Abstract
The identification of non-small cell lung cancer (NSCLC) patients potentially responsive to targeted therapies relies on a number of relevant biomarkers, including EGFR, ALK, ROS-1, and PD-L1. Biomarker identification is most commonly based on surgical sample collection. However, when tissues are difficult to reach or when multiple analyses are necessary to monitor tumor progression and treatment response, liquid biopsy is a valid noninvasive alternative. This analysis, which is preferentially performed on circulating tumor DNA (ctDNA) extracted from plasma samples, has the major advantage of reducing the inherent risks and discomfort of tissue biopsy. However, a major disadvantage is that it yields only a low number of ctDNA targets. Thus, to avoid false-positive and false-negative results, it is important to adopt and validate technologies with high sensitivity and specificity in the pre-analytical phase of sampling. This review succinctly addresses the principal methodologies for analyzing plasma-derived ctDNA in NSCLC patients.
References
1.
Lindeman
NI
, Cagle
PT
, Aisner
DL
, Arcila
ME
, Beasley
MB
, Bernicker
EH
, et al Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology
. Arch Pathol Lab Med
. 2018
Mar
;142
(3
):321
–46
.
[PubMed]
0003-99852.
Mok
TS
, Wu
YL
, Thongprasert
S
, Yang
CH
, Chu
DT
, Saijo
N
, et al Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma
. N Engl J Med
. 2009
Sep
;361
(10
):947
–57
.
[PubMed]
0028-47933.
Wu
YL
, Zhou
C
, Hu
CP
, Feng
J
, Lu
S
, Huang
Y
, et al Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): an open-label, randomised phase 3 trial
. Lancet Oncol
. 2014
Feb
;15
(2
):213
–22
.
[PubMed]
1470-20454.
Jänne
PA
, Yang
JC
, Kim
DW
, Planchard
D
, Ohe
Y
, Ramalingam
SS
, et al AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer
. N Engl J Med
. 2015
Apr
;372
(18
):1689
–99
.
[PubMed]
0028-47935.
Solomon
BJ
, Mok
T
, Kim
DW
, Wu
YL
, Nakagawa
K
, Mekhail
T
, et al; PROFILE 1014 Investigators
. First-line crizotinib versus chemotherapy in ALK-positive lung cancer
. N Engl J Med
. 2014
Dec
;371
(23
):2167
–77
.
[PubMed]
0028-47936.
Hida
T
, Nokihara
H
, Kondo
M
, Kim
YH
, Azuma
K
, Seto
T
, et al Alectinib versus crizotinib in patients with ALK-positive non-small-cell lung cancer (J-ALEX): an open-label, randomised phase 3 trial
. Lancet
. 2017
Jul
;390
(10089
):29
–39
.
[PubMed]
0140-67367.
Shaw
AT
, Ou
SH
, Bang
YJ
, Camidge
DR
, Solomon
BJ
, Salgia
R
, et al Crizotinib in ROS1-rearranged non-small-cell lung cancer
. N Engl J Med
. 2014
Nov
;371
(21
):1963
–71
.
[PubMed]
0028-47938.
Reck
M
, Rodríguez-Abreu
D
, Robinson
AG
, Hui
R
, Csőszi
T
, Fülöp
A
, et al; KEYNOTE-024 Investigators
. Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer
. N Engl J Med
. 2016
Nov
;375
(19
):1823
–33
.
[PubMed]
0028-47939.
Pisapia
P
, Pepe
F
, Smeraglio
R
, Russo
M
, Rocco
D
, Sgariglia
R
, et al Cell free DNA analysis by SiRe® next generation sequencing panel in non small cell lung cancer patients: focus on basal setting
. J Thorac Dis
. 2017
Oct
;9
(S13
Suppl 13
):S1383
–90
.
[PubMed]
2072-143910.
Ofiara
LM
, Navasakulpong
A
, Ezer
N
, Gonzalez
AV
. The importance of a satisfactory biopsy for the diagnosis of lung cancer in the era of personalized treatment
. Curr Oncol
. 2012
Jun
;19
(0
Suppl 1
):S16
–23
.
