Results of the joint pilot ctDNA scheme… and next steps

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Results of the

joint pilot ctDNA scheme…..

and next steps

Naples 2017

Dr Sandi Deans

UK NEQAS for Molecular Genetics and Molecular Pathology, Edinburgh, UK

On behalf of the IQN Path ctDNA pilot EQA Group

(a sub-group of the Liquid Biopsy Working Group)

(2)

Objective

Initiate a collaboration between four EQA providers to provide a pilot EQA to assess the standard of testing for the presence of circulating tumour DNA

(ctDNA) in plasma with the purpose of promoting high quality molecular testing.

(3)

Pilot EQA working group

Member Affiliation

Dr Sandi Deans (Chair) UK NEQAS

Dr Jenni Fairley UK NEQAS

Dr Simon Patton EMQN

Dr Melanie Cheetham EMQN

Prof Els Dequeker ESP EQA

Cleo Keppens ESP EQA

Dr Nicola Normanno AIOM

Dr Ed Schurring Scientific Advisor Mrs Rachel Butler Scientific Advisor

Dr Jacqueline Hall IQN Path

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Aims

 Investigate the feasibility of delivering a technical challenging EQA

 Assess the ability of laboratories to detect ctDNA mutations in artificial plasma samples using a range of methodologies

 Share findings with participant laboratories and the IQN Path Liquid Biopsy Working Group

 Assess the standard of reporting ctDNA testing results

 Hold a workshop to review current practice and develop consensus guidelines to promote high quality ctDNA testing

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Scope of pilot EQA

 Well-designed EQA schemes require the development and validation of distribution materials

 Harmonised between several EQA schemes to increase efficiencies and speed of access to EQA

 Include common and clinically relevant mutations/hot spots

 Challenging samples with low frequency allele mutations present at the limit of detection of the methods used should also be included to reassure

laboratories that their testing strategies can meet the clinical need.

Warning – too low too soon!!!!

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Timeline

Oct/

Nov 2015

Dec 2015 /Jan 2016

Feb /Mar 2016

Apr /May 2016

Jun /Jul 2016

Aug /Sep 2016

Oct /Nov 2016

Dec 2016 /Jan 2017

Feb /Mar 2017

Apr /May 2017

Jun/

2017Jul

Recruitment of validating laboratories

Tender for material Award tender

Material manufacture Batch 1 Material manufacture Batch 2 Sample validation Batch 1 Sample validation

Batch 2

Pilot EQA distribution Analysis of EQA results Issue of results

International workshop

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EQA material

Competitive Tender to a defined s

pecification

 Panel of 10 plasma samples supplied in 3ml aliquots with ctDNA containing either KRAS, NRAS, EGFR variants, or wild-type

 2 different allelic frequencies (5% and 1%)

 ctDNA fragmented to 150bp and ~20ng ctDNA/ml plasma

2 providers:

(8)

Validation of EQA material

 Trial transportation mode

 Stability of samples

 Ability to obtain a reportable result

 Multiple methodologies:

 Next Generation sequencing

 Droplet digital PCR

 Therascreen

 Cobas

 Beaming

 Idylla

3 1

1

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Validation of EQA material

 Therascreen

 Cobas

 Beaming

 Idylla

3 1

1

– Frozen plasma – Shipped on dry ice – Monitored

– 1 x defrosting

(10)

Validation of EQA material

 Therascreen

 Cobas

 Beaming

 Idylla

3 1

1

– Frozen plasma – Shipped on dry ice – Monitored

– 1 x defrosting

– Shipment to Arrival (~1.4 days (range 1-5 days)

– Storage time (~8.7 days)

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DNA extraction methodologies

• NGS: QIAamp Circulating Nucleic AcidKit (Qiagen)

• Cobas: Cobas cfDNA sample preparation kit

1 (UK)

• QIAamp Circulating Nucleic AcidKit (Qiagen)

2 (UK)

• QIAamp Circulating Nucleic AcidKit (Qiagen)

• Idylla integrated workflow

3 (NL)

• QIAamp Circulating Nucleic Acid Kit (Qiagen)

