ERIC TP53 Network – activities and outcomes

Sarka Pospisilova

ERIC TP53 NETWORK

Thessaloniki

Brno

Uppsala/Stockholm

Ulm

Belfast

Paris

Novara

Salamanca

Copenhagen Amsterdam

Bellinzona

11 Training Centres 3 Certifying Centres

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

Certifying Centres Stephan Stilgenbauer, Eugen Tausch, Ulm, Germany Sarka Pospisilova, Jitka Malcikova, Sarka Pavlova, Brno, Czech Rep. Richard Rosenquist, Lesley Ann Sutton, Uppsala/Stockholm, Sweden

ERIC TP53 NETWORK STRUCTURE:

Antioncogene TP53 = "the guardian of the genome"

Conserves genetic stability by preventing genome mutation

TP53 gene is located on the short arm of chromosome 17 (17p13.1)

The p53 protein is crucial for regulation of the cell cycle arrest or apoptosis

Tumors with p53 dysfunction - bad prognosis and poor treatment response

Why is TP53 dysfunction so important in CLL ?

The most important independent prognostic marker

High risk of progression within 1-2 years

Median overall survival 3-5 years

Important predictive marker (resistance to FCR, alkylating agents, purine analogues…)

Valid also in „novel mutation era“

New therapeutic options (inhibition of BcR signaling, Bcl2 inhibition)

Frequency of TP53 aberrations increases with advanced disease stage and therapy

TP53 dysfunction in CLL - del(17p) and/or TP53 mutation

Types of TP53 defects in CLL

del(17p) sole TP53

mutation sole

Del(17p) & TP53 mutation

Frequency increases during disease development

Related to previous therapy

Dicker et al. Leukemia 2009

Zenz et al. Blood 2008

Rossi et al. CCR, 2009 Malcikova et al. Blood, 2009

LRF CLL4 (Clb vs F vs FC)

Gonzalez et al. JCO, 2011

GCLLSG CLL4 trial (F vs FC)

Zenz et al. JCO, 2010

Independent prognostic impact of del(17p) and TP53 mutations

Analysis of both del(17p) by FISH and TP53 mutation recommmended!

CLL8 trial (FC vs FCR)

Stilgenbauer et al. Blood 2014

Stengel et al, Leukemia 2017

1) ERIC Recommendations on TP53 mutation analysis – Updates 2) Organization of regular Certification rounds for TP53 mutation analysis 3) Organization of workshop onTP53 analysis (Brno 2015, Stresa 2017, …) ERIC TP53 web: www.ericll.org – Diagnostics

www.ericll.org/tp53-mutational-analysis-certification/

www.ericll.org/guidance-toolstp53/ a) Manual for TP53 mutational analysis b) TP53 Analysis Template Report Forms c) TP53 Certification Requirements d) Helpdesk (ERIC office + expert in charge) e) Free Software for Sanger Analysis Data – GLASS - for detection and annotation of low - frequent somatic variants

ERIC TP53 NETWORK ACTIVITIES

ERIC Publications on TP53 analysis Comprehensive profiling and analysis of TP53 mutation in CLL TP53 mutation profile in chronic lymphocytic leukemia: evidence for a disease specific profile from a comprehensive analysis of 268 mutations. T Zenz, D Vollmer, M Trbusek, J Smardova, A Benner, T Soussi, H Helfrich, M Heuberger, P Hoth, M Fuge, T Denzel, S Häbe, J Malcikova, P Kuglik, S Truong, N Patten, L Wu, D Oscier, R Ibbotson, A Gardiner, I Tracy, K Lin, A Pettitt, S Pospisilova, J Mayer, M Hallek, H Döhner, S Stilgenbauer, European Research Initiative on CLL (ERIC): Leukemia, 2010. ERIC recommendations on TP53 mutation analysis in Chronic Lymphocytic Leukemia. Pospisilova S, Gonzalez D, Malcikova J, Trbusek M, Rossi D, Kater AP, Cymbalista F, Eichhorst B, Hallek M, Döhner H, Hillmen P, van Oers M, Gribben J, Ghia P, Montserrat E, Stilgenbauer S, Zenz T.: Leukemia, 2012. Assessment of TP53 functionality in chronic lymphocytic leukaemia by different assays; an ERIC-wide approach. Te Raa GD, Malčiková J, Mraz M, Trbusek M, Le Garff-Tavernier M, Merle-Béral H, Greil R, Merkel O, Pospisilova S, Lin K, Pettitt AR, Stankovic T, van Oers MH, Eldering E, Stilgenbauer S, Zenz T, Kater AP; European Research Initiative on CLL (ERIC): Brit. J. Haematology, 2014. Innovation in the prognostication of chronic lymphocytic leukemia: how far beyond TP53 gene analysis can we go? Pospisilova S, Sutton LA, Malcikova J, Tausch E, Rossi D, Montserrat E, Moreno C, Stamatopoulos K, Gaidano G, Rosenquist R, Ghia P; European Research Initiative on CLL (ERIC): Haematologica, 2016.

