Archives of Medical Research - (2020) -

Molecular Pathology of Primary Non-small Cell Lung Cancer

David Ilan Sustera and Mari Mino-Kenudsonb
a
Department of Pathology, Rutgers University, New Jersey Medical School, Newark, NJ, USA
b
Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
Received for publication July 31, 2020; accepted August 13, 2020 (ARCMED_2020_1382).

Lung carcinoma is one of the most common human cancers and is estimated to have an
incidence of approximately 2 million new cases per year worldwide with a 20% mortality
rate. Lung cancer represents one of the leading causes of cancer related death in the
world. Of all cancer types to affect the pulmonary system, non-small cell lung carcinoma
comprises approximately 80e85% of all tumors. In the past few decades cytogenetic and
advanced molecular techniques have helped define the genomic landscape of lung cancer,
and in the process, revolutionized the clinical management and treatment of patients with
advanced non-small cell lung cancer. The discovery of specific, recurrent genetic abnor-
malities has led to the development of targeted therapies that have extended the life ex-
pectancy of patients who develop carcinoma of the lungs. Patients are now routinely
treated with targeted therapies based on identifiable molecular alterations or other predic-
tive biomarkers which has led to a revolution in the field of pulmonary pathology and
oncology. Numerous different testing modalities, with various strengths and limitations
now exist which complicate diagnostic algorithms, however recently emerging consensus
guidelines and recommendations have begun to standardize the way to approach diag-
nostic testing of lung carcinoma. Herein we provide an overview of the molecular genetic
landscape of non-small cell lung carcinoma, with attention to those clinically relevant al-
terations which drive management, as well as review current recommendations for mo-
lecular testing. Ó 2020 IMSS. Published by Elsevier Inc.
Key Words: Molecular pathology, Thoracic, Pulmonary, Biomarkers, NSCLC.

Introduction demonstrated that most of these tumors harbor smoking-

related genetic signatures (6,7). Non-smokers are also at
Lung carcinoma is one of the most common human cancers
risk for developing lung cancer with particularly high inci-
and is estimated to have an incidence of approximately 2
dences in the East Asian female population (8e10). In the
million new cases per year worldwide with a 20% mortality
past two decades the role of adjuvant chemotherapy and ra-
rate (1). Non-small cell lung carcinoma (NSCLC) is the
diation therapy has become relatively standardized for pa-
most common type of lung cancer and represents approxi-
tients with advanced NSCLC (11e13) and more recently
mately 80e85% of all lung cancers (2,3). The category of
the clinical management and treatment of patients has
NSCLC itself is comprised predominantly of adenocarci-
begun to evolve to a more targeted approach based on the
noma (ADC), squamous cell carcinoma (SCC), and large
specific features of an individual’s lung cancer including
cell carcinoma (LCC), although some other less common
histology and molecular genetic markers (14e16).
subtypes exist (2,4,5). Smoking is the most common risk
Over the past decades the discovery that a significant
factor for the development of lung cancer and it has been
percentage of lung carcinomas harbor specific genetic alter-
ations that are amenable to targeted therapeutic agents has
Conflicts of Interest: MMK has served as a consultant/advisor for H3 increased the importance of molecular diagnostic testing in
Biomedicine and AstraZeneca and received a (institutional) research grant routine clinical practice (17e19). This has been made
from Novartis; all not related to this work. DIS has no conflicts of interest. possible by advances in molecular diagnostic techniques
Address reprint requests to: Mari Mino-Kenudson MD, Department of
Pathology, Massachusetts General Hospital, 55 Fruit Street, Boston, MA,
including the advent of massively parallel sequencing-
02114, USA; Phone: 617-726-2967; FAX: 617-726-7474; E-mail: based technologies such as whole exome sequencing
mminokenudson@partners.org (WES) and next generation sequencing (NGS) that have

