and progression-free survivalfor patients with advanced-stage NSCLC with EGFRmutations who received EGFR TKIs compared to conven-tional therapy.21–23 These clinical trials were conducted withpatients with advanced lung cancer who had significantimprovement in progression-free survival, but no demon-strated improvement in overall survival, possibly because ofthe crossover design of these studies in which many of thepatients were first treated with chemotherapy.1

Subsequent studies have demonstrated that almost all ofthe EGFR mutations are in adenocarcinomas or NSCLCswith an adenocarcinoma component, including adenosqua-mous carcinoma and solid subtypes of adenocarcinomainitially misinterpreted histologically as squamous cellcarcinomas but proven to be adenocarcinoma after furtherworkup.1,24,25 Clinical evidence also demonstrated that EGFRmutation analysis was the most reliable method ofdetermining which NSCLCs have EGFR mutations, and ofpredicting response to first-generation TKI therapy, asopposed to EGFR copy number detection by fluorescencein situ hybridization (FISH) or protein expression byimmunohistochemistry (IHC).1,4,5,25–27

In 2007, translocation of the anaplastic lymphoma kinasegene (ALK) with echinoderm microtubule-associated pro-tein-like 4 gene (EML4) was first reported in pulmonaryadenocarcinomas.28 Later studies29 showed that there weremany variants of the EML4-ALK rearrangement in additionto ALK fusion with other partners. When clinical trialsdemonstrated that crizotinib therapy improved responserate and progression-free survival for patients with ad-vanced NSCLC with ALK rearrangements, the FDA grantedaccelerated approval of the ALK TKI crizotinib (Xalkori;Pfizer, New York, New York).30,31 Concurrently, the FDAapproved a specific companion test (Vysis ALK Break-ApartFISH Probe Kit, Abbott Molecular, Des Plaines, Illinois) thathad been used to select patients for therapy with Xalkori inthe clinical trials.32 To date, cytogenetic techniques such asFISH have been generally considered a better method fordetecting chromosomal rearrangements, such as thoseinvolving ALK. It has been observed that reverse transcrip-tion–polymerase chain reaction or other methods thatrequire primers for each possible fusion variant may havefalse negatives.31,33

THE CAP/IASLC/AMP GUIDELINE ‘‘MOLECULARTESTING GUIDELINE FOR SELECTION OF LUNG

CANCER PATIENTS FOR EGFR AND ALK TYROSINEKINASE INHIBITORS’’

In the past several years, it became obvious that a subsetof patients with lung cancer have responded to the first-generation TKI therapies. However, there were no uniformguidelines for performing biomarker testing to selectpatients for treatment that was based on an in-depth reviewand grading of evidence and that incorporated input andvetting across the relevant medical disciplines on aworldwide basis. Therefore, the CAP, IASLC, and AMPset out to create standardized international guidelines for

lung cancer biomarker testing with input from multipleother stakeholders in an extended and exhaustive process asdescribed in the opening sections of the guidelines.1 Thiswork was conducted under the auspices of the CAPPathology and Laboratory Quality Center.1

The CAP/IASLC/AMP guideline focuses on the lungcancer–predictive biomarkers corresponding to the first-generation EGFR TKIs (gefitinib and erlotinib) and ALK TKI(crizotinib), which are clinically validated and FDA approvedfor treatment of advanced-stage lung cancers.1 Per theguidelines, EGFR and ALK molecular testing of advanced-stage lung cancers is recommended to select patients forEGFR or ALK TKI therapy and, after the diagnosis ofprimary lung adenocarcinoma is made (with the caveatmentioned below), tissue should be prioritized for biomark-er testing. For limited specimens, EGFR testing should beprioritized first and ALK testing should be prioritizedsecond over other molecular markers. Clinical criteria suchas sex, ethnicity, and smoking status are not recommendedfor selecting patients for lung cancer–predictive biomarkertesting, but adenocarcinoma cell type is a basis for selectingfor biomarker testing. With small biopsy or cytologyspecimens, individual judgment is permissible and bio-marker testing may be warranted in situations in which adiagnosis of adenocarcinoma cannot be excluded.1

