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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