Blood-based Biomarkers for Lung Cancer

Tony Mok MD

Li Shu Fan Medical Foundation Professor of Clinical Oncology

The Chinese University of Hong Kong

?=

How solid is liquid biopsy?

Source of plasma DNA-tumor necrosis/apotosis-tumor exosome/microvesicles-normal tissue apotosis-inflammatory cell

Technologies for tissue samples are potentially applicable to plasma cf DNA

McLarty et al MOJ Cell Science & Report 2015

Pathogenesis Symptoms treatment selection Resistance to treatment death

Potential application

Early detection

Diagnosis and

treatment selection

Monitoring

Mechanism of

resistance and

treatment

Pathogenesis Symptoms treatment selection Resistance to treatment death

Potential application

Early detection

Diagnosis and

treatment selection

Monitoring

Mechanism of

resistance and

treatment

Confirming the superior efficacy of EGFR TKI in patients with EGFR mutations

Author Study N (EGFR mut +)

RR Median PFS

Mok et al IPASS 132 71.2% vs 47.3 9.8 vs 6.4 months

Lee et al First-SIGNAL 27 84.6% vs 37.5% 8.4 vs 6.7 months

Mitsudomi et al WJTOG 3405 86 62.1% vs 32.2% 9.2 vs 6.3 months

Maemondo et al NEJGSG002 114 73.7% vs 30.7% 10.8 vs 5.4 months

Zhou et al OPTIMAL 154 83% vs 36%

13.1 vs 4.6 months

Rosell et al EURTAC 135 56% vs 18% 9.2 vs 4.8 months

Wu et al LUX Lung 6 364 67% vs 23% 11.0 vs 5.6 months

Mok et al NEJM 2009, Lee et al WCLC 2009, Mitsudomi et al Lancet Oncology 2010, Maemondo NEJM 2010Zhou et al Lancet Oncol 2010

We need to find EGFR mutation at time of diagnosis

of adenocarcinoma

PROFILE 1014: Crizotinib Superior to Pemetrexed-based Chemotherapy in Prolonging PFSa

Solomon & Mok et al NEJM 2015

Crizotinib(N=172)

Chemotherapy(N=172)

Events, n (%) 100 (58) 137 (80)

Median, months 10.9 7.0

HR (95% CI) 0.45 (0.35−0.60)

Pb <0.0001

PF

S p

roba

bili

ty (

%)

100

80

60

40

20

00 5 10 15 20 25 30 35

Time (months)

172 120 65 38 19 7 1 0171 105 36 12 2 1 0 0

No. at riskCrizotinib

Chemotherapy

We need to find ALK re-arrangement at time of

diagnosis of adenocarcinoma

Other Targetable MutationGene Alteration Histology Frequenc

y Inhibitor

BRAF Mutation, fusion

ADC 1-3% Vemurafenib, dabrafenib, trametinib

MET Amplification, and exon14

splicing

ADC 2-4% Tivantinib, cabozantinib, INC280, onartuzumab

RET Fusion ADC 1% Carbozantinib , sunitinib, sorafenib, lenvatinib, vandetanib

HER2 Mutation ADC 2-4% Neratinib, afatinib, lapatinib, dacomitinib

ROS1 Fusion ADC <1% Crizoitinib

KRAS mutation ADC 15-25% SML-8-73-1, (Selumetinib, trametinib)

FGFR1 amplification SCC 19%

Lucitanib, Nintedanib, dovitinib, AZD4547FGFR2-3 Mutation SCC 3%

FGFR1-3 Fusion SCC 3.5%

DDR2 Mutation SCC 4% Dasatinib

Debatable on the need to identify them at time for first

line treatment

EGFR mutation testing in Asia 2011

Country (N

diagnosed with

NSCLC*)

Proportion tested

for EGFR

mutations

% (95% CI†)

