Cytogenetic Testing in Acute Leukemia. June 4, 2009
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1 Cytogenetic Testing in Acute Leukemia June 4, 2009
2 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
3 WHO 2008 Classification of Acute Leukaemia
4 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
5 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
6 t(8;21)
7 inv(16)
8 t(15;17)
9 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
10 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
11 AML with Recurrent Genetic Abnormalities AML with t(6;9) Basophilia, multilineage dysplasia FLT3 mutation common Poor prognosis
12 t(6;9)
13 AML with Recurrent Genetic Abnormalities (cont d) AML with inv(3) or t(3;3) Platelet abnormalities Poor prognosis
14 AML with Recurrent Genetic Abnormalities (cont d) AML (megakaryoblastic) with t(1;22) Infants without Down syndrome Respond well to intensive chemotherapy
15 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
16 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
17 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
18 Therapy-related Myeloid Neoplasms Most have an abnormal karyotype
19 Myeloid proliferations related to Down syndrome Transient abnormal myelopoiesis Myeloid leukemia associated with Down syndrome; usually acute megakaryoblastic
20 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
21 t(9;22)
22 t(11;19)
23 Precursor lymphoid neoplasms B lymphoblastic leukemia/lymphoma, NOS B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities T lymphoblastic leukemia/lymphoma
24 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
25 MLL
26 FISH t(12;21)
27 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)
28 Hyperdiploid
29 Chromosomes 4, 10, 17
30 Cytogenetic Methods
31 Cell culture
32 Chromosome analysis
33 Karyotyping
34 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
35 Genetic analysis Chromosome analysis Molecular analysis and molecular cytogenetics FISH, CISH Southern, PCR, sequencing, etc CGH and expression arrays
36 FISH principle
37 Fluorescence in situ hybridization (FISH)
38
39 Dual colour FISH - rearrangement
40 Interphase
41 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
42 CGH and array CGH
43 Comparative genomic hybridization (CGH)
44 CGH and array CGH
45
46 CGH array
47 Array CGH data
48 Array CGH Widely used in developmental cytogenetics Research use in cancer Potential uses in acute leukemia Hyperdiploid pediatric ALL Other leukemias with imbalance
49 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
50 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
51 Other QA ACMG/CAP cytogenetic and FISH challenges include cancer images and slides for cancer FISH Rates of abnormality Failure rates
52
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