1. Blood-based Biomarkers for Lung Cancer
Tony Mok MD
Li Shu Fan Medical Foundation Professor of Clinical Oncology
The Chinese University of Hong Kong

2. How solid is liquid biopsy?=

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

4. Technologies for tissue samples are potentially applicable to plasma cf DNA
McLarty et al MOJ Cell Science & Report 2015

5. Potential application
Pathogenesis Symptoms treatment selection Resistance to treatment death
Early detection
Diagnosis and treatment selection
Monitoring
Mechanism of resistance and treatment

6. Potential application
Pathogenesis Symptoms treatment selection Resistance to treatment death
Early detection
Diagnosis and treatment selection
Monitoring
Mechanism of resistance and treatment

7. We need to find EGFR mutation at time of diagnosis of adenocarcinoma
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
We need to find EGFR mutation at time of diagnosis of adenocarcinoma
Mok et al NEJM 2009, Lee et al WCLC 2009, Mitsudomi et al Lancet Oncology 2010, Maemondo NEJM 2010
Zhou et al Lancet Oncol 2010

8. PROFILE 1014: Crizotinib Superior to Pemetrexed-based Chemotherapy in Prolonging PFSa
PFS probability (%)
100 80 60 40 20
Time (months)
No. at risk
Crizotinib Chemotherapy
Crizotinib
(N=172)
Chemotherapy
Events, n (%)
100 (58)
137 (80)
Median, months
10.9
7.0
HR (95% CI)
0.45 (0.35−0.60) Pb
<0.0001
We need to find ALK re-arrangement at time of diagnosis of adenocarcinoma
Solomon & Mok et al NEJM 2015

9. Other Targetable Mutation
Gene
Alteration
Histology
Frequency
Inhibitor
BRAF
Mutation, fusion
ADC
1-3%
Vemurafenib, dabrafenib, trametinib
MET
Amplification, and exon14 splicing
2-4%
Tivantinib, cabozantinib, INC280, onartuzumab
RET
Fusion 1%
Carbozantinib , sunitinib, sorafenib, lenvatinib, vandetanib
HER2
Mutation
Neratinib, afatinib, lapatinib, dacomitinib
ROS1
<1%
Crizoitinib
KRAS
mutation
15-25%
SML , (Selumetinib, trametinib)
FGFR1
amplification
SCC
19%
Lucitanib, Nintedanib, dovitinib, AZD4547
FGFR2-3 3%
FGFR1-3
3.5%
DDR2 4%
Dasatinib
Debatable on the need to identify them at time for first line treatment

10. 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

11. 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
Blood ctDNA Preparation Kit
Cobas EGFR_Blood
ctDNA SP
cobas cell-free DNA Preparation Kit (To be used for other blood based assays)
2 ml
Plasma
cobas EGFR _ blood Test
cobas 4800 v 2.0
cobas EGFR _ Blood
HPEA x25
PK x2
PBB x6
Additional reagents added to cobas DNA preparation Kit
Tube
FAM
HEX
JA270 1
EX 19Del
S768I 2
L858R
T790M 3
G719X
L861Q*
EX 20Ins
Blood-specific cutoffs;
Blood-specific data analysis software
*New primer and probe for L861Q
Mok et al ASCO 2013

12. FASTACT-2 (MO22201; CTONG0902) study design
Screening
Study treatment
Maintenance phase
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)
Erlotinib 150mg/day PD
Previously untreated stage IIIB/IV NSCLC, PS 0/1
(n=451)
1:1; stratified by stage, histology, smoking status and chemo regimen R
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)
Placebo PD
Erlotinib 150mg/day
Primary 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
Wu and Mok Lancet Oncology 2013 12 12

13. EGFR Mutation Analysis using cobas 4800_blood test
TISSUE SAMPLES
397 (88%) patients consented
301 (66.7%) samples available
241 (53.4%)
samples analyzable
224 patients with matched tumor and plasma samples
PLASMA SAMPLES
451 (100%) patients consented
427 (94.6%) samples available
427 (94.6%)
samples analyzable

14. 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-
Total
t-EGFR Mut+ (Tumor) 69 21 90
t-EGFR Mut- (Tumor) 5
129
134 74
150
224

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

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

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

18. Droplet digital PCR (ddPCR)
Hindson et al. Analytical chemistry 2011

19. Positive result on exon 19 deletion assay
Plasma sample 214
Mutant concentration:72 copies/ml plasma
Fraction concentration : 3.2%

20. Negative result on exon 19 deletion assay

21. Tumor sample: COBAS EGFR Mutation Test
Analyzing plasma and tumor sample from ASPIRATION study and matched control (n=197)
Tumor sample: COBAS EGFR Mutation Test
Plasma sample: Droplet digital PCR
In formalin-fixed, paraffin-embedded tissue (FFPET) specimens, the cobas® EGFR test can detect <5% mutant sequence copies in a background of wild-type DNA.* 
Lee et al WCLC 2013

