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 for

Selection of Lung Cancer Patients for EGFR and ALK
Tyrosine Kinase Inhibitors: Guideline From the College of
American Pathologists, International Association for the
Study of Lung Cancer, and Association for Molecular
Pathology has now provided a guideline for biomarker
testing for first-generation lung cancer tyrosine kinase
inhibitors. Biomarker testing has forever altered the role of
pathologists in the management of patients with lung
cancer. Current, unresolved issues in the precision
medicine of lung cancer will be addressed by the
development of new biomarker tests, new drugs, and
new test technologies and by improvement in the cost to
benefit ratio of biomarker testing.
(Arch Pathol Lab Med. 2013;137:11911198; doi:
10.5858/arpa.2013-0319-CR)

n April 2013, the College of American Pathologists (CAP),

the International Association for the Study of Lung
Cancer (IASLC), and the Association for Molecular Pathology (AMP) officially released the Molecular Testing
Guideline for Selection of Lung Cancer Patients for EGFR
[epidermal growth factor receptor] and ALK [anaplastic
lymphoma kinase] Tyrosine Kinase Inhibitors [TKIs]. 1 The
CAP/IASLC/AMP guideline provides the first standardized
evidence-based approach for performing biomarker testing
to select patients with lung cancer for EGFR or ALK TKI
therapy that is multidisciplinary and multicontinental in
scope. Subsequent to the guideline release, the CAP is also
releasing a template protocol for the reporting of lung
cancerpredictive biomarkers as an addendum to anatomic
pathology reports, and the guidelines, themselves, will be
Accepted for publication May 28, 2013.
From the Department of Pathology and Genomic Medicine, The
Methodist Hospital, Houston, Texas, and the Department of
Pathology and Laboratory Medicine, Weill Medical College of
Cornell University, New York, New York (Drs Cagle and Olsen);
and the Department of Pathology, The University of Texas Health
Science Center at Tyler (Dr Allen).
The authors have no relevant financial interest in the products or
companies described in this article.
Presented at the New Frontiers in Pathology: An Update for
Practicing Pathologists meeting; Homestead Resort; August 35,
2012; Glen Arbor, Michigan.
Reprints: Philip T. Cagle, MD, Department of Pathology and
Genomic Medicine, The Methodist Hospital, 6565 Fannin St, Main
Bldg, Room 227, Houston, Texas 77030 (e-mail: pcagle@tmhs.org).
Arch Pathol Lab MedVol 137, September 2013

regularly updated. The number of predictive biomarkers to

be tested for lung cancer, the number of targeted therapies,
and the types of test methodologies are all expected to
increase during upcoming months and years. The need for
predictive biomarker testing on pathology specimens has
forever altered the role of pathologists in the care of patients
with lung cancer. This review provides an overview of
current developments, expected developments, and related
issues for lung cancerpredictive biomarker testing.
HISTORICAL PERSPECTIVE
For many years, lung cancer has been the leading cause of
cancer deaths in the United States2 and worldwide.3 Most
patients 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% to
15%, for many decades.4,5
Approximately 85% of lung cancers are nonsmall cell
lung cancers (NSCLCs), traditionally divided into 3 major
cell types: adenocarcinoma (approximately 50%), squamous cell carcinoma (approximately 35%), and large cell
carcinoma (approximately 15%, although this is a diminishing cell type category).6,7 About 70% of NSCLCs present
with advanced disease not considered curable by surgical
resection, either locally advanced (stage IIIB) or often with
metastatic disease (stage IV). Clinical stage IIIB NSCLCs
are associated with a 5-year survival rate of 7% and stage
IV NSCLCs, with a 5-year survival rate of 2%.8 Conventional therapy for stage IV NSCLC is doublet chemotherapy that includes cisplatin or carboplatin. Affected patients
may additionally receive radiation therapy. Of the patients
with lung cancer who initially respond to first-line therapy,
nearly all subsequently experience disease progression.
These patients may receive second-line therapy, or possibly
more lines of therapy, in an attempt to control their
disease. Eventually, virtually all of these patients die from
lung cancer as reflected in the statistics mentioned
previously.916
Given the dismal prognosis and limited treatment options
for patients with advanced-stage lung cancer, it is not
surprising that the US Food and Drug Administration (FDA)
approval of targeted molecular therapies for advanced-stage
lung cancer has been a game changer for these patients.
The first generation of EGFR TKIs approved for treatment
of advanced-stage lung cancer includes gefitinib (Iressa;
AstraZeneca, London, United Kingdom) and erlotinib
(Tarceva; Genentech, South San Francisco, California, and
OSI Pharmaceuticals, Long Island, New York). Already
Lung Cancer Biomarkers: Present and FutureCagle et al 1191

