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Next Generation Sequencing in

de moleculaire diagnostiek

Dr. Marjolijn Ligtenberg,

klinisch moleculair geneticus,

Klinisch moleculair bioloog in de pathologie (in wording)

UMC St Radboud Nijmegen

Precision medicine

Pao and Hutchinson, Nature Med, 2012

Actionable targets non small cell lung cancer EGFR mutation-analysis on endobronchial ultrasound-

guided fine needle cytological aspirates.

resistance

activating

Schuurbiers et al, J Thorac Oncol, 2010

Requirements of a molecular diagnostic test

• robust performance using limited amounts of FFPE-

material

• high accuracy

• clinically relevant sensitivity

• availability of results within days

Technical requirements

• Flexible: easy to implement new markers

• Detection of all mutations in an amplicons

>> Next Generation Sequencing

PGM: 10-100 genes/patiënt

Whole genome Sanger sequencing

1-5 genes/patiënt

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Loman et al Nature Biotechnology, 2012

PacBio RS (Pacific biosciences) too low qaulity of sequence data (Quail et al BMC genomics 2012)

Our choice Ion Torrent: fast ; flexible; acceptable costs

Proces

Centraal genoom

analyse lab (CGAL) LTG LTG

LMS/ICT

QC

Monster voorbewerking

• Multiplex PCR

• End Repair

• Ligatie adapters/barcodes

• A Biotine gelabeld

complementair primer voor seq

bevat barcode

• P1 complementair ISP

• Quantificatie m.b.v. qPCR of Qubit

• Equimolair poolen

LTG

Clonale amplificatie m.b.v. Emulsie PCR (emPCR)

• Ion Sphere Particles [ISP]

• Primers (op ISP) complementair aan P1 adapter

• PCR reactie mix

• Olie op de mix omdraaien

CGAL

Emulsie PCR (emPCR)

• Waterblaasjes micro PCR reactie tubes

• 1010 blaasjes

• 1 ISP per blaas (theoretisch)

• Amplificatie plaat

clonale PCR

CGAL

Verrijking

• Automatische zuivering wegwassen lege beads

• Streptavidine gelabelde magnetic beads (overmaat)

• Sequencing: 1 ISP per well >> sequentie van 1 DNA

molecuul

CGAL

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Sequencing

CGAL

Sequencing

chip wells reads

amplicons

> 500 x

coverage

314 1,3 106 0,5 106 500

316 6,2 106 2 106 2 103

318 11 106 4,5 106 4,5 103

costs costs

Sequencing

CGAL

Ion Reporter™ Software

Trim adapter sequences

Remove poor signal reads

Split reads per barcode Assembly

Alignment Coverage analysis

Variant calling

Read

generation

Read

mapping

Variant

filtering

Variant

confirmation

Identify known SNPs

Verify variants found

Data analysis pipeline

LTG

Software validatie

1. Analyse (11 parameters)

cut off levels gevoeligheid van mutatiecalling

annotatie mutatienomenclatuur

SNP, UV, pathogene mutatie

registratie technische interpretatie en controle

werkwijze per patiënt

2. Automatisering (4 parameters)

inladen, databases, koppelingen, rechten

3. Lay-out en gebruiksvriendelijkheid (4 parameters)

LTG

Data Analyse mbv SEQNext

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Validatie multiplex PCR benadering

LTG

• BRCA1 en BRCA2

• 9 genen voor erfelijke

paragangliomen/pheochromocytomen

• Mismatch repair genen

• OncoPanel

• BRAF/EGFR/KRAS

• Melanoom panel

• TP53

• Rest van pakket

Complete BRCA1 en BRCA2 gene testing

Alain Rico Rosella Petraroli

José Luis Costa José Carlos Machado

Arjen Mensenkamp

Marjolijn Ligtenberg

Clinical research relevance

• 15.8 kb ORF

• Genes highly polymorphic, many homopolymer regions

• Mutations scattered throughout the genes (no hot spots)

• Mostly truncating mutations--nonsense, frameshifts, indels

BRCA1 (on chromosome 17)

5.6 kb, 1863 aa

BRCA2 (on chromosome 13)

10.3 kb, 3418 aa

Hereditary forms of breast and ovarian cancer

Dominantly inherited (heterozygous mutations)

Structural aspects

Present:

• 84 amplicons + MLPA

• Fully automated PCR-robot, ABI 3700 sequencing

• 50 - 60 samples per month

• Efficient data analysis using SEQPatient; TAT 3 - 5 weeks

• Expensive technique

Aim:

• 2 - 3 multiplex PCR’s

• Standard Ion Torrent workflow

• Shorter TAT

• Reduction of costs

Why Ion Torrent approach in combination with

AmpliSeq technology?

