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 pharyngitis no bleeding

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

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

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

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

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

CLASSIFICATION FAB 1976 (revised 1985)

CELL TO CHROMOSOME TO GENE morphology cytogenetic genetic biology clinical

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

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

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

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

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

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

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

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

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

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

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

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

CBFb and CBFb/MYH11 CBF  MYH11 CBF  /MYH11

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

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