Circulating tumour DNA testing in NSCLC clinical research: an international consortia study Michael Hummel Institute of Pathology

“Liquid biopsies” – cell-free (tumour) DNA: is the “hype” justified?

DNA, deoxyribonucleic acid. Bennett CW, et al. OncoTarget. 2016;7:71013-35.

Citations of “liquid biopsies”, “next generation sequencing”, and “precision/personalized medicine”

350

300

250

200

150

100

50

0

Art

icle

s (n

)

2010 2011 2012 2013 2014 2015 Time (year)

6,000

5,000

4,000

3,000

2,000

1,000

0

Art

icle

s (n

)

2010 2011 2012 2013 2014 2015 Time (year)

3,500

3,000

2,500

2,000

1,500

1,000

500

0

Art

icle

s (n

)

2010 2011 2012 2013 2014 2015 Time (year)

cfDNA release

• Localization of the tumour

• Tumour size

• Vascularization

• Therapy status

• Other

Liquid biopsy – the source of cell-free tumour DNA (cfDNA)

cfDNA, cell-free DNA. Diaz LA Jr, Bardelli A. J Clin Oncol. 2014;32:579-86.

EGFR mutations in NSCLC predict response to EGFR TKI • Target both the initial activating EGFR mutations and the dominant

acquired T790M resistance mutation

• Treatment for mutant NSCLC in individuals with the T790M mutation after progression on EGFR-directed therapy

EGFR, epidermal growth factor receptor; NSCLC, non-small-cell lung cancer; TKI, tyrosine kinase inhibitor. Walter AO, et al. Cancer Discov. 2013;3:1404-15.

For research use only assay

Analysis of cfDNA – detection of very small amounts of tumour DNA!

PCR, polymerase chain reaction. Dietz S, et al. BIOspektrum 2015;7:731-3.

No

No

No

No

Yes

No

Moderate

Low

Moderate

Low

High

Low

Low

Low

Moderate

Low

High

Low

Moderate

Easy

Moderate

Easy

Complex

Easy

Low

Moderate

High

Low

High

Low

0.02%

< 0.1%

< 0.1%

1%

1%

> 15%

Panel sequencing

Digital PCR

BEAMing

Quantitative PCR

High-throughput sequencing

Sanger sequencing

Biomarker identification Costs

Information content

Data analysis Throughput

Detection threshold Specificity

Liquid biopsy – ctDNA Mono-nucleosomal cfDNA

Di-nucleosomal cfDNA

T½ approx. 2 h

~ 180 bp < 1% ctDNA in cfDNA gDNA

cfDNA release depends on • Tumour localization • Tumour size • Vascularity • Therapy status

bp, base pair; gDNA, genomic DNA. Diaz LA Jr, Bardelli A. J Clin Oncol. 2014;32:579-86.

• Inflammation • CTC lysis • Metastasis • Surgery, etc.

Are methods sensitive enough for resistance detection?

WT, wild type.

Primary and T790M resistance mutation

Only primary mutation WT

Other mutations

Not evaluable

17%

31% 39%

5% 8%

?

Onconetwork consortium Oncomine™ cfDNA lung assay multicentre 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 (CROM), Italy

Prof. Orla Sheils St James's Hospital

Dublin, Ireland

Prof. Ian Cree UHCW

United Kingdom

Prof. Pierre Laurent Puig Université Paris Descartes, Hôpital Européen

Georges-Pompidou (HEGP) Paris, France

Prof. Aldo Scarpa ARC-NET University of Verona

Italy

Dr Henriette Kurth Viollier AG

Basel, Switzerland

Prof. Kazuto Nishio Faculty of Medicine, Kinday

University, Osaka, Japan

Cecily P. Vaughn ARUP-Institute for Clinical and

Experimental Pathology Utah, USA

Prof. Michael Hummel Institute of Pathology

Charité Berlin, Germany

Oncomine™ cfDNA lung research 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% 0.2% 0.4% 0.6%M

inim

um a

mpl

icon

cov

erag

e

Input cfDN

A (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

0 0.2 0.4 0.6

For Research Use Only. Not for use in diagnostic procedures

Workflow and validation with reference standard

HDX, highly divergent homeobox.

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

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

cfDNA input

For research use only.

Repeatability and reproducibility

n = 22 n = 22

n = 21 5%

AF

8 7 6 5 4 3 2 1 0

PIK3CA p.E545K EGFR p.E746_A750del ELREA KRAS p.G12D

n = 22 n = 22

n = 21

1%

AF

2 1.8 1.6 1.4 1.2

1 0.8

0.3

0

0.4

0.2

n = 20 n = 20

n = 15

0.1%

AF

0.25

0.2

0.15

0.1

0.05

0 For research use only.

Repeatability and reproducibility (cont.)

