Multi Institutional Evaluation of a High Sensitive NGS Assay for Liquid Biopsy Mutation Detection in Lung Cancer

Dr. Claudia Vollbrecht Charité Medical University Berlin Institute of Pathology 05 April 2017

Conflicts of interest

DISCLOSURE I have the following potential conflict(s) of interest to report

• Congress fees paid by Thermo Fisher Scientific

2

3

LIQUID BIOPSIES - A PROMISING TOOL FOR FUTURE

• Early, non-invasive detection of MRD & resistances with sensitive methods like parallel-sequencing

• Allows monitoring of the tumour evolution

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4

Liqu

id B

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Gen

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Circ

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Tum

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NA,

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l. 20

14, J

Clin

Onc

ol

Mono-nucleosomale cfDNA

Di-nucleosomale cfDNA

T ½ approx. 2 hours

~ 180bp <1% ctDNA in cfDNA

LIQUID BIOPSY – CFDNA EXTRACTION

cfDNA Release Depends on: • Localization of tumor • Tumors size

• Vascularity • Therapy status

cfDNA: cell-free DNA ctDNA: circulating tumor DNA

gDNA

5

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

6

CTDNA DETECTION – PARALLEL SEQUENCING

?

EGFR Primary and

T790M Resistance Mutation

[PROZENTSATZ]

Only Primary EGFR

Mutation [PROZENTSA

TZ]

No Activating

EGFR Mutation

[PROZENTSATZ]

Not Evaluable

[PROZENTSATZ]

7

ONCONETWORK CONSORTIUM

Oncomine cfDNA Lung Assay Multicenter Study

Prof. Harriet Feilotter Dr. Paul Park

Queen's University, Ontario Canada

Dr. Jose Costa IPATIMUP, Medical Faculty of

Porto. Portugal

Marjolijn J.L. Ligtenberg Radboud University Nijmegen Medical Centre, Netherlands

Dr. Nicola Normanno Centro Ricerche Oncologiche

Mercogliano, Italy

Prof. Orla Sheils St James's Hospital Dublin,

Ireland

Prof. Ian Cree UHCW

United Kingdom

Prof. Pierre Laurent Puig Université Paris Descartes,

Paris, France

Prof. Aldo Scarpa ARC-NET University of

Verona Italy

Dr. Henriette Kurth VIOLLIER AG Basel ,

Switzerland

Prof. Kazuto Nishio Faculty of Medicine, Kinki University Osaka, Japan

Cecily P. Vaughn ARUP- Institute for Clinical and

Experimental Pathology Utah, USA

Prof. Michael Hummel Institute of Pathology Charité

Berlin, Germany

8 *For Research use only

ONCONETWORK CONSORTIUM

Oncomine Lung cfDNA 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

imum

am

plic

on c

over

age

Inpu

t cfD

NA

(ng)

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

9

Lab-created Report

Oncomine™ Knowledgebase

Reporter*

Template Prep Sequencing

Ion S5™ and Ion S5™ XL Systems Also enabled on Ion

PGM™ and Ion Proton™ Systems

Analysis

Variant caller in Torrent Suite and Ion Reporter™ Software

Library Prep

Oncomine™ cfDNA Assays

Ion Chef™ System

Refined variant data

Oncomine cfDNA assays include library prep and analysis

ONCONETWORK CONSORTIUM

cfDNA Input

Sample EGFR E746_A750delELREA

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

All 8 mutant hotspots were detected at 0.1%, no false positives

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ONCONETWORK CONSORTIUM

Repeatability & Reproducibility n=22 n=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

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ONCONETWORK CONSORTIUM

Reapeatability & Reproducibility

0

1

2

3

4

5

6

7

Alle

le F

requ

ency

5% ARCNET

CROM

IPATIMUP

St Radboud

Queens

St James

UHCW

Charite

Viollier

HEGP

Kindai0.0

0.5

1.0

1.5

Alle

le fr

eque

ncy

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

Alle

le F

requ

ency

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

12

ONCONETWORK CONSORTIUM

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%

Charite 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%

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ONCONETWORK CONSORTIUM

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%

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ONCONETWORK CONSORTIUM

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

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CONCLUSION

• The Oncomine Lung cfDNA Assay* enables detection of primary

driver and 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

*For Research use only

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ACKNOWLEDGEMENTS

Cecily P. Vaughn ARUP- Institute for Clinical and Experimental Pathology, Utah, USA Prof. Orla Sheils St James's Hospital Dublin, Ireland Prof. Pierre Laurent Puig Université Paris Descartes, Paris, France Prof. Ian Cree UHCW, United Kingdom Dr. Jose Costa IPATIMUP, Medical Faculty of Porto, Portugal Dr. Nicola Normanno Centro Ricerche Oncologiche Mercogliano, Italy Prof. Aldo Scarpa ARC-NET University of Verona, Italy Prof. Kazuto Nishio Faculty of Medicine, Kinki University Osaka, Japan Prof. Harriet Feilotter & Dr. Paul Park Queen's University, Ontario, Canada Marjolijn J.L. Ligtenberg, Radboud University Nijmegen, Medical Centre, Netherlands Dr. Henriette Kurth VIOLLIER AG Basel, Switzerland Prof. Michael Hummel Institute of Pathology Charité Berlin, Germany

OncoNetwork Consortium

Molpath Team AG Hummel

Thermo Fisher Scientific Rosella Petraroli & Chrisopher Allen

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