A wide range of sample types, including cytologyspecimens, and fixatives (formalin-fixed paraffin-embedded,fresh, frozen, and alcohol) are allowable for biomarkertesting. Mutation analysis, using a validated method withsufficient performance characteristics, is recommended forEGFR mutation testing. KRAS mutation testing is notrecommended as a sole determinant of EGFR TKI therapybecause most lung cancers that lack KRAS mutations alsolack EGFR mutations. FISH assay using dual-labeled break-apart probes is recommended for ALK translocation testing.Carefully validated ALK IHC can be used as a screeningmethod to select specimens for ALK FISH testing. Thereader is referred to the published guideline for additionaldetails and the complete recommendations, suggestions,and expert consensus opinions.1

UNRESOLVED ISSUES, CAVEATS,AND FUTURE OBJECTIVES

The results of clinical trials with first-generation TKIs andthe release of the CAP/IASLC/AMP ‘‘Molecular TestingGuideline for Selection of Lung Cancer Patients for EGFRand ALK Tyrosine Kinase Inhibitors’’ have offered muchpromise and generated considerable excitement amongpatients, their families, their physicians, and the media.1

Looking forward, there are multiple unresolved issues,caveats, and future objectives that must be addressed:

1. The original clinical trials of first-generation EGFR andALK TKIs were performed with patients with advanced-stage lung cancer and, therefore, the approvals are forpatients with stage IIIB and IV lung cancer. Since manypatients with earlier-stage lung cancer later have lungcancer recurrence, and many ultimately die from theirdisease, this raises the question of whether or notpredictive biomarker testing should be done in theseearlier-stage lung cancers, either to have the data fortreatment when recurrence occurs or to use as anadjuvant therapy during initial treatment.1

2. EGFR mutations are identified in only approximately15% of NSCLCs in white persons in the United States

1192 Arch Pathol Lab Med—Vol 137, September 2013 Lung Cancer Biomarkers: Present and Future—Cagle et al

and in 20% of NSCLCs in African American persons.ALK rearrangements are found in only approximately 4%of adenocarcinomas in white persons in the UnitedStates.1 Therefore, targeted therapies and correspondingbiomarkers are still needed for most pulmonary adeno-carcinomas.27

3. Virtually all NSCLCs develop acquired resistance to first-generation EGFR TKIs or crizotinib after a period ofinitial response that may last for months. This acquiredor secondary resistance results from secondary mutationsin the EGFR or ALK gene or to a variety of othermechanisms.1,25 Second-line or third-line therapies forthese patients might include second-generation TKIs ordrugs directed at other actionable targets.34,35

4. EGFR mutations and ALK rearrangements are found inpulmonary adenocarcinomas, including adenosquamouscarcinomas and other variants, but are found uncom-monly, if at all, in pure squamous cell carcinomas or puresmall cell carcinomas.4,5,24,26,27,36 Therefore, targetedtherapies and corresponding biomarkers are needed forlung cancer cell types other than adenocarcinoma.25

5. Investigations into targeted therapy of lung cancer stemcells may provide a novel alternative to treating thesepatients.37

6. New technologies are altering the paradigm for predic-tive biomarker testing.1,25

7. Reimbursement for predictive biomarker testing is animportant issue if testing is going to be done and,especially, if it is going to be done on a reflex basis.1

THERAPY IN EARLY-STAGE LUNG CANCER

Only approximately 30% of NSCLCs are diagnosed in anearly stage (stage I, II, or IIIA) with limited disease. Mostaffected patients are treated with surgical resection and mayalso receive adjuvant therapy based on various proto-cols.16,38,39 However, despite treatment during early stage, alarge percentage of these patients will nevertheless haverelapse, with disease progressing to an advanced stage, andthey will eventually die from their lung cancer (5-year survivalrate by clinical stage is 50% for stage IA, 43% for stage IB,36% for stage IIA, 25% for stage IIB, and 19% for stage IIIA).8

