Re-biopsy by endobronchial ultrasound procedures for mutation analysis of non-small cell lung cancer after EGFR tyrosine kinase inhibitor treatment
Izumo et al. BMC Pulmonary Medicine
Re-biopsy by endobronchial ultrasound procedures for mutation analysis of non-small cell lung cancer after EGFR tyrosine kinase inhibitor treatment
Takehiro Izumo 0
Yuji Matsumoto 0
Christine Chavez 0
Takaaki Tsuchida 0
0 Department of Endoscopy, Respiratory Endoscopy Division, National Cancer Center Hospital , 5-1-1, Tsukiji Chou-ku, Tokyo 104-0045 , Japan
Background: Re-biopsy for resistant non-small cell lung cancer (NSCLC) after treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is important for selection of better therapy, but there have been no reports about the utility of endobronchial ultrasound (EBUS)-guided procedures for such purpose. The aim of this study was to evaluate the utility of EBUS-guided re-biopsy for resistant NSCLC after treatment with EGFR-TKIs. Methods: From January 2013 to December 2015, 53 consecutive patients who underwent EBUS-guided re-biopsy for mutation analysis of NSCLC after EGFR-TKI treatment were assessed. Results: Nine patients underwent EBUS-guided transbronchial needle aspiration (EBUS-TBNA) and 44 patients underwent EBUS with a guide sheath (EBUS-GS) transbronchial biopsy. The technical success rates were 100 %. As for mutation analysis, all 9 specimens (100 %) from EBUS-TBNA and 33 specimens (75.0 %) from EBUS-GS were adequate for gene profiling. The remaining 11 specimens from EBUS-GS procedures were inadequate for mutation analysis owing to the absence of tumor component in the sample (n = 6) or insufficient specimen (n = 5). There were no related severe complications. Conclusions: Re-biopsy by both EBUS-TBNA and EBUS-GS were useful and safe sampling procedures for mutation analysis of EGFR-TKI resistant NSCLC.
EGFR-TKI; EBUS-TBNA; EBUS-GS; T790M; Lung cancer; Re-biopsy
Epidermal growth factor receptor tyrosine kinase
inhibitors (EGFR-TKIs) have been demonstrated to be
effective in treating non-small cell lung cancer (NSCLC)
patients with EGFR mutations [
]. EGFR-TKIs offer
both good clinical response and survival benefit in
NSCLC patients who have EGFR mutation [
a majority of the responders eventually develop acquired
resistance to EGFR-TKIs [
Until now, several studies have uncovered several
mechanisms of acquired resistance, such as secondary
EGFR mutations (T790M mutation),
mesenchymal-toepithelial transition factor receptor (MET) amplification,
and human epidermal growth factor receptor 2 (HER2)
gene amplification [
]. Among these, a secondary
missense T790M mutation was observed in nearly half of all
cases that were resistant to EGFR-TKIs [
In November 2015, a third generation EGFR-TKI
(Osimertinib; Astra Zeneca, London) has been approved
by the US Food and Drug Administration (FDA) to treat
patients with a type of advanced NSCLC that has a
specific EGFR mutation, called T790M, and which has
become worse after treatment with other EGFR-TKIs.
Osimertinib has shown clinical effectiveness and
tolerability in NSCLC patients with T790M mutation of
Examining T790M is very important in treatment
selection. Because EGFR-TKI treatment is indicated for
advanced and unresectable NSCLC, examination for
T790M mutation is usually performed on small biopsy
So far, most reports on re-biopsy procedures after
EGFRTKI resistance have been on computed
tomographyguided transthoracic needle biopsy (CTNB), which is also
the common first method of choice for the initial diagnosis
of lung cancer [
]. However, complications, such as
pneumothorax and bleeding, after CTNB have been
Another biopsy approach is by bronchoscopy, but its
use has been limited by a lower diagnostic accuracy
compared with CTNB. Recently, endobronchial
ultrasound (EBUS) guidance during bronchoscopy
procedures, such as EBUS guided-transbronchial needle
aspiration (EBUS-TBNA) and EBUS with a guide sheath
(EBUS-GS) transbronchial biopsy, have been introduced
and improved the diagnostic accuracy of bronchoscopy
]. However, there have been no reports about the
utility of EBUS procedures for re-biopsy.