since the 1980s it was known that most NSCLCs

overexpress EGFR,17 but early clinical trials of EGFR TKIs
affected only a small percentage of patients with lung
cancer. In 2004, several separate investigators reported that
the presence of somatic mutations of EGFR gene in a lung
cancer predicted the likelihood of its response to EGFR
TKIs.1820 Beginning in 2009, a number of clinical trials have
reported better response rates and progression-free survival
for patients with advanced-stage NSCLC with EGFR
mutations who received EGFR TKIs compared to conventional therapy.2123 These clinical trials were conducted with
patients with advanced lung cancer who had significant
improvement in progression-free survival, but no demonstrated improvement in overall survival, possibly because of
the crossover design of these studies in which many of the
patients were first treated with chemotherapy.1
Subsequent studies have demonstrated that almost all of
the EGFR mutations are in adenocarcinomas or NSCLCs
with an adenocarcinoma component, including adenosquamous carcinoma and solid subtypes of adenocarcinoma
initially misinterpreted histologically as squamous cell
carcinomas but proven to be adenocarcinoma after further
workup.1,24,25 Clinical evidence also demonstrated that EGFR
mutation analysis was the most reliable method of
determining which NSCLCs have EGFR mutations, and of
predicting response to first-generation TKI therapy, as
opposed to EGFR copy number detection by fluorescence
in situ hybridization (FISH) or protein expression by
immunohistochemistry (IHC).1,4,5,2527
In 2007, translocation of the anaplastic lymphoma kinase
gene (ALK) with echinoderm microtubule-associated protein-like 4 gene (EML4) was first reported in pulmonary
adenocarcinomas.28 Later studies29 showed that there were
many variants of the EML4-ALK rearrangement in addition
to ALK fusion with other partners. When clinical trials
demonstrated that crizotinib therapy improved response
rate and progression-free survival for patients with advanced NSCLC with ALK rearrangements, the FDA granted
accelerated approval of the ALK TKI crizotinib (Xalkori;
Pfizer, New York, New York).30,31 Concurrently, the FDA
approved a specific companion test (Vysis ALK Break-Apart
FISH Probe Kit, Abbott Molecular, Des Plaines, Illinois) that
had been used to select patients for therapy with Xalkori in
the clinical trials.32 To date, cytogenetic techniques such as
FISH have been generally considered a better method for
detecting chromosomal rearrangements, such as those
involving ALK. It has been observed that reverse transcriptionpolymerase chain reaction or other methods that
require primers for each possible fusion variant may have
false negatives.31,33
THE CAP/IASLC/AMP GUIDELINE MOLECULAR
TESTING GUIDELINE FOR SELECTION OF LUNG
CANCER PATIENTS FOR EGFR AND ALK TYROSINE
KINASE INHIBITORS
In the past several years, it became obvious that a subset
of patients with lung cancer have responded to the firstgeneration TKI therapies. However, there were no uniform
guidelines for performing biomarker testing to select
patients for treatment that was based on an in-depth review
and grading of evidence and that incorporated input and
vetting across the relevant medical disciplines on a
worldwide basis. Therefore, the CAP, IASLC, and AMP
set out to create standardized international guidelines for
1192 Arch Pathol Lab MedVol 137, September 2013

