Journal of Pathology

J Pathol 2014; 232: 121–133 INVITED REVIEW

Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/path.4275

The evolving genomic classification of lung cancer

David S Shames1* and Ignacio I Wistuba2
1
Genentech Inc, South San Francisco, California, USA
2 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

*Correspondence to: David S Shames, Genentech Inc, Oncology Biomarker Development, 1 DNA Way, South San Francisco, CA 94080, USA.
e-mail: shames.david@gene.com

Abstract
EGFR gene mutations and ALK gene fusions are well-characterized molecular targets in NSCLC. Activating
alterations in a variety of potential oncogenic driver genes have also been identified in NSCLC, including ROS1,
RET , MET , HER2 , and BRAF . Together with EGFR and ALK , these mutations account for ∼20% of NSCLCs. The
identification of these oncogenic drivers has led to the design of rationally targeted therapies that have produced
superior clinical outcomes in tumours harbouring these mutations. Many patients, however, have de novo or
acquired resistance to these therapies. In addition, most NSCLCs are genetically complex tumours harbouring
multiple potential activating events. For these patients, disease subsets are likely to be defined by combination
strategies involving a number of targeted agents. These targets include FGFR1, PTEN, MET, MEK, PD-1/PD-L1, and
NaPi2b. In light of the myriad new biomarkers and targeted agents, multiplex testing strategies will be invaluable
in identifying the appropriate patients for each therapy and enabling targeted agents to be channelled to the
patients most likely to gain benefit. The challenge now is how best to interpret the results of these genomic tests,
in the context of other clinical data, to optimize treatment choices in NSCLC.
 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain
and Ireland.

Keywords: ALK; biomarkers; EGFR; genomic classification; molecular targets; monoclonal antibodies; multiplex testing; lung cancer;
NSCLC; tyrosine-kinase inhibitors

Received 17 September 2013; Revised 17 September 2013; Accepted 21 September 2013

Conflict of interest statement: David S Shames is an employee of Genentech Inc and owns stock in Roche Holdings. Roche/Genentech manufactures
and markets several of the drugs and diagnostic products mentioned in this review including erlotinib and the cobas EGFR mutation assay.
Ignacio I Wistuba has received a research grant from Genentech Inc, and honoraria from Roche/Genentech for work involving onartuzumab,
erlotinib, and anti-PDL-1.

Introduction In NSCLC, multiple genetic alterations have already

been identified as therapeutic targets, including muta-
Historically, lung cancers have been sub-divided by tions of the epidermal growth factor receptor (EGFR)
histology into small-cell and non-small-cell lung can- gene and rearrangements of the anaplastic lymphoma
cers (NSCLCs) [1], with NSCLC further classified kinase (ALK ) gene. Drugs designed specifically as
into squamous cell carcinoma (SCC), large-cell car- inhibitors of these molecular targets have significantly
cinoma, and adenocarcinoma. More than half of all extended the survival times for patients with NSCLC
lung cancers are adenocarcinomas [2]. While treatment whose tumours harbour these mutations [8–14].
advances have been made with the use of platinum- As novel molecular targets are discovered, and
based chemotherapy [3], lung cancer remains the most ultimately new therapies developed, we may edge ever
frequent cause of cancer-related mortality worldwide closer to a personalized treatment approach in NSCLC
[4] and has a 5-year overall survival (OS) rate of just and further extend survival for patients. Within this
16% for all stages [5]. article, we review recently identified molecular targets
Crucial to enhancing outcomes for patients with in NSCLC, new genetic techniques for classifying the
lung cancer is the ability to build a detailed profile disease, and the implications of these findings for
of the disease, to guide treatment decisions and to clinical practice and future clinical trial design.
enable the development of more effective therapeu-
tic strategies. The last decade has seen a shift to a
more molecular-based classification, in which informa- Novel molecular targets in NSCLC
tion about genetic alterations and protein expression
level is considered alongside histology in order to bet- Superior clinical outcomes have been achieved in
ter understand the pathogenesis of the disease [6,7]. NSCLC by treating molecularly selected groups of
 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
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122 DS Shames and II Wistuba