[PubMed]
1198-005211.
Wang
S
, Yu
B
, Ng
CC
, Mercorella
B
, Selinger
CI
, O’Toole
SA
, et al The suitability of small biopsy and cytology specimens for EGFR and other mutation testing in non-small cell lung cancer
. Transl Lung Cancer Res
. 2015
Apr
;4
(2
):119
–25
.
[PubMed]
2218-675112.
Malapelle
U
, Bellevicine
C
, De Luca
C
, Salatiello
M
, De Stefano
A
, Rocco
D
, et al EGFR mutations detected on cytology samples by a centralized laboratory reliably predict response to gefitinib in non-small cell lung carcinoma patients
. Cancer Cytopathol
. 2013
Oct
;121
(10
):552
–60
.
[PubMed]
1934-662X13.
Crowley
E
, Di Nicolantonio
F
, Loupakis
F
, Bardelli
A
. Liquid biopsy: monitoring cancer-genetics in the blood
. Nat Rev Clin Oncol
. 2013
Aug
;10
(8
):472
–84
.
[PubMed]
1759-477414.
Schwarzenbach
H
, Hoon
DS
, Pantel
K
. Cell-free nucleic acids as biomarkers in cancer patients
. Nat Rev Cancer
. 2011
Jun
;11
(6
):426
–37
.
[PubMed]
1474-175X15.
Karachaliou
N
, Mayo-de las Casas
C
, Queralt
C
, de Aguirre
I
, Melloni
B
, Cardenal
F
, et al; Spanish Lung Cancer Group
. Association of EGFR L858R Mutation in Circulating Free DNA With Survival in the EURTAC Trial
. JAMA Oncol
. 2015
May
;1
(2
):149
–57
.
[PubMed]
2374-243716.
Malapelle
U
, Pisapia
P
, Rocco
D
, Smeraglio
R
, di Spirito
M
, Bellevicine
C
, et al Next generation sequencing techniques in liquid biopsy: focus on non-small cell lung cancer patients
. Transl Lung Cancer Res
. 2016
Oct
;5
(5
):505
–10
.
[PubMed]
2218-675117.
Malapelle
U
, Mayo de-Las-Casas
C
, Rocco
D
, Garzon
M
, Pisapia
P
, Jordana-Ariza
N
, et al Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients
. Br J Cancer
. 2017
Mar
;116
(6
):802
–10
.
[PubMed]
0007-092018.
Pantel
K
, Speicher
MR
. The biology of circulating tumor cells
. Oncogene
. 2016
Mar
;35
(10
):1216
–24
.
[PubMed]
0950-923219.
Reclusa
P
, Taverna
S
, Pucci
M
, Durendez
E
, Calabuig
S
, Manca
P
, et al Exosomes as diagnostic and predictive biomarkers in lung cancer
. J Thorac Dis
. 2017
Oct
;9
(S13
Suppl 13
):S1373
–82
.
[PubMed]
2072-143920.
Rolfo
C
, Giallombardo
M
, Reclusa
P
, Sirera
R
, Peeters
M
. Exosomes in lung cancer liquid biopsies: two sides of the same coin?
Lung Cancer
. 2017
Feb
;104
:134
–5
.
[PubMed]
0169-500221.
Molina-Vila
MA
, de-Las-Casas
CM
, Bertran-Alamillo
J
, Jordana-Ariza
N
, González-Cao
M
, Rosell
R
. cfDNA analysis from blood in melanoma
. Ann Transl Med
. 2015
Nov
;3
(20
):309
.
[PubMed]
2305-583922.
Sacher
AG
, Komatsubara
KM
, Oxnard
GR
. Application of Plasma Genotyping Technologies in Non-Small Cell Lung Cancer: A Practical Review
. J Thorac Oncol
. 2017
Sep
;12
(9
):1344
–56
.
[PubMed]
1556-086423.