In this study, we evaluated the efficacy and safety of
EBUS procedures for re-biopsy and mutation analysis of
EGFR-TKI resistant NSCLC.
This study was approved by the National Cancer Center
Institutional Review Board. Written informed consent
for the procedure was obtained from all patients.
Consecutive patients who underwent re-biopsy by EBUS
procedures for mutation analysis of NSCLC after
EGFRTKI treatment at the hospital between January 2013 and
December 2015 were enrolled. There were 2907 patients
in whom EBUS-guided bronchoscopy procedures were
performed during the study period. Among these, 89
patients were for re-biopsy purposes. Among these
rebiopsy patients, we selected NSCLC patients who were
treated with at least one EGFR-TKI regimen. Disease
progression during chemotherapy, based on the RECIST
ver1.1 definition, was a criterion for inclusion; 36
patients were ineligible because of the absence of prior
Methods and equipment
All bronchoscopies were performed via the oral route
under local anesthesia with mild sedation by intravenous
administration of midazolam.
EBUS-GS was performed for patients with peripheral
pulmonary lesions (PPLs) without mediastinal and hilar
lymphadenopathy; a GS kit (K-201 or K-203; Olympus
Ltd, Tokyo, Japan) and a radial EBUS (R-EBUS) probe
(UM-S20-20R or UM-S20-17S; Olympus Ltd, Tokyo,
Japan) were used. The bronchial route was planned by
reviewing the chest CT images before EBUS-GS. Virtual
bronchoscopic navigation/simulation systems (Ziostation2,
Ziosoft Ltd, Tokyo, Japan; LungPoint, Bronchus Ltd,
Mountain View, CA, USA; or Bf-Navi, Olympus Ltd,
Tokyo, Japan) were used to detect the bronchial route to
the target PPLs. A PPL was defined as an abnormal
growth surrounded by normal lung parenchyma and was
bronchoscopically invisible. Upon reaching the target
bronchus, the GS together with the R-EBUS probe was
inserted through the working channel of the
bronchovideoscope and was advanced towards the PPL under
fluoroscopic guidance (VersiFlex VISTAVR; Hitachi Ltd,
Tokyo, Japan). Ultrasound scanning was performed while
manipulating the R-EBUS probe until the lesion was
localized by the corresponding EBUS image. After confirming
the location of the PPLs, the R-EBUS probe was removed
while keeping the GS in place for the usual transbronchial
sampling using forceps, brush, and/or transbronchial
needle aspiration under fluoroscopic guidance. Lesion
location was assigned based on a study of Baaklini et al. [
but with some modifications. With the area around the
hilum on CT as reference, lesions in the inner and middle
third ellipses were designated as central parenchymal
location, whereas lesions in the outer third ellipse were
designated as peripheral parenchymal location. The bronchus
sign on CT was defined as the presence of a bronchus
leading directly to a PPL [
EBUS-TBNA was performed for patients who had
mediastinal and hilar lymphadenopathy. EBUS–TBNA was
performed using either one of the following 22G needles:
Vizishot (NA-201SX-4022; Olympus Ltd, Tokyo, Japan)
or SonoTip EBUS Pro with stainless steel
(GUS-45-18022; Medi-Globe Ltd, Germany). The convex probe
EBUS (CP-EBUS; BF-UC260FW, Olympus Ltd, Tokyo,
Japan) was inserted through the oral route in the same
way as during usual bronchoscopy. The ultrasound
images were generated using a dedicated ultrasound
processor (EU-ME1 or EU-ME2; Olympus Ltd, Tokyo,
Japan). When the target lesions were visualized by
CPEBUS, the TBNA needle was inserted through the
working channel of the bronchoscope and was advanced to
penetrate the tracheobronchial wall into the target lesion
under real-time EBUS guidance. Aspiration was done by
moving the needle back and forth inside the target lesion
for 20–30 times, under negative pressure. After
sampling, suction was released before retracting the needle
from the scope.
Pathologic evaluation and mutation analysis
Cytology and histology specimens were sent for
pathologic examination. Positive diagnostic criteria suitable
for molecular analysis were defined by the presence of
malignant cells based on histologic features or based on
class IV/V on cytology examination by Papanicolaou
stain. Overall detection rate was based on a positive
diagnosis by histology and/or cytology.
