IgCLL group activities in 2009 and beyond. Kostas Stamatopoulos Hematology Department and HCT Unit George Papanicolaou Hospital, Thessaloniki, Greece

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1 IgCLL group activities in 2009 and beyond Kostas Stamatopoulos Hematology Department and HCT Unit George Papanicolaou Hospital, Thessaloniki, Greece

2 Troubleshooting Standardization

3 Standardization and Validation

4 Standardization and Validation Immunoglobulin gene mutational status

5 TROUBLE IG SEQUENCES IGHV-(IGHD)-IGHJ recombination Rearrangements carrying non functional genes Problematic IGHV-IGHD-IGHJ junctions Somatic Hypermutation Stop codons Absence of CDR3 anchors Insertion / Deletions

6 Database of trouble sequences collected since February 2007 Over 80 trouble sequences have been submitted to the review board for analysis in 2.5 years (an average of 30 per year). 1. Borderline mutated case 2. Missing HCDR3 landmarks 3. Truncated IGHV sequences 4. Insertions and deletions 5. No IGHV sequence 6. Double productive 7. Double productive with discordant mutational status 8. Single unproductive 9. Intraclonal diversity

7 Database of trouble sequences collected since February 2007 Over 80 trouble sequences have been submitted to the review board for analysis in 2.5 years (an average of 30 per year). 1. Borderline mutated case 2. Missing HCDR3 landmarks 3. Truncated IGHV sequences 4. Insertions and deletions 5. No IGHV sequence 6. Double productive 7. Double productive with discordant mutational status 8. Single unproductive 9. Intraclonal diversity

8 Significance of trouble sequences Analysis of trouble sequences provides: An opportunity to investigate clinical correlations. An opportunity to decipher the molecular mechanisms that were used to generate the IG sequences. This information can be applied to normal B cells as well as the CLL B cells.

9 New analytical tools

10 bioinformatics analysis team CERTH REFINEMENT OF ANALYTICAL TOOLS IMGT, Montpellier Marie-Paule Lefranc Veronique Giudicelli Xavier Brochet BAT, CERTH, Thessaloniki Nikos Darzentas Anastasia Hadzidimitriou Andreas Agathagelidis

11 Insertions/Duplications Deletions Frequency in CLL: ~3% An analytical nightmare (until recently ) Belessi et al. Eur J Immunol. 2006;36:

12 A paradigmatic example of bioinformatics advances meeting clinical needs!

13 Upcoming developments in 2010 IMGT V-QUEST Alerts Sequence ambiguities or troubles Short sequences leading to gene assignment problems Identification of IGHC genes Analysis of incomplete IGHD-J rearrangements Biological significance (Tsakou et al. ASH 2009) Practical significance: MRD testing

14 EDUCATION

15 3 rd Educational Workshop Immunoglobulin Gene Analysis in Chronic Lymphocytic Leukemia September 2009 Thessaloniki - Greece

16 Sponsorship/support:

17 60 participants, 17 countries Austria 2 Belgium 2 Czech Republic 2 Denmark 4 Germany 1 Greece 17 Iran 1 Ireland 1 Italy 8 Netherlands 3 Poland 2 Romania 1 Serbia 1 Spain 4 Sweden 8 UK 2 US 1

18 Workshop activities and innovations Questionnaire: current everyday practice throughout Europe Material, PCR methodology, Interpretation of PCR results, Sequencing, Reporting to the clinic Exercises and web-based assessment and self-assessment Light chain analysis

19 Next Workshop Italy 2011

20 Publications

21 The first book!

22 A new book! (in preparation)! Biological prognostic markers in CLL

23 Biological prognostic markers in CLL Stamatopoulos, Ghia, Rosenquist, eds. Foreword Section I - Clinico-biological background of CLL CLL biology Clinical prognostic markers (Rai/Binet staging, serum markers) Section II - Cytogenetics/molecular cytogenetics Genomic aberrations p53 dysfunction Section III - Immunoglobulin genes IGHV gene mutation status as prognostic marker Stereotyped B-cell receptors in CLL Section IV - Immunophenotyping CD38 onwards: the evolution of flow cytometric markers in CLL ZAP-70 From MBL to MRD: to the limits of flow cytometry Section V RNA-based methods/micro-rna RNA-based molecular markers Micro-RNA in CLL Section IV Clinical application of new prognostic markers Clinical application of new prognostic markers Conclusions Emili Montserrat, Michael Keating Nicholas Chiorazzi, Federico Caligaris-Cappio Kanti Rai, Eva Kimby Stephan Stilgenbauer, Hartmut Doehner Andrew Pettit, Sarka Pospisilova Paolo Ghia, Richard Rosenquist Chrysoula Belessi, Kostas Stamatopoulos Rajendra Damle, Silvia Deaglio Florence Cymbalista, Francesc Bosch Andy Rawstron, Paolo Ghia Frederic Davi, Anne-Mette Buhl George Adrian Calin, Carlo Croce David Oscier, Michael Hallek Daniel Catovsky, Tom Kipps Sponsored in part by Roche Hellas Wolters Kluwer - Lippincott

24 San Raffaele, Milano Paolo Ghia Uppsala University Richard Rosenquist Pitié Salpêtrière, Paris Fred Davi Nikea Hospital, Athens Chrysoula Belessi Tenovus Lab, Southampton Kathy Potter Erasmus University, Rotterdam Ton Langerak