multi-center evaluation eines hoch sensitiven ngs assays ...€¦ · 1 patient, 3 samples...

Multi-Center Evaluation eines hoch sensitiven NGS Assays zum Mutationsnachweis an Flüssigbiopsien bei Lungentumoren

Dr. Claudia Vollbrecht

Charité Universitätsmedizin Berlin

Institut für Pathologie

23. Juni 2017

DGP Erlangen

2

Liq

uid

Bio

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es: G

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NA

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20

14

. J C

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Mono-nucleosomalecfDNA

Di-nucleosomalecfDNA

T ½ approx. 2 hours

~ 180bp <1% ctDNAin cfDNA

LIQUID BIOPSY – CTDNA

cfDNA Release Depends on:

• Tumor Localization

• Tumors Size

• Vascularity

• Therapy Status

cfDNA: cell-free DNActDNA: circulating tumor DNA

gDNA

• Inflammation

• CTC Lysis

• Metastasis

• Surgery etc.

3

CTDNA DETECTION – PARALLEL SEQUENCING

Targeted Multiplex PCR Panel

CLv2 (92 Amplicons)

• Primary mutation: EGFR p.E746_A750delELREA

• LB: EGFR p.E746_A750delELREA 22% AF + p.T790M 3% AF

LB: Liquid BiopsyAF: Allele Frequency

4

ARE METHODS SENSITIVE ENOUGH FOR RESISTANCE DETECTION?

5

ONCONETWORK CONSORTIUM

Oncomine cfDNA Lung Assay Multicenter Study

Prof. Harriet Feilotter

Alexander Boag

Queen's University, Kingston

Canada

Dr. Jose Costa

Jose Carlos Machado

IPATIMUP, Medical Faculty of Porto

Portugal

Prof Marjolijn J.L. Ligtenberg

Robbert Weren

Radboud University Nijmegen

Netherlands

Dr. Nicola Normanno

Centro Ricerche Oncologiche Mercogliano

Italy

Prof. Orla Sheils

St James's Hospital Dublin

Ireland

Prof. Ian Cree

Anne Reiman

UHCW

United Kingdom

Prof. Pierre Laurent Puig

Delphine Le Corre

Université Paris Descartes Paris

France

Prof. Aldo Scarpa

PhD Andrea Mafficini

ARC-NET Research Center

University of Verona

Italy

Dr. Henriette Kurth

VIOLLIER AG Basel .

Switzerland

Prof. Kazuto Nishio

Kinki University Osaka

Japan

Cecily P. Vaughn

ARUP- Institute for Clinical and

Experimental Pathology

Utah

USA

Prof. Michael Hummel

PhD Claudia Vollbrecht

Charité Universitätsmedizin Berlin,

Germany

6

ONCOMINE CFDNA LUNG ASSAY*

• 169 hotspots, 35 amplicons, 11 genes

• Amplification-based assay

0,60%0,40%

0,25%

0,15%

0,11%

0,09%

0,05%

0

5

10

15

20

25

30

35

-

5.000

10.000

15.000

20.000

25.000

30.000

35.000

40.000

0,0% 0,1% 0,2% 0,3% 0,4% 0,5% 0,6% 0,7%

Min

imu

m a

mp

lic

on

co

ve

rag

e

Inp

ut

cfD

NA

(n

g)

Limit of detection

Genes Hotspots

ALK, BRAF, EGFR,

ERBB2, KRAS,

MAP2K1, MET,

NRAS, PIK3CA,

ROS1, TP53

>169 hotspots including:

EGFR: T790M, C797S, L848R, Exon 19 del

KRAS: G12X. G13X. Q61X

BRAF: V600E

ALK: Exon 21-25

PIK3CA: E545K. H1047R. E542K

*For research use only

7

Lab-created

Report

Oncomine™

Knowledgebase

Reporter*

Template Prep Sequencing

Ion S5™ and Ion

S5™ XL SystemsAlso enabled on Ion

PGM™ and Ion Proton™

Systems

Analysis

Variant caller in

Torrent Suite and Ion

Reporter™ Software

Library Prep

Oncomine™ cfDNA

AssaysIon Chef™ System

Refined variant data

Oncomine cfDNA assays include library prep and analysis

WORKFLOW & VALIDATION WITH REFERENCE STANDARD

cfDNA Input

Sample EGFR

E746_A750

delELREA

EGFR

L858R

EGFR

T790M

EGFR

V769_D770

insASV

KRAS

G12D

NRAS

A59T

NRAS

Q61K

PIK3CA

E545K

0.1% HDX 0.06 0.17 0.06 0.10 0.22 0.17 0.15 0.10

1% HDX 0.72 1.07 0.75 0.74 1.14 1.15 1.15 2.29

5% HDX 4.52 4.86 6.32 3.97 6.34 6.11 6.94 5.29

100% WT 0 0 0 0 0 0 0 0

8

REPEATABILITY & REPRODUCIBILITY

n=22n=22

n=21

5%n=22

n=22

n=21

1%

PIK3CA p.E545K

n=20

n=20

n=15

0.1%EGFR p.E746_A750del ELREA

KRAS p.G12D

9

REAPEATABILITY & REPRODUCIBILITY

0

1

2

3

4

5

6

7

All

ele

Fre

qu

en

cy

5%

ARCNET

CROM

IPATIMUP

St Radboud

Queens

St James

UHCW

Charite

Viollier

HEGP

Kindai0,0

0,5

1,0

1,5

All

ele

fre

qu

en

cy

1%

ARCNET

CROM

IPATIMUP

St Radboud

Queens

St James

UHCW

Charite

Viollier

HEGP

Kindai

0,00

0,05

0,10

0,15

0,20

0,25

0,30

All

ele

Fre

qu

en

cy

0.1%

ARCNET

CROM

IPATIMUP

St Radboud

Queens

St James

UHCW

Charite

Viollier

HEGP

Kindai

• EGFR p.T790M at 5%. 1%

and 0.1% allele frequency

10

SENSITIVITY & SPECIFICITY

Lab Sensitivity Specificity NPV PPV

ARCNET 89.58% 99.53% 99.61% 87.76%

CROM 95.83% 100.00% 99.84% 100.00%

IPATIMUP 95.83% 100.00% 99.84% 100.00%

Radboud 89.58% 99.84% 99.61% 95.56%

Queens 97.92% 99.92% 99.92% 97.92%

St James 95.83% 100.00% 99.84% 100.00%

UHCW 95.83% 99.76% 99.84% 93.88%

Charité 95.83% 99.76% 99.84% 93.88%

Viollier 97.50% 99.82% 99.91% 95.12%

HEGP 93.75% 99.69% 99.76% 91.84%

Kindai 95.83% 99.69% 99.84% 92.00%

Total 94.81% 99.82% 99.80% 95.93%

NPV: negativ predictive value; PPV: positive predictive value

11

SENSITIVITY & SPECIFICITY AT 0.1% ALLELE FREQUENCY

Lab Sensitivity Specificity NPV PPV

ARCNET 68.75% 100.00% 98.43% 100.00%

CROM 87.50% 100.00% 99.37% 100.00%

IPATIMUP 87.50% 100.00% 99.37% 100.00%

Radboud 68.75% 99.68% 98.43% 91.67%

Queens 93.75% 100.00% 99.68% 100.00%

St James 87.50% 100.00% 99.37% 100.00%

UHCW 87.50% 100.00% 99.37% 100.00%

Charité 87.50% 100.00% 99.37% 100.00%

Viollier 87.50% 100.00% 99.37% 100.00%

HEGP 81.25% 99.36% 99.05% 86.67%

Kindai 87.50% 99.68% 99.37% 93.33%

Total 83.93% 99.88% 99.19% 99.13%

NPV: negativ predictive value; PPV: positive predictive value

12

SENSITIVITY & SPECIFICITY

Allele Frequency Sensitivity Specificity

0.1% - 5% 94.81% 99.82%

0.1% 83.93% 99.88%

Data collected from 11 laboratories:

UHCW, CROM, ARCNET, IPATIMUP, Radboud University, Queen’s University,

St James Hospital, Violler, HEGP, Kinday University, Charité Hospital

13

0

2

4

6

8

PIK3CA p.E545K EGFRp.V769_D770insASV

EGFRp.E746_A750delELREA

EGFR p.T790M EGFR p.L858R KRAS p.G12D NRAS p.Q61K NRAS p.A59T

CLv2 Oncomine

5% AF

0

0,5

1

1,5

2




1% AF

0

0,1

0,2

0,3

0,4

0,5

0,6




0.1% AF

CLV2 VS ONCOMINE LUNG CFDNA

• 1% and 5% AF dilution in all samples detectable with both assays

• 0.1% AF dilution detectable with Oncomine in 88% of cases, with CLv2 in 50% of

cases

AF: Allele Frequency

14

EXPERIENCE FROM ROUTINE DIAGNOSTICS

1 Patient, 3 Samples

Collection Tube Streck PAXgene Streck

cfDNA/ml Plasma 18ng/ml* 17ng/ml 85ng/ml

cfDNA Input in PCR 4.22ng 3.94ng 28.74ng

Library [pM] 57pM 28pM 75pM 49pM 35pM 36pM 8,151pM

NGS CLv2 NA NA NA NA NA NA NA NA EGFR L858R 22% AFT790M 0.2%AF

*Evaluable with Oncomine Assay: EGFR L858R 14% AF & T790M 1.4% AF

AF: Allele Frequency

15

CONCLUSION

• Oncomine Lung cfDNA Assay* enables detection of primary driver &

resistance mutations to a level of 0.1%

• OncoNetwork Consortia evaluted the assay in a repeatability and

reproducibility multicenter study using Horizon cfDNA reference set

• Results from 11 laboratories demonstrated more than 94%

sensitivity and 98% specificity

More sensitive than CLv2 panel

*For research use only

0%

20%

40%

60%

80%

100%

0,1 1 5CLv2 Oncomine

16

ACKNOWLEDGEMENTS

Anna Maria RachiglioCentro di Ricerche Oncologiche di Mercogliano (CROM)-Instituto Nazionale Tumori “FondazioneG. Pascale”-IRCCS, Naples, ItalyProf. Orla SheilsSt James's Hospital Dublin, IrelandProf. Pierre Laurent Puig & Delphine Le CorreUniversité Paris Descartes, Paris, FranceProf. Ian Cree & Anne ReimanUHCW, United KingdomDr. Jose Costa & Jose Carlos MachadoIPATIMUP, Medical Faculty of Porto, PortugalDr. Nicola NormannoCentro Ricerche Oncologiche Mercogliano, ItalyProf. Aldo Scarpa & PhD Andrea MafficiniARC-NET University of Verona, ItalyProf. Kazuto NishioFaculty of Medicine. Kinki University Osaka, JapanProf. Harriet Feilotter & Alexander BoagQueen's University, Ontario, CanadaProf. Marjolijn J.L. Ligtenberg & Robbert WerenRadboud University Nijmegen, Medical Centre, NetherlandsDr. Henriette KurthVIOLLIER AG Basel, SwitzerlandProf. Michael HummelInstitute of Pathology Charité Berlin, Germany

OncoNetwork Consortium

Molpath Team AG Hummel

Thermo Fisher Scientific TeamRosella Petraroli & Christopher Allen

Cristina Guillen Rovira & Kizzie Manning-Kroeger

multi-center evaluation eines hoch sensitiven ngs assays ...€¦ · 1 patient, 3 samples...

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