HHE Pathology and diagnostics supplement 2018 | Page 17

with a median response of 17 months. This reflects
the sensitivity of NGS and the potential of denying
life-prolonging treatment for this patient cohort
due to false negative ALK FISH results. 49
Interestingly, Ali et al also identified one out of
the two NGS-positive but ALK FISH-negative cases
(which did not respond to crizotinib) actually
contained a TSC2 alteration. TSC2 alteration is
well known to be associated with acquired
resistance to targeted therapy, which could
explain the de novo resistance in this patient. 49,50
Drug resistance in patients is one of the most
important reasons for treatment failure. Primary
resistance occurs prior to treatment due to the
presence of gene alterations conveying resistance,
whereas secondary resistance occurs when initial
useful treatment loses its effectiveness after
initial success. NGS can be used to detect
resistance genes and also predict resistance at
a genetic level to guide treatment choices. 51,52
The majority of ALK-positive patients on crizotinib
will develop resistance approximately one year
from start of treatment. This is due to resistance
mutations in ALK or amplification of the ALK fusion
gene or activation of other ALK-related signalling
pathways such as c-Kit pathway through c-Kit gene
amplification and other potential bypass
mechanisms of resistance, including activating
mutation of KRAS and EGFR. 53–55 The beauty of NGS
in this setting is that it can detect all of these
resistance mutations in one test. Furthermore, the
opportunistic testing of a panel of mutations would
help to ident ify patients for clinical trials. It is
crucial for the oncologist to evaluate all treatment
options available for patients to ensure best
standard of care is offered.
EGFR and ALK testing were first recommended
in 2013; however, the College of American
Pathologists (CAP), International Association
for the Study of Lung Cancer (IASLC), Association
for Molecular Pathology (AMP) and National
Comprehensive Cancer Network (NCCN)
guidelines have endorsed testing for ROS1, MET,
RET, ERBB2, and BRAF, which further sets the
foundation of precision medicine driven
landscape in management of lung cancer
that may significantly improve cancer
mortality. 11,41,56–58 However, this can be
extremely challenging in terms of tissue
sample conservation when multiple single-gene
molecular assays are performed. Furthermore,
diagnosis is usually made with small biopsy and
cytology samples by employing minimal invasive
technique such as endobronchial ultrasound
(EBUS)-guided transbronchial needle
aspiration. 59,60 Thus, NGS technology could
maximise available information from a single
biopsy by identifying a select panel of clinically
relevant mutations. 61,62 NGS can be used in small
biopsy specimens as well as cytological
specimens. 63
Overcoming limitations
Occasionally, a tumour biopsy or cytology sample
would be insufficient for molecular testing
because most lung cancers are metastatic or
unresectable and diagnosis frequently relies on
relatively small core or fine needle aspiration. 64,65
Up to 23% of lung biopsies had insufficient
material for pathological or cytological diagnosis,
let alone molecular diagnosis, based on the report
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HHE 2018 | hospitalhealthcare.com
from UK National Lung Cancer Audit Report. 66
After initial pathology diagnosis, only
approximately 57% of biopsies had sufficient
tissue for genomic analysis. 67,68 Furthermore,
serial biopsies may be warranted for patients who
developed resistance to treatment in order to
delineate the mechanism of resistance and tailor
subsequent treatment. However, this is clinically
challenging due to the invasive nature of the
biopsy procedure with potential serious adverse
risk for patients. 69,70 Recently, NGS has been used to
test genomic changes in liquid-based biopsy,
specifically the cell-free DNA (cfDNA) from
patients. 71,72 cfDNA isolated from blood samples was
shown to contain genetic changes, which were in
concordance with primary tumour tissue DNA. 69
In a study, Schwaederle et al established 10% of
There is a growing body of
evidence that challenges FISH as
the gold standard for ALK testing
when compared with NGS
patients harbour ALK rearrangement from
sequencing 54 cancer-related genes in plasma
cfDNA. 72 This strategy could potentially overcome
serial tissue biopsy limitations.
Conclusions
In summary, it is critical to accurately identify
ALK rearrangements in a patient sample, owing
to the fact that false negative results would
deny patients from receiving effective targeted
therapies but false positive results would be equally
deleterious as patients would be subjected to
ineffective treatments. There is a growing body of
evidence that challenges FISH as the gold standard
for ALK testing when compared with NGS. This
novel testing strategy is practical and reliable to use
on tissue specimens and potentially on liquid
biopsies, which will potentially further
revolutionise the diagnostic landscape of lung
cancer. By contrast, comprehensive molecular
profiling with NGS remains controversial at present
because clinical data to support its use are
incomplete. The MOSCATO trial, which evaluates
the clinical benefit of massive parallel sequencing
approach showed that only 7% of successfully
screened patients benefited with the use of this
new technology. 73 Dalton et al also investigated the
role of this approach where the group reported
only 6% of patients had benefited clinically. 74
In addition, European Thoracic Oncology Platform
Lungscape Consortium (ETOP) has appraised the
efficacy of NGS and RT-PCR techniques in
comparison to established diagnostic assays for
diagnosis of ALK rearrangement in a large study of
96 IHC selected NSCLC cases. This working group
reported similar sensitivity and specificity for NGS,
RT-PCR and FISH in confirming ALK status, which
was contrary to earlier studies. 49,75,76 Thus, further
investigation on the role of NGS and its magnitude
of clinical impact is required; however, this ‘new
kid’ is likely to become the mainstay technology for
screening for targetable aberrations in lung cancer.