4 (IT)

• Cobas cfDNA Sample Preparation Kit (Roche)

5 (UK)

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Variant detection methodologies

• NGS: Capture SureSelect (Agilent), MiSeq (Illumina)

• Commercial: Cobas® EGFR Mutation Test v2 (Roche)

1 (UK)

• ddPCR: QX200 Droplet Digital PCR System (Bio-rad)

• NGS: Ampliseq 50 gene hotspot panel, Ion Proton (Life Technologies)

2 (UK)

• ddPCR: QX200 Droplet Digital PCR System (Bio-rad)

• Idylla ctKRAS mutation prototype test and Idylla ctNRAS mutation prototype test (R&D version, no commercial product)

3 (NL)

• BEAMing: OncoBEAM® RAS CRC IVD KIT (Sysmex-Inostics)

• Commercial: Therascreen EGFR Plasma RGQ PCR Kit (Qiagen)

4 (IT)

• Commercial: Cobas® EGFR Mutation Test v2 (Roche)

5 (UK)

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EGFR validation results

WT (Batch 1)

Variant (Batch 1)

WT (Batch 2)

ddPCR

  

^(5%) /  ^(1%)

½ labs did not detect double mutation at 1%

NGS (Illumina)

 

(5%) /  (1%)



^

NGS (Life Technologies)

 

^(5%) /  (1%) Not p.(L858R)



^⁽^5%) ^{/ } ^(1%)

Not double mutation at 1%

Therascreen^© (Qiagen)

 

^(5%) /  (1%)

Not ex19 del

 

cobas^©(Roche)

  

 (5%) /  (1%)

 ½ labs did not detect p.(L858R)

at 1%

Batch 1 Variants: (i) del in exon 19 c.2235_2249del15 and p.(T790M) (ii) p.(L858R)

Batch 2 Variants: (i) del in exon 19 c.2235_2249del15 (ii) p.(T790M) and p.(L858R)

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EGFR validation results

WT (Batch 1)

WT (Batch 2)

ddPCR

  

^(5%) /  ^(1%)

NGS (Illumina)

 

(5%) /  (1%)



^

 

^(5%) /  (1%) Not p.(L858R)



^⁽^5%) ^{/ } ^(1%)

 

^(5%) /  (1%)

Not ex19 del

 

cobas^©(Roche)

  

 (5%) /  (1%)

at 1%

Variable detection of specific variants

(15)

EGFR validation results

WT (Batch 1)

WT (Batch 2)

ddPCR

  

^(5%) /  ^(1%)

NGS (Illumina)

 

(5%) /  (1%)



^

 

^(5%) /  (1%) Not p.(L858R)



^⁽^5%) ^{/ } ^(1%)

 

^(5%) /  (1%)

Not ex19 del

 

cobas^©(Roche)

  

 (5%) /  (1%)

at 1%

Variable detection of 1% AF variants

(16)

KRAS validation results

WT (Batch 1)

WT (Batch 2)

Idylla^©(Biocartis)

   

BEAMing^©

(Sysmex-Inostics)

 (2/8)

Codon 12 mut



Codon 12 mut^ ^(1/8)



ddPCR

   

NGS (Illumina) 



^(5%) /  (1%)   (5%) /  (1%)

NGS (Life

Technologies)

 

^(5%) /  (1%)   (5%) /  (1%)

Batch 1 Variants: p.(G12D) Batch 2 Variants: p.(G12D)

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KRAS validation results

WT (Batch 1)

WT (Batch 2)

   

BEAMing^©

(Sysmex-Inostics)

 (2/8)

Codon 12 mut



Codon 12 mut^ ^(1/8)



ddPCR

   

NGS (Illumina) 



^(5%) /  (1%)   (5%) /  (1%)

NGS (Life

Technologies)

 

^(5%) /  (1%)   (5%) /  (1%)

Comparable performance

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KRAS validation results

WT (Batch 1)

WT (Batch 2)

   

BEAMing^©

(Sysmex-Inostics)

 (2/8)