Since most patients with early-stage lung cancer willeventually have relapse with disease progression, the CAP/IASLC/AMP guideline encourages EGFR and ALK testing oflung cancers at the time of diagnosis for patients presentingwith stage I, II, or III disease.1 Alternatively, if testing is notperformed in these early-stage cancers, the guidelinesencourage the retaining of cancer tissue for future biomarkertesting should the patient’s condition progress to anadvanced stage.1

Biomarker testing of the lung cancer tissue may provide abasis for TKI therapy when the patient’s conditionprogresses to an advanced stage. It may also form the basisfor TKI therapy as an adjuvant therapy at the time of initialdiagnosis and treatment in the early stage.1 Severalstudies40–42 are investigating the use of first-generationEGFR TKIs as adjuvant therapy in early-stage lung cancers,including the RADIANT and SELECT trials.

MOLECULAR TARGETED THERAPIESUNDER INVESTIGATION

Cetuximab (Erbitux; Bristol-Myers Squibb, New York,New York, and Eli Lilly and Company, Indianapolis,Indiana) has been approved by the FDA for treatment of

advanced colon adenocarcinoma and advanced head andneck squamous cell carcinoma.43–47 In 2011 and 2012,subgroup analysis of the First-Line Erbitux in Lung Cancer(FLEX) phase III clinical trial48–50 found that high expressionwith EGFR IHC (score of 200 or more) using the DakopharmDx Kit (Glostrup, Denmark) correlated with increasedoverall survival for patients with advanced NSCLC whowere receiving first-line platinum-based chemotherapy pluscetuximab, compared to chemotherapy alone, for lungsquamous cell carcinomas and adenocarcinomas. Thescoring system for EGFR IHC in this context has since beenvalidated.51 Cetuximab may offer a new therapy for bothlung adenocarcinomas and lung squamous cell carcinomas,based on a different predictive biomarker test, EGFR IHC asopposed to EGFR mutation testing.52

Patients receiving first-generation EGFR TKIs eventuallydevelop acquired resistance to their drug. Second-genera-tion EGFR TKIs are under investigation as additional lines oftherapy when acquired resistance develops or as apotentially more effective first-line therapy. These drugsare ERBB family blockers and, compared to first-generationEGFR TKIs, they typically exhibit higher affinity for thetarget, irreversible binding, and inhibition of more than 1target in the ERBB family of receptors. In addition, some ofthe second-generation drugs may also bind to EGFRreceptors with mutations of acquired resistance to thefirst-generation EGFR TKIs.53–56

Three of these ERBB family blockers under investigationare afatinib (BIBW2992; Boehringer Ingelheim, Ingelheim,Germany),54,56 dacomitinib,57 and XL647,58 Afatinib irre-versibly binds to HER2/neu, HER4, and EGFR, even whenthe most common acquired resistance mutation (T790M) ispresent. Dacomitinib is also a pan-ERBB family blocker thatcan potentially achieve response in tumors harboring theT790M mutation.

Some TKIs inhibit more than 1 kinase. Crizotinib,currently used for ALKþ adenocarcinomas, is a primeexample. In addition to ALK, crizotinib inhibits ROS1,MET, and RON.59,60 ROS1 translocations occur in 1% to 2%of NSCLCs, with several different fusion partners reported.Approval of crizotinib for treatment of ROS1þ adenocarci-nomas is generally expected in the not too distant future.60,61