patients with rationally targeted therapies (ie drugs will not therefore form the focus of this review. In
directed against activated oncogenes, such as EGFR or addition, the RAS oncogenes (KRAS and NRAS ) have
ALK gene-fusion products) as opposed to the modest been excluded from this review, as we are not aware
benefits achieved in unselected patients [15]. However, of any molecules in clinical development that directly
de novo or acquired resistance often develops, driving inhibit RAS.
the search for novel targets and treatment mechanisms.
In addition, EGFR and ALK alterations account for
only a small minority of NSCLC cases, and both Oncogenic drivers
alterations occur predominantly in adenocarcinomas
from non-smokers [11,16]. At present, the community
does not have an answer for patients who have or For the purposes of this review, a target was considered
will get lung cancer as a result of exposure to tobacco an oncogenic driver if it is genetically activated in
carcinogens. NSCLC and if there is an approved inhibitor (clinically
validated target) or convincing proof-of-concept data
Modern treatment strategies focus on the patholog-
(high response rates in a targeted population or a
ical classification of NSCLC, which includes assess-
positive randomized phase II trial).
ment of protein expression by immunohistochemistry
(IHC) to assess cell differentiation markers such as
TTF1 and p63 (the splice variant p40), as well as the EGFR
detection of molecular predictive markers, including Mutations of the EGFR gene are a well-established
validated driver mutations in genes involved in cell example of an oncogenic driver in NSCLC. EGFR
growth and survival. A variety of novel driver muta- activating mutations are present in ∼10% of NSCLCs
tions or molecular targets have recently been identified in Caucasians and ∼40% in Asian patients, and are
in NSCLC (Figure 1 and Table 1). Here, we review primarily seen in adenocarcinomas [18]. In prospec-
some of these key targets (and interventions), including tive phase III trials, patients with previously untreated
known oncogenic drivers (EGFR, ALK , ROS1 , and EGFR mutation-positive NSCLC achieved signifi-
RET ), non-driver targets [MET, fibroblast growth fac- cantly longer progression-free survival (PFS) with
tor receptor 1 (FGFR1), PTEN, and phosphatidylinos- the reversible EGFR tyrosine-kinase inhibitors (TKIs)
itol 3-kinase (PI3K)], immunotherapies [programmed erlotinib and gefitinib than with platinum-doublet
death ligand 1 (PD-L1/PD-1)], and antibody–drug chemotherapy [8,12,14]. Erlotinib has been approved
conjugates (ADCs; NaPi2b). Mutations of a number by the FDA for the first-line treatment of patients with
of other important molecular targets identified in EGFR activating mutation-positive NSCLC detected
NSCLC, such as HER2, BRAF, and MEK1 (Table 1), by the approved cobas EGFR Mutation Test. Sev-
have been described in detail elsewhere [2,15,17] and eral other platforms (mostly sequencing assays) are

Understanding Disease

Figure 1. Evolving genomic classification of NSCLC. Li T et al: J Clin Oncol 2013; 31: 1039–1049. Reprinted with permission.  2013 by
American Society of Clinical Oncology. All rights reserved [17].

 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd J Pathol 2014; 232: 121–133
on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org.uk www.thejournalofpathology.com
 10969896, 2014, 2, Downloaded from https://pathsocjournals.onlinelibrary.wiley.com/doi/10.1002/path.4275 by KIIT University, Wiley Online Library on [24/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Genomic classification of lung cancer 123