22. 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 53 80
197
Droplet digital PCR
Sensitivity
81%
Specificity
100%
Positive Predictive Value
Concordance
86%

23. 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
Sensitivity is technology dependent, eg BEAM vs Sanger
High specificity implies clinical applicability for selection of first line EGFR TKI
Qiu et al Can Epi Biomarker Prev 2015 Jan;24(1):

24. Plasma DNA positive for EGFR exon 21 L858R
Jan 7 at 5:00pm
Plasma DNA positive for EGFR exon 21 L858R
Jan 8 at 1:00pm
CT scan guided lung biopsy
Jan 8 at 7:00pm
Start EGFR TKI
Jan 6, 2015

25. Jan
Jan 15, 2015

26. Potential application
Pathogenesis Symptoms treatment selection Resistance to treatment death
Early detection
Diagnosis and treatment selection
Monitoring
Mechanism of resistance and treatment

27. Serial plasma samples at baseline, C3 and PD
BASELINE TISSUE SAMPLES
238 (52.8%) patients with matched tumour and plasma results
397 (88%) patients consented
268 (59.4%) samples available
241 (53.4%)
samples analysable
PLASMA SAMPLES
447 (99.1%) baseline samples available
447 (99.1%) baseline samples analysable
305 (67.6%) Patients with plasma results at all three time points
451 (100%) patients consented
362 (80.3%)
C3 samples available
362 (80.3%)
C3 samples analysable
376 (83.4%)
PD samples available
376 (83.4%)
PD samples analysable

28. Dynamic mutant DNA change during therapy
Patients treated with GC+P
Patients treated with GC+E
100,000
100,000
10,000
10,000
1,000
1,000
Mutant DNA copy/mL of plasma
100
Mutant DNA copy/mL of plasma
100 10 10 1 1
Not detectable
Not detectable
Baseline C3 PD
Baseline C3 PD

29. Positive versus negative pEGFR mut status at C3 (both treatment arms combined)
pEGFR mut+ at C3
(n=42)
ORR = 33%
(14/42)
OR=3.93
(95% CI: 1.78–8.66);
p=0.0007
pEGFR mut+ at baseline
(n=122)
pEGFR mut– at C3
(n=80)
ORR = 66%
(53/80)
ORR = objective response rate; OR = odds ratio

30. Association between pEGFR mut+ at C3 and PFS/OS (both treatment arms combined)
C3 mut+
C3 mut+
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
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
1.0
0.8
0.6
0.4
0.2
1.0
0.8
0.6
0.4
0.2
PFS probability
OS probability
7.2
12.0
18.2
31.9
Time (months)
Time (months)
Patients, n
Patients, n
C3 mut C3 mut–
C3 mut C3 mut–
0 0
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
OS = overall survival

31. Detection of plasma EGFR mutations: summary
Detection of EGFR mutations by UD-NGS
EGFR status
Baseline
Progression
Mutated
31 (72%)
11 (76%)
Wild type
12 (28%)
4 (24%)
Total
43 (100%)
15 (100%)
Sensitivity: 72%
Specificity: 100%
Detection of EGFR mutations by cobas® test
EGFR status
Baseline
Progression
Mutated
30 (70%)
11 (73%)
Wild type
13 (30%)
4 (27%)
Total
43 (100%)
15 (100%)
Sensitivity: 71%
Specificity: 100%
Marchetti, et al. WCLC 2015

32. EGFR SQI on erlotinib therapy in EGFR Mut+
Rapid responders
Time (days)
EGFR SQI
Slow responders
Time (days)
EGFR SQI
In 70% of patients EGFR SQI reduced by >50% at 14 days
Patients had no T790M early mutations
Two patients showed an increase in T790M mutations
Slow responders may be more prone to developing resistance
SQI, semi-quantitative index (% of mutant compared to WT)
Marchetti, et al. WCLC 2015

33. Potential application
Pathogenesis Symptoms treatment selection Resistance to treatment death
Early detection
Diagnosis and treatment selection
Monitoring
Mechanism of resistance and treatment

34. No corresponding rebiopsy tumor for T790M
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
No corresponding rebiopsy tumor for T790M
Chen et al, WCLC, 2013

35. Serial monitoring of T790M from plasma DNA
47% found to have T790M
Droplet Digital PCR
Zhang et al ASCO 2014

36. Serial change in T790M by ddPCR
Zhang et al ASCO 2014

37. Impact of post-PD treatment on plasma DNA for T790M

38. TIGER X: Plasma Testing for T790M using BEAMing
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
Total
Positive
Negative
Inadequate tissue
Plasma
155 23 12
190 37 8 57
192 35 20
247
Tissue as reference:
Positive percent agreement
T790M
81% (155/192)
Activating mutations
87% (193/221)
Sequest et al ASCO 2015

