View
4
Download
0
Category
Preview:
Citation preview
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.
Proc
edur
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)
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
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
Recommended