0188-4409/$ - see front matter. Copyright Ó 2020 IMSS. Published by Elsevier Inc.
https://doi.org/10.1016/j.arcmed.2020.08.004
2 Suster and Mino-Kenudson/ Archives of Medical Research
provided invaluable information on the molecular land-
scape of lung carcinoma (20e22). With the advent of these
technologies and newly found information, pathologists,
oncologists, and surgeons must adjust their clinical practice
to incorporate the plethora of new diagnostic information
available. The purpose of this review article is to provide
a synopsis of the general molecular genetic features of
NSCLC, with a focus on oncogenic driver alterations that
are amenable to newly developed targeted therapies. The
latest consensus guidelines from the College of American
Pathologists (CAP), the International Association for the
Study of Lung Cancer (IASLC) and Association for Molec-
ular Pathology (AMP), and recommendations from the Na-
tional Comprehensive Cancer Network (NCCN) are
reviewed as well.
General Molecular Genetic Features of NSCLC
NSCLC is characterized by a complex genomic landscape
with numerous underlying genetic and epigenetic mecha-
nisms involved in carcinogenesis. Tumors within the
NSCLC category have been identified to have a wide vari-
ety of genomic alterations including a high rate of somatic
mutations as well as numerous other aberrations such as
chromosomal rearrangements leading to various oncogenic
protein fusions and copy number alterations including gains
and losses of critical cell cycle regulatory genes and chro-
matin remodeling genes (23e25). Epigenetic regulation of
some genes through silencing via DNA methylation, his-
tone modification and nucleosome remodeling have also
been associated with lung cancer (26,27). Although the
two most common subtypes of NSCLC; ADC and SCC,
display overlap between alterations in many of the same
genes and cellular pathways, significant differences exist
enough that these tumors can generally be differentiated
by their spectrum of molecular alterations (Figure 1)
(24,25,28). It is worth noting that the molecular alterations
involved in NSCLC are known to vary depending on the
population studied and can change depending on ethnicity,
race, and sex; thus incidences of specific alterations should
be assessed in the context of a specific patient population or
data set, although general ranges or averages are helpful in
understanding the relative incidence or general landscape of
different alterations (29e31). In addition, while these tu-
mors overall are characterized by a complex molecular
background, the clinical and therapeutic managements are
driven almost entirely by a smaller group of clinically
actionable molecular alterations, the majority of which
occur more commonly in lung ADC (11,12,22).
Adenocarcinoma
Lung ADC is the most common subtype of NSCLC and is
characterized by a complex genomic landscape defined by
multiple types of mutations, a lower level of copy number
alterations than SCC, and some gene rearrangements that
- (2020) -
Figure 1. Molecular alterations in adenocarcinoma and squamous cell car-
cinoma subtypes of NSCLC. For copy number alterations-gene names
highlighted in white represent loss of copy number while genes highlighted
in black represent copy number gains or amplifications. Data is derived
from https://cbioportal.org and depicts the most common/relevant muta-
tions, copy number alterations, and gene rearrangements for adenocarci-
noma and squamous cell carcinoma of the lung by collating data from
multiple large molecular genetic studies and/or data sets on lung adenocar-
cinoma (Broad, Cell 201234; MSKCC, Science 201535; TCGA, Firehose
Legacy; TSP, Nature 200836; MSKCC, Cancer Discovery 201725) and
squamous cell carcinoma (TCGA, Nature 201224; TCGA, PanCancer
Atlas36).
provide therapeutic targets when present
(23,25,28,32e36). Somatic alterations of various types
including substitutions, insertions, deletions, and splice site
mutations have all been demonstrated to play a role in the
carcinogenesis of ADC (32e36). While TP53 and LRP1B
mutations are common in all subtypes of NSCLC, ADC ap-
pears to have higher rates of somatic mutations in KRAS,
EGFR, KEAP1, STK11, MET, and BRAF as compared to
lung SCC. Mutations in these genes tend to primarily
disrupt the RAS-MEK-ERK and PIK3CA-MTOR pathways
(23,25,33,34).
While numerous different types of somatic alterations
have been described, lung ADC harbors a group of clini-
cally relevant, targetable or potentially targetable mutations
and rearrangements (see clinically relevant molecular alter-
ations section) including alterations in EGFR, ALK, ROS1,
BRAF, HER2, RET, MET and NTRK (25,34,35). Of these
ALK, ROS1, RET, and NTRK are involved in rearrange-
ments that led to fusions with available targeted therapies
(35). Lung ADC may also show numerous chromosomal
gains and losses, albeit at a lower rate as compared to lung
SCC (|10% in lung ADC and 30e40% in lung SCC), most
commonly showing deletions of chromosome 9p21.3 con-
taining the CDKN2A and CDKN2B genes leading to dysre-
gulation of cell cycle control (23,35). Epigenetic regulation
plays a role as well with ADC being able to be stratified de-
pending on the DNA methylation profile of each tumor, or
 Molecular Pathology of NSCLC
for example the recent discovery that loss of function alter-
ations in genes and the subsequent loss of associated pro-
tein expression involved in chromatin remodeling (such
as SMARCA4) play a critical role in carcinogenesis
(23,26,27,36e38). At the transcriptomic level, mRNA
profiling studies have shown distinct transcriptional sub-
types of ADC, some of which can be correlated with spe-
cific mutational profiles, however, for now this remains
less clinically relevant than identifying actionable muta-
tions that can be targeted with specific therapies (23,25,38).
Squamous Cell Carcinoma
Lung SCC is the second most common subtype of NSCLC
and much like lung ADC is characterized by a complex mo-
lecular background which shares many of the same molec-
ular features as lung ADC but retains a group of relatively
discrete alterations that allow for some degree of differen-
tiation at the molecular level. One of the most comprehen-
sive studies by The Cancer Genome Atlas (TCGA) of 178
lung SCC’s identified numerous recurrent somatic muta-
tions primarily involving TP53, LRP1B, CDKN2A, PTEN,
PIK3CA, KEAP1, MLL2, HLA-A, NFE2L2, NOTCH1,
RB1, and PDYN (24) that are generally in keeping with
the mutational profile described in other studies (39,40).
Unlike lung ADC, lung SCCs generally do not harbor
targetable driver mutations thus driving treatment algo-
rithms primarily towards immunotherapy and chemo-
therapy, although recurrent DDR2 mutations and FGFR1
amplification have been reported as potential therapeutic
targets (41,42). Compared to lung ADC, recurrent gene re-
arrangements occur at a lower incidence in lung SCC
(24,39,40). In addition, lung SCC shows more frequent
amplification events, particularly at the region of chromo-
some 3q26-3q29. Genes within this region with frequent
copy number gains or amplification include SOX2, PIK3-
CA, TP63, MAP3K13, and KLH6 as well as many others
(43e46). In addition, lung SCC shows a higher rate of copy
number loss at chromosome 9p21.3 containing the
CDKN2A and CDKN2B genes compared to lung ADC
(27 vs. 11%) (23,35). Somatic mutations and copy number
alterations in genes involved in the oxidative stress
response (KEAP1 and NFE2L2 mutations) and squamous
differentiation (SOX2, TP63 copy number gains) appear
to be frequently altered in lung SCC (24,47). The PI3K-
RTK-RAS signaling pathway may show a variety of alter-
ations including mutations (such as PTEN and PIK3CA)
and copy number alterations leading to dysregulation in
up to 70% of SCC cases (24). Whole transcriptome mRNA
expression profiling has also allowed lung SCC to be sepa-
rated into different subtypes, although the clinical signifi-
cance of these subtypes remains to be established (24). It
is worth emphasizing again that despite the genomic
complexity of lung SCC, targetable actionable mutations
3
are rare and treatment currently consists primarily of
chemotherapy or immunotherapy for this tumor subtype.
Large Cell Carcinoma and Other Subtypes of NSCLC
Other rare cancer subtypes sometimes included within the
category of NSCLC include large cell carcinoma (LCC),
adenosquamous carcinoma and sarcomatoid carcinoma
