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ORIGINAL ARTICLE
Clinical Impact of Hybrid Capture–Based
Next-Generation Sequencing on Changes
in Treatment Decisions in Lung Cancer
Anna Belilovski Rozenblum, MD,a,b Maya Ilouze, PhD,a Elizabeth Dudnik, MD,a
Addie Dvir, MSc,c Lior Soussan-Gutman, PhD,c Smadar Geva, MSc,a
Nir Peled, MD, PhDa,b,*
a
Thoracic Cancer Service, Davidoff Cancer Center, Rabin Medical Center, Petah Tikva, Israel
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
c
Teva Pharmaceutical Industries Ltd., Shoam, Israel
b
Received 5 April 2016; revised 23 September 2016; accepted 17 October 2016
Available online - 16 November 2016
ABSTRACT
Introduction: Targeted therapy significantly prolongs survival in lung adenocarcinoma. Current diagnostic guidelines
include only EGFR and anaplastic lymphoma receptor
tyrosine kinase gene (ALK) testing. Next-generation
sequencing (NGS) reveals more actionable genomic alterations than do standard diagnostic methods. Data on the
influence of hybrid capture (HC)-based NGS on treatment
are limited, and we investigated its impact on treatment
decisions and clinical outcomes.
Methods: This retrospective study included patients with
advanced lung cancer on whom HC-based NGS was performed between November 2011 and October 2015.
Demographic and clinicopathologic characteristics, treatments, and outcome data were collected.
Results: A total of 101 patients were included (median age
63 years [53% females, 45% never-smokers, and 85% with
adenocarcinoma]). HC-based NGS was performed upfront
and after EGFR/ALK testing yielded negative or inconclusive
results in 15% and 85% of patients, respectively. In 51.5%
of patients, HC-based NGS was performed before first-line
therapy, and in 48.5%, it was performed after treatment
failure. HC-based NGS identified clinically actionable
genomic alterations in 50% of patients, most frequently in
EGFR (18%), Ret proto-oncogene (RET) (9%), ALK (8%),
Mesenchymal-epithelial transition factor (MET) receptor
tyrosine kinase gene (6%), and erb-b2 receptor tyrosine
kinase 2 gene (ERBB2) (5%). In 15 patients, it identified
EGFR/ALK aberrations after negative results of prior standard testing. Treatment strategy was changed for 43 patients (42.6%). The overall response rate in these patients
was 65% (complete response 14.7%, partial response
50%). Median survival was not reached. Immunotherapy
was administered in 33 patients, mostly without an
Journal of Thoracic Oncology
Vol. 12 No. 2: 258-268
actionable driver, with a presenting disease control rate of
32%, and with an association with tumor mutation burden.
Conclusions: HC-based NGS influenced treatment decisions
in close to half of the patients with lung adenocarcinoma
and was associated with an overall response rate of 65%,
which may translate into a survival benefit.
2016 International Association for the Study of Lung
Cancer. Published by Elsevier Inc. All rights reserved.
Keywords: Driver mutations; Next-generation sequencing;
Oncogenic drivers; Precision/personalized medicine;
Targeted therapy; Immunotherapy
Introduction
NSCLC tumors are highly genetically diverse and present a treatment challenge.1 Until a decade ago, systemic
*Corresponding author.
Drs. Rozenblum and Ilouze equally contributed to this work.
Disclosure: Mr. Dvir and Dr. Lior Soussan-Gutman are employees of Teva
Pharmaceutical Industries Ltd. Dr. Dudnik is a consultant for Boehringer
Ingelheim, Merck/Merck Sharp and Dohme, Roche Pharmaceuticals, and
Astra Zeneca and has received payments for expenses. Dr. Peled is a
consultant for Pfizer, Boehringer Ingelheim, Roche, Astra Zeneca, Merck
Sharp and Dohme, Bristol-Myers Squib, Lilly, Novartis, and NovellusDx.
This work was performed in partial fulfillment of the doctor of medicine
thesis requirements of the Sackler Faculty of Medicine, Tel Aviv University and was supported by Davidoff Cancer Center, Rabin Medical
Center (Dr. Rozenblum).
Address for correspondence: Nir Peled, MD, PhD, Thoracic Cancer
Service, Davidoff Cancer Center, Kaplan St., Petah Tikva, 49100, Israel.
E-mail: [email protected]
ª 2016 International Association for the Study of Lung Cancer.
Published by Elsevier Inc. All rights reserved.
ISSN: 1556-0864
http://dx.doi.org/10.1016/j.jtho.2016.10.021
February 2017
treatment for advanced lung cancer focused primarily on
platinum-based doublets.2 In 2002, the concept of
“oncogene addiction” was introduced by Weinstein et al.,3
and since then important advances in understanding lung
cancer cellular signal pathways have been made, leading
to the development of multiple targeted drugs.