Mead
R
, Duku
M
, Bhandari
P
, Cree
IA
. Circulating tumour markers can define patients with normal colons, benign polyps, and cancers
. Br J Cancer
. 2011
Jul
;105
(2
):239
–45
.
[PubMed]
0007-092024.
Normanno
N
, Denis
MG
, Thress
KS
, Ratcliffe
M
, Reck
M
. Guide to detecting epidermal growth factor receptor (EGFR) mutations in ctDNA of patients with advanced non-small-cell lung cancer
. Oncotarget
. 2017
Feb
;8
(7
):12501
–16
.
[PubMed]
1949-255325.
Toro
PV
, Erlanger
B
, Beaver
JA
, Cochran
RL
, VanDenBerg
DA
, Yakim
E
, et al Comparison of cell stabilizing blood collection tubes for circulating plasma tumor DNA
. Clin Biochem
. 2015
Oct
;48
(15
):993
–8
.
[PubMed]
0009-912026.
Schmidt
B
, Reinicke
D
, Reindl
I
, Bork
I
, Wollschläger
B
, Lambrecht
N
, et al Liquid biopsy - Performance of the PAXgene® Blood ccfDNA Tubes for the isolation and characterization of cell-free plasma DNA from tumor patients
. Clin Chim Acta
. 2017
Jun
;469
:94
–8
.
[PubMed]
0009-898127.
Page
K
, Guttery
DS
, Zahra
N
, Primrose
L
, Elshaw
SR
, Pringle
JH
, et al Influence of plasma processing on recovery and analysis of circulating nucleic acids
. PLoS One
. 2013
Oct
;8
(10
):e77963
.
[PubMed]
1932-620328.
Tie
J
, Kinde
I
, Wang
Y
, Wong
HL
, Roebert
J
, Christie
M
, et al Circulating tumor DNA as an early marker of therapeutic response in patients with metastatic colorectal cancer
. Ann Oncol
. 2015
Aug
;26
(8
):1715
–22
.
[PubMed]
0923-753429.
Mauger
F
, Dulary
C
, Daviaud
C
, Deleuze
JF
, Tost
J
. Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma
. Anal Bioanal Chem
. 2015
Sep
;407
(22
):6873
–8
.
[PubMed]
1618-264230.
Mayo-de-Las-Casas
C
, Garzón Ibáñez
M
, Jordana-Ariza
N
, García-Peláez
B
, Balada-Bel
A
, Villatoro
S
, et al An update on liquid biopsy analysis for diagnostic and monitoring applications in non-small cell lung cancer
. Expert Rev Mol Diagn
. 2018
Jan
;18
(1
):35
–45
.
[PubMed]
1473-715931.
Douillard
JY
, Ostoros
G
, Cobo
M
, Ciuleanu
T
, Cole
R
, McWalter
G
, et al Gefitinib treatment in EGFR mutated caucasian NSCLC: circulating-free tumor DNA as a surrogate for determination of EGFR status
. J Thorac Oncol
. 2014
Sep
;9
(9
):1345
–53
.
[PubMed]
1556-086432.
Kimura
H
, Kasahara
K
, Kawaishi
M
, Kunitoh
H
, Tamura
T
, Holloway
B
, et al Detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer
. Clin Cancer Res
. 2006
Jul
;12
(13
):3915
–21
.
[PubMed]
1078-043233.
Kim
HR
, Lee
SY
, Hyun
DS
, Lee
MK
, Lee
HK
, Choi
CM
, et al Detection of EGFR mutations in circulating free DNA by PNA-mediated PCR clamping
. J Exp Clin Cancer Res
. 2013
Aug
;32
(1
):50
.
[PubMed]
0392-907834.
Kimura
H
, Suminoe
M
, Kasahara
K
, Sone
T
, Araya
T
, Tamori
S
, et al Evaluation of epidermal growth factor receptor mutation status in serum DNA as a predictor of response to gefitinib (IRESSA)
. Br J Cancer
. 2007
Sep
;97
(6
):778
–84
.