EGFR mutation analyses of the genomic DNA extracted
from tumor samples were performed using the Scorpion
Descriptive statistics were presented as frequency,
percentage, and median (range). Univariate analyses were
performed using Fisher’s exact test. Multivariate analysis
using logistic regression was performed to determine the
factors associated with the yield. Variables selected via
univariate analyses (P value <0.10) were evaluated using
multivariate logistic regression analysis. A two-tailed P
value <0.05 was considered to indicate statistical
significance. Correlation of study variables were performed
with EZR (Saitama Medical Center, Jichi Medical
statmed.html), a graphical user interface for R (version
2.13.0, The R Project for Statistical Computing; http://
www.r-project.org) and a modified version of R commander
After exclusion, a total of 53 patients were included in
this study population. In the 53 patients, the original
diagnoses were established by CTNB in 2 cases; by
EBUS-TBNA in 5 cases; by EBUS-GS in 39 cases; and by
surgical biopsy in 7 cases. There were no cases with
positive T790M mutation upon initial diagnosis.
For re-biopsy, EBUS–TBNA was performed in 9
patients, whereas EBUS-GS was performed in 44 patients.
The technical success rates of the re-biopsy procedures
were 100 %. All 9 specimens (100 %) from EBUS-TBNA
and 33 specimens (75.0 %) from EBUS-GS were
adequate for gene profiling and mutation analysis. The
remaining 11 specimens from EBUS-GS procedures
were inadequate for mutation analysis owing to the
absence of tumor component in the sample (n = 6) or
insufficient specimen (n = 5).
The baseline characteristics of the study population
are summarized in Table 1. Initially, EGFR mutation was
detected in 53 cases (exon 19 deletion in 30, L858R
point mutation in 22, and L861Q point mutation in 1).
Among 42 patients that had adequate samples for
gene profiling testing for EGFR mutation, 22 (52.4 %)
had EGFR mutation and T790M-resistant mutation
(Table 2). One initial EGFR-mutated tissue revealed
The adequacy of the re-biopsy specimens for mutation
analysis is described in Table 3. The overall detection
rate of re-biopsy for malignant cells was 79.2 % (42 of
53); 77.4 % (41 of 53) by cytology and 77.4 % (41 of 53)
by histologic examination. The detection rate of re-biopsy
by EBUS-TBNA for malignant cells was 100 % (9 of 9),
100 % (9 of 9) by cytology and 88.9 % (8 of 9) by histologic
examination. In contrary, the detection rate of re-biopsy
by EBUS-GS for malignant cells was 75.0 % (33 of 44);
Data are presented as number
EBUS endobronchial ultrasound, EGFR epidermal growth factor receptor
72.7 % (32 of 44) by cytology and 75.0 % (33 of 44) by
histologic examination (Table 3).
The factors affecting re-biopsy by EBUS-GS are shown
in Table 4. In the multivariate analysis, central
parenchymal location and EBUS probe within were the significant
predictors of a successful EBUS-GS re-biopsy.
There were no severe complications after both
EBUSTBNA and EBUS-GS re-biopsy procedures.
Currently, the significance of re-biopsy for mutation
analysis of NSCLC has been increasing because of a
wider range of therapeutic options. The standard
cytotoxic chemotherapy for NSCLC patients has limited
therapeutic response [
]. Moreover, after treatment
with EGFR-TKIs, kinase inhibition frequently leads to
the appearance of drug-resistant mutations within the
target kinase itself [
]. Recently, a third generation
EGFR-TKI (Osimertinib) has been approved by the US
FDA to treat patients with a type of advanced NSCLC
that has a specific EGFR mutation, called T790M, and
which has become worse after treatment with other
EGFR-TKIs. Moreover, Osimertinib has shown clinical
effectiveness and tolerability in NSCLC patients with
T790M mutation of EGFR , underscoring the
importance of checking for new mutations after EGFR-TKI
therapy in advanced NSCLC patients. There have been
several reports about the utility of re-biopsy by CTNB
for such purpose.