lung cancer biomarker testing with input from multiple

other stakeholders in an extended and exhaustive process as
described in the opening sections of the guidelines.1 This
work was conducted under the auspices of the CAP
Pathology and Laboratory Quality Center.1
The CAP/IASLC/AMP guideline focuses on the lung
cancerpredictive biomarkers corresponding to the firstgeneration EGFR TKIs (gefitinib and erlotinib) and ALK TKI
(crizotinib), which are clinically validated and FDA approved
for treatment of advanced-stage lung cancers.1 Per the
guidelines, EGFR and ALK molecular testing of advancedstage lung cancers is recommended to select patients for
EGFR or ALK TKI therapy and, after the diagnosis of
primary lung adenocarcinoma is made (with the caveat
mentioned below), tissue should be prioritized for biomarker testing. For limited specimens, EGFR testing should be
prioritized first and ALK testing should be prioritized
second over other molecular markers. Clinical criteria such
as sex, ethnicity, and smoking status are not recommended
for selecting patients for lung cancerpredictive biomarker
testing, but adenocarcinoma cell type is a basis for selecting
for biomarker testing. With small biopsy or cytology
specimens, individual judgment is permissible and biomarker testing may be warranted in situations in which a
diagnosis of adenocarcinoma cannot be excluded.1
A wide range of sample types, including cytology
specimens, and fixatives (formalin-fixed paraffin-embedded,
fresh, frozen, and alcohol) are allowable for biomarker
testing. Mutation analysis, using a validated method with
sufficient performance characteristics, is recommended for
EGFR mutation testing. KRAS mutation testing is not
recommended as a sole determinant of EGFR TKI therapy
because most lung cancers that lack KRAS mutations also
lack EGFR mutations. FISH assay using dual-labeled breakapart probes is recommended for ALK translocation testing.
Carefully validated ALK IHC can be used as a screening
method to select specimens for ALK FISH testing. The
reader is referred to the published guideline for additional
details 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 and
the release of the CAP/IASLC/AMP Molecular Testing
Guideline for Selection of Lung Cancer Patients for EGFR
and ALK Tyrosine Kinase Inhibitors have offered much
promise and generated considerable excitement among
patients, 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 and
ALK TKIs were performed with patients with advancedstage lung cancer and, therefore, the approvals are for
patients with stage IIIB and IV lung cancer. Since many
patients with earlier-stage lung cancer later have lung
cancer recurrence, and many ultimately die from their
disease, this raises the question of whether or not
predictive biomarker testing should be done in these
earlier-stage lung cancers, either to have the data for
treatment when recurrence occurs or to use as an
adjuvant therapy during initial treatment.1
2. EGFR mutations are identified in only approximately
15% of NSCLCs in white persons in the United States
Lung Cancer Biomarkers: Present and FutureCagle et al

3.

4.

5.
6.
7.

and in 20% of NSCLCs in African American persons.