Table 1. Current molecular targets in adenocarcinoma The first-in-class ALK inhibitor crizotinib was
Target Prevalence (%) Therapeutic agents approved by the FDA for the treatment of ALK -positive
EGFR Asians ∼40 Caucasians ∼10 Erlotinib, gefitinib, afatinib
advanced NSCLC, with the concurrent approval of a
ALK <5 Crizotinib companion fluorescence in situ hybridization (FISH)
HER2 <3 Afatinib, neratinib, diagnostic test, based on impressive results in phase
dacomitinib I/II trials. In the subsequent phase III trial, second-
PIK3CA <5 GDC-0941, XL-147, BKM120 line crizotinib demonstrated superior PFS and response
BRAF <5 Vemurafenib, GSK2118436 rates to chemotherapy alone in patients with locally
MEK ∼1 AZD6244 advanced or metastatic ALK -positive NSCLC [13]. A
ROS1 ∼2 Crizotinib first-line phase III trial of crizotinib in newly diagnosed
RET ∼2 Sunitinib, sorafenib, ALK -positive NSCLC is currently recruiting patients
vandetanib, cabozantinib
(NCT01154140). Results of a recent study confirm that
MET 1–11 Onartuzumab, rilotumumab,
cabozantinib, tivantinib, ALK rearrangements in lung adenocarcinoma can also
crizotinib be effectively detected using IHC for ALK expression
FGFR1 ∼3 AZD4547, S49076, ponatinib, in malignant cells [32].
brivanib
PTEN < 10 Vandetanib
PD-1/PD-L1 ∼30 Nivolumab, MPDL3280A
ROS1
NaPi2b ∼70 DNIB0600A (early ROS1 is a tyrosine-kinase receptor of the insulin
development) receptor family. ROS1 gene rearrangements are
ALK, anaplastic lymphoma kinase; EGFR, epidermal growth factor receptor; known oncogenic drivers in NSCLC, and several
FGFR1, fibroblast growth factor receptor 1; PD-L1, interaction of programmed fusion partners have been identified, including CD74,
death ligand 1; PIK3CA, phosphatidylinositol 3-kinase, catalytic subunit alpha.
SLC34A2/NaPi2b, and FIG [33,34]. ROS1 fusions are
present in ∼2% of NSCLC cases and are often seen
used to study EGFR mutations in DNA extracted from in young never-smokers with adenocarcinoma, a pop-
tumour tissue specimens. Gefitinib is also approved ulation similar to those with ALK -rearranged NSCLC
as monotherapy for EGFR mutation-positive NSCLC [33]. ROS1 rearrangements rarely present simultane-
following failure of platinum- and docetaxel-based ously with EGFR, ALK or KRAS alterations [35].
chemotherapy. Crizotinib has shown inhibitory growth effects on
The second-generation irreversible EGFR TKI ROS1 -positive cell lines, and a near-complete response
afatinib recently gained FDA approval as first-line was reported in a patient with advanced ROS1 -positive
therapy for EGFR mutation-positive NSCLC in con- NSCLC treated with crizotinib in a phase I clinical trial
junction with Qiagen’s therascreen RGQ polymerase [33]. In an expansion cohort of the trial, 14 patients
chain reaction (PCR) diagnostic test. Another second- received crizotinib for ROS1 -rearranged NSCLC (as
generation irreversible EGFR TKI, dacomitinib, tested by FISH) and nine (64%) had a confirmed
demonstrated preclinical efficacy in NSCLC tumours response [36]. A further case of a complete metabolic
harbouring the T790M gatekeeper mutation [19,20], response to crizotinib was reported in a patient with
which is present in ∼50% of NSCLCs that have advanced ROS1 -positive NSCLC [37]. A ROS1 mon-
acquired resistance to erlotinib or gefitinib [21,22]. oclonal antibody (D4D6) has recently been developed
In a randomized phase II study, dacomitinib demon- and validated for use in IHC assays [34].
strated significantly improved PFS versus erlotinib in
patients with advanced NSCLC [23]. A phase III study RET
of dacomitinib versus erlotinib as second-/third-line
therapy for advanced NSCLC is currently underway The tyrosine-kinase receptor RET is involved in cell
(NCT01360554) [24]. proliferation, migration, and differentiation. A novel
fusion oncogene between the RET gene and KIF5B
was recently described in a young never-smoker with
ALK adenocarcinoma and no family history of lung cancer
Rearrangements of the ALK gene are another recent [38,39]. Fusions between the RET gene and CCDC6
example of oncogenic drivers in NSCLC. ALK is a have since been identified [40]. RET fusions are known
transmembrane tyrosine-kinase receptor expressed in to occur in ∼2% of lung adenocarcinomas [38], are
the small intestine, testes, and brain, but not normally usually independent of other oncogenic drivers [35],
in the lung. In NSCLC, ALK signalling is activated and can be targeted with TKIs such as sunitinib,
by the creation of oncogenic fusions of the ALK gene sorafenib, vandetanib, and cabozantinib [15,41]. Pre-
with an upstream partner, EML4 [25], although other liminary data have been published for the first three
fusion partners exist [26]. EML4–ALK rearrangements patients with RET fusion-positive NSCLC enrolled
occur in 2–7% of NSCLC patients [11,27], usually in in a phase II trial of cabozantinib; confirmed partial
young never-smokers with adenocarcinoma [28–31]. responses occurred in two patients (one with a novel
ALK -rearranged tumours are resistant to the EGFR TRIM33–RET fusion), with prolonged disease stabi-
TKIs gefitinib and erlotinib [28]. lization (31 weeks) in the third patient [42].
 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd J Pathol 2014; 232: 121–133
on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org.uk www.thejournalofpathology.com
 10969896, 2014, 2, Downloaded from https://pathsocjournals.onlinelibrary.wiley.com/doi/10.1002/path.4275 by KIIT University, Wiley Online Library on [24/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
124 DS Shames and II Wistuba