(48e50). Pulmonary large cell carcinoma is the third most
common subtype of NSCLC accounting for 3e9% of
NSCLCs and is defined as a NSCLC with no overt morpho-
logical or immunohistochemical features of lung ADC or
SCC (48,51). Studies have identified that the majority of
historical LCCs expressed ADC or SCC markers by immu-
nohistochemistry (IHC) when re-examined and that many
tumors previously diagnosed as large cell type would now
be reclassified as LCC-ADC or LCC-SCC (51e53). In
these studies, the reclassification of LCCeADC or SCC
was almost always supported by an ADC or SCC muta-
tional profile. LCC-NULL, that is large cell carcinomas
without (or with unclear) expression of adeno- or squamous
specific lineage markers by IHC, have been reported to
have conflicting genetic profiles. While multiple studies
have suggested that LCC-NULL may represent poorly
differentiated TTF-1 negative ADC (51,54e56), some
studies have identified squamous lineage mutations in
LCC-NULL as well as demonstrating that a large propor-
tion of LCC-NULL do not show any lineage specific ge-
netic profile (52). LCC-NULL tumors without lineage
specific genetic profiles however have been shown to harbor
cell cycle regulatory gene mutations, WNT pathway muta-
tions and micro-RNA expression profiles that may be seen
in adenocarcinoma or squamous cell carcinoma suggesting
that while they may not contain a lung ADC or SCC ‘‘pro-
file’’, they may still harbor isolated alterations seen in those
tumor types and may represent poorly differentiated
NSCLCs that only display isolated genetic alterations
(51,57,58). When actionable mutations such as EGFR are
present in an LCC they may be amenable to targeted ther-
apies (51,52).
Pulmonary adenosquamous carcinoma (ADSQ) is a rare
tumor defined by the presence of distinct adenocarcinoma
and squamous cell carcinoma components. The current
WHO classification states that each component must consti-
tute at least 10% of the tumor by light microscopy to
qualify for a diagnosis of ADSQ (2). These tumors
comprise approximately 4% of NSCLC (50). Molecular
characterization of ADSQ has identified the presence of
many mutations also described in lung ADC and SCC,
including TP53, CDKN2A, PIK3CA, RB1, PTEN, and
EGFR mutations with EGFR mutations being the most
common (50). Interestingly, examination of the ADC and
SCC components via microdissection techniques in one
study identified mutations shared between the two histolog-
ic types suggesting that ADSQ derive of a monoclonal
4 Suster and Mino-Kenudson/ Archives of Medical Research
origin (50). The presence of targetable mutations such as
EGFR also indicate that some of these tumors may be
amenable to targeted therapies (50).
Pulmonary sarcomatoid carcinoma is a diverse category
within the World Health Organization (WHO) classification
that contains multiple tumor subtypes including pleomor-
phic carcinoma, spindle cell carcinoma, giant cell carci-
noma, pulmonary blastoma and carcinosarcoma (2).
These tumors are incredibly rare, representing less than
3% of primary lung malignancies and they may be difficult
to classify as the definitions are based on proportions of the
different tumor cell components (2). Of the sarcomatoid
carcinomas, pleomorphic carcinoma represents approxi-
mately 70e80% of cases and in most cases the conven-
tional NSCLC component can easily be identified (59).
Molecular studies of sarcomatoid carcinoma of the lung
have identified many shared mutations with conventional
NSCLC including frequent mutations in TP53, KRAS,
KMT2, EGFR, STK11, NOTCH1, NRAS, and PI3KCA
(59e61). KRAS mutations have been described to be a
marker of poor prognosis in pulmonary sarcomatoid carci-
noma (62). Some studies have also reported a relatively
high frequency of MET exon 14 mutations, although the to-
tal number of cases examined was small (63,64). Sarcoma-
toid carcinomas with mixed morphology where the spindle,
giant and conventional epithelial cell components have
been micro-dissected and subjected to molecular testing
have shown a high concordance between the mutations
identified within the different histological areas supporting
the idea that the sarcomatoid areas represent poorly differ-
entiated components of NSCLCs (60). The presence of
actionable mutations found in conventional NSCLCs in sar-
comatoid carcinomas with minimal conventional epithelial
components also supports the idea that many of the tumors
in the sarcomatoid carcinoma category represent poorly
differentiated NSCLCs. Sarcomatoid carcinomas have also
been demonstrated to harbor chromosomal-level changes
such as amplification events similar to NSCLC (64). Iden-
tification of an actionable mutation in a sarcomatoid carci-
noma or poorly differentiated carcinoma also suggests that
there may be some role for targeted therapies in the treat-
ment of this tumor type (64,65).
Clinically Relevant Molecular Alterations with Available
Targeted Therapies
While NSCLC shows a high degree of genomic complexity
as described above, current treatment regimens rely primar-
ily on targeted therapies directed at a much smaller group
of so-called clinically relevant (sometimes called actionable
or targetable) genetic alterations (Table 1). Specific gene
names are sometimes colloquially used as umbrella terms,
but it is worth noting that multiple different types of genetic
alterations within one gene can lead to similar downstream
effects within a cell. It is also worth mentioning that
- (2020) -
alterations in specific genes can often be associated with
specific clinical or histological characteristics but that they
can also occur independently of those associations.
EGFR
The prototypical gene amenable to targeted therapy in lung
adenocarcinoma is the epidermal growth factor receptor
(EGFR) gene. The EGFR gene encodes for the epidermal
growth factor receptor (EGFR) protein, which is a member
of the receptor tyrosine kinase (RTKs) family and is
involved in numerous cellular pathways including the
PI3K-AKT-mTOR and RAS-RAF-MEK-ERK pathways
(Figure 2) (66). EGFR mutations in lung adenocarcinoma
occur in 10e50% of NSCLC depending on the population
studied, tend to be more common in female, Asian, non-
smokers with lepidic and/or papillary histology, and cluster
around exons 18, 19, 20, and 21 (67). Some mutations, such
as the EGFR L858R point mutation on exon 21 or deletions
of exon 19 (del 19), confer sensitivity to targeted therapies
with tyrosine kinase inhibitors (TKI), while other muta-
tions, such as the EGFR T790M point mutation on exon
20 or exon 20 insertions, confer resistance (68,69). Patients
with sensitizing mutations may also respond differentially
to TKI therapy depending on the specific mutation present
(70,71). EGFR mutations are typically mutually exclusive
with KRAS mutations and some other driver alterations,
although they can co-occur in some populations, particu-
larly as resistance mechanisms in patients being treated
with TKIs (72,73). While the clinical significance of the
co-occurrence of other gene mutations or even between
specific variants within a single gene remains to be
completely defined, many changes related to resistance
mechanisms have been shown to have great clinical impact.
Genetic changes related to TKI therapy serve as a rela-
tively well described model of resistance mechanisms to
targeted therapies. Resistance to a targeted therapy may
be primary, resulting from concomitant alterations present
at the time of therapy initiation, or acquired, resulting from
mutations that manifest after treatment with a particular
therapy has occurred. Concurrent resistance alterations
are varied and can be generally separated into gene-
dependent (that is occurring within the original gene tar-
geted by treatment, aka ‘‘on target’’) and gene-
independent (that is alterations in genes other than the orig-
inal gene targeted by treatment, aka ‘‘off target’’). One of
the first, and currently most common, gene-dependent alter-
ations discovered was the EGFR T790M point mutation on
exon 20 (73,74). This alteration substitutes a methionine for
a threonine at amino acid position 790 that leads to disrup-
tion of drug binding and increases the ATP affinity within
the EGFR ATP-binding pocket (74). This secondary on-
target resistance mutation has been identified in approxi-
mately 60% of EGFR-mutant NSCLC treated with 1st and
2nd generation TKI’s (erlotinib, gefitinib and afatinib)
 Molecular Pathology of NSCLC 5