The new treatment paradigm in lung cancer focuses
on personalizing treatment on the basis of tumor molecular properties, specifically, on targeting driver genomic
alterations (GAs).4–9 Tumor genotyping allows detection
of oncogenic drivers in approximately 60% of patients
with lung adenocarcinoma, and using the appropriate targeted treatment has a significant impact on
survival.10–12 The most frequently altered genes are
KRAS, EGFR, and anaplastic lymphoma receptor tyrosine
kinase gene (ALK), which are detected in 15% to 25%,
10% to 35%, and 3% to 7% of patients, respectively.13–16
At present, the KRAS driver cannot be effectively targeted;
thus, the current treatment strategy focuses on targeting
GAs in the EGFR and ALK genes. However, current typical
technologies for identifying GAs (i.e., polymerase chain
reaction [PCR], immunohistochemistry, and fluorescence
in situ hybridization [FISH]) cannot identify all druggable
alterations in EGFR exons and/or introns or variants of
ALK rearrangement.5,7,10–12 Other targets that are not
routinely tested in lung adenocarcinoma include ret
proto-oncogine (RET) (1%), ROS1 (1%), Mesenchymalepithelial transition factor (MET) receptor tyrosine
kinase gene (2%), and erb-b2 receptor tyrosine kinase
2 gene (ERBB2) (2%).13
Massive parallel sequencing (i.e., next-generation
sequencing [NGS]), is a new platform that allows detection of numerous GAs in tens to hundreds of genes
simultaneously, as well as detection of rare somatic
mutations. One NGS approach uses PCR amplification
of candidate regions followed by NGS (amplicon
sequencing) and offers sequencing of a narrow gene
spectrum, focusing on point mutations in several hotspot
regions. Other, more comprehensive methods such as
hybrid capture (HC)-based NGS offer broad gene
sequencing and provide extensive genetic information
regarding the broad aberration repertoire, including
information on exon and intron mutations, gene rearrangements, amplifications, etc.
Clinical utilization of NGS began in 2011. Since then,
NGS assays have improved considerably, with respect to
the bioinformatics involved, as well as with respect to
translational/clinical implementation.17 A study from
2013 investigated the genetic repertoire of 2221 clinical
specimens by using HC-based NGS and detected actionable GAs in 76% of tumors, which is three times the
number detected by traditional diagnostic tests.18
NGS is also applicable to liquid biopsies, a novel
strategy for cancer diagnosis. Since circulating tumor
HC-Based NGS and Treatment in Lung Cancer
259
cell-free DNA (cfDNA) was discovered by Stroun et al. in
1987,19 numerous technologies have been developed to
detect and use these genetic materials as an alternative
to invasive tissue biopsy.20–22 Notably, cfDNA may
mirror in real time the spatial genomic repertoire of the
tumor in contrast to pathologic specimens that reflect
the genomic status at the time of biopsy.
Although HC-based NGS allows elaborate molecular
characterization of the tumor, many of the GAs are not
druggable (i.e., without an associated therapy that is
approved and commercially available). The cost of
HC-based NGS, as well as treatment reimbursement
issues, should be considered. Therefore, the role of
HC-based NGS in disease management is unclear. Here,
we assessed the contribution of HC-based NGS to clinical
decision making and clinical outcomes in real-life clinical
practice while also considering other diagnostic tests
carried out according to physicians’ decisions.
Materials and Methods
Patients
This retrospective cohort study included 101
sequential patients with advanced lung cancer who were
treated at the Davidoff Cancer Center at Rabin Medical
Center (Petah Tikva, Israel) between November 2011
and October 2015 and underwent HC-based NGS with
broad gene panels. HC-based NGS was performed upon
the recommendation of the treating physician, mostly on
the basis of young age and smoking history. The results
of standard molecular testing for EGFR mutations and
ALK rearrangements (using validated and approved
diagnostic kits) were negative before HC-based NGS in
80.2% (81 of 101) and 70.3% (71 of 101) of the patients,
respectively. Upfront HC-based NGS was performed on
15 patients because of very little biopsy material. Bloodbased cfDNA analysis served as a salvage method for
gene analysis in cases of tissue exhaustion. The study
was approved by the ethical committee of Rabin Medical
Center (approval no. 0391-14 RMC). The clinicopathologic data were collected from patients’ medical and
electronic charts.
HC-Based NGS Cancer Gene Tests
HC-based NGS was performed off-site on tumor
samples with FoundationOne (Foundation Medicine,
Inc., Cambridge, MA)18 or on blood samples using a
liquid biopsy approach with Guardant360 (Guardant
Health Inc., Redwood City, CA) if the tissue sample had
been exhausted.20 (For extended information on these
HC-based NGS technologies, see the Supplementary
Data). Both assays were paid either out of pocket or by
private insurance coverage. An effort was made to
financially assist patients who could not fund tests.
260 Rozenblum et al
Standard Molecular Pathology Tests
EGFR mutations were assessed with real-time PCR or
narrow-spectrum NGS assays (amplicon-based hotspot
NGS). ALK rearrangements were assessed with immunohistochemistry and/or FISH (for extended information
on these methods, see the Supplementary Data).
Gene Analysis
This study focused on GAs with potential clinical
relevance. Initial analysis (level 1) included GAs associated with U.S. Food and Drug Administration–approved
anticancer therapies (including off-label drugs) for all
cancer types. A subsequent analysis (level 2) included
GAs with appropriate evidence-based targeted agents
with antidriver activity in lung cancer, as recommended
by the National Comprehensive Cancer Network (NCCN)
guidelines for NSCLC.23 GAs associated with investigational treatments were not included in the current
analysis, although one patient went on to participate in a
driver-based clinical trial.
Results
Patient Characteristics
The analysis included 101 patients with advanced
lung cancer. Patient characteristics are summarized in
Table 1. Patients’ median age at diagnosis was 63 years
(range 20–84), 94% of patients had their lung cancer
diagnosed at stage III to IV, 53% were women, 45%
were never-smokers, and 85% had adenocarcinoma.
