pathology and diagnostics
The new kid on the block
Next generation sequencing is practical and reliable to use on tissue
specimens and potentially on liquid biopsies, which potentially will further
revolutionise the diagnostic landscape of lung cancer
Marvin Lim Chang Jui
MBBCh BAO BA
MRCPUK
Anne-Marie Baird
BSc (Hons) PG Dip
(Statistics) PhD
Stephen Finn
MB BAO BCh FDS PhD
FRCPath FFPATH
Trinity College Dublin,
School of Medicine,
Dublin, Ireland
Lung cancer continues to be the major cause
of cancer-related death globally. 1 More than
two-thirds of lung cancer patients present with
advanced disease, 2,3 which excludes the option
of potentially curative treatments. Another
important reason accounting for the high
mortality rate is excessive mutational load in
patients with smoking history, a phenomenon
central to the pathogenesis of lung cancer
progression, compared with patients with
age-related cancers. 4 Five-year survival of all
patients with lung cancer is only 18%. 5 Recently,
great advances have been made in terms of
screening, minimally invasive techniques for
diagnosis and new treatments. 6–9
The recognition of genetic driver mutations
in NSCLC has paved the way for the development
of targeted therapies, which often provides
outstanding responses in patients harbouring
specific genetic mutations. 10,11 Approximately
two thirds of lung adenocarcinomas contain
actionable driver mutations, which can be
detected using comprehensive molecular
profiling. 11–13 ALK gene rearrangement in NSCLC
was first discovered by Japanese researchers
a decade ago. 14 This gene rearrangement, which
precipitates expression of oncogenic fusion
proteins, is found in approximately 3–7% of
patients with metastatic lung carcinoma based
on early studies using reverse transcription-
polymerase chain reaction (RT-PCR) and
fluorescence in situ hybridisation (FISH). 14,15
ALK gene fusion with echinoderm microtubule-
associated protein like 4 (EML 4) represents the
most frequent rearrangement among the ALK
alterations. 12,16 Other
fusion partners have also
been reported such as
TPR, HIP 1, FAM 179 A
and COL25A1. 17–20
compared with standard platinum doublet
chemotherapy laid the foundation for targeted
therapy as the first-line treatment for ALK-positive
NSCLC. 21 Second-generation ALK inhibitors such
as ceritinib and alectinib have not only been
shown to be effective in the first-line treatment
setting but are also effective in patients who
develop crizotinib resistance. 22–24 In 2013,
FDA granted the approval of crizotinib for
treatment of metastatic ALK-positive NSCLC
with FISH as companion diagnostic based
on efficacy and safety data of Phase II and III
studies. 21,25
Benchmark technique
FISH is currently the benchmark technique for
diagnosis of ALK rearrangements; however,
meticulous preparation and skillful interpretation
according to guidelines is necessary for achieving
accurate results. Thus, it is expensive, labour-
intensive and requires a high level of pathology
expertise. 26–29 On rare occasions, FISH may
produce equivocal results because in 5–10% of
NSCLC, the rate of rearrangement of positive cells
falls within the range of 10–20%; however, the
current accepted cut-off for positive cells is 15% or
more. 30,31 Immunohistochemistry (IHC) is another
method that can be used for ALK diagnosis in lung
cancer. An IHC companion diagnostic assay was
approved in 2015 based on its ability to accurately
identify patients with ALK-rearranged NSCLC. 32,33
Although IHC has been extensively used in
laboratories due to the cost effectiveness, its
interpretation requires experience and rarely
protein expression may be absent in cases with
atypical ALK
rearrangement. 34,35
Despite these
limitations, ALK IHC
is gaining momentum
The recognition of genetic
in Europe as the
driver mutations in NSCLC has
Drug discovery
primary test usually
The discovery of ALK
paved the way for the development in a two-step approach
rearrangement led to
with FISH being
of targeted therapies
advent of crizotinib,
performed only to
a tyrosine kinase
confirm positive or
inhibitor (TKI) with powerful activity against ALK.
equivocal IHC results. 36–38 However,
a few studies have reported false negative results
Crizotinib showed a response rate of 74% with
using IHC, which potentially risk excluding
progression-free survival (PFS) of 10.9 months
patients from receiving standard of care
compared with a response rate of 45% with PFS
treatment. 39,40,41 Molecular diagnosis could surpass
of 7 months in standard platinum doublet
the limitations of both FISH and IHC either as a
chemotherapy (either carboplatin or cisplatin
stand alone assay or in concert with either FISH
plus pemetrexed). 21 This superior outcome
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