Cytogenetic Testing in Acute Leukemia June 4, 2009
Objectives Describe genetic aspects of the 2008 WHO classification of acute leukemia Understand cytogenetic and molecular cytogenetic techniques Understand advantages and limitations of cytogenetic techniques
WHO 2008 Classification of Acute Leukaemia
AML and related precursor neoplasms AML with recurrent genetic abnormalities AML with myelodysplasia-related changes Therapy-related myeloid neoplasms Acute myeloid leukemia, NOS Myeloid sarcoma Myeloid proliferations related to Down syndrome Blastic plasmacytoid dendritic cell neoplasm
AML with recurrent genetic abnormalities AML with t(8;21)(q22;q22);(runx1/runx1t1) Good prognosis AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);cbfb-myh11 Good prognosis Acute promyelocytic leukemia with t(15;17)(q22;q12);pml-rara Good prognosis
t(8;21)
inv(16)
t(15;17)
AML with Recurrent Genetic Abnormalities (cont d) AML with t(9;11)(p22;q23);mllt3-mll AML with t(6;9)(p23;q34); DEK-NUP214 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); PRN1-EVI1 AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 AML with mutated NPM1 AML with mutated CEBPA
AML with Recurrent Genetic Abnormalities (cont d) AML with t(9;11)(p22;q23);mllt3-mll Monocytic and myelomonocytic Intermediate prognosis, better than other MLL rearrangements
AML with Recurrent Genetic Abnormalities AML with t(6;9) Basophilia, multilineage dysplasia FLT3 mutation common Poor prognosis
t(6;9)
AML with Recurrent Genetic Abnormalities (cont d) AML with inv(3) or t(3;3) Platelet abnormalities Poor prognosis
AML with Recurrent Genetic Abnormalities (cont d) AML (megakaryoblastic) with t(1;22) Infants without Down syndrome Respond well to intensive chemotherapy
AML with gene mutations Not detectable by cytogenetics or FISH, but by PCR AML with mutated FLT3 AML with mutated NPM1 AML with mutated CEBPA Others: KIT, MLL, WT1, NRAS, KRAS
AML and related precursor neoplasms AML with recurrent genetic abnormalities AML with myelodysplasia-related changes Therapy-related myeloid neoplasms Acute myeloid leukemia, NOS Myeloid sarcoma Myeloid proliferations related to Down syndrome Blastic plasmacytoid dendritic cell neoplasm
AML with myelodysplasia- related changes 20% or more blood or bone marrow blasts Specific cytogenetic abnormalities Unbalanced, e.g. 7/del(7q), -5/del(5q), -13/del(13q), del(12p) Balanced, e.g. t(3;21), t(5;12), t(3;5) No abnormalities described in AML with recurrent genetic abnormalities
Therapy-related Myeloid Neoplasms Most have an abnormal karyotype
Myeloid proliferations related to Down syndrome Transient abnormal myelopoiesis Myeloid leukemia associated with Down syndrome; usually acute megakaryoblastic
Acute Leukemia of Ambiguous Lineage Acute undifferentiated leukemia Mixed phenotype acute leukemia with t(9;22) Poor prognosis Mixed phenotype acute leukemia t(v;11q23); MLL rearranged Poor prognosis Mixed phenotype acute leukemia, B/myeloid, NOS Mixed phenotype acute leukemia, T/myeloid, NOS Mixed phenotype acute leukemia, NOS-rare types Other ambiguous lineage leukemia
t(9;22)
t(11;19)
Precursor lymphoid neoplasms B lymphoblastic leukemia/lymphoma, NOS B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities T lymphoblastic leukemia/lymphoma
B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities B lymphoblastic leukemia/lymphoma with t(9:22)(q34;q11.2); BCR-ABL1 Poor prognosis B lymphoblastic leukemia/lymphoma with t(v;11q23); MLL rearranged Poor prognosis B lymphoblastic leukemia/lymphoma with t(12;21) (p13;q22); TEL-AML1 (ETV6-RUNX1) Good prognosis
MLL
FISH t(12;21)
B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities (cont d) B lymphoblastic leukemia/lymphoma with hyperdiploidy good prognosis B lymphoblastic leukemia/lymphoma with hypodiploidy poor prognosis B lymphoblastic leukemia/lymphoma with t(5;14)(q31;q32);il3-igh B lymphoblastic leukemia/lymphoma with t(1;19)(q23;p13.3); E2A-PBX1(TCF3-PBX1)
Hyperdiploid
Chromosomes 4, 10, 17
Cytogenetic Methods
Cell culture
Chromosome analysis
Karyotyping
Chromosome analysis Turnaround time 24hr + Sensitivity: variable; theoretically 14% Advantage: a good genome screen Disadvantages: labor-intensive, some malignant cells are difficult to find and to analyze
Genetic analysis Chromosome analysis Molecular analysis and molecular cytogenetics FISH, CISH Southern, PCR, sequencing, etc CGH and expression arrays
FISH principle
Fluorescence in situ hybridization (FISH)
Dual colour FISH - rearrangement
Interphase
FISH analysis Turnaround time: overnight after harvest Sensitivity: <1% to 8% Advantages: sensitivity, interphase analysis, small sample Disadvantages: need to know the abnormality being looked for; availability of probes
CGH and array CGH
Comparative genomic hybridization (CGH)
CGH and array CGH
CGH array
Array CGH data
Array CGH Widely used in developmental cytogenetics Research use in cancer Potential uses in acute leukemia Hyperdiploid pediatric ALL Other leukemias with imbalance
Children s Oncology Group (COG) Calgary cancer cytogenetics lab approved since 1999 Approval process Requirements Chromosome and FISH capability Abnormality rate >55% to maintain approval
COG analysis and submission All patients on COG acute leukemia protocols Flow cytometry information essential Chromosome analysis required for AML and ALL FISH analysis required for ALL: 4, 10, 17, t(9;22), MLL, t(12;21) Submit analysis data and images for central review
Other QA ACMG/CAP cytogenetic and FISH challenges include cancer images and slides for cancer FISH Rates of abnormality Failure rates