ERIC Recommendations UPDATE 2017

Currently recommended methods: Sanger sequencing with or without prescreening Next generation sequencing

Updated Recommendations

Detailed recommendations Which material? Which exons? Methods – Sanger sequencing, Next Generation sequencing… Interpretation and reporting

Manual for TP53 mutational analysis

ERIC Recommendations for TP53 Mutation Analysis in Chronic Lymphocytic Leukemia – Update on

Methodological Approaches and Results Interpretation

J. Malcikova, E. Tausch, D. Rossi, L. A. Sutton, T. Soussi, T. Zenz, A.P. Kater, C. U. Niemann, D. Gonzalez, F. Davi, M. Gonzalez Diaz, C. Moreno, G. Gaidano, K. Stamatopoulos, R.

Rosenquist, S. Stilgenbauer, P. Ghia, S. Pospisilova

on behalf of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) – TP53 network

Supplementary files: Report forms for reporting results of TP53 mutation analysis

ERIC recommendation Notes and alternatives

Patients Sampling Always when deciding about treatment.

Material

Type of material Peripheral blood (PB) Bone marrow, lymph nodes – suitable alternatives if PB lymphocyte count is <10x109/l, e.g. in SLL/CLL. Fresh/frozen tissues are strongly preferred.

Anticoagulant EDTA or heparin (in case of PB)

Cells Mononuclear cells

When PB or BM contains <60-70% of lymphocytes, separation of CD19+ cells or using deep-NGS with low detection limit is recommended.

Nucleic acid DNA RNA analysis carries a risk of omitting truncating/splice site variants.

Covered region Optimum: exons 2-11 (coding region), Minimum: exons 4-10, Always include splice sites (+/-2 intronic bp)

Variants found in introns at positions +2/-2 impair splicing.

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e

Sanger sequencing

Primers and PCR protocol Available in the IARC TP53 database: http://p53.iarc.fr/ProtocolsAndTools.aspx

Sequencing Both strands (forward + reverse) Data analysis Use software designed for somatic variant detection Free web-based software available on the ERIC website.

NGS

Protocol Amplicon or capture-based approaches are applicable. DNA input should be calculated with respect to the limit of detection. Testing the integrity of DNA is recommended

Several ready-to use kits involving TP53 analysis are commercially available.

Sequencing Minimum of 100 reads per each position. Number of variant reads for reliable variant calling should be at least 10. ≥99% minimum coverage percentage should be reported.

Data analysis Use software designed for somatic variant detection. Validated minimal limit of detection should be 10% VAF.

Proper bioinformatics approach represents the most challenging part of NGS and no universal tool is currently available.

Interpretation and clinical reporting

Variant description

Use HGVS nomenclature: http://varnomen.hgvs.org/ Report the cDNA and protein level including reference sequence.

Interpretation

Check the detected variants using locus-specific database: IARC TP53 database: http://p53.iarc.fr/TP53GeneVariations.aspx or TP53 web site: http://p53.fr/

If a rare variant or variant with preserved functionality is detected: 1) Repeat the analysis by starting from PCR to exclude analytical errors. 2) If the variant is confirmed, test the paired germline DNA.

Polymorphisms and benign variants It is preferred not to include common polymorphisms and benign variants in the report to physicians.

