Research Institute for Disease of the Chest,
Kyushu University, Fukuoka, Japan
Eiji Iwama
Tissue or Liquid Biopsy?
~For Diagnosis, Monitoring mutations~
16th Nov. 2017. ESMO Preceptorship ProgramNon-Small-Cell Lung Cancer @Singapore
1
Haber DA, et al. Cancer Discovery, 2014 Jun 4(6);650-61.
Circulating tumor DNA
Circulating free DNA (cfDNA)
≒ Circulating tumor DNA (ctDNA)
2
Invasive methodImaging
<Chest X-ray>
<Chest CT>
<Bronchoscopy>
<CT-guided biopsy>
Pathological diagnosis
Genetic testing
Treatment
Diagnostic process for genetic signature of NSCLC
Recurrence 3
Imaging
<Chest X-ray>
<Chest CT>
Genetic testing
TreatmentRecurrence
Liquid Biopsy
Advantage:
• Useful for patients whose tumor is
inaccessible
• Monitoring during treatment
• Potentially provide a genetic signature
of the patient’s entire tumor
(information for heterogeneity)
Diagnostic process for genetic signature of NSCLC
4
Cut-off is determined with the ΔCt value. ΔCt = Ct (Target allele) - Ct (Control allele)
Allele specific PCR-based method(Cobas ®, ARMS ®)
(Detection limit = 1%)
5
1 mutant40 wild
5 mutant1,000 wild
Digital PCR
= 2.5%= 0.5%
✓Fluidigm Corporation’s BioMark HD System for digital PCR and qPCR
Detection limit; 0.1%~0.01%
(Allele Specific-PCRcannot detect)
(Allele Specific-PCR can detect !!)
✓Bio-Rad’s QX100 droplet digital PCR System
6
Next Generation Sequencing (NGS)
(http://www.slideshare.net/ueb52/introduction-to-next-generation-sequencing-v2)7
Sensitivity and Specificity of liquid biopsy for detecting T790M in plasma
Tissue testing (usually used with comas®)
Positive Negative
Liquid
biopsy
Positive Both positive
Negative Both negative
・Sensitivity (True positive rate) of Liquid biopsy
= (Both positive) / Tissue positive
・Specificity (True negative rate) of Liquid biopsy
= (Both negative) / Tissue negative
8
0%
20%
40%
60%
80%
100%
Sensitivity Specificity
Series1
Series2
Detection of EGFR activating mutations in cfDNA
(Yung TKF, CCR, 2009; Karlovich C, CCR, 2016,
http://blog.aacr.org/fda-approval-liquid-biopsy-test-lung-cancer/)
(Compared with tissue DNA using an allele specific PCR)
Sensitivity and Specificity for detection of
EGFR activating mutations in cfDNA
80%
95%98%
77%
“Cobas® EGFR Mutation Test v2” for plasma sample was approved for companion diagnostics of TARCEVA®
(Erlotinib).
Cobas ® (Allele specific PCR)
dPCR, BEAMing (Highly sensitive method)
9
(Sacher AG, JAMA Oncol. 2016)
Se
nsitiv
ity w
ith
alle
le
sp
ecific
PC
RSensitivity of plasma analysis compared with tissue
analysis according to metastatic status
0
20
40
60
80
100
IIIb or IV-M1a IV-M1b
23.8% (5/21)
78.0% (32/41)
With or without extra-thoracic
metastases
According to the number of
metastases
Se
nsitiv
ity w
ith
dd
PC
R
(Tseng JS, J Thorac Oncol. 2015)
More extensive disease can shed more amount of cfDNA and determines upper limit of the detection. 10
Clearance of EGFR activating mutations in cfDNA
during treatment predicts a good outcome
Treatment with platinum/GEM+Erlotinib for EGFR mutation positive patients(Subset of FASTACT-2 study)
(Mok T. Clin Cancer Res. 2015)
Monitoring for copy number of EGFR activating mutations in cfDNA (allele specific PCR)
PFS OS
11
Clearance of EGFR activating mutations in cfDNA
during treatment predicts a good outcome
・PFS according to the detection of EGFR activating mutations in
cfDNA at 4 weeks after the initiation of afatinib treatment
(Iwama E, Ann Oncol. 2017)
Treatment with Afatinib for EGFR mutation positive patients
・Monitoring for copy number of EGFR activating mutations in cfDNA (dPCR)
12
(Yu HA, Clin Cancer Res., 2013)