ALK rearrangements are found in only approximately 4%
of adenocarcinomas in white persons in the United
States.1 Therefore, targeted therapies and corresponding
biomarkers are still needed for most pulmonary adenocarcinomas.27
Virtually all NSCLCs develop acquired resistance to firstgeneration EGFR TKIs or crizotinib after a period of
initial response that may last for months. This acquired
or secondary resistance results from secondary mutations
in the EGFR or ALK gene or to a variety of other
mechanisms.1,25 Second-line or third-line therapies for
these patients might include second-generation TKIs or
drugs directed at other actionable targets.34,35
EGFR mutations and ALK rearrangements are found in
pulmonary adenocarcinomas, including adenosquamous
carcinomas and other variants, but are found uncommonly, if at all, in pure squamous cell carcinomas or pure
small cell carcinomas.4,5,24,26,27,36 Therefore, targeted
therapies and corresponding biomarkers are needed for
lung cancer cell types other than adenocarcinoma.25
Investigations into targeted therapy of lung cancer stem
cells may provide a novel alternative to treating these
patients.37
New technologies are altering the paradigm for predictive biomarker testing.1,25
Reimbursement for predictive biomarker testing is an
important 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 an
early stage (stage I, II, or IIIA) with limited disease. Most
affected patients are treated with surgical resection and may
also receive adjuvant therapy based on various protocols.16,38,39 However, despite treatment during early stage, a
large percentage of these patients will nevertheless have
relapse, with disease progressing to an advanced stage, and
they will eventually die from their lung cancer (5-year survival
rate 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 will
eventually have relapse with disease progression, the CAP/
IASLC/AMP guideline encourages EGFR and ALK testing of
lung cancers at the time of diagnosis for patients presenting
with stage I, II, or III disease.1 Alternatively, if testing is not
performed in these early-stage cancers, the guidelines
encourage the retaining of cancer tissue for future biomarker
testing should the patients condition progress to an
advanced stage.1
Biomarker testing of the lung cancer tissue may provide a
basis for TKI therapy when the patients condition
progresses to an advanced stage. It may also form the basis
for TKI therapy as an adjuvant therapy at the time of initial
diagnosis and treatment in the early stage.1 Several
studies4042 are investigating the use of first-generation
EGFR TKIs as adjuvant therapy in early-stage lung cancers,
including the RADIANT and SELECT trials.
MOLECULAR TARGETED THERAPIES
UNDER 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
Arch Pathol Lab MedVol 137, September 2013

advanced colon adenocarcinoma and advanced head and

neck squamous cell carcinoma.4347 In 2011 and 2012,
subgroup analysis of the First-Line Erbitux in Lung Cancer
(FLEX) phase III clinical trial4850 found that high expression
with EGFR IHC (score of 200 or more) using the Dako
pharmDx Kit (Glostrup, Denmark) correlated with increased
overall survival for patients with advanced NSCLC who
were receiving first-line platinum-based chemotherapy plus
cetuximab, compared to chemotherapy alone, for lung
squamous cell carcinomas and adenocarcinomas. The
scoring system for EGFR IHC in this context has since been
validated.51 Cetuximab may offer a new therapy for both
lung adenocarcinomas and lung squamous cell carcinomas,
based on a different predictive biomarker test, EGFR IHC as
opposed to EGFR mutation testing.52
Patients receiving first-generation EGFR TKIs eventually
develop acquired resistance to their drug. Second-generation EGFR TKIs are under investigation as additional lines of
therapy when acquired resistance develops or as a
potentially more effective first-line therapy. These drugs
are ERBB family blockers and, compared to first-generation
EGFR TKIs, they typically exhibit higher affinity for the
target, irreversible binding, and inhibition of more than 1
target in the ERBB family of receptors. In addition, some of
the second-generation drugs may also bind to EGFR
receptors with mutations of acquired resistance to the
first-generation EGFR TKIs.5356
Three of these ERBB family blockers under investigation
are afatinib (BIBW2992; Boehringer Ingelheim, Ingelheim,
Germany),54,56 dacomitinib,57 and XL647,58 Afatinib irreversibly binds to HER2/neu, HER4, and EGFR, even when
the most common acquired resistance mutation (T790M) is
present. Dacomitinib is also a pan-ERBB family blocker that
can potentially achieve response in tumors harboring the
T790M mutation.
Some TKIs inhibit more than 1 kinase. Crizotinib,
currently used for ALK adenocarcinomas, is a prime
example. 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 ROS1 adenocarcinomas is generally expected in the not too distant future.60,61
There are multiple other potentially druggable targets
with corresponding predictive biomarkers in lung adenocarcinomas, including in the signaling pathways downstream of the ERRB receptors. Other drugs undergoing
clinical trials include (1) the mammalian target of rapamycin
(mTOR) inhibitor everolimus, the phosphoinositide-3 kinase (PI3K) and mTOR inhibitor BEZ235, the PI3K
inhibitors GDC-0941 and XL147, and the AKT inhibitor
MK-22066265; (2) inhibitors and antibodies for c-MET and
its ligand, hepatocyte growth factor66; tivantinib as secondline therapy in patients with advanced nonsquamous
NSCLC in the MARQUEE (Met Inhibitor ARQ 197 plus
Erlotinib versus Erlotinib plus placebo in NSCLC) trial67,68;
the JAK (Janus kinase) inhibitors enzastaurin and AZD1480
and the STAT inhibitor NSC-7433806971; inhibitors of MEK
(mitogen-activated protein kinase kinase),7274 vandetanib
for RET (rearranged during transfection),75,76 and dasatinib
for Src.77,78
KRAS is the most frequently mutated oncogene in lung
adenocarcinomas, occurring in about 30% of cases, mostly
in smokers. There are currently no direct inhibitors of KRAS,
although there are inhibitors of targets downstream to
KRAS.79
Lung Cancer Biomarkers: Present and FutureCagle et al 1193