Other targets a selective inhibitor of FGFR1/2/3 [58,59], and

S49076, an ATP-competitive TKI of MET, AXL,
and FGFR1/2/3, which demonstrated marked anti-
MET
tumour activity in MET- and FGFR-dependent tumour
Binding of the hepatocyte growth factor (HGF) to the xenografts [56]. In addition, the novel FGFR inhibitor
transmembrane tyrosine-kinase receptor MET activates ponatinib suppressed the growth of NSCLC cells
multiple signalling pathways involved in cell prolifer- overexpressing FGFR1, and significantly inhibited
ation, survival, motility, and invasion [43]. Dysregula- the growth of primary lung cancer cultures in vitro,
tion of the MET/HGF pathway can occur via several suggesting that ponatinib may also be effective in
mechanisms and is observed in many human malig- patients whose tumours overexpress FGFR1 [60].
nancies, including NSCLC [43]. Mutations in MET
are rare, but high MET gene copy number has been
detected in 1–11% of NSCLC cases and is often asso- PTEN
ciated with high MET protein expression and poor Many cancers are associated with deletions or muta-
prognosis [44–46]. MET amplifications have also been tions of the PTEN tumour suppressor gene, which
linked with secondary resistance to EGFR TKIs in plays a significant role in cell cycle progression, apop-
patients with EGFR mutation-positive NSCLC [47,48]; tosis, growth, proliferation, and migration via negative
MET amplifications can be found in up to 20% of these control of the PI3K/Akt pathway [61]. PTEN muta-
patients [45]. tions [62] and loss of PTEN protein expression are
A number of therapeutic agents targeting the relatively common in SCC of the lung [63]. How-
MET/HGF pathway are in clinical development, ever, in many cases, the functional consequences of
including small molecule MET inhibitors (eg cabozan- PTEN mutations remain to be elucidated. PTEN loss
tinib), specific MET TKIs (eg crizotinib), antagonistic has also been linked with acquired resistance to EGFR
antibodies against MET (eg onartuzumab), and neu- TKIs in EGFR mutation-positive NSCLC [18]. In a
tralizing antibodies against HGF (eg rilotumumab). meta-analysis of mutation incidence in NSCLC, PTEN
Although tivantinib was initially believed to be a mutations were influenced by ethnicity, with a higher
MET inhibitor, several recent reports suggest that, at frequency amongst Asian patients with SCC (9.8%)
least preclinically, tivantinib does not appear to inhibit versus adenocarcinoma (1.6%), and in western patients
MET signalling [49,50]. with adenocarcinoma (6.0%) versus SCC (0%) [64].
Onartuzumab is a humanized monovalent (one- The TKI vandetanib has shown efficacy against EGFR
armed) monoclonal antibody that binds to the extra- mutation-positive lung cancer cell lines showing loss
cellular domain of MET to prevent HGF binding and of PTEN, suggesting that it may also be effective in
activation [51,52]. In a randomized phase II trial, onar- patients with EGFR mutation-positive NSCLC whose
tuzumab plus erlotinib improved PFS and OS versus tumours lack PTEN expression [65].
placebo plus erlotinib in patients with tumours pre-
defined as MET-positive by IHC (≥ 50% of tumour
cells expressing moderate-to-strong staining intensity) PI3K
[53]. Clinical outcomes were worse in MET-negative PI3Ks are lipid kinases involved in the regulation
patients treated with onartuzumab plus erlotinib, exem- of cell growth, proliferation, and survival. Mutations
plifying the need for parallel diagnostic testing in in the PIK3CA gene that encodes the catalytic sub-
drug development [54]. A randomized phase III study unit of PI3Kα have been identified in several can-
is investigating the combination of onartuzumab and cers [66]. Furthermore, aberrant signalling through the
erlotinib in patients with MET-positive advanced or PI3K/Akt/mTOR pathway has been observed in a num-
metastatic NSCLC (NCT01456325). The MET IHC ber of human cancers, including NSCLC [67]. PIK3CA
assay is being developed as a companion diagnostic mutations occur in less than 5% of NSCLCs [15] and
for onartuzumab within this study, based on the 50% often co-exist with other oncogenic mutations, particu-
cut-off used in the phase II trial [54]. larly EGFR, KRAS or ALK [68]. PTEN loss is thought
to be a marker for PI3K dependency in some tumours
FGFR1 [69].
FGFR1 is a membrane-bound tyrosine-kinase receptor Several PI3K inhibitors are in clinical development
involved in the regulation of cell proliferation and (eg GDC-0941, BKM120, BEZ235, XL-147, XL-765,
angiogenesis [15]. FGFR1 amplification occurs more perifosine), but the response rate to single agents
frequently in SCC (21%) than in adenocarcinoma (3%) has been low [2,70]. Ongoing phase II trials in lung
[55]. Activation of FGFR2 and FGFR3 has also cancer are examining combinations of PI3K inhibitors
been reported in NSCLC cell lines treated with EGFR and chemotherapy (NCT01297491) or targeted agents
inhibitors [56]. Recently, high FGFR1 gene copy (NCT01493843, NCT01487265). In human NSCLC
number was reported to be an independent favourable lines, the novel PI3K inhibitor imidazopyridine
prognostic factor in NSCLC [57]. demonstrated anti-proliferative effects, including the
Several small molecule FGFR TKIs are currently induction of apoptosis, in a dose-dependent manner
under clinical investigation, including AZD4547, [71].
 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd J Pathol 2014; 232: 121–133
on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org.uk www.thejournalofpathology.com
 10969896, 2014, 2, Downloaded from https://pathsocjournals.onlinelibrary.wiley.com/doi/10.1002/path.4275 by KIIT University, Wiley Online Library on [24/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Genomic classification of lung cancer 125