[PubMed]
0007-092035.
Goto
K
, Ichinose
Y
, Ohe
Y
, Yamamoto
N
, Negoro
S
, Nishio
K
, et al Epidermal growth factor receptor mutation status in circulating free DNA in serum: from IPASS, a phase III study of gefitinib or carboplatin/paclitaxel in non-small cell lung cancer
. J Thorac Oncol
. 2012
Jan
;7
(1
):115
–21
.
[PubMed]
1556-086436.
Douillard
JY
, Ostoros
G
, Cobo
M
, Ciuleanu
T
, McCormack
R
, Webster
A
, et al First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study
. Br J Cancer
. 2014
Jan
;110
(1
):55
–62
.
[PubMed]
0007-092037.
Pasquale
R
, Fenizia
F
, Esposito Abate
R
, Sacco
A
, Esposito
C
, Forgione
L
, et al Assessment of high-sensitive methods for the detection of EGFR mutations in circulating free tumor DNA from NSCLC patients
. Pharmacogenomics
. 2015
;16
(10
):1135
–48
.
[PubMed]
1462-241638.
Wu
YL
, Zhou
C
, Liam
CK
, Wu
G
, Liu
X
, Zhong
Z
, et al First-line erlotinib versus gemcitabine/cisplatin in patients with advanced EGFR mutation-positive non-small-cell lung cancer: analyses from the phase III, randomized, open-label, ENSURE study
. Ann Oncol
. 2015
Sep
;26
(9
):1883
–9
.
[PubMed]
0923-753439.
Malapelle
U
, Sirera
R
, Jantus-Lewintre
E
, Reclusa
P
, Calabuig-Fariñas
S
, Blasco
A
, et al Profile of the Roche cobas® EGFR mutation test v2 for non-small cell lung cancer
. Expert Rev Mol Diagn
. 2017
Mar
;17
(3
):209
–15
.
[PubMed]
1473-715940.
Thress
KS
, Brant
R
, Carr
TH
, Dearden
S
, Jenkins
S
, Brown
H
, et al EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291
. Lung Cancer
. 2015
Dec
;90
(3
):509
–15
.
[PubMed]
0169-500241.
Malapelle
U
, de Luca
C
, Vigliar
E
, Ambrosio
F
, Rocco
D
, Pisapia
P
, et al EGFR mutation detection on routine cytological smears of non-small cell lung cancer by digital PCR: a validation study
. J Clin Pathol
. 2016
May
;69
(5
):454
–7
.
[PubMed]
0021-974642.
Vendrell
JA
, Mau-Them
FT
, Béganton
B
, Godreuil
S
, Coopman
P
, Solassol
J
. Circulating Cell Free Tumor DNA Detection as a Routine Tool forLung Cancer Patient Management
. Int J Mol Sci
. 2017
Jan
;18
(2
):18
.
[PubMed]
1661-659643.
Yung
TK
, Chan
KC
, Mok
TS
, Tong
J
, To
KF
, Lo
YM
. Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients
. Clin Cancer Res
. 2009
Mar
;15
(6
):2076
–84
.
[PubMed]
1078-043244.
Sacher
AG
, Paweletz
C
, Dahlberg
SE
, Alden
RS
, O’Connell
A
, Feeney
N
, et al Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer
. JAMA Oncol
. 2016
Aug
;2
(8
):1014
–22
.
[PubMed]
2374-243745.
Oxnard
GR
, Thress
KS
, Alden
RS
, Lawrance
R
, Paweletz
CP
, Cantarini
M
, et al Association Between Plasma Genotyping and Outcomes of Treatment With Osimertinib (AZD9291) in Advanced Non-Small-Cell Lung Cancer
. J Clin Oncol
. 2016
Oct
;34
(28
):3375
–82
.
[PubMed]
0732-183X46.
Mok
TS
, Wu
YL
, Ahn
MJ
, Garassino
MC
, Kim
HR
, Ramalingam
SS
, et al; AURA3 Investigators
. Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer
. N Engl J Med
. 2017
Feb
;376
(7
):629
–40
.
