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