Clinical implication of ctDNA Analysis in advanced lung carcinoma patients using different technologies.

Dr Pierre-Jean Lamy

Institut d'Analyse Génomique, Montpellier, France,

Disclosures

• Grant/Research Support: Agena Bioscience, ID-Solution, AstraZeneca, Cepheid

• Consultant: Agena Bioscience, AstraZeneca, Beckman Coulter, Bristol-Myers Squibb, Roche, Roche diagnostics, VitaDX, Novartis

The era of precision medicine The right treatment to the right person thanks to molecular tests

NSCLC: TKI in EGFR mt patients

Molecular Analysis

Lung biopsy

PJ Lamy 2017

Lung Biopsy carry risks : blood clots, bleeding, pneumothorax, Infections, fever, pain, pneumoria

Difficult for patient with lung diseases (stop breathing, no coughing)

Not always informative : low number of cells. Insufficient for molecular analyses

NSCLC mutation profile and targeted therapies

PJ Lamy 2017

EG

FR

Inhib

itors

1st

and 2

nd G

3rd

T

AG

RIS

SO

(T790M

)

C797S

mutation

1st line treatment

Oxnard GRJ Thorac Oncol.

2019 Aug 1

NSCLC mutation profile and EGFR inhibitors Initial diagnostic of NSCLC

EGFR + EGFR -

TKI Alternate therapy

Reccurence

Testing for resistance mutation

T790M – C797S

Adtaped TKI regimen Stop TKI regimen

Other mutations

Molecular reflex testing

CAP/IASLC/AMP: Molecular Testing

Guidelines

« ctDNA could be used for clinical

settings in which tissue is limited and/or

insufficient for molecular testing”

Neal I. Lindeman JT0 2018

Challenges for cf-tumor-DNA analysis

LAMY 2018

-High background -Low level of specific tumor DNA

LAMY 2018

Circulating cell-free tumor DNA: pre-analytical issues

-Classical EDTA-blood tubes

Advantage: commonly used

Drawback: high risk of contamination with genomic DNA due to white

blood cells lysis

Blood should centrifugated before 3h after sample collection

- Preservative tubes

- Roche DNA ct

- Streck

- Paxgene

LAMY 2018

Circulating cell-free tumor DNA: pre-analytical issues

-Extraction: -QIAamp Circulating Nucleic Acid Kit (Qiagen) -Cobas® cfDNA Sample Preparation (Roche Diag) -IDXtract-MAG (ID-Solution)

-Quantification & qualification : small fragments (160 pb peak)

‐ qPCR is better than spectrophotometry,

‐ LabChip GX Touch, (PerkinElmer): Microfluidic capillary

electrophoresis system, Quantitation and size analysis

‐ LiquidIQ Panel (Agena Bioscience) for fragmentation,

amplifiable copy number (quantity), WBC contamination, identity.

-

-

LAMY 2018

Challenge LiquIDIQTM Solution

Disruption of highly plentiful white blood

cells can dilute cfDNA template

High variability in the amount of cfDNA

in plasma

The presence of PCR inhibitors in sample

may reduce assay sensitivity

PCR reactions may be biased to larger

fragments, thereby enriching wild-type

template

Sample mix-ups can complicate results

>

>

>

>

>

Determine the level of white blood cell

contamination

Determine total amount of cfDNA in

sample

Determine the total amplifiable copies

of cfDNA

Determine the presence of high

molecular weight fragments in sample

Authenticate samples using a

comprehensive set of SNPs

Liquid-IQ™ pre-analytical control

Paul van der Leest et al. Validation Study: Pre-Analytical QC for plasma DNA

with LiquidIQ™ Panel. Agena Corporate Poster AMP 2019 (at booth #3023)

Analytical issues

11

From Saliou et al, Exp Rev Mol Diag 16(1):39-50.

ctDNA fraction

100%

10%

1%

0.1%

0.01%

Sanger

RT-PCR

NGS

• PCR (ARMS, PNA-LNA)

• BEAMing

• dPCR

• NGS for LB

• MassARRAY Ultraseek

Methods of detection

LOD

Advanced metastatic

cancers

Diagnostic, follow-up,

residual disease,

early reccurences

Clinical setting

Volume tumoral

~ 10 cm3 LOD

LOD

cfDNA Copy Number and Level of Detection Calculations

10 ng x 1000 (convert to picograms)/3.3 picograms (10-12 g) of

DNA per haploid genome = 3030 total copies of DNA

www.seracare.com

UltraSEEK™ Biochemistry 1. PCR

0.1% 99.9%

2. Mutant specific single base extension

4. MassArray Analysis

Single base extension

specific for the mutant

allele using biotinylated

terminating nucleotides

3. Capture of SBE primer extended for

the mutant allele only

Mass

Inte

nsi

ty

Analytical performance of the UltraSEEK™ Lung Panel: a ring trial study

• The Seraseq ctDNA Mutation Mix v2 reference materials (9 cancer-relevant somatic variants) at variant allele frequency of 2%, 1%, 0.5%, 0.25%, 0.125%, and Wild-Type (WT) were used in 5 testing laboratories.

