1. MOLECULAR GENETICS and LEUKEMIA Clive S. Zent M.D. Division of Hematology/Oncology

2. CASE PRESENTATION 45 year old WF Hx x 1 week fever dyspnea on exertion malaise. Examination temp. 100.8 pharyngitis no bleeding

3. INVESTIGATION CBC WCC 14.2 Hgb 6.5 platelets 22 000 smear = BLASTS Chemistry LDH 335 creatinine 0.1 Coagulation normal

4. BONE MARROW Aspirate and biopsy increased myeloblasts M4Eo morphology Flow cytometry CD34+, CD33+, CD13+, HLA-DR+ population Genetic analysis Karyotype inv (16) FISH trisomy 22 Molecular CBF  /MHY11

5. MANAGEMENT Induction therapy daunorubicin + cytarabine x2 Consolidation High Dose Ara-C (HDAC) Follow up CBC BM –karyotype –molecular

6. WHAT IS MYELOID LEUKEMIA ? Clonal progeny of single malignant precursor Myeloid blood forming elements Proliferation failure of differentiation and apoptosis

7. ETIOLOGY Environmental toxin Benzene Smoking Chemotherapy Alkylating Topoisomerase II inhibitors Radiation Congenital syndromes Downs

8. CLASSIFICATION FAB 1976 (revised 1985)

9. CELL TO CHROMOSOME TO GENE morphology cytogenetic genetic biology clinical

10. CYTOGENETICS Ph chromosome (1960) Non random chromosome rearrangements (1973) –translocation –inversion –deletion, insertion, reduplication clinical significance –diagnosis –follow up –prognosis

11. FISH Fluorescent labeled DNA probe Hybridize Metaphase Interphase Advantage More sensitive than karyotype Numerical Non dividing cells Limitations Operator dependent Target specific

12. MOLECULAR GENETICS breakpoint cluster regions (BCR) clone breakpoints identify genes determine function & role in leukemogenesis transcription factors oncogenes

13. MOLECULAR GENETICS Southern blot Detects 1:100 malignant cells RT-PCR More sensitive 1:1,000 -10,000 BCR specific Gene expression microarrays

14. GENETIC CLASSIFICATION Good prognosis CBF PML/RAR  Intermediate Normal cytogenetics Poor prognosis Deletion Trisomy MLL …….

15. CBF (AML/ETO) M2 t(8;21) = 20% 5 % adult AML AML/ETO Good prognosis

16. CBF (CBFB/MYH11) M4Eo = 25% M4 inv (16;16), t(16;16) CBF  /MYH11 good prognosis

17. CBF a transcription factor CBF = AML1 + CBF  Runt homology region core binding site TGTGGTT Target gene regulatory regions –viruses: MMLV, polyoma –cell surface proteins: CSF-1R, TCR, IL-1R –cytokines: IL-1,3,5, GM-CSF, G-CSF –myeloid specific genes: MPO, NE

18. CBF Normal Function Expressed in hematopoietic tissue In vitro - transcription factor Knockout mice: –no fetal hematopoiesis –die at E 12.5 –CBF  = CBF 

19. CBF CHIMERIC GENES The partners CBF  –AML1/ETO –AML1/EAP/MDS/EVI1 –TEL/AML1 CBF  –CBF  /MYH11

20. AML1 ETO AML/ETO Transactivation domain 2 x Zn fingersPEST Runt AML1 and AML1/ETO

21. CBF  and leukemogenesis Dominant negative –expressed –Runt intact –binds DNA and CBF  –inhibits transactivation Knock in mice: –AML1/ETO

22. CBFb and CBFb/MYH11 CBF  MYH11 CBF  /MYH11

23. CBF  and leukemogenesis Dominant negative –myosin –nuclear localization Knock- in mouse –CBF  /MYH11

24. SUMMARY Chromosome translocation Chimeric gene Transcriptional dysregulation Pathway convergence Molecular characterization Pathology Diagnosis Prognosis Treatment