Mechanisms of acquired
resistance to 1st gen. EGFR-TKIs
Detection of T790M in tissue samples
Not underwe
nt re-biopsy
38%Underwent re-biopsy
62%
Success79%
Failure21%
・139 consecutive NSCLC pts after the failure of
treatment with EGFR-TKI
・Success or failure in diagnosis of malignancy at
re-biopsy for 395 NSCLC pts after the failure of
treatment with EGFR-TKI
(Kawamura T, Cancer Sci. 2016)
(Nosaki K, Lung Cancer. 2016)13
0%
20%
40%
60%
80%
100%
Sensitivity Specificity
Detection of EGFR T790M mutation in cfDNA
Sensitivity and Specificity for detection of
T790M in cfDNA by Cobas ®
(, compared with tissue DNA)
61%
79%
(Jenkins S, J Thorac Oncol. 2017)
“Cobas® EGFR Mutation Test v2” for plasma sample was approved for companion diagnostics of TAGRISSO®
(Osimertinib).14
Efficacy of Osimertinib according to T790M status in patients with acquired resistance to 1st gen. EGFR-TKI
<Tissue assessed by Cobas®> <cfDNA assessed by BEAMing>
<T790M negative in cfDNA assessed by BEAMing>
(Oxnard GR, JCO, 2016)
0%
20%
40%
60%
80%
100%
Sensitivity Specificity
Sensitivity and Specificity for detection of
T790M in cfDNA by Beaming
69%70%
15
Combination use of liquid and tissue biopsy for detection of T790M
T790M assessment with cfDNA
Acquired resistance to 1st or 2nd gen. EGFR-TKI
3rd gen. TKI
Positive Negative
Positive Negative
T790M assessment with Tissue
Chemotherapy
Strategy 1
T790M assessment with Tissue
Acquired resistance to 1st or 2nd gen. EGFR-TKI
3rd gen. TKI
Positive
Unknown
Positive Negative
T790M assessment with cfDNA
Chemotherapy
Strategy 2
Negative
Liquid biopsy reduces the invasion, cost and time caused by tissue biopsy.
Liquid biopsy may salvage the patients whose tissue samples are not obtained or insufficient
(Oxnard GR, JCO, 2016) (Tan DSW, JTO, 2016)
16
【Case】 71 y.o. Female, EGFR mutation positive (Exon19 deletion)
・(1st line) Afatinib・Biopsy from the primary lesion ⇒T790M negative・Liquid Biopsy ⇒T790M positive・(2nd line) Nov. 2016.〜Aug. 2017:Osimertinib
Before osimertinib After 6 months, PR
Primary lesion
(Burrell. Nature. 2013)
Intratumor heterogeneity
17
Summary of sensitivity and specificity in the detection of T790M in
plasma compared with that in tissue (Cobas ®)
0%
20%
40%
60%
80%
100%
Sensitivity Specificity
Series1Series2
Cobas (before optimization)
Cobas (after optimization)
BEAMing
ddPCR
41%
70%
61%
77%
100%
69%
79%
63%
(Thress KS. Lung Cancer. 2015; Jenkins S. J Thorac Oncol. 2017;
Oxnard GR. J Clin Oncol. 2016; Sacher AG, JAMA Oncol. 2016)
18
Detection of EGFR T790M mutation in cfDNA from various individuals by cSMART (NGS)
(Wang Z, WCLC 2016)
What is true positive for T790M?19
What is true negative for T790M?
“The concurrent presence of activating mutation helps improve the diagnostic
usefulness of T790M testing in plasma; informative patients with T790M mutation-
negative and activating mutations-positive plasma samples are more likely to be
true negative.” (Mok T, J Clin Oncol. 2017)
(Oxnard GR, JCO, 2016)
Plasma T790M +
mPFS 9.7 months
Plasma T790M- /Activating+
mPFS 4.4 months
Plasma T790M- /Activating-
mPFS 15.2 months
PFS in the treatment with osimertinib
20
➢ cfDNA potentially has the information including primary and metastatic
tumors (inter-tumor heterogeneity)
What causes low sensitivity and specificity in the detection of T790M in cfDNA?
( ⇒ low specificity)
➢ Highly sensitive method may detect the T790M mutation which is not
responsible for the resistance to EGFR-TKIs as detected in healthy
volunteers.
( ⇒ low specificity)
➢ Alleles with T790M are fewer
than that with activating
mutations.