There are multiple other potentially druggable targetswith corresponding predictive biomarkers in lung adeno-carcinomas, including in the signaling pathways down-stream of the ERRB receptors. Other drugs undergoingclinical trials include (1) the mammalian target of rapamycin(mTOR) inhibitor everolimus, the phosphoinositide-3 ki-nase (PI3K) and mTOR inhibitor BEZ235, the PI3Kinhibitors GDC-0941 and XL147, and the AKT inhibitorMK-220662–65; (2) inhibitors and antibodies for c-MET andits ligand, hepatocyte growth factor66; tivantinib as second-line therapy in patients with advanced nonsquamousNSCLC in the MARQUEE (Met Inhibitor ARQ 197 plusErlotinib versus Erlotinib plus placebo in NSCLC) trial67,68;the JAK (Janus kinase) inhibitors enzastaurin and AZD1480and the STAT inhibitor NSC-74338069–71; inhibitors of MEK(mitogen-activated protein kinase kinase),72–74 vandetanibfor RET (rearranged during transfection),75,76 and dasatinibfor Src.77,78

KRAS is the most frequently mutated oncogene in lungadenocarcinomas, occurring in about 30% of cases, mostlyin smokers. There are currently no direct inhibitors of KRAS,although there are inhibitors of targets downstream toKRAS.79


Cetuximab in combination with first-line platinum-basedchemotherapy has been reported to improve overall survivalfor patients with lung squamous cell carcinomas that havehigh total EGFR expression by IHC (score of 200 or more)using the Dako pharmDx kit, compared to chemotherapyalone.48–52

Potential targets for squamous cell carcinoma underinvestigation include members of the PI3K pathway, thefibroblast growth factor receptor, and the discoidin domainreceptor.80–83 Potential drugs under investigation for smallcell lung cancer include the mTOR inhibitors everolimusand temsirolimus.84–86

MOLECULAR TARGETINGOF LUNG CANCER STEM CELLS

Stem cells in lung cancer have been postulated for morethan 3 decades,87 and in the last decade they have beenincreasingly examined for the presence of possible diagnos-tic and therapeutic benefits. Lung cancer stem cells havespecific characteristics of all stem cells, including chemore-sistance and radioresistance, the ability to self-renew, theability to produce a large number of multilineage progeny,and slow proliferation.88,89 Postchemoradiotherapy expres-sion of lung cancer stem cell markers correlates with poorprognosis in patients with lung cancer.88 Research iscontinuing to clarity the critical role lung cancer stem cellsplay in metastases, drug resistance, and tumor regenera-tion.37 The predominant use of mouse models, as well aslung cancer stem cells’ slow cycling and asymmetric celldivision, make their identification difficult; and studies ofpotential treatments targeting lung cancer stem cells are allthe more challenging.37 Identification of molecular targetsfor effective lung cancer stem cell therapy requires thedifferentiation of lung cancer stem cells from normalmultipotent stem cells. This differentiation might beaccomplished by using magnetic bead isolation or flowcytometry to identify unique cell type markers.90

Treatment regimens for cancers have traditionally as-sumed that all cancer cells have equal malignant potential,so the concept that cancers are driven by cancer stem cellshas significant clinical implications.91 Researchers are nowactively seeking therapies that target stem cells, and cancerstem cell inhibitory mechanisms, such as blocking stem cellfactor, antagonists of ABCG2 pumping activity, and Notchinhibitors, are becoming increasingly studied.91 Hedgehog,Wng, and Notch pathways have been found to be critical tostem cell regulation, and might provide appropriate therapytargets.37 The Hedgehog signaling pathway is essential fordetermining whether a cell undergoes self-renewal ordifferentiation.92 Wnt signaling is necessary for embryogen-esis and homeostatic maintenance of adult tissues, and itsactivation has been shown to significantly enhance lungcancer proliferation, migration, colony formation, and drugresistance.37,92 Approximately one-third of lung cancershave an activated Notch pathway, and its presence has beenshown to indicate a significantly worse prognosis in patientswho concomitantly lack TP53 mutations.37 Studies of lungcancer stem cell–targeted therapy based on these pathwaysis ongoing.92 Ongoing studies of potential lung cancer stemcell–targeted therapies are also ongoing. For example, astudy showing significantly increased Rac1 guanosinetriphosphatase activity in lung cancer cells that haveundergone epithelial-mesenchymal transition suggests thattargeting Rac1 might provide a more effective lung cancer