Cetuximab in combination with first-line platinum-based

chemotherapy has been reported to improve overall survival
for patients with lung squamous cell carcinomas that have
high total EGFR expression by IHC (score of 200 or more)
using the Dako pharmDx kit, compared to chemotherapy
alone.4852
Potential targets for squamous cell carcinoma under
investigation include members of the PI3K pathway, the
fibroblast growth factor receptor, and the discoidin domain
receptor.8083 Potential drugs under investigation for small
cell lung cancer include the mTOR inhibitors everolimus
and temsirolimus.8486
MOLECULAR TARGETING
OF LUNG CANCER STEM CELLS
Stem cells in lung cancer have been postulated for more
than 3 decades,87 and in the last decade they have been
increasingly examined for the presence of possible diagnostic and therapeutic benefits. Lung cancer stem cells have
specific characteristics of all stem cells, including chemoresistance and radioresistance, the ability to self-renew, the
ability to produce a large number of multilineage progeny,
and slow proliferation.88,89 Postchemoradiotherapy expression of lung cancer stem cell markers correlates with poor
prognosis in patients with lung cancer.88 Research is
continuing to clarity the critical role lung cancer stem cells
play in metastases, drug resistance, and tumor regeneration.37 The predominant use of mouse models, as well as
lung cancer stem cells slow cycling and asymmetric cell
division, make their identification difficult; and studies of
potential treatments targeting lung cancer stem cells are all
the more challenging.37 Identification of molecular targets
for effective lung cancer stem cell therapy requires the
differentiation of lung cancer stem cells from normal
multipotent stem cells. This differentiation might be
accomplished by using magnetic bead isolation or flow
cytometry to identify unique cell type markers.90
Treatment regimens for cancers have traditionally assumed that all cancer cells have equal malignant potential,
so the concept that cancers are driven by cancer stem cells
has significant clinical implications.91 Researchers are now
actively seeking therapies that target stem cells, and cancer
stem cell inhibitory mechanisms, such as blocking stem cell
factor, antagonists of ABCG2 pumping activity, and Notch
inhibitors, are becoming increasingly studied.91 Hedgehog,
Wng, and Notch pathways have been found to be critical to
stem cell regulation, and might provide appropriate therapy
targets.37 The Hedgehog signaling pathway is essential for
determining whether a cell undergoes self-renewal or
differentiation.92 Wnt signaling is necessary for embryogenesis and homeostatic maintenance of adult tissues, and its
activation has been shown to significantly enhance lung
cancer proliferation, migration, colony formation, and drug
resistance.37,92 Approximately one-third of lung cancers
have an activated Notch pathway, and its presence has been
shown to indicate a significantly worse prognosis in patients
who concomitantly lack TP53 mutations.37 Studies of lung
cancer stem celltargeted therapy based on these pathways
is ongoing.92 Ongoing studies of potential lung cancer stem
celltargeted therapies are also ongoing. For example, a
study showing significantly increased Rac1 guanosine
triphosphatase activity in lung cancer cells that have
undergone epithelial-mesenchymal transition suggests that
targeting Rac1 might provide a more effective lung cancer
1194 Arch Pathol Lab MedVol 137, September 2013