( ⇒ low sensitivity)
Detection of EGFR T790M mutation in cfDNA
21
EGFR C797S10%
HER2 exon20 ins
11%
KRAS G12D and amp
11%
MET amp5%EGFR amp
5%MEK1 G128V
5%
Unknown53%
Putative resistance mechanisms to 3rd
generation TKI detected in cfDNA by NGS
<T790M positive ⇒ Rociletinib ⇒ PD> <1st line Osimertinib ⇒ PD>
(Chabon JJ, Nature communications. 2016) (Ramalingam SS, J Clin Oncol. 2017)22
A nationwide Lung Cancer Genomic Screening Project for
Individualized Medicine in Japan (LC-SCRUM-Japan)
Frequencies of gene mutations and fusions
detected in tumor samples by NGS in LC-
SCRUM-Japan
1,000 pts who will be enrolled in LC-SCRUM-Japan will also be able to take the blood screening by GUARDANT health.These studies take no charge for pts!!
(Yokoyama T, ASCO 2017)
Guardant360 (GUARDANT health)
23
Atezolizumab versus docetaxel in pts with
previously treated NSCLC (OAK)
(Fabrizio DA. ESMO 2017)
OS (ITT population)
PFS according to the value of bTMBEvaluation of tumor mutation burden
from blood (bTMB)
(Gandara DR. ESMO 2017)
(Rittmeyer A. Lancet. 2017)
24
bTMB ≥ 10 or 16
Treatment naïve
Advanced NSCLC
N = 3500
ALK positive
Alectinib
(n=78)
RET positive
Alectinib
(n=52-62)
cfDNA analysis
by NGS
Primary End Point: PFS
Atezolizumab
Platinum-based
chemotherapy
R
n=440
(1 : 1)
(Mok T. WCLC 2017)
Blood first assay screening trial (BFAST)
25
➢ Liquid biopsy cannot replace tissue biopsy, but these two methods can
complement each other with regard to detection of EGFR (especially
T790M) mutations.
➢ Liquid biopsy will probably provide us with more information about
resistance mechanisms to EGFR-TKIs.
➢ Liquid biopsy has been under investigation for biomarker analysis to
assess the efficacy of immunotherapy.
Conclusions
26
Target sample size: 60 patients
(Feb.2016 – Dec.2017)
Primary End Point: ORR
EGFR-TKI failured
NSCLC
PS 0-1
Osimertinib
80 mg p.o
T790M
positive
case
A phase II study to assess the efficacy of AZD9291 in patients with NSCLC positive for T790M mutation detected by liquid biopsy
(WJOG8815L)
Screening of T790M in plasma with
2 methods (Cobas ® and ddPCR)
PD
Monitoring of EGFR mutations in plasma
with 3 methods (Cobas ® and ddPCR)
Analysis of cfDNA with NGS
30
Efficacy of EGFR-Tyrosine Kinase Inhibitors
➢ EGFR-Tyrosine Kinase Inhibitors (EGFR-TKIs) for EGFR active mutations positive NSCLC pts.
1st gen. EGFR-TKI: Gefitinib, Erlotinib
2nd gen. EGFR-TKI: Afatinib
– ORR:60~80%
– MST:2 years ~
EGFR40-55%
KRAS10%ALK
5%
ROS13%
RET2%
HER23%
BRAF1%
Others
Genetic mutations in adeno ca. of lung cancer (Japan)
Kohno et al. Cancer Sci. 2013
➢ Cytotoxic chemotherapy for NSCLC pts.
– ORR:30~40%
– MST:1 year
31
Detection frequency of T790M in tissue samples obtained before treatment with EGFR-TKIs
TechniqueDetection frequency of
T790M
Sanger Sequencing 2.2% (11/503) OxnardGR, JTO,2012
Scorpion-ARMS(ARMS)
1.4% (21/1450) ShiY,JTO,2014
MALDI-TOF MS 31.5% (23/73) SuKY,JCO,2012
Colony hybridization 78.9% (30/38) Fujita Y, JTO,2012
Microdissection + PCR-based method
65.3% (62/95) Costa C, CCR. 2014
Digital PCR 79.9% (298/373) WatanabeM,CCR,2015
Digital PCR 100% (13/13) IwamaE,Oncotarget,2015
The detection frequency of T790M is dependent
on the sensitivity of techniques.33
Model of the emergence of T790M
Only highly sensitive
methods such as dPCR
can detect T790M.
Sanger sequencing or
allele specific PCR can
detect T790M.
T790M detected by these methods is responsible for the resistance to EGFR-TKIs !!
34
Are there any solutions to improve the specificity for detecting T790M in cfDNA??
(Quantification with allele specific PCR or NGS)
<Efficacy of rociletinib according to the T/A ratio>
Tissue Plasma
Piotrowska Z, Cancer Discovery, 2015
(BEAMing)
Karlovich C, Clin. Can. Res. 2016
➢ Evaluate the T/A (T790M/Activating mutation) ratio with a highly
sensitive and quantitative manner.
35
Sensitivity to erlotinib according to the frequency of T790M positive cells
Chmielecki J, Science Translational Med., 201136