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• 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)
M0 agranular myeloblast 2-3%
M1 myeloblast 20%
M2 myeloblast with maturation 25-30%
M3 promyelocyte 8 - 15%
M4M4Eo
myelomonocytic+ eosinophils
20 - 25%5%
M5 monocytic 10%
M6
M7
erythroblastic
megakaryocytic
5%
1%
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 -10,000• 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
CBFa 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
CBFNormal Function
• Expressed in hematopoietic tissue
• In vitro - transcription factor
• Knockout mice: – no fetal hematopoiesis– die at E 12.5– CBF = CBF
CBF CHIMERIC GENESThe partners
• CBF– AML1/ETO– AML1/EAP/MDS/EVI1– TEL/AML1
• CBF– CBF/MYH11
AML1
ETO
AML/ETO
Transactivation domain
2 x Zn fingers PEST
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