therapy by eliminating cancer stem cell subpopulations and

by blocking noncancer stem cell to cancer stem cell
transition and eliminating cancer stem cell subpopulations.93 It has also been suggested that ALDH1A1 lung
cancer stem cells may cause EGFR TKI resistance.94 Also,
another study95 suggests that an existing phenothiazine-like
antipsychotic drug, trifluoperazine, has the ability to downregulate cancer stem cell markers CD44 and CD133 and as
such might possess antilung cancer stem cell properties.
Finally, the concept of a single lung cancer stem cell has
been complicated by the possibility of an associated
mesenchymal stem cell that might play a role in lung
cancer tumorigenesis and progression by producing or
maintaining a cancer-permissive microenvironment.96
ADVANCES IN NEXT-GENERATION TECHNOLOGY
Before the debut of benchtop next-generation sequencing
instruments, clinical laboratories had limited options for
identifying the presence or absence of gene mutations in
tumors. Many molecular pathologists developed tests using
traditional technologies, such as pyrosequencing, Sanger
dideoxynucleotide chain termination sequencing, or realtime polymerase chain reaction.97 Although these assays
generate the results needed for patient care, they are limited
by a single-target-gene approach.98 That is, a separate
assayand 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 be
the standard of care for lung cancer, and additional tests will
be needed as new predictive biomarkers and targeted
therapies are discovered. Thus, when specimen volumes
are limited, such as commonly occurs with small biopsies or
cytopathology procedures, there may be insufficient diagnostic material available to perform all molecular diagnostics testing.6,99 For this reason, many laboratories are now
developing strategies to optimally triage tissue for histopathology examination, immunohistochemistry, FISH, and
molecular testing with direction provided by the CAP/
IASLC/AMP guidelines.98 In this context, next-generation
sequencing technologies are significantly advancing molecular diagnostics by enabling the design of highly multiplexed assays.100,101
Several next-generation sequencing platforms are commercially available. The technologies vary in the type of
DNA molecule used as a sequencing template, the
chemistry underlying the sequencing reaction, and the
amount of sequence data generated per run. The instruments most commonly used in clinical laboratories are the
454 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 the
market. It uses emulsion polymerase chain reaction to
clonally amplify DNA fragments that are sequenced via
sequencing-by-synthesis technology.102 The Ion Torrent is
based on a similar technology, but rather than using
fluorescence to determine the nucleotide sequence, it
detects H ions that are released during base incorporation.103 In comparison, the MiSeq uses bridge amplification
to clonally amplify DNA fragments before sequencing.103
The MassARRAY Analyzer (Sequenom Inc, San Diego,
California) is another innovative technology. It uses mass
spectrometry to determine the sequence of amplified gene
Lung Cancer Biomarkers: Present and FutureCagle et al

