French Initiative and NGS extension in molecular pathology
Dr Etienne Rouleau
Gustave Roussy, Villejuif- France
6
thMeeting on External Quality Assessment in Molecular Pathology
May 13, 2017, Naples, Italy
Gen&tiss,
A French EQA program
EQA organization
Leuven Nijmegen
AFAQAP
Partner Platforms Joint partners
Reference Platforms
EQA organization Strict validation
Tumor
Slides HES
Slides HES
Histology validation Neoplastic cells % Molecular validation NGS validation
Histology validation Neoplastic cells % Molecular validation NGS validation
Side B Side A
EQA organization
Number of samples per scheme
Colorectal scheme Lung scheme
Melanoma scheme Ovarian scheme
Total
10 10 - - 20 2012
10 10 - - 20 2013
5 5 - - 10 2014
5 5 5 - 15 2015
5 5 5 5 20 2016
+3 educational samples
+4 educational samples +2 educational
samples
EQA organization
Practice improvement
National meeting
– Educational meeting – Final report
– Technical discussion to standardize practices nationally – 4 workshops / every session – Conclusions in plenary session and guidelines
14/20 BAD
14/20 BAD
Individual and personalized comments
Guidelines (INCa, GFCO) Method validation guidelines
NGS guidelines for somatic mutations cfDNA guidelines (in review)
KRAS (1) EGFR (3) BRAF (1)
KRAS (4) EGFR (4) BRAF (2) NRAS (4) MET KIT
PIK3CA (2) ERBB2
ABL1 EZH2 JAK3 PTEN AKT1 FBXW7 IDH2 PTPN11 ALK FGFR1 KDR RB1 APC FGFR2 KIT RET ATM FGFR3 KRAS SMAD4 BRAF FLT3 MET SMARCB1 CDH1 GNA11 MLH1 SMO CDKN2A GNAS MPL SRC CSF1R GNAQ NOTCH1 STK11
CTNNB1 HNF1A NPM1 TP53 EGFR HRAS NRAS VHL ERBB2 IDH1 PDGFRA ERBB4 JAK2 PIK3CA
CDKN2C CDKN1A CCND1 CDKN1B CDK12 RB1 CDKN2A FGFR3 KIT FGFR2 EPHB6 PDGFRA ERBB4 EPHA3 FLT3 EGFR ERCC2 RAD21 CHEK2 SMC3 SMC1A BRCA1 BAP1 STAG2 ATR BRCA2 ATRX ATM TP53 EZH2 ASXL1 ARID5B MLL4 KDM6A KDM5C SETBP1 NSD1 SETD2 PBRM1 ARID1A MLL2 MLL3 FOXA2 CEBPA VEZF1 ELF3 SOX9 CBFB PHF6 FOXA1 EIF4A2 WT1 SIN3A TBX3 MECOM RUNX1 TSHZ2 TAF1 CTCF EP300 TSHZ3 GATA3 VHL MIR142 B4GALT3 EGR3 CRIPAK PRX LIFR AR EPPK1 HGF NPM1 USP9X NCOR1 POLQ ARHGAP35 MALAT1 LRRK2 NOTCH1 NAV3 STK11 MTOR RPL5 RPL22 PTPN11 PPP2R1A NFE2L3 NFE2L2 IDH2 IDH1 TET2 DNMT3A AJUBA CDH1 PCBP1 U2AF1 SF3B1 SPOP KEAP1 FBXW7 HIST1H2BD H3F3C HIST1H1C SOX17 TBL1XR1 AXIN2 CTNNB1 APC ACVR2A SMAD2 ACVR1B TGFBR2 SMAD4 AKT1 PIK3CG TLR4 PIK3R1 PTEN PIK3CA MAPK8IP1 MAP2K4 NRAS BRAF MAP3K1 NF1
EXOMES
Kandoth C, et al. Mutational landscape and significance across 12 major cancer types. Nature. 2013 Oct 17;502(7471):333-9.
Scarpa, A., Sikora, K., Fassan, M., Rachiglio, A. M., Cappellesso, R., Antonello, D., ... & Normanno, N. (2013).