Proportion of males/females, smokers and non-

smokers, and histological subtypes that were tested for

EGFR mutations‡

Gender Smoking status Histology

Males / Females

(%)

Current + ex-

smoker / Never

smoker (%)

ADC /

All non-ADC / Only

SCC (%)

Total (22,193) 31.8 (31.2–32.5) 26.9 / 40.2 47.0 / 57.4 50.4 / 12.5 / 12.5

China (12,086§) 18.3 (17.6–19.0) 15.2 / 25.3 N.D. 30.3 / 8.0 / 9.4

Hong Kong

(795)42.0 (38.6–45.5) 36.2 / 52.3 34.1 / 52.1 55.4 / 9.0 / 6.4

Japan (2,379) 64.8 (62.9–66.7) 63.6 / 67.0 68.8 / 68.3 69.2 / 55.0 / 50.3

Korea (3,794) 33.5 (32.0–35.0) 26.1 / 38.1 27.1 / 42.9 62.7 / 9.8 / 8.3

Taiwan (2,890) 54.3 (52.5–56.1) 47.1 / 64.3 37.0 / 56.8 69.3 / 15.5 / 8.5

Thailand (249§) 57.8 (51.6–63.8) 51.6 / 69.3 49.5 / 84.7 83.6 / 7.1 / 6.9

cobas® EGFR _ blood Test _ Kit Components

Utilizing most of the reagents in the cobas EGFR_FFPET test and requiring additional reagents and the blood-specific data analysis software

2 mlPlasma

cobas cell-free DNA Preparation Kit (To be used for other blood based assays)

cobas EGFR _ blood Testcobas 4800 v 2.0

cobas EGFR _ Blood

Blood-specific cutoffs; Blood-specific data analysis software

Blood ctDNA Preparation Kit

ctDNA SP

Tube FAM HEX JA270

1 EX 19Del S768I

2 L858R T790M

3 G719X L861Q* EX 20Ins

Cobas EGFR_Blood

HPEA x25

PK x2 PBB x6

Additional reagents added to cobas DNA preparation Kit

*New primer and probe for L861Q

Mok et al ASCO 2013

Placebo

Erlotinib 150mg/day

Previously untreated stage IIIB/IV NSCLC,

PS 0/1(n=451)

R

PD

Gemcitabine 1,250mg/m2 (d1, 8) + carboplatin AUC=5 or cisplatin

75mg/m2 (d1) + placebo (d15–28); q4wks x 6 cycles

GC-placebo (n=225)

Gemcitabine 1,250mg/m2 (d1, 8) + carboplatin AUC=5 or cisplatin

75mg/m2 (d1) + erlotinib 150mg/day (d15–28); q4wks x 6 cycles

GC-erlotinib (n=226)

PD

Study treatment Maintenance phaseScreening

Erlotinib 150mg/dayPrimary endpoint: PFS with IRC confirmation

Secondary endpoints: subgroup analyses, OS in all patients and subgroups, ORR, duration of response, TTP, NPR at 16 weeks, safety, QoL

FASTACT-2 (MO22201; CTONG0902) study design

1:1; stratified by stage, histology, smoking status and chemo regimen

Wu and Mok Lancet Oncology 2013

EGFR Mutation Analysis using cobas 4800_blood test

397 (88%) patients

consented

301 (66.7%) samples available

241 (53.4%)samples

analyzable

451 (100%) patients

consented

427 (94.6%) samples available

427 (94.6%)samples

analyzable

PLASMA SAMPLES

TISSUE SAMPLES

224 patients with

matched tumor and

plasma samples

Concordance between tumor and plasma samples

• Total of 224 patients had both tumor and baseline plasma samples with available EGFR mutation analysis results (Table 3)

– Sensitivity: 77% (69/90)

– Specificity: 96% (129/134)

– Positive predictive value: 93% (69/74)

– Negative predictive value: 86% (129/150)

– Overall concordance: 88% (198/224)