Coverage of targets:

• 100% coverage of all coding exons and exon-intron boundaries

(-20 to +20; at least -10 to +10)

• Amplicons covering exons are overlapping

Primers:

• Primers do not overlap

• No validated SNPs in the last five nucleotides of primer

• Max 3 validated SNPs per primer

>1 nt

F1 F2

R2 R1

+20

Primer design Validation experiments multiplex AmpliSeq design

65 mutations

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Validatie Ampliseq multiplex PCR tests

• 3 multiplex PCR primer sets (50 - 55 primerparen)

• 65 verschillende pathogene mutaties

• In homopolymeren ✓

• Deleties / inserties ✓

• Puntmutaties ✓

• Exon deleties (✓)

• Data-analyse in 2 centra

BRCA2: c.2197_2201del

OncoNetwork Consortium 8 labs experienced in colon & lung cancer screening

Dr. Nicola Normanno Centro Ricerche

Oncologiche Mercogliano,

Italy

Prof. Orla Sheils Trinity College Dublin,

Ireland

Dr. Marjolijn Ligtenberg & Dr. Bastiaan Tops Radboud University

Nijmegen Medical Centre

The Netherlands

Prof. Ian Cree Warwick Medical School

United Kingdom

Prof. Pierre Laurent Puig Université Paris Descartes,

France

Dr. Ludovic Lacroix Institut Gustave Roussy

Paris, France

Prof. Aldo Scarpa ARC-NET University of

Verona Italy

Dr. Cristoph Noppen & Dr. Henriette Kurth

VIOLLIER AG Basle,

Switzerland

Alain Rico Rosella Petraroli

Proof of principle

• Optimization of workflow

• Validation of oncopanel

• 109 amplicons; 22 genes:

• EGFR, ERBB2, ERBB4, MET

FGFR1, FGFR2, FGFR3, DDR2

ALK

KRAS, NRAS PIK3CA,

BRAF PTEN

MAP2K1 AKT1

TP53, STK11, CTNNB1, SMAD4, FBXW7, NOTCH1

10 ng FFPE material in 1 reaction

N.B. no complete gene coverage

Sequence 155 samples by 6 labs

Phase 1

• 5 control FFPE samples ✓

• 2 KRAS AcroMetrix® cell line controls, 1 lung adenocarcinoma, 2 Xenograft colon cancer

Phase 2

• 60 Blind FFPE samples (60 samples ✓)

• 10 colon and lung cancer

• Each lab sent in 10 previously tested samples & received back 10 blind samples for sequencing

Phase 3

• 90 additional FFPE samples (90 samples ✓)

• Each lab sequences 10 lung & 5 colon samples

• Samples vary greatly in tumor content levels (heterogeneity)

155 samples tested in 180 experiments in 6 labs

Validation experiments FFPE

Sample

Type

Gene RefSeq Protein Lab1 Lab2 Lab3 Lab4 Lab5 Lab6

A12 KRAS NM_033360.2 ✓ ✓ ✓ ✓ ✓ ✓

A13 KRAS NM_033360.2 ✓ ✓ ✓ ✓ ✓ ✓

X32 PIK3CA NM_006218.2 ✓ ✓ ✓ ✓ ✓ ✓

X32 KRAS NM_033360.2 ✓ ✓ ✓ ✓ ✓ ✓

X32 TP53 NM_000546.4 ✓ ✓ ✓ ✓ ✓ ✓

X23 PIK3CA NM_006218.2 ✓ ✓ ✓ ✓ ✓ ✓

X23 KRAS NM_033360.2 ✓ ✓ ✓ ✓ ✓ ✓

X23 FBXW7 NM_033632.2 ✓ ✓ ✓ ✓ ✓ ✓

L1 KRAS NM_033360.2 ✓ ✓ ✓ ✓ ✓ ✓

Sequence Phase 1 optimizing calling parameters

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Sequence Phase 2 Blind Samples

Lab Variant

Type KRAS EGFR BRAF CTNNB1

Expected Variants

FOUND Variants

Variant Detection

Rate

Lab 1 SNVs 5 1 2 8 ✓

100 % Indel 2 2 ✓

LAB 2 SNVs 4 2 1 7 ✓

100 % indel

LAB 3 SNVs 5 1 6 ✓

100 % indel 1 1 ✓

LAB 4 SNVs 3 2 5 ✓

100 % indel

LAB 5 SNVs 4 1 2 7 ✓

100 % indel 1 1 ✓

LAB 6 SNVs 10 10 ✓

100 % indel

Sequence Phase 3 15 additional samples (Nijmegen)

