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MOLECULAR GENETICS and LEUKEMIA

MOLECULAR GENETICS and LEUKEMIA. Clive S. Zent M.D. Division of Hematology/Oncology. CASE PRESENTATION. 45 year old WF Hx x 1 week fever dyspnea on exertion malaise. Examination temp. 100.8 pharyngitis no bleeding. INVESTIGATION. CBC WCC 14.2 Hgb 6.5 platelets 22 000

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MOLECULAR GENETICS and LEUKEMIA

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  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 CBFb/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/RARa • 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) • CBFb/MYH11 • good prognosis

  17. CBFa transcription factor • CBF = AML1 + CBFb • 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. CBFNormal Function • Expressed in hematopoietic tissue • In vitro - transcription factor • Knockout mice: • no fetal hematopoiesis • die at E 12.5 • CBFa = CBFb

  19. CBF CHIMERIC GENESThe partners • CBFa • AML1/ETO • AML1/EAP/MDS/EVI1 • TEL/AML1 • CBFb • CBFb/MYH11

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

  21. CBFa and leukemogenesis • Dominant negative • expressed • Runt intact • binds DNA and CBFb • inhibits transactivation • Knock in mice: • AML1/ETO

  22. CBFb and CBFb/MYH11 CBFb MYH11 CBFb/MYH11

  23. CBFb and leukemogenesis • Dominant negative • myosin • nuclear localization • Knock- in mouse • CBFb/MYH11

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

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