Update on the ERIC TP53 Network Activities and

TP53 Certification – Overview and Results of Round 4

Sarka Pospisilova Jitka Malcikova

CEITEC, Masaryk University and University Hospital Brno, Czech Republic

ERIC meeting, Mannheim, January 31st, 2017

Thessaloniki

Brno

Uppsala

Ulm

U.K.

Paris

Novara

Salamanca

Copenhagen

Amsterdam

ERIC TP53 NETWORK

Bellinzona

Training Centres (11) ● Sarka Pospisilova, Jitka Malcikova, Brno, Czech Republic ● Stephan Stilgenbauer, Eugen Tausch, Ulm, Germany ● Fred Davi', Paris, France ● Kostas Stamatopoulos, Thessaloniki, Greece ● Gianluca Gaidano, Novara, Italy ● Ramon Garcia Sanz, Salamanca, Spain ● Richard Rosenquist, Uppsala, Sweden and Nordic countries ● Carsten Niemann, Copenhagen, Denmark ● Arnon Kater, Amsterdam, Netherlands ● Davide Rossi, Bellinzona, Switzerland ● David Gonzalez, Mark Catherwood, Belfast, United Kingdom

Certifying Centres Sarka Pospisilova, Jitka Malcikova, Brno, Czech Republic Stephan Stilgenbauer, Eugen Tausch, Ulm, Germany

ERIC TP53 NETWORK STRUCTURE:

1) Organization of regular TP53 certification rounds (twice a year) 2) Organization of 2nd TP53 workshop (autumn 2017, date and venue to be announced) 3) CLL - TP53 mutation database (in collaboration with T. Soussi, in connection with ERIC CLL Database) 4) Update on ERIC Recommendations on TP53 mutation analysis – planned for 2017 5) ERIC TP53 web tools: www.ericll.org – Networks – TP53 Network a) FAQ on TP53 certification b) Helpdesk (ERIC office + expert in charge) c) Manual for TP53 mutational analysis d) Sanger data analysis software for detection and annotation of low - frequent somatic variants „GLASS“

ERIC TP53 NETWORK - ACTIVITIES

GLASS standardized assessment of gene variations from Sanger sequences

Pal, […], Ghia & Stamatopoulos, Pospisilova & Malcikova, Darzentas also on behalf of the European Research Initiative on CLL (ERIC) – TP53 Network (paper in revision)

GLASS = online bioinformatics tool to discover and report gene variants from Sanger ABI files Available for these genes, more on request:

TP53, ATM, NOTCH1… in preparation genebank files uploading TP53: validated ERIC’s TP53 Analysis Certification samples Assisted detection of: minor variant allele

small insertion/deletion

Output: standardised table of variants genomic coordinates proper HGVS (varnomen.hgvs.org) nomenclature (on both DNA and protein, when appl.) dbSNP annotation w link

Freely available online - also through ERIC’s website: http://www.ericll.org/pages/networks/tp53network http://shiny.bat.infspire.org/glass/

ERIC Certified Centres from all 4 rounds

Round 1: 30 Round 2: 16 Round 3: 29 Round 4: 35 (3 centres participated in 2 rounds with different techniques) ______________________________________________________

Total Number of Certified Centers: 107 (total number of applicants 135, 25 failed)

ERIC Certified Centres - 4 Rounds

19

14 12

9 7

6 6 5

4 3

2 2 2 2 2 2 2 2 1 1 1 1 1 1

107 Centres from 24 countries

TP53 Certification Update

Jitka Malcikova

on behalf of ERIC TP53 Certification Centers:

Brno, Czech Republic (Sarka Pospisilova, Jitka Malcikova) Ulm, Germany (Stephan Stilgenbauer, Eugen Tausch)

TP53 Certification – Round 4 University Hospital Brno and CEITEC MU

Czech Republic Application deadline: September 15, 2016

Samples Sent to Participants: October 10, 2016

Deadline to Submit Reports: December 15, 2016

Number of Applications: 40 Samples sent: 38 Results delivered: 37 Number of Participating Countries: 17

Round 4 - Participants by Country

14

3 3 2 2 2 2

1 1 1 1 1 1 1 1 1

Number of Participants

Belgium • AZ Sint-Jan Brugge-Oostende AV (Brugge) Brasil • University of Sao Paulo (Sao Paulo) Cyprus • Karaiskakio Foundation (Nicosia) Czech Republic • Faculty Hospital (Hradec Králové) France • (EFSBFC) (Besançon) • CHU Nancy Hôpitaux de Brabois (Vandœuvre-lès-Nancy) • L'Hôpital Robert Debré - CHU de Reims (Reims) Germany • University Hospital Cologne (Cologne) • IHO GmbH (Mannheim) Ireland • St James´s Hospital (Dublin) Israel • Tel-Aviv Sourasky Medical Centre (Tel-Aviv) • Rambam Medical Centre (Haifa) Italy • Azienda Ospedaliero – Universitaria Policlinico de Modena

(Modena) Poland • Medical University of Lublin (Lublin) Portugal • Hospital de São João (Porto)

The Netherlands • Erasmus University Medical Center (Rotterdam). • Sanquin Diagnostics (Amsterdam) • University Medical Centre Groningen (Groningen) Spain • Hospital Clinic de Barcelona (Barcelona) • ICO - Hospital Germans Trias i Pujol, IJC (Barcelona) • Hospital del Mar (Barcelona) • Hospital Universitario de Gran Canaria Dr. Negrín Hospital (Las

Palmas de Gran Canaria) • Hospital Universitario Central de Asturias (Asturias) • Universidad de Navarra - CIMA (Navarra) • Complejo Hospitalario de Navarra (Navarra) • LABCO (Madrid) • H.G.U. Gregorio Marañón (Madrid) • Hospital Clínico San Carlos (Madrid) • Hospital Universitario Fundació Jiménez Díaz (Madrid) • NIM Genetics (Madrid) • Hospital La Fé (Valencia) • Hospital General Universitario Reino Sofia (Murcia) Sweden • Akademiska University Hospital (Uppsala) • Karolinska University Hospital (Stockholm) Switzerland • University Hospital Basel (Basel) • Unilabs (Lausanne) United Kingdom • Wessex Regional Genetics Laboratory (Salisbury)

Participating Centers:

Methods used

Sanger; 16

HRM +Sanger; 3

Sanger +MLPA; 1

NGS; 6

Sanger; 18

HRM +Sanger; 2

dHPLC +Sanger; 1

Sanger +MLPA; 2

NGS; 14

Round 3

Round 2 Sanger;

23 Sanger+MLPA; 1

NGS; 13

Round 4 - sample selection

2 3 4 5 6 7 8 9 10 11 Sample 2

c.320_331del p.(Y107_R110del); VAF=73%

2 3 4 5 6 7 8 9 10 11 Sample 5

c.541C>T p.R181C; VAF=26% c.817C>T p.R273C; VAF=4,5%

2 3 4 5 6 7 8 9 10 11 Sample 3

c.503A>G p.(H168R); VAF=5%

2 3 4 5 6 7 8 9 10 11 Sample 4

c.734G>A p.(G245D); VAF=50% c.949C>T p.(Q317*); VAF=50%

3 4 5 6 7 8 9 10 11 Sample 1 2

Polymorphism c.108G>A p.P36=; VAF: 50%

Common polymorphism c.215C>G p.Pro72Arg not reported

Round 4 Certification requirements

2 3 4 5 6 7 8 9 10 11 Sample 2

c.320_331del p.(Y107_R110del); VAF=73%

2 3 4 5 6 7 8 9 10 11 Sample 5

c.541C>T p.R181C; VAF=26% c.817C>T p.R273C; VAF=4,5%

2 3 4 5 6 7 8 9 10 11 Sample 3

c.503A>G p.(H168R); VAF=5%

2 3 4 5 6 7 8 9 10 11 Sample 4

c.734G>A p.(G245D); VAF=50% c.949C>T p.(Q317*); VAF=50%

3 4 5 6 7 8 9 10 11 Sample 1 2

Polymorphism c.108G>A p.P36=; VAF: 50%

Wild type

Mutated

Mutated

Mutated

Wild type/Mutated based on detection

sensitivity

Common polymorphism c.215C>G p.Pro72Arg not reported

Minimal

requirements

Good laboratory

practice

requirements

Identification of sample 1 as unmutated / TP53 wt

Identification of sample 2, 4 and 5 as mutated

Correct identification of sample 3

Wild type with detection sensitivity >5%VAF

Mutated with detection sensitivity <5% VAF

Identification of both mutations in sample 4

Provided lab specific report

Covering at least exon 4-9

Covering at least exon 4-10

Correct variant annotation

Finished and submitted analysis within 28 days

Certification requirements

Interpretation not evaluated…

Results Failed; 2

Passed; 28

Passed - good

laboratory

practice; 7

Did not identify 12nt deletion in sample 2

Individual feedback

Your result

Minimal

requirements

Good laboratory

practice requirements

Identification of sample 1 as unmutated / TP53 wt

Identification of sample 2, 4 and 5 as mutated

Correct identification of sample 3

Wild type with detection sensitivity >5%VAF

Mutated with detection sensitivi ty <5% VAF

Identification of both mutations in sample 4

Provided lab specific report

Covering at least exon 4-9

Covering at least exon 4-10

Correct variant annotation

Finished and submitted analysis within 28 days

Feedback sent last week Certification valid for 3 years ˣ Centers that failed can re-apply in round 5

TP53 Certification – Round 5

Deadline for applications : February 15 2017 Samples to be sent to participants: March 15 2017 Deadline to receive reports: May 15, 2017 Results Available: June 2017 (report at ERIC meeting during EHA)

• Number of participants in each round will be restricted to 40

• Material to be sent: DNA • Certified methods: both Sanger sequencing and NGS,

Not two methods in one round

TP53 Certification – Round 6

Expected deadline for applications : September 15st

Centers Certified in the round 1 should re-apply in round 6

TP53 Certification –APPLICATION Questionnaire at www.ericll.org – Services – TP53 analysis Certification Applications are collected continuously

Acknowledgements

University of Ulm Eugen Tausch

ERIC Office Natalie Sorolla

Martin Fox

Project initiators Sarka Pospisilova

Paolo Ghia Stephan Stilgenbauer Kostas Stamatopoulos

Emili Montserrat