Table 1. Clinically relevant molecular alterations in non-small cell lung carcinoma

Most common molecular Available targeted

Full name Gene abbreviation alterations Resistance mechanisms therapies

Epidermal growth EGFR - Exon 21 L858R mutation - EGFR T790M mutation Erlotinib
factor receptor - Exon 19 deletions - EGFR C797S mutation Gefitinib
- Exon 18 mutations - EGFR Exon 20 insertion Afatinib
(various)
- Exon 20 mutations - Various other EGFR mutations Dacomitinib
- Mutations and CNV in other path- Osimertinib
ways (including P53, PIK3CA/KRAS,
cell cycle)
- Small cell transformation
- Secondary fusion events

Anaplastic ALK - ALK-EML4 fusion - ALK L1196M mutation Crizotinib

Lymphoma Kinase - Multiple other fusion - ALK G1269A mutation Brigatinib
partners
- ALK G1202R mutation Certinib
- Various other ALK mutations Alectinib
- ALK copy number gains Lorlatinib
- Alternative pathway activation
(EGFR, MET, AXL, SRC and others)
- Off target mutations
- Small cell transformation

ROS Proto-oncogene ROS1 - ROS1 fusions - ROS1 G2032R mutation Crizotinib

1, Receptor - Multiple fusion partners - Various other ROS1 mutations Ceritinib
Tyrosine Kinase - Off target mutations (TP53) Entrectinib

MET Proto-oncogene MET - Exon 14 skipping muta- - MET Y1230 C mutation CrizotinibCabozantinib
receptor Tyrosine tions-MET amplification - MET D1228 N mutation Capmatinib
Kinase - Other MET alterations
- Off target alterations

Human Epidermal HER2 (aka ERBB2) - Exon 20 insertions and - HER2 C805S mutation Trastuzumab
Growth Factor duplications
Receptor 2 - PIK3CA mutations
- HER2 copy number gain
- Off target alterations