Using dbSNP for filtering out polymorphisms and neutral variants is strongly discouraged.

Limit of detection and clinical reporting

Report variants detectable by Sanger sequencing and variants present in >10% VAF if tested by NGS. Reporting variants between 5-10% VAF is acceptable only if explicitly stating that the clinical impact of minor subclonal mutations has not been conclusively documented.

There is not enough evidence for making therapeutic decisions based on the detection of mutations present in low variant allele frequency.

Report form Template report form is available on the ERIC website.

Table: Overview of ERIC recommendations for TP53 analysis.

For all laboratories performing the TP53 analysis in CLL – validation of different techniques on TP53 mutation analysis

Support the 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

ERIC Certification on the assessment of TP53 mutations AIMS:

ERIC Certified Centres from 4 rounds (2014 - 2016)

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

107 Centres from 24 countries

TP53 Certification Requirements

ERIC Certification on the assessment of TP53 mutations

Institution

ROUND 5 (2017): 23 participating centres from 16 countries

ERIC Certification

on the assessment of TP53 mutations

Austria Medical University of Vienna (Vienna) Belgium • UZ Leuven (Leuven) • Hopital Erasme (Brussels) Brasil • Instituto Nacional de Cancer-INCA (Rio de Janeiro) Czech Republic • University Hospital Brno (Brno)

Denmark Odense University Hospital (Odense) Finland Tykslab (Torku) France • (EFSBFC) (Besançon) • CHU Bordeaux (Pessac) Germany AgenDix GmbH (Dresden) Labor Dr. Wisplinghoff (Cologne) MVZ Dr Eberhard & Partner Dortmund (Dortmund)

Hungary Semmelweis University (Budapest) Italy University of Bari (Bari) Asst Papa Giovanni XXIII (Bergamo) Poland • The Maria Skłodowska Curie Memorial Cancer Centre and

Institute of Oncology – MCMCC (Warsaw) Sweden Sahlgrenska University Hospital (Goteborg) Spain • Hospital La Fé (Valencia)

Switzerland Kantonsspital St.Gallen (St Gallen)

The Netherlands UMC Utrecht - 2 departments (Utrecht) The United Kingdom John Radcliffe Hospital (Oxford) King´s College Hospital London (London)

Round 5 – List of Participating Centres:

Round 5 Results: Available in Dec 2017 (ASH meeting)

Round 6 - Expected deadline for applications: December 15, 2017

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

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

Not two methods in one round

ERIC Certification

on the assessment of TP53 mutations

1st ERIC Workshop on TP53 analysis in CLL

(Brno, 2015)

131 participants from 23 countries

ERIC TP53 Network Activity: Organization of workshops on TP53 analysis

2nd ERIC Workshop on TP53 analysis in CLL

(Stresa, 2017)

134 participants (26 invited speakers, chairs, tutors)

58%

19%

19%

3% 1%

Sanger

NGS

Sanger + NGS

FISH

Functional

94% Sanger only 4% Sanger + MLPA 2% Sanger + Functional

ERIC TP53 Workshop Stresa – Registration Questionnaire Output

96% 38%

ERIC TP53 Survey

Includes both clinical and laboratory questions

Aim: better understanding of TP53 analysis and

treatment implications of TP53 variants in CLL patients in clinical and laboratory practice

Supported by Gilead

More information on survey results will be presented in several lectures this afternoon

N=81

ERIC TP53 Survey

N=81

ERIC TP53 Survey - PARTICIPANTS

N=33 N=33

ERIC TP53 Survey - CLINICIANS

N=55

ERIC TP53 Survey - DIAGNOSTICIANS

Jitka Malčíková

Šárka Pavlová

Karla Plevová

Karol Pál

Boris Tichý

Michael Doubek

THANK YOU FOR YOUR ATTENTION !

Paolo Ghia

Kostas Stamatopoulos

Arnon Kater

Carol Moreno

Emili Montserrat

Natalie Sorolla - ERIC office

ACKNOWLEDGEMENTS

Certifying Centers:

Stephan Stilgenbauer

Eugen Tausch

Richard Rosenquist

Lesley Ann Sutton

Jitka Malčíková

Natalie Sorolla

Training Centers Representatives