CURRENT MOLECULAR DIAGNOSTICS IN HEMATOONCOLOGY

Transcription

1 Third Slovenian congress of hematology and transfusion medicne, Podcetrtek, April 08 Third Slovenian congress of hematology and transfusion medicne, Podcetrtek, April 08 CURRENT MOLECULAR DIAGNOSTICS IN HEMATOONCOLOGY Rajko Kusec, MD, PhD Departments of Haematology and Clinical chemistry Dubrava University hospital Zagreb

2 Clinical Value Of Molecular Diagnostic Markers R.K., Portoroz 2004 LEUKEMIA BCR/ABL (Q-PCR)! PML/RARα RUNX1/ETO TEL/RUNX1 CBFβ-SMMHC E2A/PBX1 MLL/AF4 FLT3-ITD NPM mutations? WT1 expression? FIP1L1-PDGFRα JAK2 V617F (Q-PCR)! LYMPHOMA: BCL2/IgH (Follicular) BCL1/IgH (Mantle) (FISH>PCR) NPM/ALK (ALCL) (FISH>PCR) API1/MALT? (MALT) B-,T-lymph CLONALITY TESTS:! IgHorL TCRγ MYELOMA: IgH/MMSET?!

3 Genetic And Molecular Testing In Ph + CML Consensus Mayo Clinic- Portland - M.D.Anderson STANDARD CYTOGENETICS FISH Q-RT-PCR At diagnosis BM PB Imatinib or IFNα therapy BM PB YES NO Every 3-6 mo to CCR, THEN NO NO YES NO NO NO OR NO YES, 3-6 mo YES, 3-6 mo After HSCT BM Every 3-6 mo as indicated NO NO PB NO YES, 6 mo-2y YES, 6 mo-2y Tefferi et al, Mayo Clin Proc, 2005

4 CML tumour mass size and diagnostic limits for MRD Clinical disease Partial cytogenetic remission leukem. cells Complete cytog. remission (CCR) Q-RT-PCR positiv nested Molec. remission 10 0

5 1 0,1 LEVELS OF MOLECULAR RESPONSE FISH 0,01 0,001 0,0001 0,00001 (Bcr-Abl/Abl) Minimal Molecular Response Major Moleculari Response PCR negative PCR start mo Bcr/Abl stand. curve Bcr/Abl First sample Abl Bcr/Abl Abl Follow up sample Bcr/Abl Abl stand. curve Abl Relative quantification- log response absolute quantification

6 MRD levels in Dubrava Uni hospital patients on Imatinib Not cytg or molec resp. Cytog. remiss bcr-abl/abl=1% Major mol. R. bcr.abl/abl<0,1% PCR negat mo

7 More than 40 different Abl KD mutations ~10% 22 GIMEMA WP-CML n = 477 pts resistant to imatinib P-loop catalytic domain activation loop V289A M343T H396R/P M244V L364I D276G E281A/K M351T L387M/F L248V T277A L384M E279K F359V/I S417Y/F G250E E255K/V T315I E355G/D F382L Q252R/H Y253F/H E292V E453G/K V379I F311L/I F317L E459K/Q A380T F486S

8 Which mutations to test? 1,8 1,6 Gašparović bcr-abl FT increasing 1,4 1,2 Bcr-Abl/Abl KS 1 0,8 Skup1 0,6 0,4 0, Br. uzorka DdeI RFLP-PCR for T315I (this case - no mutation) Hochhaus et al. Leukemia 2002;16;2190

9 Mutations in the MPDs JAK2 Exon 12 mutations V617F V617F + + PV PV V617F - MPL - ET MPL W515 V617F + ET V617F + IMF V617F - MPL - IMF MPLW515

10 JAK2 Exon14 point mutation detected V617F FERM SH2 JH2 Kinase Hs Cf Mm Rn Gg (G T) FFEAASMMSKLSHKHLVLNYGVCVCGDENILVQEFV FFEAASMMSQLSHKHLVLNYGVCVCGEENILVQEFV FFEAASMMSQLSHKHLVLNYGVCVCGEENILVQEFV FFEAASMMSQLSHKHLVLNYGVCVCGEENILVQEFV FFEAASMMSQLSYKHLVLNYGVCVCGEENILVQEYV

11 SINGLE GENE FOR THREE DISEASES?!? JAK2 V617F >95% Polycythaemia vera JAK2 V617F ~50% IMF/OMS JAK2 V617F ~50% Essential thrombocythaemia

12 Disproportion of 20q deleted and JAK2 mutated cells del(20q) 100% JAK2 14% Two patients (1 PV and 1ET): V617F+ granulocytes = 14% 20q deleted grans = 100% Kralovics et al, Blood, 2006

13 Fusion gene FIP1L1-PDGFRα is novel genetic marker of Chronic Eosinophilic Leukemia FIP1L1 PDGFRα intr.7-10 eks.12 4q12 Cen Tel deletion 800 Kpb fusion gene FIP1L1-PDGFRα

14 RT-PCR test for FIP1L1-PDGFRα transcript M EOL bp 24y, BM: 34% mbl-eo PB: WBC 29,4 (eo 6,0); Hb 91; Plts 182

15 Male, 50 years, sent to the haematologist for absolute eosinophilia of 2,58 x10 9 /L (WBC 15,58), haemoglobin 172 g/l, Hct 0,49, plts 300x10 9 /L spleen not palpable scarse clinical symptoms RT-PCR for H4-PDGFRβ assay after Schwaller J.et al. Blood 2001;97:3910

16 PROPOSAL FOR DIAGNOSTIC WORKUP AND THERAPY OF HYPEREOSINOPHILIA Gotlib,et al. Blood 2004;103:2879

17 M. Myeloma associated cytogenetic/genetic mutations Fonseca, R. Blood 2007;109:

18 Clinical Characteristics of MMSET-IgH + Patients (N=10) Age 64,5 (52-75y) Gender M=5 F=5 D-S stage CS1= 2 CS2=1 CS3=5 (B=1) 1 PCL Paraprotein type IgA=2, IgG=5 LC=3(2λ) Skeletal disease 0=2, 1=2, 2=2, 3=3 Extramedulary plasmacytic tumour Metabolic (Ca ) 1/ PCL (3/10) & 1 MDS/AML Dubrava Univ hosp

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20 MOLECULAR TESTING OF T- AND B-CELL CLONALITY REACTIVE LYMPHADENOPATHY LYMPHOMA Ig or TCR genes PCR = POLYCLONAL = MONOCLONAL

21 DIAGNOSTIC EXAMPLE Clinical problem: Persisting gastric mucosa lesion, PHD: suspect lymphoid neoplasm - Maltoma TEST 1 IgH gene rearrangement CD3 region TEST 2 IgH gene rearrangement BIOMED2 design Additional molecular tests: FISH t(11;18)(q21;q21) RT-PCR (API2-MALT) polyclonal clonal Merkur Uni Hosp Zagreb 05