• Two runs performed per site

• The LoD was determined yielding a variant detected rate of at least 95% for the targeted variant (as defined in CLSI Guideline EP17-A2).

-The lowest limit of detection achieved was 0.125% for the EGFR Deletion and the highest of 1% with the EGFR Insertion. -The overall LoD for the SNV class was 0.5% MAF. - This study generated an overall specificity of 99.8% across 8180 known negative variant positions. - No significant differences in LoD or Sensitivity were observed across the 5 participating

laboratories. Demetric et al; Establishing the Sensitivity, Specificity, Interlaboratory Reproducibility, and Analytical Limit Of Detection of The UltraSEEK™ Liquid Biopsy Application Using Well-Defined Seraseq Reference Material. Poster #TT040 AMP2019

O-22: PJ Lamy et al. “Clinical implication of

ctDNA analysis in advanced lung

carcinoma patients using

different technologies: real-time PCR and

MALDI-TOF”

Oral presentation

Friday,18th May 10:45

USK Lung Panel study

• 100 plasma of advanced lung carcinoma patients were analyzed for somatic mutations detection

at time of diagnostic or after recurrence under EGFR inhibitors therapy.

• CtDNA extraction was performed with the QiaAMP Circulating Nucleic Acid Kit, (Qiagen)

• Quality control of ctDNA : Novel LiquidIQ™ Panel on MassARRAY (Agena Bioscience)

Results n= 100 samples USK

Lung Panel Mutation 1 Mutation 2 Mutation 3

WT 35 / /

EGFR MT 58

EGFR EX19 DEL 32 14 EGFR T790M

1 KRAS Q61H 0

EGFR L858R 18 2 EGFR T790M 1 BRAF V600E

EGFR S768I 1 1 EGFR

G719A/R

EGFR INS X20 1 0

EGFR L861Q/R 4 1 EGFR T790M 2 EGFR G719X

EGFR G719X 1 0

EGFR T790M 1 0

KRAS 7 2 BRAF V600E

Invalid 0 0 0

UltraSEEKTM Lung Panel n=63

Gene Coverage (Missense mutations) 1 well PCR LOD 0.1 25 to 1%

67 mutations

BRAF Codon 469 (exon 11) and codons 594, 600 (exon 15) 4

EGFR E709A, E709G, E709K, E709V, G719A, G719D, G719S, G719C, S768I, T790M, L858R, L861Q, L861R, C797S, Exon 19 indels, Exon 20 insertions

43

KRAS G12A, G12C, G12D, G12R, G12S, G12V, G13C, G13D, Q61H, Q61K, Q61E, Q61P, Q61R, Q61L

14

ERBB2 A775_G776insYVMA, G776>VC 2

PIK3CA Codons 542, 545 (exon 9), codon 1047 of (exon 20) 4

Cobas® EGFR Mutation

Gene Coverage (Missense mutations) 3 wells PCR LOD 1.4 to 13%

42 mutations

EGFR Exon 18 : G719X (G719A, G719C et G719S) · Exon 19 : deletions · Exon 20 : S768I, T790M and insertions · Exon 21 : L858R , L861Q

42

10 ng cfDNA were analyzed for somatic mutations for Cobas and USK

.

.

+ Controls 0.5% EGFR cfDNA positive/negative (Horizon Discovery)

+NTC

Comparison Cobas EGFR mutation Test V2 vs USK Lung Panel (n=63)

54% 40%

6%

COBAS EGFR V2

WT = 34

EGFR = 25

KRAS/BRAF = 0

INV = 4

41%

49%

10%

USK LUNG PANEL

WT = 26

EGFR = 31

KRAS/BRAF = 6

INV = 0

Concordant results were achieved for 72% of patients (43/59) with

overall concordance between EGFR mutant and wildtype reaching 83%.