therapy by eliminating cancer stem cell subpopulations andby blocking non–cancer stem cell to cancer stem celltransition and eliminating cancer stem cell subpopula-tions.93 It has also been suggested that ALDH1A1þ lungcancer stem cells may cause EGFR TKI resistance.94 Also,another study95 suggests that an existing phenothiazine-likeantipsychotic drug, trifluoperazine, has the ability to down-regulate cancer stem cell markers CD44 and CD133 and assuch might possess anti–lung cancer stem cell properties.Finally, the concept of a single lung cancer stem cell hasbeen complicated by the possibility of an associatedmesenchymal stem cell that might play a role in lungcancer tumorigenesis and progression by producing ormaintaining a cancer-permissive microenvironment.96

ADVANCES IN NEXT-GENERATION TECHNOLOGY

Before the debut of benchtop next-generation sequencinginstruments, clinical laboratories had limited options foridentifying the presence or absence of gene mutations intumors. Many molecular pathologists developed tests usingtraditional technologies, such as pyrosequencing, Sangerdideoxynucleotide chain termination sequencing, or real-time polymerase chain reaction.97 Although these assaysgenerate the results needed for patient care, they are limitedby a single-target-gene approach.98 That is, a separateassay—and in some cases, such as EGFR, multiple assays—must be performed for each biomarker. As outlined above,testing for multiple biomarkers is already considered to bethe standard of care for lung cancer, and additional tests willbe needed as new predictive biomarkers and targetedtherapies are discovered. Thus, when specimen volumesare limited, such as commonly occurs with small biopsies orcytopathology procedures, there may be insufficient diag-nostic material available to perform all molecular diagnos-tics testing.6,99 For this reason, many laboratories are nowdeveloping strategies to optimally triage tissue for histopa-thology examination, immunohistochemistry, FISH, andmolecular testing with direction provided by the CAP/IASLC/AMP guidelines.98 In this context, next-generationsequencing technologies are significantly advancing molec-ular diagnostics by enabling the design of highly multi-plexed assays.100,101

Several next-generation sequencing platforms are com-mercially available. The technologies vary in the type ofDNA molecule used as a sequencing template, thechemistry underlying the sequencing reaction, and theamount of sequence data generated per run. The instru-ments most commonly used in clinical laboratories are the454 GS Junior (454 Life Sciences, Roche, Branford,Connecticut), Ion Torrent Personal Genome Machine(Ion Torrent Systems, Life Technologies, San Francisco,California), and MiSeq Personal Sequencer (Illumina Inc,San Diego, California). The 454 was first to enter themarket. It uses emulsion polymerase chain reaction toclonally amplify DNA fragments that are sequenced viasequencing-by-synthesis technology.102 The Ion Torrent isbased on a similar technology, but rather than usingfluorescence to determine the nucleotide sequence, itdetects Hþ ions that are released during base incorpora-tion.103 In comparison, the MiSeq uses bridge amplificationto clonally amplify DNA fragments before sequencing.103

The MassARRAY Analyzer (Sequenom Inc, San Diego,California) is another innovative technology. It uses massspectrometry to determine the sequence of amplified gene


fragments.104 Of note, investigators have now developednovel strategies to detect EML4-ALK gene rearrangementson the MassARRAY Analyzer, making it possible toconcurrently evaluate EGFR gene mutations and ALK generearrangements during a single run.105 A similar strategycould be designed to also detect ROS1 or other generearrangements. More recently, single-molecule sequenc-ing platforms, such as the PacBio RS (Pacific Biosciences,Menlo Park, California), have entered the researchenvironment as powerful tools for whole genome se-quencing.106 Although these instruments generate high-quality sequence data, each varies in key performancecharacteristics such as throughput, read length, error rate,hands-on time, run time, instrument cost, and reagentcost.107,108 We recommend that potential users carefullyevaluate their options, considering the intended clinicalapplication and individual laboratory needs. To assistpathologists in designing highly multiplexed gene muta-tion assays, reagent kits licensed for research use only arenow available. Also, an expert panel for next-generationsequencing recently suggested guidelines for test valida-tion, quality control, proficiency testing, and referencematerials,109 and the CAP has published a next-generationsequencing checklist (www.cap.org, June 21, 2013).