fragments.104 Of note, investigators have now developed

novel strategies to detect EML4-ALK gene rearrangements
on the MassARRAY Analyzer, making it possible to
concurrently evaluate EGFR gene mutations and ALK gene
rearrangements during a single run.105 A similar strategy
could be designed to also detect ROS1 or other gene
rearrangements. More recently, single-molecule sequencing platforms, such as the PacBio RS (Pacific Biosciences,
Menlo Park, California), have entered the research
environment as powerful tools for whole genome sequencing.106 Although these instruments generate highquality sequence data, each varies in key performance
characteristics such as throughput, read length, error rate,
hands-on time, run time, instrument cost, and reagent
cost.107,108 We recommend that potential users carefully
evaluate their options, considering the intended clinical
application and individual laboratory needs. To assist
pathologists in designing highly multiplexed gene mutation assays, reagent kits licensed for research use only are
now available. Also, an expert panel for next-generation
sequencing recently suggested guidelines for test validation, quality control, proficiency testing, and reference
materials,109 and the CAP has published a next-generation
sequencing checklist (www.cap.org, June 21, 2013).
Although still in its infancy as a diagnostic tool, whole
genome sequencing has the potential to truly personalize
patient care. Importantly, the long-sought $1000 genome
will soon be a reality, and advances in automation and
bioinformatics are making cancer genomics an increasingly
tractable tool in the molecular diagnostics laboratory.110 Two
investigations80,111 recently reported the whole genome
sequences of 178 lung squamous cell carcinomas and of
183 lung adenocarcinomas. These incredibly rich data sets
clearly reveal the genomic complexity of lung cancer. For
example, the frequent presence of EGFR, KRAS, and BRAF
mutations was confirmed in lung adenocarcinoma.111
Unexpectedly though, mutations were also identified in
several other genes including U2AF1, RBM10, and ARID1A.
In total, 25 genes were shown to have a statistically
significant number of mutations in lung adenocarcinoma.111
Each represents a possible target for new therapeutics. In
comparison, the squamous cell carcinoma investigation
identified only 11 genes with recurrent mutations.80
However, multiple signaling pathways were significantly
altered. These include gene pathways implicated in oxidative
stress response, apoptotic signaling, and squamous cell
differentiation.80 Of note, many of the alterations identified
in squamous cell carcinoma of the lung are shared with
those occurring in squamous cell carcinoma of the head and
neck, suggesting a possible common molecular biology.
Taken together, these genomic investigations show that
potential therapeutic targets can be found in nearly every
lung tumor. Furthermore, researchers are expanding our
genomic understanding of lung cancer to include epigenetic,
transcriptomic, proteomic, and metabolomic signatures.112
When clinical phenotypes based on genomic information
can be reliably predicted, then increasingly personalized
therapeutic strategies will become possible.
Inasmuch as a molecular diagnostics laboratory can generate
tremendous amounts of data by using next-generation
sequencing, information management and interpretation is a
significant challenge. Given the magnitude of the human
genome (approximately 3 billion base pairs encoding approximately 20 000 genes), some investigative pathologists are now
building bioinformatics and statistics support into their clinical
Arch Pathol Lab MedVol 137, September 2013