Routine molecular analysis for EGFR mutations and ALK
rearrangement was performed in 86 and 72 patients,
respectively. In general, no alterations were detected
except in five cases of inconclusive EGFR alterations and
one case of inconclusive ALK rearrangement. In 15 patients (14.9%), HC-based NGS testing was performed
before routine molecular testing. In 52 patients (51.5%),
HC-based NGS was performed before initiation of firstline therapy, and in 49 (48.5%) it was performed after
treatment failure.
Genomic Findings
Tissue HC-based NGS was performed in 82 patients
(81.2%) and liquid HC-based NGS was performed in 19
(18.8%). For the tissue assay, biopsy specimens were
obtained from the lungs of 40 patients and from
metastases of 42 patients. At level 1 analysis, at least one
actionable GA was detected in 84 patients (83.2%),
including in 73 of the 82 patients with tissue HC-based
NGS (89.0%), and in 11 of the 19 patients with liquid
HC-based NGS (57.9%). Overall, in 23 patients (22.8%)
HC-based NGS detected two actionable GAs, and in six
patients (5.9%) it detected three or more actionable GAs.
At level 2 analysis, actionable driver GAs were identified
Journal of Thoracic Oncology
Vol. 12 No. 2
Table 1. Patient, Tumor, and Molecular Testing
Characteristics
Characteristics of the Study Population
(N ¼ 101)
Sex, n (%)
Female
Male
Age at diagnosis, y
Median (range)
Stage at diagnosis, n (%)
IV
III (A/B)
I (A/B) or II (A/B)
Cigarette smoking status, n (%)
Never
Ever
Average pack-years
Unknown
Histopathologic type, n (%)
Adenocarcinoma
Other
Source of sample for HC-based NGS
analysis, n (%)
Tumor sample (solid tissue)
Blood circulating cell-free DNA
Prior testing using standard molecular
methods,a n (%)
EGFR
ALK
Timing of HC-based NGS testing, n (%)
Before first-line therapy
After treatment failure
Numerical
Value
54 (53.5)
47 (46.5)
63 (20–84)
79 (78.2)
16 (15.8)
6 (5.9)
45 (44.6)
53 (52.5)
38
3 (3.0)
86 (85.1)
15 (14.9)
82 (81.2)
19 (18.8)
86 (85.2)
72 (71.3)
52 (51.5)
49 (48.5)
a
The results of testing of all patients were found to be negative except in
five cases in which they were inconclusive for EGFR mutation and in one case
in which they were inconclusive for ALK rearrangement.
ALK, anaplastic lymphoma receptor tyrosine kinase gene; HC, hybrid
capture; NGS, next-generation sequencing.
in 50 patients (49.5%). The most common genes with
mutational or structural change (level 1) involved in the
121 GAs detected in 101 patients were KRAS (18.2%),
EGFR (16.5%), RET (7.4%), serine/threonine kinase 11
gene (STK11) (7.4%), ALK (6.6%), ERBB2 (5.8%), and
MET (5.8%). At level 2 analysis (Table 2), the most
common actionable GAs in 101 patients were sensitizing
EGFR mutations (15%), RET rearrangements (9%), ALK
rearrangements (8%), and MET amplifications and/or
exon 14 mutations (6%). The proportion of level 2
actionable GAs detected in patients who underwent
HC-based NGS before first-line therapy (n ¼ 52) was
54% and that detected in patients who underwent
HC-based NGS after treatment failure 47% (n ¼ 49).
Interestingly, in 15 patients (14.9%), HC-based NGS
detected a GA in EGFR or ALK after negative results of
standard molecular testing. Moreover, four additional
cases were identified as EGFR/ALK positive by previous
standard methods, whereas different drivers were
eventually identified by HC-based NGS. The possible
Pts Receiving
Targeted Therapy
Based on Tissue
HC-Based NGS, n (%)
Pts Receiving
Targeted Therapy
Based on Liquid
HC-Based NGS, n (%)
Total Pts
Receiving
Targeted
Therapy, n (%)
Treated Pts Evaluable
for Tumor Response
Who Had an Objective
Response, n (% ORR)
Median Duration
of Targeted
Treatment,
wk (Range)
Pts with
Ongoing
Targeted
Therapy, n (%)
n (%)
Study population
No drivers with FDA-approved
treatments
Any driver with FDA-approved
treatment
No NCCN-recommended drivers
Any NCCN-recommended driver
EGFR sensitizingb
Exon19 del
L858R
Otherc
RET rearrangement
KIF5B-RET rearrangement
CCDC6-RET rearrangement
PICALM-RET rearrangement