Molecular typing of lung adenocarcinoma on cytological samples using a multigene next generation sequencing panel. PloS one, 8(11), e80478.
EQA organization
Multiparametric approach
TECHNIQUEMOLECULAR TARGET
Description of NGS
practices
Network
Number of platforms is not number of labs
28 platforms
48 labs for mCRC EQA schemes
46 labs for lung EQA schemes
45 labs for melanoma schemes
42 labs have all the schemes
Accreditation
Number of platforms accredited
EQA2016
Accreditation : 29/51 NGS accredited : 3/51
NGS for EEQ : 39/51
Extraction process
Automatization
Analytical approach Many techniques
As many cheeses as molecular methods
Toward a unique approach NGS diffusion in France
Pricing Reform
Figure : Number of participants with a routine application of NGS
Techniques
NGS techniques
Figure : NGS diffusion and its routine application within 48 labs from 2013 to 2017
Techniques
Enrichment approach
Figure : Enrichment methods for somatic mutations analysis.
Techniques Sequencers
Figure : NGS machines available for sequencing.
Figure : bioinformatic analysis and data visualization.
Techniques
Bioinformatic analysis
Parameters
Pre-analytical quality assessment
Criteria for quality n Amplification (Cp/Ct) 9 Amplification (Cp/Ct)
quantity (ng) 3 Fragment analysis 2 Postsequencing 12
Quantity (ng) 16
Bloc 3-6 slides 6µ
HES
Extraction
Analytical phase Post-analytical phase
Parameters
Deepness and limit of detection
1000X 4
600X 1
500X 7
400x 1
300X
24200X 2
100X 2
50X 1
1% 2
2% 10
3% 3
4% 1
5% 18
7% 1
10% 2
Minimal deepness
Limit of detection
Validation : 27 (deepness + allelic frequency), 5 (deepness), 11 (D + AF + strand bias)
Parameters
Checking and validation
No validation 6
Other technique(Sanger, pyro, qPCR, HRM) 13
SNP validation 7
Duplicate NGS 10
Random validation 6
EGFR MUTANT
Alamut 17
Other 5
IGV 19
No visualization 2
Quality
Intern quality control
Commercial IQ 31
Horizon Diagnostics 27
Accrometrix 2
Autre 2
Home made IQ 8
Autre 1
Patient samples 4
Cell lines 3
Use of an IQ : 39/43
One IQ every run : 33/43
Pilot NGS scheme
Genotyping Principle
• Three “educational” samples
• Loading of the vcf and bam files
• Common Bed and Calling Analysis
• Outliers identification
– False positives – hotspots – False negatives - hotspots
– Discrepancy on allelic frequency
BWA 0.7.5a;TMAP 3.4.1;Samtools v-0.1.19;
Picard-tools v-1.127;GATK HaplotypeCaller/UnifiedGenotyper v-3.3-0;
MutaCaller-1.6;LifeScope-2.5.1;snpEff v-4.0
NGS pilot
Organisation
NGS implementation n=44
Routine application n=34
Pilot NGS scheme Lung control 24 Melanoma control 19 Colon control 24
n=27
« Lung »
EGFR:c.2361G>A;p.Gln787Gln EGFR:c.2369C>T;p.Thr790Met FGFR3:c.1953G>A;p.Thr651Thr EGFR:c.2573T>G;p.Leu858Arg
KRAS:c.38G>A;p.Gly13Asp PIK3CA:c.3140A>G;p.His1047Arg CTNNB1:c.131_133delCTT;p.Pro44_Ser45del
TP53:c.818G>A;p.Arg273His
« Colon »
NRAS:c.181C>A;p.Gln61Lys EGFR:c.2155G>A;p.Gly719Ser FGFR3:c.1953G>A;p.Thr651Thr
CTNNB1:c.98C>A;p.Ser33Tyr
« Melanoma »
EGFR:c.2361G>A;p.Gln787Gln FGFR3:c.1953G>A;p.Thr651Thr
KIT:c.2447A>T;p.Asp816Val
Coverage Bed file
Figure : coverage of the common bed and number of BAM files covering the locus (n=87).