[PubMed]
0028-479347.
Bentley
DR
, Balasubramanian
S
, Swerdlow
HP
, Smith
GP
, Milton
J
, Brown
CG
, et al Accurate whole human genome sequencing using reversible terminator chemistry
. Nature
. 2008
Nov
;456
(7218
):53
–9
.
[PubMed]
0028-083648.
Rothberg
JM
, Hinz
W
, Rearick
TM
, Schultz
J
, Mileski
W
, Davey
M
, et al An integrated semiconductor device enabling non-optical genome sequencing
. Nature
. 2011
Jul
;475
(7356
):348
–52
.
[PubMed]
0028-083649.
Li
J
, Batcha
AM
, Grüning
B
, Mansmann
UR
. An NGS Workflow Blueprint for DNA Sequencing Data and Its Application in Individualized Molecular Oncology
. Cancer Inform
. 2016
Apr
;14
Suppl 5
:87
–107
.
[PubMed]
1176-935150.
Clinical and Laboratory Standards Institute
. Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved Guideline. (CLSI document MM09-A2)
. 2nd ed. Wayne (PA)
: Clinical and Laboratory Standards Institute
; 2014
.51.
Couraud
S
, Vaca-Paniagua
F
, Villar
S
, Oliver
J
, Schuster
T
, Blanché
H
, et al; BioCAST/IFCT-1002 investigators
. Noninvasive diagnosis of actionable mutations by deep sequencing of circulating free DNA in lung cancer from never-smokers: a proof-of-concept study from BioCAST/IFCT-1002
. Clin Cancer Res
. 2014
Sep
;20
(17
):4613
–24
.
[PubMed]
1078-043252.
Reckamp
KL
, Melnikova
VO
, Karlovich
C
, Sequist
LV
, Camidge
DR
, Wakelee
H
, et al A Highly Sensitive and Quantitative Test Platform for Detection of NSCLC EGFR Mutations in Urine and Plasma
. J Thorac Oncol
. 2016
Oct
;11
(10
):1690
–700
.
[PubMed]
1556-086453.
Paweletz
CP
, Sacher
AG
, Raymond
CK
, Alden
RS
, O’Connell
A
, Mach
SL
, et al Bias-Corrected Targeted Next-Generation Sequencing for Rapid, Multiplexed Detection of Actionable Alterations in Cell-Free DNA from Advanced Lung Cancer Patients
. Clin Cancer Res
. 2016
Feb
;22
(4
):915
–22
.
[PubMed]
1078-043254.
Newman
AM
, Bratman
SV
, To
J
, Wynne
JF
, Eclov
NC
, Modlin
LA
, et al An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage
. Nat Med
. 2014
May
;20
(5
):548
–54
.
[PubMed]
1078-895655.
Normanno
N
, Maiello
MR
, Chicchinelli
N
, Iannaccone
A
, Esposito
C
, De Cecio
R
, et al Targeting the EGFR T790M mutation in non-small-cell lung cancer
. Expert Opin Ther Targets
. 2017
Feb
;21
(2
):159
–65
.
[PubMed]
1472-822256.
Piotrowska
Z
, Niederst
MJ
, Karlovich
CA
, Wakelee
HA
, Neal
JW
, Mino-Kenudson
M
, et al Heterogeneity Underlies the Emergence of EGFRT790 Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third-Generation EGFR Inhibitor
. Cancer Discov
. 2015
Jul
;5
(7
):713
–22
.
[PubMed]
2159-827457.
Remon
J
, Menis
J
, Hasan
B
, Peric
A
, De Maio
E
, Novello
S
, et al The APPLE Trial: Feasibility and Activity of AZD9291 (Osimertinib) Treatment on Positive PLasma T790M in EGFR-mutant NSCLC Patients. EORTC 1613
. Clin Lung Cancer
. 2017
Sep
;18
(5
):583
–8
.
[PubMed]
1525-7304© 2018 S. Karger AG, Basel
2018
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