ALK rearrangement
EML4-ALK rearrangement
Intron 19 rearrangement
MET amplification and/or exon
14 mutation
ERBB2 mutation
EGFR (nonsensitizing)
ROS1 rearrangement
CD74-ROS1 rearrangement
MYH9-ROS1 rearrangement
SDC4-ROS1 rearrangement
BRAF V600E mutation
101 (100) 37 of 82 (45)
17 (17)
1 of 9 (11)
6 of 19 (32)
0 of 8 (0)
43 of 101 (43)
1 of 17 (6)
22 of 34a (65)
1 of 1 (100)
26 (1–227)
34 (34)
14 of 43 (33)
0 of 1 (0)
84 (83)
36 of 73 (49)
6 of 11 (55)
42 of 84 (50)
21 of 33 (64)
26 (1–227)
14 of 42 (33)
34 (34)
50 (50)
15 (15)
6 (6)
5 (5)
7 (7)
9 (9)
7 (7)
1 (1)
1 (1)
8 (8)
6 (6)
2 (2)
6 (6)
5 of 30 (17)
31 of 43 (72)
9 of 13 (69)
3 of 5 (60)
3 of 4 (75)
4 of 6 (67)
4 of 7 (57)
4 of 6 (67)
0 of 0 (0)
0 of 1 (0)
7 of 8 (88)
5 of 6 (86)
2 of 2 (100)
3 of 4 (75)
0
6
2
1
1
1
2
1
1
0
0
0
0
1
of
of
of
of
of
of
of
of
of
of
of
of
of
of
4
7
2
1
1
1
2
1
1
0
0
0
0
2
(0)
(86)
(100)
(100)
(100)
(100)
(100)
(100)
(100)
(0)
(0)
(0)
(0)
(50)
5 of 34 (15)
37 of 50 (74)
11 of 15 (73)
4 of 6 (67)
4 of 5 (80)
5 of 7 (71)
6 of 9 (67)
5 of 7 (71)
1 of 1 (100)
0 of 1 (0)
7 of 8 (88)
5 of 6 (83)
2 of 2 (100)
4 of 6 (67)
0 of 2 (0)
21 of 31 (68)
9 of 10 (90)
4 of 4 (100)
4 of 4 (100)
3 of 4 (75)
1 of 4 (25)
1 of 4 (25)
NA
—
5 of 6 (83)
3 of 4 (75)
2 of 2 (100)
3 of 4 (75)
6 (4–26)
30 (1–227)
63 (22–124)
78 (47–124)
63 (27–115)
47 (22–115)
11 (2–30)
13 (4–30)
2 (2)
—
93 (14–227)
32 (14–139)
160 (93–227)
20 (13–32)
0 of 5 (0)
14 of 37 (38)
6 of 11 (55)
3 of 4 (75)
1 of 4 (25)
4 of 5 (80)
1 of 6 (17)
0 of 5 (0)
1 of 1 (100)
—
4 of 7 (57)
2 of 5 (40)
2 of 2 (100)
1 of 4 (25)
5
3
3
1
1
1
1
4
1
2
1
1
0
1
1
0
0
0
0
0
0
of
of
of
of
of
of
of
1
0
0
0
0
0
0
(100)
(100)
(100)
(100)
(100)
(100)
(100)
5
1
2
1
1
0
1
1 of
0 of
2 of
1 of
1 of
—
NA
10 (1–58)
12 (12)
97 (97)
97 (97)
NA
—
2 (2)
1 of
0 of
1 of
1 of
0 of
—
0 of
(5)
(3)
(3)
(1)
(1)
(1)
(1)
of
of
of
of
of
of
of
4
3
3
1
1
1
1
(100)
(33)
(67)
(100)
(100)
(0)
(100)
of
of
of
of
of
of
of
5
3
3
1
1
1
1
(100)
(33)
(67)
(100)
(100)
(0)
(100)
4
1
2
1
1
(25)
(0)
(100)
(100)
(100)
5
1
2
1
1
(20)
(0)
(50)
(100)
(0)
1 (0)
Note: Of 43 patients treated with targeted therapy on the basis of the results of HC-based NGS, 37 were harboring NCCN-recommended drivers for lung cancer. The other six were treated on the basis of other genomic
alterations: NF1 mutation, a high-volume EGFR amplification, KRAS mutation, an NTRK1 variant of unknown significance other than exon 14–activating MET mutation, and a MET variant of unknown significance.
a
The ORR in patients previously tested for EGFR/ALK who are evaluable for tumor response is 62% (18 of 29).
b
The sum of counts for the three EGFR categories differs from the head count of total EGFR sensitizing mutations because of three patients each harboring two EGFR mutations from different EGFR categories. For full
details, please refer to Supplementary Table 1.
c
Two patients who were each harboring two mutations of this category were counted by number of patients (n ¼ 2).
Pts, patients; HC, hybrid capture; NGS, next-generation sequencing; ORR, objective response rate; FDA, U.S. Food and Drug Administration; NCCN, National Comprehensive Cancer Network; del, deletion; RET, ret
proto-oncogene; KIF5B, kinesin family member 5B gene; CCDC6, coiled-coil domain containing 6 gene; NA, not available; PICALM, phosphatidylinositol binding clathrin assembly protein gene; ALK, anaplastic
lymphoma receptor tyrosine kinase gene; EML4, echinoderm microtubule associated protein like 4 gene; MET, Mesenchymal-epithelial transition factor receptor tyrosine kinase gene; ERBB2, erb-b2 receptor tyrosine
kinase 2 gene; CD74, CD74 molecule gene; MYH9, myosin heavy chain 9 gene; SDC4, syndecan 4 gene; NF1, neurofibromic 1; NTKR1, neurotrophic tyrosine kinase, receptor, type 1 gene.
HC-Based NGS and Treatment in Lung Cancer
Characteristic
February 2017
Table 2. Prevalence of Genomic Drivers and Impact on Treatment Decision
261
262 Rozenblum et al
reasons for these findings are discussed in the
Discussion section.