Although still in its infancy as a diagnostic tool, wholegenome sequencing has the potential to truly personalizepatient care. Importantly, the long-sought $1000 genomewill soon be a reality, and advances in automation andbioinformatics are making cancer genomics an increasinglytractable tool in the molecular diagnostics laboratory.110 Twoinvestigations80,111 recently reported the whole genomesequences of 178 lung squamous cell carcinomas and of183 lung adenocarcinomas. These incredibly rich data setsclearly reveal the genomic complexity of lung cancer. Forexample, the frequent presence of EGFR, KRAS, and BRAFmutations was confirmed in lung adenocarcinoma.111

Unexpectedly though, mutations were also identified inseveral other genes including U2AF1, RBM10, and ARID1A.In total, 25 genes were shown to have a statisticallysignificant number of mutations in lung adenocarcinoma.111

Each represents a possible target for new therapeutics. Incomparison, the squamous cell carcinoma investigationidentified only 11 genes with recurrent mutations.80

However, multiple signaling pathways were significantlyaltered. These include gene pathways implicated in oxidativestress response, apoptotic signaling, and squamous celldifferentiation.80 Of note, many of the alterations identifiedin squamous cell carcinoma of the lung are shared withthose occurring in squamous cell carcinoma of the head andneck, suggesting a possible common molecular biology.Taken together, these genomic investigations show thatpotential therapeutic targets can be found in nearly everylung tumor. Furthermore, researchers are expanding ourgenomic understanding of lung cancer to include epigenetic,transcriptomic, proteomic, and metabolomic signatures.112

When clinical phenotypes based on genomic informationcan be reliably predicted, then increasingly personalizedtherapeutic strategies will become possible.

Inasmuch as a molecular diagnostics laboratory can generatetremendous amounts of data by using next-generationsequencing, information management and interpretation is asignificant challenge. Given the magnitude of the humangenome (approximately 3 billion base pairs encoding approx-imately 20 000 genes), some investigative pathologists are nowbuilding bioinformatics and statistics support into their clinical

laboratories. However, before genomics can be performed as aroutine test, automated bioinformatics pipelines and analysisalgorithms must be developed. Currently, whole genomesequence data cannot be interpreted within a time frame that isoptimal for patient care. Furthermore, the functional conse-quence of many gene polymorphisms remains unknown.Molecular pathogenesis researchers must investigate allcommon gene alterations, defining which are driver mutationsand which are bystander mutations. Coordinated efforts togenerate a comprehensive reference human genome sequenceand cancer genome database (http://cancergenome.nih.gov,http://cancercommons.org, and http://icgc.org; each accessedMay 27, 2013) have begun filling these knowledge gaps.

ADVANCES IN BIOMARKERIMMUNOHISTOCHEMISTRY

Traditional EGFR IHC detects total EGFR proteinexpression, but does not differentiate between wild typeand mutations so it is not recommended as a basis forselecting patients for EGFR TKI therapy.98 Investigationshave examined antibodies to proteins associated with the 2primary mutations, which represent 90% of EGFR mutationsthat are associated with response to first-generation EGFRTKIs. These antibodies may be useful in screening for mostEGFR mutations but will not detect the less frequent EGFRmutations.113–115

As discussed previously, total EGFR protein expression byIHC may be the predictive biomarker used if cetuximab isapproved for lung cancer therapy. High expression withEGFR IHC (score of 200 or more) using the Dako pharmDxkit correlated with increased overall survival for patientswith lung cancer—specifically, adenocarcinoma and squa-mous cell carcinoma—receiving first-line platinum-basedchemotherapy plus cetuximab compared to chemotherapyalone.48–50 The EGFR expression scoring system has alreadybeen validated for this particular setting.51,52

