ngs, a suitable approach for tp53 screening in cll? - eric · ngs, a suitable approach for tp53...

NGS, a suitable approach forTP53 screening in CLL?

Ferran Nadeu

2nd ERIC WORKSHOP ON TP53 ANALYSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA7-8 November 2017, Stresa (Italy)

1 patient

TP53

4 PCR/case*

+

* Exon 4, exons 5-6, exon 7, exons 8-9.

=

4 PCRs

20 patients

TP53

4 PCR/case*

+

=

100 PCRs

400 patients

TP53

4 PCR/case*

+

=

1600 PCRs

The Sanger sequencing bottleneck

1 patient

TP53

4 PCR/case*

+

=

4 PCRs

20 patients

TP53

4 PCR/case*

+

=

100 PCRs

400 patients

TP53

4 PCR/case*

+

=

1600 PCRs

TP53, SF3B1, BIRC3,

NOTCH1 and ATM

≈75 PCR/case

75 PCRs 1500 PCRs

TP53, SF3B1, BIRC3,

NOTCH1 and ATM

≈75 PCR/case

30000 PCRs

TP53, SF3B1, BIRC3,

NOTCH1 and ATM

≈75 PCR/case

The Sanger sequencing bottleneck

Nadeu et al Blood 2016

Outline

1. NGS overcomes this Sanger sequencing bottleneck (focus on the Access-Array system)

2. NGS for TP53 analysis:

a) Access-Array system

b) Comparison of NGS libraries

3. Pros and cons of the use of NGS:

a) Sensitivity

b) Bioinformatic analysis

c) Accuracy

d) Possibility to study several genes simultaneously (i.e. panel of genes)

Access-Array system (Fluidigm) – Quick overview

Amplicon-based

15 amplicons of 180bp

4-Primer Amplicon Tagging


48.48 Access Array IFC (Integrated Fluidic Circuit)

IFC controller AX

2304 individual reactions

chambers (30 nL)



IFC controller AX BioMark HD



IFC controller AX



Clean-up

Access-Array system – Up to 480 amplicons

Multiplexing

Figures adapted from Access-Array System User Guide (Fluidigm).

2nd PCR done in a

96-well plate in a

or al ther ocycler

Outline

1. NGS overcomes this Sanger sequencing bottleneck (Access-Array system)





a) Sensitivity


c) Accuracy

d) Possibility to study several genes simultaneously

Coverage/depth in NGS

A T G C C T G A T G

Spencer et al J Mol Diagn 20142 4 6 6 6 6 6 4 2 0Coverage

T G C C T G A

A T G C C T G

G C C T G A T

T G C C T G A

G C C T G A T

A T G C C T G

Access-Array system for TP53 analysis

Access-Array system (Fluidigm) – Coverage


Access-Array system (Fluidigm) – Coverage


A coverage >1000x was obtained in >85% of the sequence in 95% of the samples

Capture hybridization-based

Amplicon-based

• Acces-Array - Flluidigm

• AmpliSeq - ThermoFisher (Ion Torrent)

• Genereadv2/QIASeq - QIAgen

•Multiplicom - Agilent

• TruSeq Custom Amplicon - Agilent

• TruSeq Custom Amplicon DS – Agilent

• NexteraXT - Illumina

•SureSelect - Agilent

• SeqCap EZ System -Nimblegen/Roche

• xGen Lockdown - IDT

• Haloplex – Agilent

• Nextera XT - Illumina

Comparison of NGS libraries for TP53 screening

Comparison of NGS libraries for TP53 screening

Amplicon-based Capture hybridization-based

Outline

1. NGS overcomes this Sanger sequencing bottleneck (Access-Array system)





a) Sensitivity


c) Accuracy

d) Possibility to study several genes simultaneously

Pros and cons of NGS: Sensitivity


Pros and cons of NGS: Sensitivity

Rossi et al Blood 2014:

• 30% (15/50) patients carried only subclonal TP53 mutations

• 8% (4/50) patients carried isolated del(17p)

• NGS: 92% (46/50) of patients with TP53 alterations

• Sanger + FISH: 70% (35/50) of patients with TP53 alterations

Nadeu et al Blood 2016:

• 34% (16/47) patients carried only subclonal TP53 mutations

• 9% (4/47) patients carried isolated del(17p)

• NGS: 91% (43/47) of patients with TP53 alterations

• Sanger + SNP array: 66% (31/47) of patients with TP53 alterations

Pros and cons of NGS: Bioinformatic analysis

Sequencing error rate:

Platform

Chemistry

Read length

Genomic context

...

Spencer et al J Mol Diagn 2014

Pros and cons of NGS: Bioinformatic analysis

SVC


Pros and cons of NGS: Accuracy


VAF ≥

%

VA

F <

12

%

(Sanger sequencing on

302 regions/genes detected 69 mutations)

100% specificity and

100% sensitivity

(43 cases carrying high frequency mutations

were subjected to a second round of NGS)

100% reproducibility

88 mutations verified by AS-PCR

77 mutations verified by a 2nd round of NGS

13/13 were also verified by AS-PCR

Verification: AS-PCR or 2nd run of NGS

After a non-stringent custom filtering ≈75% specificity < % of VAF

Pros and cons of NGS: Scalability

Genes targeted

Cost

Hands-on

Precision medicine

1 nNumber of genes targeted

n

0

Tim

eE

uro

sB

en

efi

t

Pros and cons of NGS: Panel of genes

Nadeu et al Leukemia 2017

Pros and cons of NGS: Panel of genes

Nadeu et al Leukemia 2017 Guièze et al Blood 2015

NGS libraries and TP53

• Easy design

• Short hands-on time

• Compatible with multiple sequencing platforms (Illumina, Ion Torrent, etc.)

• Percentage of amplification success ≥95%, and good accuracy (>90% mapping to target)

• Uniform coverage

• Highly sensitive and reproducible approach to detect mutations in TP53 and other genes in clinical samples

Take home messages

NGS in the clinics

• Many different library approaches work well.

• We may face a CLL (or lymphoma) panel rather than analyzing an individual gene

• Which genes? How we validate it in larger cohorts?

• Should we sequence the normal DNA? It could be useful for the study of ATM mutations and for the variant calling.

Bioinformatic approach (still not well defined)

• A gold-standard bioinformatic pipeline for deep-targeted NGS is not yet stablished.

• Limited accuracy in the detection of clinically relevant very low VAF mutations. Verification may be needed.

• Feasible identification of CNA [del(17p), del(11q)] from the same NGS data is still pending.

…should all this be uniform for the different centers?

Elías Campo

Cristina Capdevila

Sara Guijarro

Julio Delgado

Guillem Clot

Thank you for your attention!

Acknowledgment

Anna Enjuanes

Magda Pinyol

Helena Suárez-Cisneros

Montse Sanchez

Laura Pla

ngs, a suitable approach for tp53 screening in cll? - eric · ngs, a suitable approach for tp53...

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