HC-Based NGS and Changes in Treatment
Decisions
After HC-based NGS, 43 patients (42.6%) received
targeted therapies accordingly (see Table 2). Of these 43
patients, six were excluded from the decision impact
analysis as they elected to undergo HC-based NGS before
standard EGFR/ALK testing and their HC-based NGS
assays detected EGFR/ALK GAs that could have been
identified by standard testing. Thus, the calculated
exclusive impact of post-PCR/FISH HC-based NGS testing
was 36.6%, representing 37 of 101 patients with drivers
that could not have been detected otherwise. In 19% of
patients (seven of 37), targeted therapy was administered with a concurrent drug (e.g., erb-b2 receptor tyrosine kinase 2 tyrosine kinase inhibitors (TKIs) with
chemotherapy [see Supplementary Table 1]). Tissue
HC-based NGS had an impact rate of 45.1% (37 of 82
patients), and liquid HC-based NGS had an impact rate of
31.6% (six of 19). In addition to the 43 patients who
received targeted therapy, two were prescribed targeted
therapy but died before initiation of the treatment and
two more died shortly before receiving the HC-based NGS
report indicating a possible targeted therapy. Of the 43
patients treated with targeted therapy after HC-based
NGS, 56% underwent HC-based NGS before first-line
therapy and 44% underwent it after treatment failure.
Twelve patients had EGFR mutations and were
treated with EGFR TKIs. Another 13 patients were
treated with crizotinib: seven had alterations in the ALK
gene, four had exon 14 mutations or amplifications in
the MET gene, and two had ROS1 rearrangements. Six
patients were treated for RET rearrangements with
cabozantinib (n ¼ 5) or alectinib (n ¼ 1). Five patients
had ERBB2 mutations and were treated with erb-b2
receptor tyrosine kinase 2 TKIs (trastuzumab, trastuzumab emtansine, or pertuzumab). One patient was
treated with vemurafenib because of a V600E mutation
in the BRAF gene. Another six patients were treated for
GAs that were not considered druggable: a neurofibromin 1 gene (NF1) mutation (treated with everolimus), a high-volume EGFR amplification (17,
erlotinib), non–exon 14 MET mutation of an activating
nature (crizotinib), MET (exon 13, E999K) and NTRK1
(S396L) variants of unknown significance (crizotinib),
and a KRAS mutation. The patient with the KRAS mutation was enrolled in the SELECT-1 trial and received
selumetinib (or placebo) together with docetaxel. This
patient was omitted from the response analysis because
the nature of the administered treatment was unknown.
A full description of the treated patients and the
administered drugs is provided in Supplementary
Journal of Thoracic Oncology
Vol. 12 No. 2
Table 1. Of the 43 patients who were treated with targeted therapy, the 19 with ALK/EGFR alterations had
their treatment reimbursed by the National Health Insurance Law. Another 23 patients received targeted
treatment under off-label use and paid for it individually,
were assisted through donations from pharmaceutical
companies, or received reimbursement from private insurance companies. As already mentioned, one patient
participated in a clinical trial.
Time to Treatment and HC-Based NGS
Median turnaround time for HC-based NGS, excluding
shipment time, was 13 days (range 8–460), and treatment started a median of 8 days after receipt of the
report (range 0–364). First-line treatment was started
within a median of 44 days from diagnosis (range
32–48) in the group of patients who underwent
HC-based NGS before their first-line treatment and
within a median of 39 days (range 12–122) in the group
that underwent HC-based NGS after first-line treatment.
The difference between the two groups was found to be
not significant (t test p ¼ 0.202). In the latter group, the
median time between initiation of first-line treatment
and HC-based NGS was 169 days (range 22–1612).
Response to Targeted Therapy and Treatment
Duration
Best response to targeted therapy after HC-based
NGS, as measured using the Response Evaluation
Criteria in Solid Tumors, is summarized in Table 2 and
Figure 1. Of the 43 patients treated upon receipt of the
results of HC-based NGS, 34 were evaluable for tumor
response. The overall response rate was 64.7% (62% if
excluding patients not previously tested). Five patients
(14.7%) experienced a systemic complete response (CR);
17 (50%) experienced a partial response (PR), three of
whom achieved a metabolic CR; nine (26.5%) experienced stable disease; and three (8.8%) experienced
progressive disease. For 13 patients, tumor response
evaluation was also available for previous lines of therapy before targeted treatment (Fig. 2). In these cases,
targeted therapy achieved better disease control than
did previous lines (77% versus 54%, respectively).
Among the 15 patients in whom HC-based NGS detected
a GA in EGFR or ALK after negative results of standard
molecular testing, 12 were treated with targeted therapies, and CR or PR was reported in eight (67%). Duration
of targeted treatment ranged from 1 to 227 weeks at
time of analysis (Fig. 3). Median survival was not
reached at the time of analysis; 21 of 43 patients treated
with targeted therapy were still alive, with a mean
follow-up from diagnosis of stage IV disease of
18 months (range 1–58 months).
February 2017
HC-Based NGS and Treatment in Lung Cancer
263
Figure 1. Percentage of best response in 34 patients who received targeted therapy according to hybrid capture (HC)-based
next-generation sequencing (NGS) results. Each bar represents one patient. Number of previous lines and genomic information guiding the targeted therapy in each case are elaborated. Nine of 43 patients not evaluable for tumor response at the
time of tumor response were omitted. *Nonsensitizing EGFR mutation. NTRK1, neurotrophic tyrosine kinase receptor type 1
gene; VUS, variant of unknown sequence; ERBB2, erb-b2 receptor tyrosine kinase 2 gene; RET, ret proto-oncogene;
MET, Mesenchymal-epithelial transition factor receptor tyrosine kinase gene; NF1, neurofibromin 1 gene; ALK, anaplastic
lymphoma receptor tyrosine kinase gene.
TMB and Response to Immunotherapy
During the study period, 33 patients received
immunotherapy (either nivolumab [n ¼ 20] or pembrolizumab [n ¼ 13]), 13 of whom were carrying a KRAS
mutation. Disease control rate for the whole group was
32% (eight of 25 patients evaluable for tumor response).
Data on tumor mutation burden (TMB) was available for
80 patients on whom tissue HC-based NGS was
Figure 2. Percentage of best response for 13 patients in previous treatment lines versus in targeted therapy. Of 43 patients
treated with targeted therapy after hybrid capture–based next-generation sequencing results, 23 had previous lines of
treatment and 13 were evaluable for tumor response. Each vertical section represents one patient, encompassing one or two
previous lines of treatment. Patient number and genomic information guiding the targeted therapy in each case is elaborated. The results of standard testing of all patients for EGFR/ALK alterations before hybrid capture–based next-generation
sequencing were negative. ALK, anaplastic lymphoma receptor tyrosine kinase gene; ERBB2, erb-b2 receptor tyrosine kinase
2 gene; MET, Mesenchymal-epithelial transition factor receptor tyrosine kinase gene; NTRK1, neurotrophic tyrosine kinase
receptor type 1 gene; RET, ret proto-oncogene; PLD, platinum doublet; PLT, platinum triplet (i.e., platinum doublet with
bevacizumab); Peme., pemetrexed; Nivo., Nivolumab; Tra., trastuzumab; Per., pertuzumab; Vin., vinorelbine; Pac., paclitaxel; TDM1, trastuzumab emtansine, NA, not available.
264 Rozenblum et al
Journal of Thoracic Oncology
Vol. 12 No. 2
Figure 3. Treatment duration with targeted therapy for hybrid capture (HC)-based next-generation sequencing (NGS)identified drivers (weeks). Of 43 patients treated with targeted therapy after receipt of HC-based NGS results, 41 had
available data on duration of targeted treatment. Each bar represents one patient, and the number of weeks of treatment is
stated to the right of the bar. If initial targeted therapy was immediately followed by another targeted therapy addressing
the same gene, the duration was measured as a whole for the sum of the durations of the treatment. *Other than exon 14
activating MET mutation. **Nonsensitizing EGFR mutation. ***EGFR high-volume amplification (17). ALK, anaplastic lymphoma receptor tyrosine kinase gene; ERBB2, erb-b2 receptor tyrosine kinase 2 gene; MET, Mesenchymal-epithelial transition
factor receptor tyrosine kinase gene; VUS, variation of unknown sequence; NTRK1, neurotrophic tyrosine kinase receptor
type 1 gene; RET, ret proto-oncogene; NF1, neurofibromin 1 gene.
performed. Patients who were identified by HC-based
NGS as not carrying any treatment-associated driver
(n ¼ 17) (level 1 analysis) had the highest mean TMB
(11.8 ± 5 mutations/MB]) and the highest overall
response rate to immunotherapy (33%). Among KRAS
mutation carriers, the average TMB was 6.7 ± 4, and the
immunotherapy objective response rate was 10%. As
demonstrated by Rizvi et al.,24 our results also showed
that a higher TMB was associated, although not
significantly, with improved objective response to
immunotherapy, with a mean TMB of 9.6 ± 0.8 in patients with a PR as compared with a mean TMB of 5.8
± 5 in patients with stable or progressive disease
(p ¼ 0.27). Immunotherapy response rates according to
driver type and TMB data are listed in Table 3.
Discussion
The diagnostic landscape in NSCLC is evolving
rapidly, with new tumor profiling tests becoming available every year. The importance of performing multiplex
genetic testing is already well established, and although
it is recommended by the National Comprehensive
Cancer Network (NCCN), it has yet to be widely adopted.
Despite its strengths,25,26 HC-based NGS has several
limitations, and its efficacy as a practical tool in therapeutic decision making is yet to be thoroughly evaluated.