Immunohistochemistry does not demonstrate ALK expres-sion in most tissues that lack an ALK fusion gene. When anALK fusion gene is present, IHC usually reveals increasedALK expressions. Well-known examples include lymphomaand inflammatory myofibroblastic tumor. Probably because ofthe relatively low expression of ALK protein in lung cancerswith ALK translocations, false-negative IHC results have beenobserved in lung cancers that were shown to have ALK fusiongenes by other methods.1,25,116 Several procedures have beenused to enhance the sensitivity of IHC for ALKþ lung cancers,but ALK antibodies that are highly specific and sensitive toALK expression in lung cancers have been developed. WhileALK expression is known to correlate with ALK transloca-tions with these antibodies, clinical trials validating ALK IHCas a predictive biomarker for patient outcomes with crizotinibtherapy have not been performed.1,25,116–120 The CAP/IASLC/AMP guidelines note that screening for ALK translocationswith the new sensitive ALK antibodies may potentially beused to identify lung cancers that should then have ALKtranslocations confirmed with FISH.1

PAYMENT FOR PREDICTIVE BIOMARKER TESTSAND THE FUTURE OF TARGETED THERAPY

A common and important, but as yet unanswered,question is how pathologists and laboratories will be paidfor performing lung cancer biomarker tests. Many oncolo-gists and patients are now requesting biomarker testing and,in some hospitals, reflex testing has been implemented as a


policy. The number of requests is expected to increase andthe addition of new biomarkers for which testing may bedone is also expected to increase. For patients with lungcancer with a very bleak prognosis, for their families, and fortheir oncologists, it is difficult to put any price on potentialadditional months of life, particularly based on taking a pillwithout the significant inconveniences and side effects ofchemotherapy, even if ultimately there is relapse. Currently,pathologists and laboratories have the option of billingMedicare or private payers for each individual test that isordered separately, or of billing for testing as part of a reflextesting protocol at their institution. Some payers areconsidering bundled payments for biomarker testing withresulting treatment based on the average cost of care, forwhich providers accept an increased risk for overutiliza-tion.121

Whether, under what circumstances, and to what extentMedicare and private payers will pay for these tests,however, is currently unknown. No standard billing strategyexists for lung cancer biomarker test payment by Medicareor private payers. Cost-effectiveness analysis, evaluatingboth the clinical and economic effect of a test or treatment,is currently used by the United Kingdom’s National HealthService and, with looming health care reform, may soon beused in the United States to determine whether moleculartherapies and biomarkers are covered.122,123 If cost-effec-tiveness analyses become an accepted method in the UnitedStates for payers to determine coverage and price of lungcancer biomarker tests, evidence-based literature regardingthe value of these tests in patients with lung cancer will beneeded to influence payers’ payment determinations.However, advances in testing and treatment discussed,including less costly tests with improvements in turnaroundtime (eg, IHC or next-generation technology) and betteroutcomes from new therapies (new drugs and new targets)and treatment strategies (combined or sequential targets,adjuvant therapy in earlier stages), may significantly alterthe cost-benefit dynamic in the future.

CONCLUSION

The advent of predictive biomarker testing on tissuesamples for targeted therapy has forever altered the role ofthe pathologist in the management of patients with lungcancer. The role of the pathologist in biomarker testing,including very direct participation with IHC, has potential togrow. Patients with lung cancer and their families are nowmore aware of who pathologists are and the critical role thatthey have in their health care. New tests, new testtechnologies, and overall advances in precision medicinewill likely improve the cost to benefit ratio for biomarkertesting which, along with the powerful desire from patientsand physicians to improve survival and quality of life,suggest that predictive biomarker testing will expand as aroutine component of cancer care.

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