EGFR Activating Mutations

p-EGFR Mut+(Plasma)

p-EGFR Mut-(Plasma) Total

t-EGFR Mut+ (Tumor) 69 21 90

t-EGFR Mut- (Tumor) 5 129 134

Total 74 150 224

PFS of p-EGFR and t-EGFR mut+ patients

PFS of p-EGFR and t-EGFR mut- patients

Therascreen in plasma/serum samples from LUX Lung 3 and 6

Droplet digital PCR (ddPCR)

Hindson et al. Analytical chemistry 2011

Plasma sample 214 Mutant concentration:72 copies/ml plasma Fraction concentration : 3.2%

Positive result on exon 19 deletion assay

Negative result on exon 19 deletion assay

Analyzing plasma and tumor sample from ASPIRATION study and matched control (n=197)

• Tumor sample: COBAS EGFR Mutation Test

• Plasma sample: Droplet digital PCR

Lee et al WCLC 2013

Diagnostic utility of digital PCR for detection of EGFR mutation

POS in plasma NEG in plasma

POS in tumor 117 27 144

NEG in tumor 0 53 53

117 80 197

Droplet digital PCR

Sensitivity 81%

Specificity 100%

Positive Predictive Value 100%

Concordance 86%

Meta-analysis on cf DNA for EGFR mutation

• 3110 subjects (27 studies)• Tissue EGFR mutation status as gold

standard• Pooled Analysis

– Sensitivity 62%– Specificity 96%– Diagnostic odd ratio– Area under summary ROC 0.91

Qiu et al Can Epi Biomarker Prev 2015 Jan;24(1):206-12.

Sensitivity is technology

dependent, eg BEAM vs Sanger

High specificity implies clinical applicability for selection of first line EGFR TKI

Jan 6, 2015

Jan 7 at 5:00pmPlasma DNA positive for EGFR

exon 21 L858R

Jan 8 at 1:00pmCT scan guided lung biopsy

Jan 8 at 7:00pmStart EGFR TKI

Jan 8 2015 Jan 15, 2015

Pathogenesis Symptoms treatment selection Resistance to treatment death

Potential application

Early detection

Diagnosis and

treatment selection

Monitoring

Mechanism of

resistance and

treatment

241 (53.4%)samples

analysable

447 (99.1%) baseline samples

analysable

PLASMA SAMPLES

BASELINE TISSUE SAMPLES

362 (80.3%) C3 samples analysable

376 (83.4%) PD samples analysable

Serial plasma samples at baseline, C3 and PD

238 (52.8%) patients with

matched tumour and

plasma results

305 (67.6%) Patients with

plasma results at all three time

points

397 (88%) patients

consented

268 (59.4%) samples available

451 (100%) patients

consented

447 (99.1%) baseline samples

available

362 (80.3%) C3 samples

available

376 (83.4%) PD samples

available

Dynamic mutant DNA change during therapy

C3

Mut

ant

DN

A c

opy/

mL

of p

lasm

a

100,000

1,000

100

10

1

Not detectable

10,000

Mut

ant

DN

A c

opy/

mL

of p

lasm

a

100,000

1,000

100

10

1

Not detectable

10,000

Baseline PD C3Baseline PD

Patients treated with GC+P Patients treated with GC+E

Positive versus negative pEGFR mut status at C3 (both treatment arms combined)

ORR = 33%(14/42)

ORR = 66%(53/80)

pEGFR mut+ at C3(n=42)

pEGFR mut– at C3(n=80)

pEGFR mut+ at baseline

(n=122)

OR=3.93 (95% CI:

1.78–8.66);p=0.0007

ORR = objective response rate; OR = odds ratio

Association between pEGFR mut+ at C3 and PFS/OS (both treatment arms combined)

OS = overall survival

OSPFS

18.2 31.9

C3 mut+C3 mut–

Median=18.2 months(95% CI: 14.2–27.4)

Median=31.9 months(95% CI: 23.5–undefined)

HR=0.51 (95% CI: 0.31–0.84);

p=0.0066

7.2 12.0

C3 mut+

C3 mut–

Median=7.2 months(95% CI: 6.0–7.8)

Median=12.0 months(95% CI: 9.6–16.5)

HR=0.32(95% CI: 0.21–0.48);

p<0.0001

Time (months)

OS

pro

babi

lity

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36

Time (months)

PF

S p

roba

bilit

y

C3 mut+ 42 42 35 28 14 7 6 4 1 1 1 1 0 0 0 0 0C3 mut– 80 80 77 65 59 47 40 34 32 28 23 19 13 10 7 3 0

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32

1.0

0.8

0.6

0.4

0.2

0

1.0

0.8

0.6

0.4

0.2

0

C3 mut+ 42 42 42 41 37 32 30 28 23 21 18 14 14 12 9 4 3 2C3 mut– 80 80 80 77 77 77 76 71 68 64 59 52 38 29 22 12 3 1

00

Patients, n Patients, n

Positive pEGFR at baseline followed by negative pEGFR at C3 is associated with improved outcomes; patients positive at baseline and still positive at C3 experienced worse outcomes

Detection of plasma EGFR mutations: summary

EGFR status Baseline Progression

Mutated 31 (72%) 11 (76%)

Wild type 12 (28%) 4 (24%)

Total 43 (100%) 15 (100%)

EGFR status Baseline Progression

Mutated 30 (70%) 11 (73%)

Wild type 13 (30%) 4 (27%)

Total 43 (100%) 15 (100%)

Detection of EGFR mutations by UD-NGS

Detection of EGFR mutations by cobas® test

Sensitivity: 72%Specificity: 100%

Sensitivity: 71%Specificity: 100%

Marchetti, et al. WCLC 2015

EGFR SQI on erlotinib therapy in EGFR Mut+

Rapid responders

Time (days)

EG

FR

SQ

I

• In 70% of patients EGFR SQI reduced by >50% at 14 days

• Patients had no T790M early mutations

Slow responders

Time (days)

EG

FR

SQ

I

• Two patients showed an increase in T790M mutations

• Slow responders may be more prone to developing resistance

Marchetti, et al. WCLC 2015SQI, semi-quantitative index (% of mutant compared to WT)

Pathogenesis Symptoms treatment selection Resistance to treatment death

Potential application

Early detection

Diagnosis and

treatment selection

Monitoring

Mechanism of

resistance and

treatment

cfDNA for T790M• Digital PCR (Rui Chen et al):

• Studied 135 patients with acquired resistance to TKI

ARMS Digital PCR

Pre-TKI (N=109) 5% T790M pos 30% T790M pos

Post-TKI (N=135) 25% T790M pos 43% T790M pos

Chen et al, WCLC, 2013

No corresponding rebiopsy tumor

for T790M

Serial monitoring of T790M from plasma DNA

Zhang et al ASCO 2014

Droplet Digital PCR

47% found to

have T790M

Serial change in T790M by ddPCR

Zhang et al ASCO 2014

Impact of post-PD treatment on plasma DNA for T790M

TIGER X: Plasma Testing for T790M using BEAMing

Tissue

TotalPositive Negative

Inadequate

tissue

PlasmaPositive 155 23 12 190

Negative

37 12 8 57

Total 192 35 20 247

• When inadequate tissue specimens are factored in, plasma testing identifies as many patients as T790M+ as tissue testing

• T790M tissue-plasma+ are not false positives – T790M confirmed in plasma on subsequent testing in 5/7 samples studies

Tissue as reference:Positive percent agreement

T790M81% (155/192)

Activating mutations87% (193/221)

Sequest et al ASCO 2015

T790M Plasma Testing is a Viable Alternative to Tissue Testing

Plasma T790M

Tissue T790M

+ -

+55%

(72/130)43%

(13/30)53%

(85/160)

−35%

(6/17)27%(3/11)

32%(9/28)

53%(78/147)

39%(16/41)

Objective response rate for 188 evaluable patients with both central T790M tissue test result and plasma T790M result

• Similar ORR observed when detecting T790M in either tissue or plasma

• Not all patients with progression on first-line TKI are candidates for tissue re-biopsy

Targeting EGFR mutation versus NGS

Guardant 360Gene selection based on clinical utility - actionability

2014: 54 Genes2015 Complete* or Critical Exon Coverage in 68 Genes

All Exons and partial introns Covered (Copy Number Variations in Bold/ INDELS)

EGFR exon 19 deletions 38 patient samples were Analyzed using this panel

Partial Coverage (“Hot” Exons):

POINT MUTATIONS AMPLIFICATIONS

FUSIONS

INDELS

Valifor et al WCLC 2015

Mutations were detected in majority of patients

0 alte

ratio

ns

1 alte

ratio

n

2 alte

ratio

ns

3 alte

ratio

ns

4 alte

ratio

ns

5 alte

ratio

ns

6 alte

ratio

ns0%

5%

10%

15%

20%

25%

22%

19%

22%

7%

11%

9% 9%

Number of alterations identified in blood

Per

cen

tag

e o

f al

l p

atie

nts

(N

= 5

4)

78% (n=42/54) patients had at least 1 alteration

Are these alternation clinically relevant?

Distribution of alterations in 42 mutation positive* patients (n mutations=115)

TP53

KRAS AL

KATM AR

GNAS ME

TSTK11

ARID1A

CCNE1FGFR1

JAK2

PIK3CA

RB1

VHL

0

5

10

15

20

25

30

3 2 1 1 1

259

116

5 43 3 2 2 2 2

22 2 1 1 1

11 1

11 1 1 1 1 1 1

5

1 1 1

5

SNVNonsenseIndelAMP

Genes

Nu

mb

er

of

mu

tati

on

s p

er

ge

ne

*Non-synonymous only

54 lung cancer cases with

ctDNA analysis

42 with 1 or more alteration

7/42 (17%) FDA approved therapy

5 exon 19 deletion -2 with

concurrent T790M

mutations

2 L858R mutation

17/42 (40%) Therapy

approved in other disease

11/42 (26%) Clinical trials

available

12 no alterations identified

ALK 5MET 2

BRAF 1

CDKNA2 5KIT 2

BRCA1 2

Patients who really benefited from molecular testingEGFR 7/ALK 5/MET 2/BRAF 1 = 15

NGS is to eat the whole genomic pie in one go

Alternative approach is to rule out EGFR mutation first

Prospective study of plasma genotyping in advanced NSCLC

Cohort 2 – EGFR resistancePlasma ddPCR for EGFR T790M/

exon 19 del/L858Rn=59

EGFR exon 19/L858R Tissue genotyping complete

Cohort 1: n=115Cohort 2: n=59

(87 EGFR mutant)

Excluded – missed blood drawCohort 1: n=3Cohort 2: n=1

KRAS G12XTissue genotyping complete

n=87(26 KRAS G12X mutant)

Excluded – failed tissue genotyping

T790M: n=5

Cohort 1 – newly diagnosedPlasma ddPCR for EGFR exon 19 del/L858R and KRAS G12X

n=117

EGFR T790M Tissue genotyping complete

n=54(35 T790M mutant)

Excluded – failed tissue genotyping

EGFR: n=2KRAS: n=30

Pilot study of sequential plasma NGS, n=46

Eligible patients – adv. NSCLCNewly diagnosed (Cohort 1) n=120

EGFR resistance (cohort 2) n=60

Sacher et al WCLC 2015

ddPCR for EGFR and KRAS

AssaySpecificity

Positive Predictive

Value Sensitivity

EGFR exon 19 del & L858R

Cohort 1 99% 95% 77%Cohort 2 100% 100% 80%Overall 99% 98% 79%

EGFR exon 19 del

Cohort 1 99% 92% 85%

Cohort 2 100% 100% 81%

Overall 99% 98% 82%

EGFR L858R

Cohort 1 100% 100% 69%

Cohort 2 100% 100% 78%

Overall 100% 100% 74%

EGFR T790M63% 79% 77%

KRAS G12X100% 100% 64%

ddPCR from plasma cfDNA in ASPIRATIOM studyPark et al ESMO 2014

Sequential plasma ddPCR and NGS

Plasma NGS detected a diversity of complex genomic alterations in patients with negative plasma ddPCR (n=19) with 74% sensitivity. This assay was able to detect EGFR/KRAS mutations confirmed by orthogonal plasma ddPCR with 78% sensitivity (n=27).

Study ID Tissue Genotype Plasma ddPCR Plasma NGS15 ROS1 Negative Detected

18 ALK Negative Detected22 EGFR G719A Negative Detected36 EGFR G719A Negative Detected81 PIK3CA E545K Negative Detected

107 RET, p53 Negative Detected127 ROS1 Negative Detected130 EGFR exon 20 ins, p53 Negative Detected133 KRAS Q61H Negative Detected137 RET Negative Detected145 EGFR exon 20 ins Negative Detected202 ALK Negative Detected250 EGFR exon 19 del/ins Negative Detected258 None Negative None89 KRAS G13D Negative Negative

108 BRAF V600E Negative Negative115 KRAS Q61L Negative Negative169 RET Negative Negative209 ALK Negative Negative

Study ID Tissue Genotype Plasma ddPCR Plasma NGS1 KRAS G12C Detected Detected4 EGFR exon 19 del Detected Detected

11 EGFR exon 19 del Detected Detected17 EGFR exon 19 del Detected Detected39 EGFR exon 19 del Detected Detected44 EGFR exon 19 del Detected Detected53 EGFR exon 19 del Detected Detected70 KRAS G12C Detected Detected74 EGFR exon 19 del Detected Detected81 KRAS G12C Detected Detected91 EGFR exon 19 del Detected Detected95 EGFR exon 19 del Detected Detected

105 EGFR exon 19 del Detected Detected116 EGFR exon 19 Detected Detected120 EGFR exon 19/T790M Detected Detected179 KRAS G12C Detected Detected195 EGFR exon 19 Detected Detected200 KRAS G12C Detected Detected232 EGFR L858R/T790M Detected Detected48 EGFR L858R Negative Detected

244 EGFR exon 19 Negative Detected8 KRAS G12C Detected Negative

28 EGFR L858R Detected Negative61 KRAS G12C Detected Negative45 EGFR exon 19 del Negative Negative94 KRAS G12V Negative Negative

109 EGFR exon 19 del Negative Negative

Complex genomic alterations EGFR/KRAS mutant

Plasma NGS detected 13/19 (74%) complex genomic alternation and 8/13 patients had FDA approved treatment

This approach is to take a fast big bite of the genomic pie with ddPCR

Possible paradigm

Patient with newly diagnosed adenocarcinoma

ddPCR for EGFR

NGS panel (tissue or blood)

EGFR TKI+

-ive

3 to 4 days

10 to 14 days

Summary• Finding EGFR mutation from cfDNA

– Finding EGFR mutation from cfDNA is feasible– Sensitivity 70 to 80%, highly specific– ddPCR or BEAM

• Monitoring progress– Potentially could be used to monitor the EGFR mutation status,

but clinical relevance is lacking

• Detecting T790M– Digital PCR is relatively sensitive– BEAMing for T790M is associated with clinical response

• NGS– Multiple technology platform is being developed– Sensible to perform NGS only in patients without EGFR mutation

in plasma

A liquid can be very solid

Liquid Crystal