EGFR p.T790M at 5%, 1%, and 0.1% AF

0

2

4

6

8

AF

5% ARCNETCROMIPATIMUPSt RadboudQueensSt JamesUHCWChariteViollierHEGPKindai

ARC-NET CROM IPATIMUP Radboud Queen’s St James’s UHCW Charité Viollier HEGP Kindai

0.00

0.05

0.10

0.15

0.20

0.25

0.30

AF

0.1% ARCNETCROMIPATIMUPSt RadboudQueensSt JamesUHCWChariteViollierHEGPKindai

ARC-NET CROM IPATIMUP Radboud Queen’s St James’s UHCW Charité Viollier HEGP Kindai

0.0

0.5

1.0

1.5

AF

1% ARCNETCROMIPATIMUPSt RadboudQueensSt JamesUHCWChariteViollierHEGPKindai0

0

ARC-NET CROM IPATIMUP Radboud Queen’s St James’s UHCW Charité Viollier HEGP Kindai

For research use only.

Sensitivity and specificity (cont.)

Allele frequency (%) Sensitivity (%) Specificity (%)

0.1–5 94.81 99.82

0.1 83.93 99.88

Data collected from 11 laboratories

UHCW, CROM, ARC-NET, IPATIMUP, Radboud University, Queen’s University,

St James’s Hospital, Viollier, HEGP, Kindai University, Charité Hospital

For research use only.

Oncomine™ cfDNA research assays content

35 amplicon panel for lung Covering key hotspot mutations in 11 genes 169 hotspot SNVs and indels

49 amplicon panel targeting multiple cancer types Covering key hotspot mutations in 14 genes 236 hotspot SNVs and indels

26 amplicon panel for breast Covering key hotspot mutations in 10 genes 152 hotspot SNVs and indels

• Designed to detect primary tumour drivers and resistance mutations • Reagents, primers, and analysis software to analyse mutations from a single tube of blood • Flexible input amounts and tolerance of sample input variability to achieve 0.1% limit of detection

of SNV hotspots and indels from cfDNA

Oncomine™ lung cfDNA assay

ALK BRAF EGFR ERBB2 KRAS MAP2K1

MET NRAS PIK3CA ROS1 TP53

Oncomine™ colon cfDNA assay

AKT1 BRAF CTNNB1 EGFR ERBB2 FBXW7 GNAS

KRAS MAP2K1 NRAS PIK3CA SMAD4 TP53 APC

Oncomine™ breast cfDNA assay

AKT1 EGFR ERBB2 ERBB3 ESR1

FBXW7 KRAS PIK3CA SF3B1 TP53

*For research use only.

There is more to come!

Oncomine™ lung cell free total nucleic acid assay ALK

BRAF EGFR ERBB2

KRAS MAP2K1 MET NRAS

PIK3CA ROS1 TP53

NEW Oncomine™ lung and breast cell-free research assays

The content provided herein may relate to products that have not been officially released and are subject to change without notice. CNV, copy number variation; CCND1, cyclin D1; FGFR1, fibroblast growth factor receptor 1.

• Designed to detect primary tumour drivers and resistance mutations • Containing fusions and CNVs

Enhanced Oncomine™ breast cfDNA assay AKT1

EGFR ERBB2 ERBB3

ESR1 FBXW7 KRAS PIK3CA

SF3B1 TP53

Amplicons: 76 Covering • Key hotspot mutations in 10 genes • CNVs – CCND1, ERBB2, FGFR1 • More complete coverage of TP53 (~ 80%) • Single library to detect SNVs and CNVs SNV LOD down to 0.1% with 20 ng input Same sensitivity and specificity

58 amplicon + 49 fusion assays + 3 MET exon skipping assays for new lung assay Covering • Key hotspot mutations in 11 genes • Fusions – ALK, RET, ROS1 • CNV – MET • MET skipping SNV LOD down to 0.1% with 20 ng input Same sensitivity and specificity for SNVs Single library from both DNA and RNA

*For research use only.

Conclusions

• Liquid biopsies represent a sensitive approach for tumour detection in body fluids

• Technical pitfalls (including pre-analytics!) need to be considered

• Amplicon-based panel NGS is very well suited for cfDNA analysis

• Very well designed and validated panels are a prerequisite for reliable and sensitive use

*For research use only.