Codon 12 mut



Codon 12 mut^ ^(1/8)



ddPCR

   

NGS (Illumina) 



^(5%) /  (1%)   (5%) /  (1%)

NGS (Life

Technologies)

 

^(5%) /  (1%)   (5%) /  (1%)

Comparable performance

(19)

NRAS validation results

WT (Batch 1)

WT (Batch 2)

 

^(5%) /  (1%)

 

^(5%) /  (1%)

BEAMing^© (Sysmex- Inostics)

 (3/8)

False positives 

 

ddPCR

   

^(5%) /  (1%)

NGS (Illumina)

 

^(5%) /  ^(1%)



^ ^(5%) ^{/ } ^(1%)

NGS (Life

Technologies)

   

^(5%) /  ^(1%)

Batch 1 Variants: p.(G12D) Batch 2 Variants: p.(Q61R)

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NRAS validation results

WT (Batch 1)

WT (Batch 2)

 

^(5%) /  (1%)

 

^(5%) /  (1%)

 (3/8)

False positives 

 

ddPCR

   

^(5%) /  (1%)

NGS (Illumina)

 

^(5%) /  ^(1%)



^ ^(5%) ^{/ } ^(1%)

NGS (Life

Technologies)

   

^(5%) /  ^(1%)

Batch 1 Variants: p.(G12D) Batch 2 Variants: p.(Q61R)

False positives reported

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Validation conclusions

• Verified manufacturers results

• Shipping and storage conditions confirm stability

Stability and homogeneity

• Expected genotypes confirmed

• Method limitations confirmed (LOD)

Assigned results and uncertainties

• Reliable results if:

• Shipment on dry ice

• Storage at -80°C

• Transportation ≤ 5 days

• Storage ≤ 14 days

Preparation, handling and distribution of EQA materials

• Both batches of material were stable and gave reportable results – to a degree

• Batch 1 gave false positive NRAS results

• Inconsistent detection of EGFR variants in Batch 1

Selection of pilot

EQA material

(22)

Validation conclusions

• Verified manufacturers results

• Shipping and storage conditions confirm stability

Stability and homogeneity

• Expected genotypes confirmed

• Method limitations confirmed (LOD)

Assigned results and uncertainties

• Reliable results if:

• Shipment on dry ice

• Storage at -80°C

• Transportation ≤ 5 days

• Storage ≤ 14 days

Preparation, handling and distribution of EQA materials

• Both batches of material were stable and gave reportable results – to a degree

• Batch 1 gave false positive NRAS results

• Inconsistent detection of EGFR variants in Batch 1

Selection of pilot EQA material

Batch 2 selected by working group for EQA pilot run 2017

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Sample distribution

8 labs 8 labs 8 labs 8 labs

32 Participants world-wide

0 1 2 3 4 5 6 7 8 9

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Sample distribution

EGFR mutation testing

Lung cancer case scenarios

• Panel of 5 plasma samples

• Range of EGFR mutations…or not!

• At different allelic frequencies 7 EGFR only laboratories

KRAS/NRAS mutation testing Colorectal cancer case scenarios

• Panel of 5 plasma samples

• Range of RAS mutations…or not!

• At different allelic frequencies 0 RAS only laboratories

AND/OR

25 EGFR and RAS laboratories

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Participants

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Next Steps

FOR EQA PROVIDERS

 T/C to agree marking criteria

 Assessment meeting to finalise scoring and feedback comments to laboratories Review issues arisen from pilot run and draft scheme report

 Discussion on addressing challenges:

• Dry ice shipment not economic ~£500 per package

• Shipment at room temperature

• Freeze drying/lyophilisation of plasma

• Use of artificial plasma

 Planning to for open participation to all interested laboratories

(27)

Next Steps

FOR PILOT PARTICIPANTS,

INTERESTED LABORATORIES

& KEY STAKEHOLDERS

 Host workshop on 23^rd June 2017 in Florence

 Generate consensus ctDNA testing guidelines

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Next Steps

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Acknowledgements

• Liquid Biopsy Working Group

• Validation Laboratories

• The Sponsors

• The EQA providers: