Update on TP53 analysis accreditation project

Sarka Pospisilova, Eugen Tausch,

Jitka Malcikova, Martin Fox and Stephan Stilgenbauer

For all laboratories performing the TP53 analysis in CLL - validate different

techniques on TP53 mutation analysis

Support introduction of TP53 analysis in labs with less experience

(to provide assurance of the results quality).

Discuss the problematic issues and give individual recommendations.

Improve analysis harmonization and interaction between centers.

Provide an official document on the quality control assessment.

Aims of the project

Participating Laboratories :

49 centers participated in the study and asked for samples

> 47 centers asked for gDNA

> 6 centers asked for RNA (and gDNA)

> 1 center asked for viable cells (not delivered)

39 centers delivered results within 3 months after sample arrival

3 centers notified not to able to perform analysis

3 centers (2x Russia, Venezuela) did not receive samples (logistic reasons)

4 centers did not reply to our reminders

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

Participating Labs (49) Applicant Center:

E. Eldering Amsterdam, Academic Medical Center P. Panayiotidis Athens, University of Athens

J. Nomdedeu Barcelona, Hospital de la Santa Creu i Sant Pau

M. Catherwood Belfast, Belfast City Hospital

F. Baran-Marszak Bobigny, Hopital Avicenne P. Piccaluga Bologna, University of Bologna

Sottini Brescia, lab. Biotecnologie

J. Malcikova Brno, University Hospital Brno D. Naguib Caen, CHU Côte de Nacre

L. Veronese/P. Combes Clermont Ferrand, CHU ESTAING I. Praulich/S. Prall Cologne, University at Cologne

L. Pedersen / C. Geisler Copenhagen, Rigshospitalet S. GIRAUDIER Créteil, Hopital Henri Mondor

M. McBean East Melbourne, Peter MacCallum Cancer Centre

J. Bryon Edgbaston, Birmingham Women's Hospital J. Lindgren Gothenburg, Sahlgrenska university Hospital

N. Halbsgut Göttingen, Klinik für Hämatologie und Onkologie T. Tadmor Haifa, Bnai Zion Medical Cenzer

N. von Neuhoff Hannover, Hannover Medical School

N. Bilous Kiev, Research Center for Radiation Medicine K. Filonenko Kyiv, National Cancer Insitute

S. Jayne Leicester, University of Leicester S. Franke/F. Lambert Liege, CHU Liege

K. Lin Liverpool, University of Liverpool, Sanger

Dr. Nicholas Lea London, Kings College Hospital

Applicant Center: Veronese Silvio Milano, Molecular Pathology Unit

Cristina Tresoldi Milano, Ospedale San Raffaele Andrey Sudarikov Moscow, National Hematology Center

Davide Rossi Novara, University of Eastern Piedmont Miss Helene DREAU Oxford, Oxford University Hospitals NHS Trust

Fred Davi Paris, Hopital Pitié-Salpêtrière Silvia ZIbellini Pavia, Fondazione IRCCS Policlinico San Matteo

Cristina Mecucci Perugia, University of Perugia

M. González, E. Sebastián Salamanca, University Hospital of Salamanca Dimovski Skopje, Faculty of Pharmacy

F. Forconi Southampton, University of Southampton Rumyanciev St. Petersburg, V.A.A. Federal Centre of Heart

A. N. Tran Stockholm , Karolinska University Hospital

U. Tostar Stockholm, Karolinska University Hospital, D. Wren/D. de Castro Sutton, The Royal Marsden NHS Foundation Trust

K. Stamatopoulos Thessaloniki, G. Papanicolaou Hospital A. Makris/ K. Stamatopoulos Thessaloniki, INAB-CERTH

S. Niki Thessaloniki, Papanikolaou Hospital

E. Delabesse Toulouse, CHU M. Hermanson Uppsala, Clinical Genetics

M. Hernandez Valencia, Universidad de Carabobo M. Fritzer-Szekeres/R. Ferdig

Sunder-Plassmann Vienna, Clinical Institute for Laboratory Medicine

E. Novella Vinzenca, S. Bortolo Hospital

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

Participating Labs

49 centers participated in the study and asked for samples

without result with result

BRNO ULM

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide

50 labs worldwide 49 labs in Europe* Sample shipping

Correspondence

Project work flow

Results

* and Australia and Venezuela

Online Survey Participation

Exon1 Exon2 Exon3 Exon4 Exon5 Exon6 Exon7 Exon8 Exon9 Exon10 Exon11

3 centers 2 centers

19 centers

5 centers 3 centers

8 centers

5 centers

45 labs

23 labs

45 labs

23 labs

IAR

C

4 5 6 7 8 9 10 11 3 2

40

cen

ters

21

cen

ters

N=2

87

17

Covered TP53 Exons in participating labs

3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Disomic Somatic

Deleted

Somatic

Disomic Somatic

Disomic Somatic

Deleted

Somatic

FISH IARC

2

TP53 accreditation - Sample Selection

c.517G>T VF=47%

c.715A>G VF=44%

c.811G>T VF=41% c.370T>C VF=38%

c.1028delA VF=93%

Accreditation requirements - exon 4-9

3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

c.517G>T VF=47%

c.715A>G VF=44%

c.811G>T VF=41% c.370T>C VF=38%

2

Accreditation Requirements

WT

Mutated

Mutated

Mutated

Mutated

Result

c.517G>T VF=47%

c.715A>G VF=44%

c.811G>T VF=41% c.370T>C VF=38%

c.1028delA VF=93%

3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

2 3 4 5 6 7 8 9 10 11

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

2

Accreditation requirements - exon 4-10 Accreditation Requirements

WT

Mutated

Mutated

Mutated

Mutated

Result

c.1028delA VF=93%

c.517G>T VF=47%

c.715A>G VF=44%

c.811G>T VF=41% c.370T>C VF=38%

Results of sample analysis

80%

8%

6%

6%

Not able to send results

(4)

No answer (3)

No sample received (3)

Results provided

failed Exons 4-9

n=49 n=39 (34 succeeded, 5 failed)

Exons 4-10

<28d

>28d >28d

<28d

single

single

<28d

>28d *

*

*

*

0

5

10

15

20

25

<28d >28d: 3

<28d: 8

>28d

* Only 1 of 2 mutations in sample 4 detected

n=23

n=11

Minimal requirements

Validate sample 2-4 as mutated and sample 1 as unmutated.

Perform analysis within 3 months after sample receipt.

Good laboratory practice

Detect all mutations in samples 2-5.

Finish analysis and confirmation assay within 28 days.

Provide a clinical report including minimal required informations.

Accreditation requirements

Centers with successful TP53 mutation analysis

Applicant Center: F. Baran-Marszak Bobigny, Hopital Avicenne

P. Piccaluga Bologna, University of Bologna Sottini Brescia, lab. Biotecnologie

J. Malcikova Brno, University Hospital Brno

L. Veronese/P. Combes Clermont Ferrand, CHU ESTAING I. Praulich/S. Prall Cologne, University at Cologne

L. Pedersen / C. Geisler Copenhagen, Rigshospitalet S Giraudier Créteil, Hopital Henri Mondor

M. McBean East Melbourne, Peter MacCallum Cancer Centre J. Bryon Birmingham Women's Hospital

N. Halbsgut Göttingen, Klinik für Hämatologie und Onkologie

N. von Neuhoff Hannover, Hannover Medical School N. Bilous Kiev, Research Center for Radiation Medicine

S. Jayne Leicester, University of Leicester K. Lin Liverpool, University of Liverpool, Sanger

M. McBean Melbourne, PeterMac

Veronese Silvio Milano, Molecular Pathology Unit

Applicant Center: Cristina Tresoldi Milano, Ospedale San Raffaele

Davide Rossi Novara, University of Eastern Piedmont Miss Helene DREAU Oxford, Oxford University Hospitals NHS Trust

Silvia ZIbellini Pavia, Fondazione IRCCS Policlinico San Matteo

Cristina Mecucci Perugia, University of Perugia M. González, E. Sebastián Salamanca, University Hospital of Salamanca

Dimovski Skopje, Faculty of Pharmacy

A. N. Tran Stockholm, Karolinska University Hospital

U. Tostar Stockholm, Karolinska University Hospital D. Wren/D. de Castro Sutton, The Royal Marsden NHS Foundation Trust

K. Stamatopoulos Thessaloniki, G. Papanicolaou Hospital A. Makris/ K. Stamatopoulos Thessaloniki, INAB-CERTH

N. Stovroyianni Thessaloniki, Papanikolaou Hospital

E. Delabesse Toulouse, CHU M. Hermanson Uppsala, Clinical Genetics

Sunder-Plassmann Vienna, Clinical Institute for Laboratory Medicine

E. Novella Vinzenca, S. Bortolo Hospital

Examples of Reports

Reports

n=39

Report provided

46%

54%

0% 0%

Yes

Reports Lab name

Type of analysis

Result & Interpretation

Date of sample acquisition

Patient Identifier

Type of material and technique

Responsible Person

Next Steps

Brief report of survey and analysis results

Contact each lab with individual feedback / specific recommendations

ERIC Certificate to 34 successfull participants

Initiation of annual sample exchange for re-accreditation

Sample exchange for NGS based analysis

TP53 analysis technique harmonization approach

Acknowledgements Ulm lab team Eugen Tausch Doris Winter Melanie Flauger Julia Sempf Sabrina Kless Christina Galler

Project initiators

Sarka Pospisilova Stephan Stilgenbauer

Kostas Stamatopoulos Emili Montserrat ERIC Office

Martin Fox