Immunotherapies stably linked to an active cytotoxic drug. Due to their

targeted nature, ADCs offer the potential for fewer side
effects than standard chemotherapies.
PD-L1/PD-1
Interaction of PD-L1 with the PD-1 and B7.1 receptor
NaPi2b
on activated T cells plays a key role in tumour evasion
of the host immune system [72–75]. Whether PD- The human sodium-dependent phosphate transporter
L1 is overexpressed in solid tumours and associated NaPi2b is encoded by the SLC34A2 gene and belongs
with increased tumour aggressiveness remains unclear. to the type II family of sodium-dependent phosphate
However, high levels of PD-L1 were recently reported transporters [81]. In normal tissue, NaPi2b is expressed
in patients with sarcomatoid lung cancer, a rare, high- at the intestinal brush border membrane and plays
grade, poorly differentiated form of NSCLC [76]. a role in the synthesis of surfactant in lung alveoli.
Furthermore, in a 5-year follow-up study in patients Immunohistochemical analysis of NaPi2b using the
with NSCLC, PD-L1 was a significant independent monoclonal antibody MX35 revealed heterogeneous
poor prognostic factor, with PD-L1-positive patients NaPi2b expression in lung cancer tissues, ranging
having a shorter 5-year OS than PD-L1-negative from no immunoreactivity to 100% positively stained
patients [77]. cells [82]. An ADC targeting NaPi2b (DNIB0600A)
Nivolumab (BMS-936558), an anti-PD-1 mono- is currently in clinical development, with preliminary
clonal antibody, has shown anti-tumour activity in a evidence of anti-tumour activity in NSCLC; of the 16
phase I clinical trial in patients with advanced solid patients with NSCLC enrolled in the phase I trial, 70%
tumours, including NSCLC [78,79]. Of 129 patients expressed high levels of NaPi2b by IHC [83].
with NSCLC treated with nivolumab, 22 (17.1%)
achieved an objective response (13 with non-squamous
cell histology and nine with squamous cell histology) The impact of molecular testing on lung cancer
and 13 patients (10.1%) had disease stabilization for at management
least 24 weeks [78]. The 24-week PFS rate was 33%
[79]. Patients are currently being enrolled in a phase The identification and characterization of molecular
II study of nivolumab in SCC (NCT01721759) and a targets are having a growing impact on the manage-
phase III study in non-SCC (NCT01673867). ment of patients with lung cancer. Several clinical
MPDL3280A, an engineered anti-PD-L1 monoclonal practice guidelines, including those published by the
antibody, has also demonstrated anti-tumour activity National Comprehensive Cancer Network (NCCN), the
in a phase I NSCLC clinical trial. MPDL3280A treat- American Society of Clinical Oncology (ASCO), and
ment was associated with a 22% objective response the International Association for the Study of Lung
rate in 41 heavily pretreated NSCLC patients, with Cancer (IASLC)/College of American Pathologists
12% of patients having disease stabilization for at (CAP)/Association of Molecular Pathology (AMP),
least 24 weeks [80]. A correlation between PD-L1 now recommend that all patients with NSCLC contain-
status and efficacy was reported: 4/5 patients (80%) ing an adenocarcinoma component undergo biomarker
with PD-L1-positive tumours achieved an objective testing for EGFR mutations and ALK rearrangements
response and 0/4 had disease progression, while 4/28 [6,84,85]. The French National Cancer Institute, in col-
patients (14%) with PD-L1-negative tumours had an laboration with the French Ministry of Health, recently
objective response and 15/26 experienced disease pro- introduced a programme of free molecular diagnos-
gression [80]. The 24-week PFS rate was 46% [80]. tic testing for all patients with solid tumours, which
Clinical studies of MPDL3280A in PD-L1-selected includes testing for EGFR mutations and EML4–ALK
or unselected locally advanced or metastatic NSCLC rearrangements in lung cancer [86]. Not only has this
are currently open for recruitment (NCT01846416, mandate granted equal access to molecular tests and
NCT01903993, NCT01375842). appropriate targeted therapies for all patients across
MK-3475, a humanized monoclonal antibody against France, but it has also reduced the unnecessary treat-
PD-1, has not presented any clinical data in the ment of unselected patients.
setting of NSCLC at the time of this publication. Across the USA, the Lung Cancer Mutation Con-
Clinical studies of MK-3475 are currently recruiting sortium (LCMC), a collaborative approach between
patients with NSCLC (NCT01295827, NCT01840579, 16 academic centres, is actively promoting molec-
NCT01905657). ular mutation testing in lung cancer in order to
match patients to optimal treatment strategies [87].
The LCMC is conducting an observational study with
ADCs the aim of genotyping ten driver mutations in tumour
specimens from 1000 patients with advanced lung ade-
ADCs are a new class of targeted therapy being nocarcinoma (NCT01014286); results of EGFR test-
investigated for use against a number of cancers. ing are passed to treating physicians, while patients
They consist of a monoclonal antibody, or antibody with other driver mutations are offered enrolment into
fragment, against a known molecular target that is LCMC-linked clinical trials of various targeted agents
 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd J Pathol 2014; 232: 121–133
on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org.uk www.thejournalofpathology.com
 10969896, 2014, 2, Downloaded from https://pathsocjournals.onlinelibrary.wiley.com/doi/10.1002/path.4275 by KIIT University, Wiley Online Library on [24/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
126 DS Shames and II Wistuba

[88]. Preliminary reports from the first LCMC study availability, as well as lack of validation [92]. Using
confirm the presence of several key target genes, samples from the JBR.10 clinical trial, Zhu et al devel-
including KRAS mutations (24%), EGFR mutations oped a 15-gene expression signature that demonstrated
(20%), and ALK rearrangements (8%) [88–90]. the potential to select patients with stage IB/II NSCLC
most likely to benefit from adjuvant chemotherapy
with cisplatin/vinorelbine [91]. Kratz et al developed
New techniques in the genomic classification a prognostic gene signature that was able to iden-
of lung cancer tify patients with early-stage, non-squamous NSCLC at
high risk for mortality after surgical resection [92]. The
14-gene mRNA expression assay was based on quan-
The landmark studies that led to the approval of the first titative PCR using formalin-fixed, paraffin-embedded
targeted agents in NSCLC used gene-based molecular (FFPE) tissue samples and improved prognostic accu-
tests that were focused on single biomarkers. How- racy beyond NCCN criteria for stage I high-risk
ever, with the advent of many more potential molecular tumours (p < 0.0001). Blinded and independent vali-
targets, and the challenges associated with obtaining dation of the assay was confirmed in a cohort of 433
tissue from patients with late-stage NSCLC, there is a patients from the USA and in a larger sample of 1006
growing need to develop and utilize molecular tech- patients from China [92].
nologies that can determine the expression or mutation More recently, Tang et al developed an 18-gene
status of several genes simultaneously, so-called multi- prognostic signature in resectable NSCLC, which was
plex testing, in order to obtain the maximum diagnostic then integrated with genome-wide functional data
information from the limited tumour tissue available and genetic aberration data to derive a 12-gene pre-
(Figure 2). dictive signature for survival benefits with adjuvant
chemotherapy [93]. The prognostic signature predicted
the prognosis of patients with adenocarcinoma in all
Predictive and prognostic gene signatures validation datasets across four microarray platforms,
including Illumina (Illumina Inc, San Diego, CA,
A number of research groups have developed pre- USA), Affymetrix (Affymetrix, Santa Clara, CA,
dictive and prognostic gene signatures in surgically USA), and Agilent (Agilent Technologies Inc, Santa
resected lung cancer [91–93]. However, the use of Clara, CA, USA). The predictive signature was suc-
these signatures in clinical practice is often hampered cessfully validated in two independent datasets in 266
by issues such as reproducibility, cost, and limited patients. Prospective clinical trials are needed to further

Understanding Disease

Figure 2. Lung diagnostic testing today and in the future.

 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd J Pathol 2014; 232: 121–133
on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org.uk www.thejournalofpathology.com
 10969896, 2014, 2, Downloaded from https://pathsocjournals.onlinelibrary.wiley.com/doi/10.1002/path.4275 by KIIT University, Wiley Online Library on [24/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Genomic classification of lung cancer 127

validate the use of prognostic and predictive signatures a specific investment in computational analysis and
in lung cancer [93,94]. bioinformatic support to produce and interpret the data,
and the assays are expensive and often cumbersome
[99]. Over the next few years, the cost and complexity
of NGS-based testing will continue to decrease rapidly
Multiplex PCR (mutation detection and gene
and testing is likely to become even more widespread.
expression) In the USA, NGS-based clinical assays are already
being offered as laboratory-developed tests (LDTs)
Multiplex PCR involves the simultaneous amplifica- in several Clinical Laboratory Improvement Amend-
tion of two or more cDNA/DNA targets in a single ments (CLIA)-approved laboratories (eg Foundation
reaction vessel with uniquely labelled probes for each Medicine and laboratories in academic institutions).
target [95]. A number of multiplexed PCR-based assays However, there are currently no NGS-based FDA-
are available, including SNaPshot (Applied Biosys- approved companion diagnostic tests. Given the
tems, Foster City, CA, USA), which detects hotspot high-resolution data that NGS can provide, traditional
mutation sites in key cancer genes using fluorescently- prospective clinical validation can be challenging, par-
labelled primer extension products [96], and Sequenom ticularly for rare genomic alterations. Overcoming this
MassARRAY (Sequenom Inc, San Diego, CA, USA), challenge, and/or refining the definition of prospective
which analyses primer extension products using mass clinical validation, will require the cooperation of
spectrometry [97]. A high-throughput microfluidics clinical scientists, regulatory authorities, and payers.
method (Fluidigm, South San Francisco, CA, USA) Collaboration between institutions and patient referral
has been developed for mutation detection [mutation centres are necessary to identify these rare patients, as
multi-analyte panel (MUT-MAP)] based on quantita- are innovative clinical trial designs, as described later.
tive PCR, which includes ∼120 hotspot mutations and NGS-based companion diagnostics will also require the
works effectively with less than 100 ng of FFPE tissue flexibility to be updated as additional clinical informa-
[98]. tion emerges. As the cost of NGS-based tests continues
Similar assays and formats are widely used in the to drop to the point where whole genome sequenc-
cancer research community and are starting to be ing becomes routine clinical practice, the test update
applied in clinical trials. For example, the LCMC is required may simply be a software update that changes
predominantly utilizing the SNaPshot and Sequenom the clinical report to include, for example, detection of
MassARRAY platforms, together with the FDA- a newly validated rare EGFR mutation or ROS1 fusion
approved FISH test for ALK gene rearrangement in partner.
their ongoing genotyping trial. Multiplex PCR has the Clearly, other important platforms for identifying
advantage of needing only a small sample of tumour molecular targets, such as FISH (as exemplified with
compared with conventional tests, but it is restricted crizotinib and ALK rearrangements) and IHC (as
to codons previously determined as mutation hotspots, exemplified with onartuzumab and MET expression),
and is unable to detect chromosomal rearrangements or should be considered alongside these newer techniques.
determine gene copy number [70]. The key for clinicians and pathologists, therefore, will
be to determine the optimal method for molecularly
classifying lung cancers moving forward.
Next-generation sequencing (NGS)

High-throughput NGS technology has been commer- Challenges of widespread genetic testing
cially available since 2004 and offers the ability to
analyse DNA, mRNA, transcription factor regions, The merits of molecular testing in lung cancer are
and DNA methylation patterns throughout the entire clear; however, there are a number of challenges to
genome [99]. Several NGS platforms are available, overcome in the widespread use of these tests. Firstly,
including Illumina HiSeq 2500 (Illumina Inc, San the community will need to come to some consen-
Diego, CA, USA), SOLID4 System (Applied Biosys- sus as to what an actionable test result might be. A
tems, Foster City, CA, USA), and Ion Torrent valid test result can mean very different things depend-
(Applied Biosystems, Foster City, CA, USA) [17,99]. ing on the technology, bioinformatics pipeline, and
NGS has been applied to clinical settings in almost all what the investigator or treating physician perceives
tumour types and is being used as a research tool, as to be clinically relevant, and there are obvious poten-
well as to screen patients for clinical trial enrolment. tial dangers in this. Additionally, the quantity, quality,
NGS can detect chromosomal rearrangements and gene and type of tumour tissue available for testing vary
copy number alterations at a very high resolution [70]. extensively between different centres and countries.
Indeed, identification of the KIF5B–RET fusion was One of the greatest challenges is obtaining adequate
made possible through the application of NGS [38]. tumour samples for all genomic tests, while avoid-
NGS has huge potential over traditional sequencing ing contamination with normal and necrotic cells, in
techniques; however, currently each platform requires a minimally invasive manner [70]. Substrates derived
 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd J Pathol 2014; 232: 121–133
on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org.uk www.thejournalofpathology.com
 10969896, 2014, 2, Downloaded from https://pathsocjournals.onlinelibrary.wiley.com/doi/10.1002/path.4275 by KIIT University, Wiley Online Library on [24/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
128 DS Shames and II Wistuba

from peripheral tissues, such as circulating tumour cells failure, genomic testing of NSCLCs must be included
and circulating tumour DNA, are less invasive alterna- alongside histological testing, such that candidate
tives to surgical or biopsy specimens and have yielded patients can be identified and treatment choices opti-
comparable results in molecular tests [100], although mized. For example, non-mucinous bronchoalveolar
further research in this field is required [70]. Intratu- carcinoma (BAC) is now classified as lepidic predom-
mour heterogeneity can result in a mixed response to inant adenocarcinoma (100% TTF1, ∼45% EGFR
a molecularly targeted agent in different tumour sites, mutation-positive, 5% BRAF mutation-positive), while
and throughout the course of successive treatment lines, mucinous BAC is classified as mucinous invasive
due to alterations in the genetic make-up of the tumour carcinoma (15% TTF1, ∼80–100% KRAS mutation-
as the disease progresses or in response to therapy [17]. positive, 0% EGFR mutation-positive) [7,104]. The
Such heterogeneity may represent a major treatment potential impact of targeting different molecular targets
challenge if the therapy choice is based on genomic or histologies within the context of historical ‘all-
analysis of a single tumour biopsy sample at a spe- comer’ studies is illustrated in Figure 4; not only do
cific time point [101]. Thus, serial biopsy or cytology the specific targeted subsets need to be considered in
sampling during the course of the disease may pro- terms of their particular prognostic behaviour (EGFR
vide a more accurate genomic analysis of the tumour mutation-positive patients do much better on both
(Figure 3). chemotherapy and EGFR TKIs than EGFR wild-type
Another challenge with genomic testing will be patients), but the impact of removing these patients
for clinicians to decide which of the genomic data from the remaining pool should also be considered if
is of relevance to an individual patient’s treatment spurious interpretations of trial data are to be avoided.
choice [102]. Importantly, in cases where a patient Furthermore, it is likely that most patients with
has more than one activating alteration, the physician NSCLC will test positive for at least two potential
will need to decide which lesion to treat first. An molecular targets when IHC and genetic tests are
additional consideration will be the time it takes to considered together. Thus, there is a real need to under-
perform the tests – should physicians start treating a stand how these molecular targets fit into current and
patient with the current standard of care for unselected future treatment algorithms, especially for patients with
patients while testing is taking place, and then switch multiple biomarkers.
the patient once a positive result is identified? What Earlier clinical trials were not designed adequately
decisions should be made in the event that a patient for the testing of multiple molecular targets, but rather
has a mutation in a gene for which there is no restricted enrolment to patients with a known single
currently approved therapy in NSCLC, but where a mutation. Evaluating biomarker combinations or over-
targeted treatment is approved in other indications? lap (ie between mutations, gene expression, and IHC)
At a minimum, a multidisciplinary team approach could inform rational drug combinations or sequencing
will be required to accurately interpret the results of trials in the future. Ongoing programmes, such as the
the tests, and central to this will be engaging patients LCMC study and the MD Anderson BATTLE trials,
to help them realize the importance of molecular are already utilizing novel designs to evaluate mul-
testing in the first instance [17]. Several institutions tiple targeted therapies in NSCLC [70,106]. Careful
have implemented multidisciplinary molecular tumour ethical consideration must also be given to the design
boards to discuss the management of patients whose of control arms in clinical trials of biomarker-selected
lung tumours harbour rare genetic abnormalities with patients. From the patients’ and treating physicians’
no validated targeted therapy available. perspective, strong arguments can be made to permit
The costs of widespread genetic testing will also crossover in biomarker enabled trials, such that patients
come into question, in terms of the cost–benefit ratio of whose tumours have the relevant biomarker can gain
the newer platforms versus the more conventional tests, benefit from the targeted agent at some point; how-
and the challenges faced by diagnostic laboratories in ever, from regulatory and payer perspectives, similarly
keeping up with the costs of buying new equipment and strong arguments are made to prevent crossover, to
validating new assays as the sequencing platforms con- demonstrate differences in OS. In addition, recently
tinually evolve [103]. Finally, as the current regulatory obtained tumour samples should be used, rather than
environment does not allow for the rapid adoption of archival tissue from surgical resection, as the tumour
new technology, we may face unavoidable delays in the profile can change considerably over time, and trial
implementation of genomic testing and, ultimately, the enrolment should be based on the current disease pro-
optimization of treatment for patients with lung cancer. file, rather than that at the initial diagnosis.

Implications for clinical trial design Conclusions

Lung cancer is a competitive landscape with many A deeper understanding of the molecular classifica-
new drugs in development and several failed phase III tion of lung cancer may ultimately lead to personal-
clinical trials. To reduce the risk of further clinical trial ized treatment strategies, which will improve care for
 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd J Pathol 2014; 232: 121–133
on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org.uk www.thejournalofpathology.com
 10969896, 2014, 2, Downloaded from https://pathsocjournals.onlinelibrary.wiley.com/doi/10.1002/path.4275 by KIIT University, Wiley Online Library on [24/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Genomic classification of lung cancer 129

Understanding Disease

Figure 3. The future of oncology testing in NSCLC. BC, breast cancer; CTCs, circulating tumour cells; qRT, real-time reverse transcription;
WGS, whole genome sequencing.

 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd J Pathol 2014; 232: 121–133
on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org.uk www.thejournalofpathology.com
 10969896, 2014, 2, Downloaded from https://pathsocjournals.onlinelibrary.wiley.com/doi/10.1002/path.4275 by KIIT University, Wiley Online Library on [24/03/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
130 DS Shames and II Wistuba

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on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org.uk www.thejournalofpathology.com