39. T790M Plasma Testing is a Viable Alternative to Tissue Testing
Objective response rate for 188 evaluable patients with both central T790M tissue test result and plasma T790M result
Plasma T790M
Tissue
T790M + -
55%
(72/130)
43%
(13/30)
53%
(85/160) −
35%
(6/17)
27%
(3/11)
32%
(9/28)
(78/147)
39%
(16/41)
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

40. Targeting EGFR mutation versus NGS

41. Guardant 360 Gene selection based on clinical utility - actionability
2015 Complete* or Critical Exon Coverage in 68 Genes
All Exons and partial introns Covered (Copy Number Variations in Bold/ INDELS)
Point Mutations
AMPLIFICATIONs
Fusions
EGFR exon 19 deletions
38 patient samples were
Analyzed using this panel
Partial Coverage (“Hot” Exons):
INDELS
Valifor et al WCLC 2015

42. Mutations were detected in majority of patients
Are these alternation clinically relevant?
78% (n=42/54) patients had at least 1 alteration

43. Distribution of alterations in 42 mutation positive
Distribution of alterations in 42 mutation positive* patients (n mutations=115)
*Non-synonymous only

44. Patients who really benefited from molecular testing
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
Patients who really benefited from molecular testing
EGFR 7/ALK 5/MET 2/BRAF 1 = 15
ALK 5
MET 2
BRAF 1
CDKNA2 5
KIT 2
BRCA1 2

45. NGS is to eat the whole genomic pie in one go

46. Alternative approach is to rule out EGFR mutation first

47. Prospective study of plasma genotyping in advanced NSCLC
Eligible patients – adv. NSCLC
Newly diagnosed (Cohort 1) n=120
EGFR resistance (cohort 2) n=60
Excluded – missed blood draw
Cohort 1: n=3
Cohort 2: n=1
Cohort 1 – newly diagnosed
Plasma ddPCR for EGFR exon 19 del/L858R and KRAS G12X
n=117
Cohort 2 – EGFR resistance
Plasma ddPCR for EGFR T790M/ exon 19 del/L858R
n=59
Excluded – failed tissue genotyping
EGFR: n=2
KRAS: n=30
Excluded – failed tissue genotyping
T790M: n=5
KRAS G12X
Tissue genotyping complete
n=87
(26 KRAS G12X mutant)
EGFR exon 19/L858R
Tissue genotyping complete
Cohort 1: n=115
Cohort 2: n=59
(87 EGFR mutant)
EGFR T790M
Tissue genotyping complete
n=54
(35 T790M mutant)
Pilot study of sequential plasma NGS, n=46
Sacher et al WCLC 2015

48. Positive Predictive Value
ddPCR for EGFR and KRAS
Assay
Specificity
Positive Predictive Value
Sensitivity
EGFR
exon 19 del & L858R
Cohort 1
99%
95%
77%
Cohort 2
100%
80%
Overall
98%
79%
exon 19 del
92%
85%
81%
82%
L858R
69%
78%
74%
EGFR T790M
63%
KRAS G12X
64%
ddPCR from plasma cfDNA in ASPIRATIOM study
Park et al ESMO 2014

49. Sequential plasma ddPCR and NGS
Complex genomic alterations
EGFR/KRAS mutant
Study ID
Tissue Genotype
Plasma ddPCR
Plasma NGS 15
ROS1
Negative
Detected 18
ALK 22
EGFR G719A 36 81
PIK3CA E545K
107
RET, p53
127
130
EGFR exon 20 ins, p53
133
KRAS Q61H
137
RET
145
EGFR exon 20 ins
202
250
EGFR exon 19 del/ins
258
None 89
KRAS G13D
108
BRAF V600E
115
KRAS Q61L
169
209
Study ID
Tissue Genotype
Plasma ddPCR
Plasma NGS 1
KRAS G12C
Detected 4
EGFR exon 19 del 11 17 39 44 53 70 74 81 91 95
105
116
EGFR exon 19
120
EGFR exon 19/T790M
179
195
200
232
EGFR L858R/T790M 48
EGFR L858R
Negative
244 8 28 61 45 94
KRAS G12V
109
Table of sensitivity in rare genotypes and overall cohort.
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).
Plasma NGS detected 13/19 (74%) complex genomic alternation and 8/13 patients had FDA approved treatment

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

51. Possible paradigm -ive + Patient with newly diagnosed adenocarcinoma
3 to 4 days +
ddPCR for EGFR
EGFR TKI
10 to 14 days
-ive
NGS panel (tissue or blood)

52. Summary Finding EGFR mutation from cfDNA Monitoring progress
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

53. A liquid can be very solid
Liquid Crystal