February 2017
Table 3. Best Response in 33 Patients Treated with Immunotherapy
Disease Control Rate
(Treated Pts Evaluable
for Tumor Response
Whose Disease Was
Controlled), n (%)
Median
Treatment
Duration
(Range) wk
Pts with
Ongoing
Therapy, n (%)
Pts Treated with
Immunotherapy, n (%)
Total
No level 1 drivers (with FDA-approved
treatments)
7.1 ± 7.0
11.8 ± 5.1
33 of 101 (33)
6 of 17 (35)
3 of 25 (12)
2 of 6 (33)
8 of 25 (32)
4 of 6 (67)
15 (1–31)
19 (1–30)
6 of 33 (18)
3 of 6 (50)
No level 2 drivers
(NCCN-recommended)
KRAS muts only, amps excluded
9.8 ± 8.2
27 of 51 (53)
3 of 21 (14)
8 of 21 (38)
16 (1–31)
6 of 27 (22)
6.7 ± 4.1
13b of 22 (59)
1 of 10 (10)
4 of 10 (40)
15 (1–31)
1 of 13 (8)
BRAF all muts
EGFR sensitizing and other muts, amps
excluded
6.7 ± 5.9
6.1 ± 6.6
0 of 2 (0)
1 of 18 (6)
—
NA
—
NA
—
2 (2)
—
0 of 0 (0)
ROS1
MET, including all types of known muts
and amps
5.0 ± 5.6
4.5 ± 2.8
0 of 3 (0)
0 of 7 (0)
—
—
—
—
—
—
—
—
ERBB2 muts only, amps excluded
RET
3.8 ± 1.2
3.5 ± 3.6
2 of 5 (40)
4b of 9 (44)
0 of 1 (0)
0 of 4 (0)
0 of 1 (0)
1 of 4 (25)
NA
11 (1–26)
0 of 0 (0)
0 of 0 (0)
ALK
Response type
3.3 ± 3.4
Average
TMB mut/MB
9.6 ± 0.8
3.6 ± 2.5
6.1 ± 4.8
0 of 8 (0)
n (with available
TMB data), n (%)
2 of 18 (11)
2 of 18 (11)
14 of 18 (78)
—
—
—
—
Characteristic
PR
Stable disease
PD
Note: Types of immunotherapy administered are nivolumab (13 of 33 patients) and pembrolizumab (20 of 33 patients); two patients received nivolumab with a concomitant drug: crizotinib (KRAS þ NTRK1 VUS carrier,
response not evaluated because of patient death) and neratinib (ERBB2 carrier, PD). Upper part of table represents response rates with regard to driver status. Lower part of table shows mean TMB with respect to
response type.
a
Average TMB was calculated on the basis of tissue hybrid capture–based next-generation sequencing data, with two patients from this group lacking TMB data (n ¼ 80).
b
One patient was a carrier of two concomitant drivers: a RET fusion and a KRAS mutation. He was counted twice as part of the treated patients in the KRAS and RET groups.
TMB, tumor mutation burden; mut, mutation; amp, amplification; MB, megabyte; Pts, patients; ORR, objective response rate; FDA, U.S. Food and Drug Administration; NCCN, National Comprehensive Cancer
Network; MET, Mesenchymal-epithelial transition factor receptor tyrosine kinase gene; ERBB2, erb-b2 receptor tyrosine kinase 2 gene; RET, RET proto-oncogene; ALK, anaplastic lymphoma receptor tyrosine kinase
gene; PR, partial response; PD, progressive disease.
HC-Based NGS and Treatment in Lung Cancer
Average TMB
mut/MBa
Treated Pts Evaluable
for Tumor Response
Who Had an Objective
Response, n (% ORR)
265
266 Rozenblum et al
At present, the technological power of HC-based NGS has
surpassed the bioinformatic capabilities required to fully
understand the genetic findings. Moreover, many GAs
are not clinically applicable in the absence of specific
targeted treatments that can block the involved oncogenic pathway. In this study, GAs associated with U.S.
Food and Drug Administration–approved therapies were
assessed, and although presumably targetable drivers
were identified in 83% of patients (in level 1 analysis),
only half of those patients were actually treated with
targeted therapy. A leading example contributing to the
creation of this gap is the KRAS gene, which was the most
abundant driver. Another limitation is the difficulty of
prioritizing drivers for targeted therapy when multiple
drivers are present. However, compared with other
molecular diagnostic methods, including amplicon-based
NGS, HC-based NGS offers the ability to detect the full
spectrum of clinically relevant GAs (point mutations,
small insertions and deletions, copy number alterations,
and genomic rearrangements/fusions) in a single assay,
avoiding the need for FISH or other techniques and
thereby saving precious tumor tissue and time. In addition, HC-based NGS permits the capture of large intronic
regions in which rearrangements or fusions can be
detected, and it offers statistically meaningful representation of gene amplifications and deletions, so that
numerous types of cancer genome alterations can be
enriched from a single sample.26 The possibility of
identifying and potentially treating previously unknown
oncogenic GAs is another advantage of HC-based NGS, as
performed in our study. Compared with data from
amplicon-based NGS, the data on the impact of HC–based
NGS on clinical decisions in lung cancer are limited, and
further investigation is warranted. Recently, Takeda
et al. prospectively applied amplicon-based NGS panels
that cover both mutational hotspots in 22 genes related
to lung and colon tumorigenesis, and 72 major variants
of ALK, RET, ROS1, and NTRK1 fusion transcripts.27
Actionable genetic alterations were identified in 40%
of the 110 study patients, and 21% received targeted
therapy, most of whom (83%) bore an EGFR or ALK
driver. Notably, as in the Takeda et al. study,27 prior
standard EGFR and ALK testing was not performed,
(or used for comparison), and the real advantage of NGS
over standard genomic testing is yet to be evaluated.
Other groups have also developed amplicon-based NGS
methods,28–30 as compared with the more complex and
expensive HC-based NGS. Drilon et al. used HC-based
NGS to evaluate 31 patients in whom previous testing
for alterations in 11 oncogenic genes by non-NGS
methods had yielded negative results.31 Actionable GAs
associated with a targeted drug according to NCCN
guidelines were identified in eight patients (26%), six of
whom eventually received targeted therapy.