NGS pilot
BAM results
BAM filesn=87
Correct BAM n=75
BAM raw n=6
Inversion n=6
Error n=3 Success
n=65
Success with FP at 1%
n=7
FP n=1
KRAS c.182A>G p.Gln61Arg
2%
FN n=2 NRAS
PIK3CA
PIK3CA à 43%
c.3140A>G p.His1047Arg
NRAS at 2%
c.181C>A p.Gln61Lys
NGS pilot
Participant results
Participants n=27
At least 1 failure n=3
Success n=24
Success with FP at 1%
n=3
AF outsider n=6
NGS pilot Deepness
Figure : Medium deepness for the NGS pilot for the selected bed
NGS pilot
Technology and enrichment kits
Technique Failure (FP / FN) Success Success but FP inferior to 1%
PGM IonTorrent (Life Technologies) 29
Ion S5 system (Life Technologies) 1 13
Ion Proton (Life Technologies) 3
MiSeq (Illumina) 1 17 6
Miseq + PGM 1 1
MiSeqDx (Illumina) 2 1
Total 3 65 7
NGS pilot
Technology and enrichment kits
Technique Failure
(FP / FN) Success Success FP <1% AF outsiders Ion Ampliseq Custom panel (Life Technologies) 1 15
Ion AmpliSeq Colon and Lung Cancer Panel (Life technologies) 15 (1)
Oncomine Solid Tumour DNA kit (Life Technologies) 15 (1)
Tumor Hotspot MASTR Plus (Multiplicom) 5 (1)
TruSeq Amplicon Cancer Panel (Illumina) 1 4 2 (4)
TruSeq Custom Amplicon (Illumina) 5 1 (1)
Access Array System (Fluidigm) 2 4
Home-made primer design 3 (1)
Other (Life/Multiplicom/Illumina) 1 1
Total général 3 65 7 (9)
111+48 -11-94
Same story with new method KRAS in-house primer design
tggtggagtatttgatagtgtattaaccttatgtgtgacatgttctaatatagtcacattttcattattttta ttataaggCCTGCTGAAAATGACTGAATATAAACTTGtGGTAGTTGGAGC TGGTGGCGTAgGCAAgAGTGcCTTGACGATACAGCTAATTCAGAATCA TTTTGTGGACgAATATGATCCAACAATAGAGgtaaatcttgttttaatatgcatatt actggtgcaggaccattctttgatacagataaaggtttctctgaccattttcatgagtacttattacaa g
To conclude
Conclusion
• French EQA program
– Follow a country evolution during the 5 last years and the implementation of NGS
– Recommandation based on the real data
• Extension of NGS will change the way to propose EEQ
– Future of univariable molecular EQA ?
• NGS pilot scheme
– Need for common guidelines to assess the results – Identification of key markers for NGS quality
– Common samples and common pipeline
Thanks
Steering CommitteeJean-Christophe SABOURIN Aude LAMY et Paul GUEGEN Céline GARREC
Frederique PENAULT-LLORCA Cécile AUBE
Anne CAYRE
Cédric LEMARECHAL Laurent DOUCET
Clotilde DESCARPENTRIES Isabelle QUINTIN-ROUE Hélène BLONS
Jean-François EMILE Jean-François COTE Antoinette LEMOINE
Valérie DURANTON-TANNEUR Yves DENOUX
Farid ZERIMECH Karen LEROY
Isabelle SOUBEYRAN Véronique HADDAD Paul HOFMAN
Florence PEDEUTOUR Alexandre HARLE Ludovic LACROIX Alexander VALENT
Marc-Antoine BELAUD-ROTUREAU Pierre-Jean LAMY
AFAQAP
Caroline Egele Jean-Pierre Bellocq Dominique Fetique
Gustave Roussy Jean-Yves Scoazec Isabelle Miran
Catherine Richon Ludovic Lacroix Sophie Cotteret Birama Ndiaye
Biomedical Quality Assurance Research unit of the University
of Leuven Cleo Keppens Els Dequeker Lien Tembuyser Veronique Tack
Departement of Pathology of the Radboud University Nijmegen Medical Centre Marjolijn Ligtenberg
Han van Krieken
Institut National du Cancer Frédérique Nowak
Etienne Lonchamp
Funding support :