Comparison Cobas EGFRV2 vs USK Lung Panel (n=63)

13

10

1 0

1 0

4 5

1 1 1

0

2

4

6

8

10

12

14

EGFR EX19DEL

EGFR L858R EGFR S768I EGFR INS X20 EGFR L861Q KRAS INV

COBAS EGFR V2

16

11

1 1 2

6

0

8

1 1 1 1

0

2

4

6

8

10

12

14

16

18

EGFR EX19DEL

EGFRL858R

EGFRS768I

EGFR INSEX20

EGFRL861Q

KRAS INV

USK Lung Panel

Mutation detected and listed by techniques Blue bars: Total of EGFR mutations. Red bars EGFR T790M. Green bars: G719X. Yellow bar: KRAS mutation. Violet

bar: BRAF V600E. Orange bar: invalid.

The second bar per group shows the number of samples with a secondary mutations.

UltraSEEK Lung Panel detected 10 additional EGFR mutations (4*exon 19 deletion,

3*T790M, 1*exon 20 insertion, 1*G719A, 1*L861QR) not detected by Cobas.

Only 1 EGFR Exon 19 del was detected by Cobas and not by UltraSEEK.

Allele frequency N° USK lung panel VAF USK

LOD COBAS

EGFR

MUTATIONUSK-1 EGFR_pE746_A750del 1,04% 1,40%

EGFR_pE746_A750del 0,29% 1,40%

PIK3CA_pE545K 0,85%

USK-4 EGFR_pE746_A750del 0,36% 1,40%

USK-5 EGFR_pG719S 1,03% 2,50%

USK-6 EGFR_pE746_A750del 0,53% 0,014

USK-7 EGFR_pL747_P753toS 8,60% 1,40%

EGFR_pE746_A750del 1,34% 1,40%

PIK3CA_pH1047R 0,42%

EGFR_T790M 0,31% 2%

USK-10 EGFR_L858R 0,28% 4%

USK-11 EGFR_G719S 0,22% 2,50%

EGFR_pL747_A750toP 6,06% 1,40%

EGFR_T790M 1,92% 2%

USK-16 EGFR_L858R 5,67% 4%

USK-17 EGFR_L858R 4,48% 4%

USK-18 EGFR_L858R 2,88% 4%

USK-20 EGFR_pH773_V774insH 3,56%

EGFR_pE746_S752toV 6,70% 4%

EGFR_pT790M 0,25% 2%

USK-28 EGFR_pD770_N771insG 0,88%

EGFR_L858R 6,58% 4%

EGFR_T790M 4,50% 2%

EGFR_pE746_S752toV 0,51% 1,40%

EGFR_pT790M 0,12% 2%

USK-31 EGFR G719A 0,30% 2%

USK-2

T790M

EX19DEL

T790M

Not analysed

EX19DEL

WT

Cobas EGFR V2

Not analysed

EX19DEL

T790M

L858R

T790M

L858R

L858R

L858R

WT

WT

WT

EX19DEL

EX19DEL

EX19DEL

T790M

WT

Not analysed

WT

WT

Not analysed

USK-30

USK-29

USK-27

USK-15

USK-8

All mutation detected

by USK only had an

allele frequency below

the LOD of the Cobas

Cobas test: LOD : 1.4 to 13.4 % (50 ng/well)

USK lung panel: 0.1-0.5% (10-20 ng/well)

UltraSEEKTM Condordance with Tissue

• Tissue mutation analysis for EGFR mutations were available for 80/100 samples analysed in this study.

• In 38/47 tissue positive samples a corresponding EGFR mutation was detected in cfDNA by UltraSEEK, resulting in a PPV of 80%.

• 11/33 tissue negative samples identified an EGFR mutation in cfDNA using UltraSEEK.

• 10/11 were confirmed using the same cfDNA sample on the Cobas EGFR v2 Panel (Roche).

Sartori et al. Application of a Staged Testing Model for cfDNA Samples From NSCLC Patients Progressing on EGFR Tyrosine Kinase Inhibitor Therapy. Poster#ST065 AMP 2019

Conclusions (1)

1) High specificity and High sensitivity

2) Good concordance with tissue analysis

3) Higher level of mutation at time of diagnostic that allows

TKI treatment for mote patients: + 14% EGFR mutant

were detected

4) Evaluation of resistance with T790M/C797S

5) Another mechanism of resistance (PIK3CA, KRAS,

BRAF mutations)

6) Treatment response prediction - VAF could be use for

monitoring (immunotherapy)

Ultra Seek Lung Panel

presented:

-a higher sensitivity

-a broader panel

Gracias por su atencion Thank you for your attention

Nicolas Lozano

Leila Gambier

Bastien Pasquier

Florian Almela

Dr S Richebourg

Dr Sartori