Sample* Tissue Tumor cell % Expected variant Verified Frequency

1 lung 60% EGFR ✓ 69%, 13%

2 lung 65% - ✓

3 lung <10% EGFR ✓ 13%, 13%

4 lung 80% KRAS ✓ 44%

5 lung 50% - ✓

6 lung 30% KRAS ✓ 18%

7 lung 65% KRAS ✗ Coverage too low

8 lung 90% - ✓

9 lung 70% - ✓

10 lung 40% - ✓

11 colon 0-40% KRAS ✓ 2%

12 colon 40% KRAS ✓ 46%

13 colon 10% KRAS ✓ 9%

14 colon 60% BRAF ✓ 43%

15 colon 50% BRAF ✓ 47%

*Unpurified proteinase K extracts of small amounts of tissue / FNA

Amplicon Coverage Amplicon Coverage Oncopanel version 2

• Further optimization of primer set >> more equal coverage

• 8 in stead of 5 samples on 316 chip

• Novel validation

All amplicons winthin ~ 10-fold range

98 samples tested in 138 experiments in 6 labs

Validation experiments Oncopanel version 2

✓

✓

BRAF / EGFR/ KRAS (BEK)

• Genstatus nu gevraagd door kliniek

• 7 amplicons

• Minder chip capaciteit dus goedkoper

• Kostenneutrale vervanger van huidige test (alleen indien

gecombineerd met andere testen)

Redenen vervanging huidige test

• Input slechts 10 ng FFPE DNA

• Sensitiever

• Expertise en logistiek gereed voor nabije toekomst

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Sample* Tissue Tumor cell % Expected variant Verified Frequency

1 lung 90% EGFR ✓ 38%

39%

2 lung 90% EGFR ✓ 57%

c.2369C>T; p.Thr790Met: 11%

3 lung 60% EGFR ✓ 60%

18%

4 lung 60% EGFR ✓ 55%

5 lung 40% EGFR ✓ 23%

6 lung 45% EGFR ✓ 80%

80%

7 lung 60% KRAS ✓ 30%

8 colon 0-40% KRAS ✓ 2%

9 colon 45% KRAS ✓ 57%

10 colon 10% KRAS ✓ 9%

11 colon 50% KRAS ✓ 38%

12 lung 30% KRAS ✓ 25%

13 colon 60% KRAS ✓ 64%

14 lung 50% - - ✓ -

15 colon 40% - - ✓ -

*Unpurified proteinase K extracts of small amounts of tissue / FNA

Validation BRAF, EGFR, KRAS (BEK) mutation detection Benefit of clonal sequence analysis

c.2235_2249del; p.Glu746_Ala750del

c.2102A>T; p.Gln701Leu c.2156G>C; p.Gly719Ala

Sample* Tissue Tumor cell

% Expected variant Verified Frequency

1 melanoma 95% BRAF ✓ 48%

2 melanoma 70% BRAF ✓ 62%

3 colon 40% BRAF ✓ 14%

4 thyroid 100% BRAF ✓ 18%

5 melanoma 80% BRAF ✓ 22%

6 melanoma 80% BRAF ✓ 34%

7 melanoma 60% BRAF no data

8 melanoma 45% - - ✓

9 melanoma 55% - - ✓

*Unpurified proteinase K extracts

of small amounts of tissue / FNA

Validation BRAF, EGFR, KRAS (BEK) mutation detection

c.1798_1799delinsAA

p.Val600Lys

Requirements of a molecular diagnostic test

• robust performance using limited amounts of FFPE-

material (10 ng)

• high accuracy

• clinically relevant sensitivity

• Hot spot mutations

• Loss of function mutations

• availability of results within days

Technical requirements

• Flexible: easy to implement new markers

• Detection of all mutations in an amplicons

✓ ✓

✓

✓

✓ ✓

(multiplex) PCR

IonTorrent sequencing

✓

Op weg naar implementatie

• Inrichten LMS

• Optimalisatie software settings

• Validatie combi-runs

• Afronden SOPs

• Afronden validatierapporten

• Uitvoeren 2e lijns controles

• Inwerken overig personeel

Doel:

• 1 maart in productie BRCA1 & BRCA2

• 15 maart BRAF / EGFR / KRAS

• Eind 2013: alle mutatie analyses moleculaire pathologie

• Eind 2013: multiplex PCR en library prep geautomatiseerd

Diagnostische flow / weekplanning

TAT: 3-5 werkdagen

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Acknowledgements

LTG

BRCA1/BRCA2

Hicham Ouchene

Marloes Tychon

Arjen Mensenkamp

Oncopanel / BEK

Annemiek van Raaij

Teun Kuijper

Marian Verdijk

Bastiaan Tops

CGAL

Ronny Derks

Michiel Oorsprong

Michael Kwint

Marcel Nelen

LMS / ICT

Ermanno Bosgoed

QC

Danielle Bodmer

Kornelia Neveling

consortia members