RET Proto-oncogene RET - RET-KIF5B fusion - RET V804L mutation Cabozantinib

Tyrosine-Protein - Multiple fusion partners - RET G810A mutation Alectinib
Kinase Receptor - Various other RET mutations Apatinib
- EGFR, AXL bypass signaling Vandetanib

Selpercatinib
Serine/Threonine- BRAF - V600E mutation - MAPK signaling reactivation Dabrafenib/trametinib
Protein Kinase - Non V600E mutations - EGFR signaling Vemurafenib/trametinib
B-Raf - Elevated YAP1 expression

Neutrophic Receptor NTRK NTRK1-fusions - IGF1R pathway activation Larotrectinib

Tyrosine Kinase - Multiple partner genes - Various NTRK mutations Etrectinib
- NTRK3 fusions

Modified and reprinted with permission from Suster D, Suster S. Biopsy Interpretation of the Lung, 2nd Edition. Philadelphia: Lippincott Williams and Wil-
kins, 2020. Chapter 12: Epithelial Neoplasms; pp. 285-349.
^aka 5 also known as, CNV 5 copy number variations.

(73e75). Resistance to this mutation was overcome with the resistance (76e78). Off-target resistance mechanisms are
development of newer TKIs such as osimertinib, however it numerous and include alterations of several genes including
was found that tertiary EGFR mutations such as the EGFR TP53 and PIK3CA amongst others, secondary fusion events,
C797S mutation could then develop that provided additional copy number variations such as MET amplification,
6 Suster and Mino-Kenudson/ Archives of Medical Research - (2020) -

Figure 2. Simplified schematic of EGFR cellular signaling and resistance mechanisms. Green arrows indicate alterations that support cell proliferation, red
arrows indicate inhibitory signals. The epidermal growth factor family of receptor tyrosine kinases consists of four genes, EGFR (erb1), HER2 (erb2), HER3
(erb3) and HER4 (erb4). Intracellular signaling is mediated by a cytoplasmic tyrosine kinase domain. Activation of EGFR to leads to the formation of homo-
or heterodimers with other receptor tyrosine kinase family members which activates the kinase domain leading to downstream cellular signaling with various
functions. Tyrosine kinase inhibitor therapy can block EGFR signaling, although numerous resistance mechanisms may develop; including secondary mu-
tations (EGFR, PTEN, BRAF mutations etc), secondary gene amplifications (MET, HER2), secondary fusion events (ALK ), and small cell transformation as
well as other less common resistance mechanisms.

dysregulation of cell cycle genes, and alterations of some nu-
clear transcription factors (such as MYC and NKX2-1)
(79e89). Another, less common, resistance mechanism that
occurs in TKI treated NSCLC is transformation to different
histologies, such as small cell lung carcinoma (SCLC)
morphology (90) that is likely driven by specific underlying
genetic alterations (91e93). These resistance mechanisms
may occur in isolation or in combination with each other
and therapeutic management of patients must be constantly
updated to accommodate changes occurring within a specific
tumor, furthermore, as new therapies are developed, the
landscape of resistance mechanisms will shift with them
and alterations that were once prominent, such as T790M,
will occur with less frequency.
ALK
Genetic alterations in the anaplastic lymphoma receptor
tyrosine kinase (ALK ) occur in approximately 2e7% of
NSCLC patients (94,95). These occur primarily in the form
of chromosomal rearrangements; the most common being
an EML4-ALK rearrangement that leads to an oncogenic
fusion protein with the downstream effect of increased tyro-
sine kinase activity (94). Studies have reported several
 Molecular Pathology of NSCLC
associated clinical features in tumors with ALK rearrange-
ments including a younger, never or slight smoking patient
population and advanced tumors with more aggressive dis-
ease (96,97). Tumors with ALK alterations have also been
associated with mucinous cribriform histology or solid
pattern with abundant signet ring cells (98e101). ALK rear-
ranged tumors are sensitive to multiple generations of TKIs,
although like EGFR-mutated NSCLC, ALK-rearranged
NSCLC is known to harbor multiple resistance mecha-
nisms. Secondary on-target ALK resistance mutations
include the ALK L1196M and G1269A variants that
develop following crizotinib therapy, although these two
variants only develop in a low percentage of crizotinib-
treated tumors, likely due to the fact that crizotinib is not
a potent inhibitor of ALK (102). However, other resistance
mutations in ALK, such as the G1202R mutation, occur at a
higher frequency following treatment with newer genera-
tion ALK inhibitors (ceritinib, alectinib, brigatinib)
(102,103). Copy number variations in ALK and off-
target alterations in other genes may also occur
(104,105). Small cell transformation as a resistance mech-
anism in ALK-rearranged NSCLC has also been described
(106,107).
ROS1
Chromosomal rearrangements in the c-ROS proto-
oncogene 1 (ROS1) gene occur approximately 1-2% of
NSCLC patients (108,109). The ROS1 gene encodes a re-
ceptor tyrosine kinase that is part of the insulin receptor
family with downstream RAS-MAPK pathway signaling
function (109). ROS1 can become rearranged with multiple
other genes including CD74, FIG, SLC34A2 and SDC4
(110,111). ROS1 rearranged NSCLC shows a similar clin-
ical phenotype to ALK rearranged carcinomas, although
some studies have reported lower rates of extrathoracic
metastasis, including fewer brain metastases (112). Tumors
with this alteration may also display solid pattern with sig-
net ring cell morphology and/or mucinous cribriform
pattern and have been found to be responsive to crizotinib
(107,113e115). The predominant resistance mechanism
to crizotinib treatment occurs in the form point mutations
in ROS1, with the G2032R variant being the most common.
The G2032R variant is thought to result in steric hindrance
to crizotinib (116). Newer generation kinase inhibitors are
becoming available (ceritinib, entrectinib) or under devel-
opment (DS-6051b) that will allow for treatment of crizoti-
nib resistant ALK-rearranged NSCLC (117,118).
MET
Alterations in the Met proto-oncogene (MET) occur in
approximately 1e5% of NSCLC (119,120). The MET gene
encodes for the MET receptor tyrosine kinase (also known
as mesenchymal epithelial transition factor and hepatocyte
growth factor receptor). Constitutive MET activation leads
7
to induction of downstream cellular pathways including
PI3K-AKT-mTOR, STAT, and MAPK (121). In NSCLC
this most commonly occurs through intronic splice donor
site alterations, including substitutions and insertion-
deletions, that affect the normal MET gene splicing mech-
anism and lead to the loss of MET exon 14 (referred to as
MET Exon 14 ‘‘skipping’’ mutations) (119). MET amplifi-
cation and increased copy number, in part as a mechanism
for resistance to TKIs, in NSCLC has also been described
(121). MET alterations are generally mutually exclusive
with other driver alterations. Studies examining MET alter-
ations have associated them with older patient populations,
tobacco use, and a poor prognosis when MET amplification
is present (although not with isolated MET exon 14 ‘‘skip-
ping’’ mutations) (122e126). These studies have also re-
vealed that MET alterations can be identified in nearly all
histologic subtypes of NSCLC, but that they appear to be
enriched in the sarcomatoid carcinoma subgroup, particu-
larly MET exon 14 ‘‘skipping’’ mutations (63). MET alter-
ations are amenable to therapy with MET inhibitors such
as crizotinib and capmatinib, although like other genes,
numerous ancillary resistance mechanisms have been docu-
mented (Table 1) (127e129).
HER2
Human epidermal growth factor receptor 2 (HER2) alter-
ations are found in approximately 1e5% of NSCLC and
include mutations and amplification events. HER2 is also
known as Erb-B2 receptor tyrosine kinase 2 (ERBB2) and
is a member of the RTK gene family (130,131). HER2 al-
terations in NSCLC have been described to occur in an old-
er patient population (O60 years old) the majority of which
are never smokers and occur almost exclusively with
adenocarcinoma histology (132,133). The most common
HER2 alteration appears to be an exon 20 insertion,
although other mutations and amplification events, some-
times in combination with alterations in other genes, have
been observed (134). HER2 altered NSCLC is known to
be sensitive to chemotherapy regimens as well as some tar-
geted therapies (135). As with other genes, both on-target
and off-target resistance mechanisms have been described
and HER2 amplification itself is a reported resistance
mechanism in EGFR mutated NSCLC treated with TKI
therapy (Table 1) (134,135).
RET
Ret proto-oncogene tyrosine-protein kinase receptor (RET )
rearrangements occur in 1e2% of NSCLC (136). More
than 10 different fusion partners have been identified,
although the most common appears to be KIF5B (137).
The resulting fusion protein affects numerous downstream
cellular pathways, many of which are shared with other
genes mutated across NSCLC (137). RET rearrangements
are found almost exclusively in the adenocarcinoma
8 Suster and Mino-Kenudson/ Archives of Medical Research
subtype of NSCLC and tend to correlate with never-
smoking status, younger age and more aggressive disease
(137,138). Studies have shown RET-rearranged NSCLC to
be sensitive to a number of inhibitor agents, although it is
unclear whether there is variation in treatment response de-
pending on the fusion partner present (137e141). Resis-
tance mechanisms to inhibitor therapy in RET-rearranged
NSCLC consist of various on-target secondary RET muta-
tions as well as off-target bypass signaling mechanisms that
result in RAS-MAPK pathway activation (142,143).
BRAF
B-raf proto-oncogene serine/threonine kinase (BRAF ) mu-
tations are found in 1e3.5% of NSCLC (144). Mutations
occur at both the well characterized amino acid 600 posi-
tion (BRAF V600E) and at other positions on the BRAF
gene (144). This has clinical implications as V600E
mutated NSCLCs are sensitive to inhibitor therapy with
dabrafenib and vemurafenib, while non-V600E tumors are
resistant (145,146). While pre-clinical evidence suggests
that non-V600E mutations may be targetable with alterna-
tive therapies, such as MEK inhibitors, there is currently
no clear consensus guidelines of the management of non-
V600E mutated NSCLC (147,148). Studies have reported
conflicting clinicopathological characteristics for BRAF-
mutated NSCLC that appear to shift depending on the pop-
ulation examined and whether the mutation present is
V600E or non-V600E (149,150). Various resistance mech-
anisms have also been identified including off-target muta-
tions and alternative pathway signaling (151e155).
NTRK
The neutrophic receptor tyrosine kinase gene (NTRK ) is a
member of the tropomyosin-receptor kinase family of
RTKs that includes NTRK1, NTRK2 and NTRK3 (156).
NTRK alterations, primarily rearrangements in NTRK1
and NTRK3, have been identified in NSCLC and are present
in 0.2e3% of cases (157,158). Studies examining the clin-
icopathologic characteristics are limited by the small num-
ber of reported cases with NTRK fusions, however it
appears that NTRK fusions tend to be mutually exclusive
with other driver alterations and may be enriched in mini-
mal or never smoker patient populations (158). Fusions
can occur within the squamous cell and adenocarcinoma
subtypes of NSCLC and have also been described in a pul-
monary neuroendocrine carcinoma (158). Although rare,
NTRK rearranged NSCLCs are amenable to targeted ther-
apy with the tyrosine kinase inhibitors entrectinib and laro-
trectinib (159e161). Resistance mechanisms in NTRK
rearranged cancers have already begun to be identified
and include on-target NTRK mutations as well as alternative
pathway signaling, although some early data suggests that
resistance can be overcome with alternate drugs such as ca-
bozantinib (162).
- (2020) -
Genetic Alterations in NSCLC without Targeted Therapies
While targetable alterations in NSCLC have increased the
median overall survival of patients with advanced disease,
a large proportion of patients remain who have no action-
able driver alterations and who must receive alternative
therapies such as chemotherapy or immunotherapy. Howev-
er, continued research into the molecular landscape of
NSCLC has the potential to uncover additional targetable
driver alterations and many known alterations currently
have ongoing clinical trials. Because they are sometimes
associated with specific clinical or histopathologic charac-
teristics and because some may one day become actionable
mutations, it is worth becoming familiar with the currently
non-targetable alterations that occur in NSCLC. Some al-
terations, such as TP53 and LRP1B occur at a high rate
across many of the NSCLC subtypes and can be co-
altered with other genes (23,24,163). Other alterations have
been shown to have specific characteristics such as NF1 and
RASA1 preferentially co-mutating and showing some sensi-
tivity to MEK inhibitors or the presence of certain patient
characteristics in KEAP1 and NFE2L2 mutated NSCLC
(164e172). Some alterations such as KRAS and SMARCA4
have been associated with specific histology and may be of
benefit with regards to the diagnosis of NSCLC
(36,173,174). KRAS mutations, particularly KRAS G12C,
provide an excellent example of how currently untargetable
gene alterations may one day provide future avenues for
targeted therapy; currently numerous clinical trials are un-
derway (AMG 510 and MRTX849) with additional trials
for novel inhibitors (JNJ-74699157 and LY3499446)
entering phase I (175e177). See Table 2 for an expanded
list of molecular alterations without currently approved tar-
geted treatments.
Molecular Diagnostics in NSCLC
Molecular diagnostic testing has rapidly advanced in the
past decade with the advent of new massively parallel
sequencing technologies such as next generation
sequencing and whole exome sequencing. In 2013 the Col-
lege of American Pathologists (CAP), the International As-
sociation for the Study of Lung Cancer (IASLC), and the
Association for Molecular Pathology (AMP) created a set
of guidelines for the molecular testing of lung carcinoma
specimens (178). In 2018 the guidelines were updated
and then endorsed by the American Society of Clinical
Oncology (ASCO) (179,180). The guidelines state that mo-
lecular testing should be available at centers where patients
with lung cancer are treated and recommend that EGFR,
ALK, and ROS1, at a minimum, should be tested on non-
squamous NSCLC as well as SCC with clinical features
associated with a higher probability of an oncogenic driver
mutation being present (young age, never or light smoking
history). BRAF, MET, RET, ERBB2 (HER2), and KRAS
should be offered as well if possible, with ASCO stating

Table 2. Additional molecular alterations in non-small cell lung carcinoma without currently approved targeted therapy
Full name
Tumor Protein P53
Low-density Lipoprotein
Receptor-related Protein 1B
Kirsten Rat Sarcoma Viral
Oncogene Homolog
Kelch-like ECH Associated
Protein 1
Serine/threonine Kinase 11
SWI/SNF-related, Matrix
Associated, Actin Dependent
Regulator of Chromatin,
Subfamily A, Member 4
Lysine Methyltransferase 2 C/D
Phosphatidylinositol-4,5-
bisphosphate 3-kinase
Catalytic Subunit a
Nuclear Factor Erythroid 2 e
Related Factor 2
RAS GTPase Activating
Protein 1
Molecular Pathology of NSCLC 9
Gene Most common molecular
abbreviation alterations Notes
TP53 - Multiple frameshift, nonsense - Most common mutation present in lung adenocarcinoma
and missense mutations (|50%) and squamous cell carcinoma (|80%) subtypes of
NSCLC
- Commonly co-mutated with other driver alterations in
NSCLC
LRP1B - Multiple missense mutations - Commonly identified in lung adenocarcinoma and squa-
mous cell carcinoma subtypes of NSCLC
- Some frameshift or nonsense - LRP1B mutated tumors associated with increased muta-
mutations tional burdens and early evidence suggests increased
response to immunotherapy and better patient survival
KRAS - G12C, G12V, G12D missense - KRAS co-mutation with KEAP1/NFE2L2 reported be an
mutations independent prognostic factor, predicting shorter survival and
response to chemotherapy and immune therapy.
- KRAS mutations associated with mucinous histology
(particularly invasive mucinous adenocarcinoma)
- Mutually exclusive with EGFR mutations
KEAP1 - Loss of function mutations - Common in lung adenocarcinoma subtype of NSCLC
(various) - Alterations in KEAP1 co-occur with mutations in other
genes
- Less prevalent in non-smokers
STK11 - Loss of function missense - Common in lung adenocarcinoma subtype of NSCLC
mutations
- Some deletions and trun- - Commonly co-mutated with KRAS and TP53 and may
cating mutations portend worse prognosis and suppression of immune
surveillance
SMARCA4 - Loss of function mutations - SMARCA4
(various)
- Deletions - deficiency associated with a distinct clinicopathological
group of poorly differentiated NSCLC with aggressive
behavior
- Loss of heterozygosity
KMT2C/D - Loss of function f rameshift - KMT2C and KMT2D mutations more common in (various)
and nonsense mutations squamous cell carcinoma subtypes of NSCLC
- KMT2D mutations reported to be associated with poor
prognosis in NSCLC compared to wild type KMT2D
PIK3CA - Exon 20 mutations - Occurs in both adenocarcinoma and squamous cell carci-
noma subtypes of NSCLC
- Limited data exists suggesting the presence of PIK3CA
mutations may confer a survival advantage
NFE2L2 - Gain of function missense - Commonly co-mutated with KEAP1-Enriched in a
mutations (various) subgroup of Male Japanese patients with squamous cell
carcinoma subtype of NSCLC
- Associated with chemotherapy resistance and rapid pro-
gression in lung adenocarcinoma subtype of NSCLC
RASA1 - Loss of function mutations - Preferentially commutated with NF1 in a subset of NSCLC
(various) - May confer sensitivity to MEK inhibition
(continued on next page)
10 Suster and Mino-Kenudson/ Archives of Medical Research - (2020) -

Table 2 (continued )
Gene Most common molecular
Full name abbreviation alterations Notes

Discoidin Domain Receptor DDR2 - Gain of function missense - DDR2 mutations identified in approximately 4% of squa-
Tyrosine Kinase 2 mutations (various) mous cell carcinoma

- DDR2 mutations may confer sensitivity to dasatinib

Neuregulin-1 NRG1 - NRG1-CD74 fusion - Described in 25-30% of invasive mucinous lung adenocar-
cinoma subtype
- Other fusion partners - May respond to ERBB3 targeted therapy
described

Fibroblast Growth Factor
Receptor/Transforming Acidic
Coiled-coil Containing
Protein 3
FGFR3/TACC3 - FGFR3-TACC3 fusion - Described in 3-4% of squamous cell carcinoma and rarely
in some adenocarcinoma samples
- FGFR signaling is involved in the RAS/MAPK pathway
- Numerous ongoing clinical trials underway for FGFR
pathway inhibitors

that BRAF should be offered as a stand-alone test on all
lung cancer patients. The National Comprehensive Cancer
Network (NCCN) maintains similar recommendations and
also recommends PD-L1 testing to guide potential immuno-
therapy (181). In general, testing can be accomplished
either by comprehensive molecular panels (usually
sequencing based panels) or with sequential testing using
smaller panels (including smaller targeted sequencing as-
says, fluorescent in-situ hybridization [FISH] and/or poly-
merase chain reaction-based assays). The advantage of
larger comprehensive sequencing panels is the ability to
identify fewer common alterations not tested for by smaller
directed panels, but which have available therapies, such as
NTRK rearrangements. In addition, large panels have the
ability to provide information on tumor mutational burden
which is an emerging biomarker that may indicate patient
responsiveness to immunotherapy (182) The advantage of
smaller directed panels is that they tend to be more rapid
(some small targeted panels such as the PCR-based Idylla
assay can return results in as little as a few hours) and allow
for faster clinical decision making (183).
Multiple diagnostic modalities are required to arrive at a
complete diagnosis of advanced NSCLC including histo-
logical assessment, primary IHC testing for the diagnosis
of NSCLC, NOS morphology, secondary IHC testing
including PD-L1, and ALK and ROS1 in the appropriate
setting, as well as molecular testing. Given that these all
require tissue from the patient sample, specimen handling

Figure 3. Suggested simple schematic for appropriate triage of biopsy specimens from advanced NSCLC patients. When limited tissue is available the min-
imal amount of IHC required for primary diagnosis and PD-L1 testing should be ordered followed by molecular studies. According to current guidelines
EGFR, ALK, ROS1 and BRAF should be included in the first round of molecular testing.
 Molecular Pathology of NSCLC
is of utmost importance, particularly when dealing with
small core needle biopsy specimens. If feasible, multiple
tissue cores from the tumor should be procured and ideally
processed in separate tissue fixation cassettes. In addition,
care should be taken when triaging material procured from
bone as standard decalcification procedures with hydro-
chloric acid degrade nucleic acids and prevent many types
of molecular testing (184). If tissue is limited, care should
be taken to use the minimum number of immunohistochem-
ical stains needed for the initial diagnosis and the remaining
tissue should be preserved for ancillary IHC testing and
molecular testing (Figure 3).
Conclusion
Non-small cell lung carcinoma is characterized by a com-
plex genomic background with numerous genetic alter-
ations involved in various genes. As the molecular
landscape of NSCLC continues to advance there will likely
be further refinements in the molecular subclassification of
NSCLC, some of which will have prognostic and/or thera-
nostic implications. Although current targeted therapies are
directed at a small group of genes, numerous clinical trials
are currently underway that will potentially expand the list
of targetable candidate genes and continue to advance the
management of lung cancer patients.
Acknowledgments
The authors would like to thank Christopher and Dana Green at
CGMotion for their graphical design work on Figures 1, 2 and 3.
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