laboratories. However, before genomics can be performed as a

routine test, automated bioinformatics pipelines and analysis
algorithms must be developed. Currently, whole genome
sequence data cannot be interpreted within a time frame that is
optimal for patient care. Furthermore, the functional consequence of many gene polymorphisms remains unknown.
Molecular pathogenesis researchers must investigate all
common gene alterations, defining which are driver mutations
and which are bystander mutations. Coordinated efforts to
generate a comprehensive reference human genome sequence
and cancer genome database (http://cancergenome.nih.gov,
http://cancercommons.org, and http://icgc.org; each accessed
May 27, 2013) have begun filling these knowledge gaps.
ADVANCES IN BIOMARKER
IMMUNOHISTOCHEMISTRY
Traditional EGFR IHC detects total EGFR protein
expression, but does not differentiate between wild type
and mutations so it is not recommended as a basis for
selecting patients for EGFR TKI therapy.98 Investigations
have examined antibodies to proteins associated with the 2
primary mutations, which represent 90% of EGFR mutations
that are associated with response to first-generation EGFR
TKIs. These antibodies may be useful in screening for most
EGFR mutations but will not detect the less frequent EGFR
mutations.113115
As discussed previously, total EGFR protein expression by
IHC may be the predictive biomarker used if cetuximab is
approved for lung cancer therapy. High expression with
EGFR IHC (score of 200 or more) using the Dako pharmDx
kit correlated with increased overall survival for patients
with lung cancerspecifically, adenocarcinoma and squamous cell carcinomareceiving first-line platinum-based
chemotherapy plus cetuximab compared to chemotherapy
alone.4850 The EGFR expression scoring system has already
been validated for this particular setting.51,52
Immunohistochemistry does not demonstrate ALK expression in most tissues that lack an ALK fusion gene. When an
ALK fusion gene is present, IHC usually reveals increased
ALK expressions. Well-known examples include lymphoma
and inflammatory myofibroblastic tumor. Probably because of
the relatively low expression of ALK protein in lung cancers
with ALK translocations, false-negative IHC results have been
observed in lung cancers that were shown to have ALK fusion
genes by other methods.1,25,116 Several procedures have been
used to enhance the sensitivity of IHC for ALK lung cancers,
but ALK antibodies that are highly specific and sensitive to
ALK expression in lung cancers have been developed. While
ALK expression is known to correlate with ALK translocations with these antibodies, clinical trials validating ALK IHC
as a predictive biomarker for patient outcomes with crizotinib
therapy have not been performed.1,25,116120 The CAP/IASLC/
AMP guidelines note that screening for ALK translocations
with the new sensitive ALK antibodies may potentially be
used to identify lung cancers that should then have ALK
translocations confirmed with FISH.1
PAYMENT FOR PREDICTIVE BIOMARKER TESTS
AND THE FUTURE OF TARGETED THERAPY
A common and important, but as yet unanswered,
question is how pathologists and laboratories will be paid
for performing lung cancer biomarker tests. Many oncologists and patients are now requesting biomarker testing and,
in some hospitals, reflex testing has been implemented as a
Lung Cancer Biomarkers: Present and FutureCagle et al 1195

policy. The number of requests is expected to increase and

the addition of new biomarkers for which testing may be
done is also expected to increase. For patients with lung
cancer with a very bleak prognosis, for their families, and for
their oncologists, it is difficult to put any price on potential
additional months of life, particularly based on taking a pill
without the significant inconveniences and side effects of
chemotherapy, even if ultimately there is relapse. Currently,
pathologists and laboratories have the option of billing
Medicare or private payers for each individual test that is
ordered separately, or of billing for testing as part of a reflex
testing protocol at their institution. Some payers are
considering bundled payments for biomarker testing with
resulting treatment based on the average cost of care, for
which providers accept an increased risk for overutilization.121
Whether, under what circumstances, and to what extent
Medicare and private payers will pay for these tests,
however, is currently unknown. No standard billing strategy
exists for lung cancer biomarker test payment by Medicare
or private payers. Cost-effectiveness analysis, evaluating
both the clinical and economic effect of a test or treatment,
is currently used by the United Kingdoms National Health
Service and, with looming health care reform, may soon be
used in the United States to determine whether molecular
therapies and biomarkers are covered.122,123 If cost-effectiveness analyses become an accepted method in the United
States for payers to determine coverage and price of lung
cancer biomarker tests, evidence-based literature regarding
the value of these tests in patients with lung cancer will be
needed to influence payers payment determinations.
However, advances in testing and treatment discussed,
including less costly tests with improvements in turnaround
time (eg, IHC or next-generation technology) and better
outcomes from new therapies (new drugs and new targets)
and treatment strategies (combined or sequential targets,
adjuvant therapy in earlier stages), may significantly alter
the cost-benefit dynamic in the future.
CONCLUSION
The advent of predictive biomarker testing on tissue
samples for targeted therapy has forever altered the role of
the pathologist in the management of patients with lung
cancer. The role of the pathologist in biomarker testing,
including very direct participation with IHC, has potential to
grow. Patients with lung cancer and their families are now
more aware of who pathologists are and the critical role that
they have in their health care. New tests, new test
technologies, and overall advances in precision medicine
will likely improve the cost to benefit ratio for biomarker
testing which, along with the powerful desire from patients
and physicians to improve survival and quality of life,
suggest that predictive biomarker testing will expand as a
routine component of cancer care.
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