Journal of Thoracic Oncology
Vol. 12 No. 2
This study provides a retrospective view on the
clinical use of HC-based NGS technologies for lung cancer
diagnosis in the real-life setting. Special emphasis was
put on patients with EGFR/ALK-negative results and a
clinical presentation suggesting that they might have a
genomic driver. HC-based NGS before standard molecular testing was preferred in the case of a limited amount
of tissue sample, as was the case for 15% of patients in
our study, in which the treating physician used HC-based
NGS upfront to prevent tissue exhaustion and the
potential need for another biopsy. We also used
HC-based NGS of cfDNA for 19 tissue-exhausted patients,
alleviating the need for an additional invasive
biopsy.20,32 In this study, we demonstrated that in
addition to standard molecular testing (for EGFR/ALK
mutation), HC-based NGS resulted in a changed treatment strategy for 37% of patients (n ¼ 37). In 32 of
these 37 cases (86%), the chosen treatment replaced
chemotherapy, thus being more effective and less toxic
and offering potential for improved quality of life and
survival.7,10,12,33 Although defining clinical benefit from
treatment in lung cancer may be challenging, in our
study, 65% of the patients treated with targeted therapy
achieved an objective CR or PR (62% if patients not
previously tested are excluded). This rate is evidence of
the high accuracy of HC-based NGS, and it is even more
impressive considering that almost half of the treated
patients (21 of 43 [48.8%]) received targeted therapy
after failure of first-line treatment. As expected, patients
with EGFR/ALK mutation had better response rates than
did patients with other drivers, with rates of CR or PR of
82% (14 of 17) and 47% (eight of 17), respectively. The
use of tissue for HC-based NGS is presumably preferable
if available. In our cohort, a higher fraction of tissue
biopsies resulted in changed treatment strategies (37 of
82 [45%]) compared with liquid biopsies (six of 19
[32%]), although the difference was not significant.
Our results suggest that broad use of HC-based NGS
in lung cancer may provide a key for therapeutic decision making and that negative results by standard
molecular tests (e.g., PCR and FISH) should not preclude
HC-based NGS testing when the probability of identifying
a targetable driver is high (e.g., adenocarcinoma in a
young patient with a history of a small amount of or
never smoking). However, guidelines from the College of
American Pathologists, the International Association for
the Study of Lung Cancer, and the Association for
Molecular Pathology recommend performing molecular
testing based on histologic type, irrespective of clinical
characteristics (e.g., smoking history, sex, and race).34
Our study demonstrated a high rate of false-negative
results of standard molecular testing for EGFR and ALK
mutations (15% [n ¼ 15], with 12 of those patients
(80%) treated with targeted therapy after HC-based NGS).
February 2017
This could be explained in a number of ways. First, technical failures and sensitivity issues can explain seven of
the 15 GAs that were covered by standard assays but
missed. Second, eight of 15 patients carried an EGFR/ALK
mutation that could not have been picked up by
the standard real-time PCR/FISH assays used (see
Supplementary Table 1). For example, two of five patients
who were identified by HC-based NGS as having an ALK
rearrangement after previous negative FISH results had
intron 19 rearrangement (i.e., lack of translocation).
One patient, who had a high-volume EGFR amplification
(17), was exceptionally treated by EGFR TKIs and was
included in this group. It is also noteworthy that four
patients who were identified as EGFR/ALK positive by
standard testing experienced progressive disease when
treated with EGFR TKIs or an ALK TKI. In two of these four
patients, HC-based NGS detected ALK and MET GAs, and
both responded to the appropriate targeted drug.
One of the current challenges in clinically deploying
HC-based NGS is financial. In our clinical setting, the cost
of one assay of tissue HC-based NGS in 2015 was
approximately $5274, whereas liquid HC-based NGS cost
$4838. The compared total cost of testing individually
for other drivers in addition to EGFR/ALK in cases in
which a CR or PR was reached (ERBB2, ROS1, RET, and
MET) was approximately $4300. Aside from cost, parallel gene testing using HC-based NGS might be more
modest in terms of tissue consumption compared with
sequential driver testing. In our setting, the amount of
tissue required for tissue HC-based NGS is roughly
10 times greater than the amount required for standard
testing of one driver (e.g., EGFR testing using real-time
PCR). An HC-based NGS assay testing for more than
10 drivers would therefore be more tissue economic
than testing for each of the drivers separately.
Another potential use of HC-based NGS is in estimating response to immunotherapy. Our results confirm
previous data24 showing a possible association between
response to immunotherapy and TMB. Interestingly
enough, we show here a higher TMB within the group
that did not have treatment-associated drivers and in
patients with KRAS mutations. These groups were
possibly also associated with better response to
immunotherapy.
Our study is restricted by its retrospective nature, by
its relatively small sample size, and by its being a singlecenter study. In addition, the high percentage of neversmokers, the preponderance of female patients, and
the relatively young median age of our patient group
represent a selection bias with a high pretest probability
for the existence of driver mutation. The results of large
future prospective trials such as the National Lung
Matrix Trial in the United Kingdom35 and the Molecular
Analysis for Therapy Choice Program, which is being led
HC-Based NGS and Treatment in Lung Cancer
267
by the U.S. National Cancer Institute,36 are thus
eagerly anticipated. Nevertheless, the high impact of
HC-based NGS on treatment strategy, and the high
overall response rate observed in this study, highlight
the need for identifying molecular drivers and support
the implementation of HC-based NGS in lung cancer.
Supplementary Data
Note: To access the supplementary material accompanying this article, visit the online version of the Journal of
Thoracic Oncology at www.jto.org and at http://dx.doi.
org/10.1016/j.jtho.2016.10.021.
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