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

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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%）

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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

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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

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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)

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(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

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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)

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(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%

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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)

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【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

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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)

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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

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➢ 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

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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)

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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)

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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)

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➢ 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

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Please come to Japan!!

Kobe

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Thank you for your attention

Kyushu University Hospital 28

Back Up Slide

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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

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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

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Heitzer E, et al. Clin Chem., 2015 Jan;61(1):112-23

Circulating free DNA (cfDNA)

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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 !!

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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.

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Sensitivity to erlotinib according to the frequency of T790M positive cells

Chmielecki J, Science Translational Med., 201136

各検査法の検出限界

Diaz LA Jr, et al. J Clin Oncol. 2014;32(6):579-86

検出限界

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I declare no conflicts of interest.

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