To our best knowledge, this study was the first to
demonstrate the utility of bronchoscopic procedures,
especially with EBUS guidance, for mutation analysis of
NSCLC after EGFR-TKI therapy. EBUS is a very
important procedure to determine and collect samples from
target sites in the mediastinal, hilar, and peripheral
locations under real-time ultrasound [
sampling of histologic specimens is necessary for the
development of new treatment options for cancer, especially
chemotherapy and gene-targeted therapy; therefore,
further improvements of the histologic sampling yield
is essential [
EBUS-TBNA is an established minimally invasive
procedure for proper staging and diagnosis of lung cancer
]. In this study, EBUS-TBNA was performed
successfully and was able to obtain adequate samples in all
cases. EBUS-TBNA is useful not only for proper staging
and diagnosis of lung cancer, but also to obtain samples
for mutation analysis of NSCLC after EGFR-TKI, as
demonstrated in this study. Furthermore, EBUS-TBNA
was a safe re-biopsy procedure and had no associated
In this study, EBUS-GS was able to obtain samples for
mutation analysis of NSCLC after EGFR-TKI. However,
when compared with EBUS-TBNA, the detection rate
for malignancy was only 75 %. The yield of EBUS-GS for
primary diagnosis of PPLs has been reported to be about
70–80 % [
], which is similar to the yield for re-biopsy
for mutation analysis in this study. The factors that
influence the diagnostic yield of EBUS-GS for PPLs have
been reported to be the location of the PPL (central
parenchymal or peripheral parenchymal), detected EBUS
images (within or adjacent to/invisible), and the presence
of a bronchus sign. Although central parenchymal
location and detection of EBUS image within were significant
factors that predicted a successful yield, the number of
peripheral parenchymal cases in this study was small.
Further study is needed to confirm the usefulness of
EBUSGS according to the location of the lesion. Although there
is need for further technical improvement, EBUS-GS was
useful to get samples for mutation analysis of NSCLC
after EGFR TKI treatment. In addition, EBUS-GS had no
CTNB is another diagnostic procedure for PPLs, with
a relatively high rate of both diagnostic accuracy and
complications, such as pneumothorax. On the contrary,
EBUS-GS was a safe method [
]. However, it is
important to note that although EBUS-GS might be considered
one of the sampling procedures to safely obtain tissues
from PPLs for mutation analysis of NSCLC after
EGFRTKI treatment, the detection rate for malignant cells
was significantly lower for peripherally located lesions,
as shown in this study. In this regard, EBUS-GS should
be performed to get samples for mutation analysis of
lesions that are in a central parenchymal location.
Another alternative approach, specifically liquid
biopsy, now present as a crucial point in the field [
Liquid biopsy has grown in importance because the
genetic profile of tumors can affect how well they respond
to a certain treatment. A recent paper showed that the
concordance between re-biopsy and liquid biopsy,
including plasma DNA and circulating tumor cell, was
57–60 % [
]. The usefulness of monitoring T790M
status in liquid biopsy was already reported [
liquid biopsy has the potential to detect new mutations
after chemotherapy, several reports have demonstrated
some difficulties in detecting tumor-derived mutations
in plasma [
]. Therefore, liquid biopsy and re-biopsy
may be considered to be complementary methods of
The limitations of this study were its retrospective and
single-institution design. Prospective, multi-center trials
are ideal and recommended in the future.
EBUS procedures for re-biopsy were useful sampling
methods for mutation analysis of NSCLC after
EGFRTKI treatment. This could play an important role in
the choice of better targeted therapy and for the
development of a novel treatment for advanced lung
CP-EBUS, convex probe endobronchial ultrasound; CTNB, computed
tomography-guided transthoracic needle biopsy; EBUS, endobronchial
ultrasound; EBUS-GS, endobronchial ultrasound with a guide sheath;
EBUS-TBNA, endobronchial ultrasound guided-transbronchial needle aspiration;
EGFR-TKIs, epidermal growth factor receptor-tyrosine kinase inhibitors; NSCLC,
non-small cell lung cancer; FDA, Food and Drug Administration; HER2, human
epidermal growth factor receptor 2; MET, mesenchymal-to-epithelial transition
factor receptor; PPLs, peripheral pulmonary lesions; R-EBUS, radial endobronchial
This work was supported in part by The National Cancer Center Research
and Development Fund (28-K-1).
Availability of data and materials
The dataset supporting the conclusions of this article is presented within the
article. The detailed clinical data set is not publically available to protect
research subject privacy and confidentiality.
TI designed the overall study and performed the statistical analysis. All
authors substantially contributed to the acquisition, interpretation, and
consolidation of data. All authors wrote the manuscript and approved the
The authors declare that they have no competing interests.
Ethics approval and consent to participate
The Institutional Review Board of National Cancer Center approved this
study without the need to obtain informed consent.
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