Hematology 23 Diagnostic Tests in Leukemia Lymphoma Myeloma

Hematology 23 Diagnostic Tests in Leukemia, Lymphoma & Myeloma Lee Woo In, MD, Ph. D Department of Laboratory Medicine

Definition • Blast – 정상 골수 <5% of ANC(all nucleated cell) – Acute leukemia ≥ 20% of ANC • FAB classification vs. WHO classification – MDS < 20% • Multiple myeloma – plasma cell • MPD-lineage proliferation • Lymphoma

Characteristics of Hematological Malignancies • Proliferation of malignant clone – * Clonality ? • Proliferation of daughter cells originating from a single precursor cell, • Identical phenotypes, growth characteristics as that originating precursor cells • Frozen of the maturation stage – Except : CML – Lymphoid • No stage specific marker

Formation of a clone Normal tissue Clone Mutation Possible outcomes. Apoptosis. Neoplastic growth. Chromosome evolution. Metastatic potential

Diagnostic Tests 1. Morphology PBS, Bone Marrow aspirates & biopsy 2. Cytochemical Stain 3. Immunophenotyping 1) Immunocytochemistry 2) Flow Cytometry

4. Cytogenetics 1) Karyotyping 2) Fluorescent In Situ Hybridization(FISH) 5. Molecular Methods 1) Southern blotting 2) PCR, RT-PCR, Multiplex PCR, Competitive PCR 3) Quantitative Real-Time PCR 4) Gene Scan

I. Morphology - PBS and BM - Myeloblast (Auer rod) Blast Lymphoblast

II. 급성 백혈병의 세포 화학적 염색 양상 Acute Leuk Peroxidase/SBB ANBE AP ALL - AML, M 0 - - AML, M 1 + - AML, M 2 ++ - + + AML, M 3 +++ - -/+ ++ AML, M 4 ++ ++ -/++ ++ AML, M 5 - +++ ++ + AML, M 6 - + ++ - AML, M 7 - - ++ + *sudan black B /MPO. . myeloperoxidase Alpha-naphtylbutyrate esterase - PAS +/++ -/+

Sudan black B

ANBE

III. Immunophenotyping • 세포의 특이한 항원결정인자(epitope) 를 인지하는 항체를 이용하는 면역학적 방법 – CD (Cluster designation/Cluster of differentiation) • Monoclonal antibody • 세포를 인지하는 특이도가 높다

각 세포별 CD 표지자 • T cell – CD 2, CD 7, CD 3(이상 pan-T cell), CD 4, CD 8 • B cell – CD 10(CALLA), CD 19, CD 20, CD 21, CD 22, CD 23, CD 40 • NK cell – CD 16, CD 57 • Granulocytic lineage – HLA-DR, CD 34, CD 38(non-comm. Stem cell) – Myeloblast : HLA-DR, CD 13, CD 33, CD 11 b • Monoblast : CD 14, CD 15, CD 11 b • Platelet – CD 41, CD 42, CD 61, CD 62, CD 63 • RBCs – CD 45, CD 71, glycophorin A

Immunophenotyping by flow cytometry 세포의 크기 (forward scatter) 세포내의 복잡성 (side scatter) 형광염색

AML M 0 -* M 1 M 2 M 3 M 4 M 5 M 6 -* M 7 -*

Scoring system for Dx of Acute Leukemia Score B cell lineage T cell lineage Myeloid lineage 2 m/cyt CD 22, cyt CD 79 a, cyt Ig. M, surf Ig m/cyt CD 3, m/cyt TCR MPO, cyt CD 13 1 CD 19, CD 20, CD 10 CD 2, CD 5, CD 8 CD 13, CD 33, CD 117, CD 65 Td. T, CD 24 Td. T, CD 7, CD 1 a CD 14, CD 15, CD 64 0. 5 Dx … Lineage score > 2. 5 1) Acute biphenotypic leukemia, 2) aberrant antigen axpression *m: membranous, cyt : cytoplasmic

Immunophenotyping of B lineage ALL Marker CD 34 Null ALL Common ALL Pre-B ALL B-ALL + + + - Tdt ++ ++ ++ - HLA-DR ++ ++ CD 10 - ++ ++ + CD 19 ++ ++ CD 20 - + + ++ CD 22 ++ ++ Cyt CD 79 a ++ ++ Pre-B cyt Ig - - + - s. Ig-CD 79 - - - ++

IV. Cytogenetic tests • Cytogenetic characteristics of Hematologic malignancies – Non random style – Subgroup • Conventional cytogenetics • Cytogenetics + Molecular =FISH

Cytogenetics =Study of cell + Genetics

* 급성 백혈병에서 흔히 관찰되는 염색체 이상 소견 Disease Translocation Genes involved AML t(6; 9)(p 23; q 34) DEK-CAN AML-M 2 t(8; 21)(q 22; q 22) AML 1 -ETO AML-M 3 t(15; 17)(q 22; q 21) PML-RARa AML-M 4 Eo inv(16)(p 13; q 22) CBFbeta-MYH AML-M 4/M 5 t(11; 19)(q 23; p 13) MLL-ENL AML-M 5 t(9; 11)(p 21; q 23) MLL-AF 9 CML t(9; 22)(q 34; q 11) BCR-ABL B-ALL t(9; 22)(q 34; q 11) BCR-ABL Precursor Pre-B-ALL t(1; 19)(q 23; p 13) E 2 A-PBX 1 B-ALL t(8; 14)(q 24; q 32) MYCㅡIg. H

* Myelodysplastic syndrome +8, 5, 7번 염색체 이상 * 비호즈킨 림프종에서 흔히 관찰되는 염색체 이상 Disease SLL Translocation t(14; 19)(q 32; q 13) Genes BLC-3 Follicular center cell t(14; 18)(q 32; q 31) BCL-2 Burkitt t(8; 14)(q 24; q 32) C- MYC

Fluorescent In Situ Hybridization(FISH) Metaphase, interphase, paraffin, frozen, cytospin, touch print, BM aspiration smears, etc

t(8; 21)… AML ETO AML 1

2 O 2 G : no rearrangement of AML 1/ETO 1 O 1 G 2 F : Rearrangement of AML 1/ETO

Chromosome Enumeration Probe: X/Y probe

V. Molecular study • Clonality – Clonal process … a proliferation of daughter cells originating from a single precursor cell , each having identical phenotypes & growth characteristics • Normal B & T cell – Polyclonal process

Monoclonal protein confirm & quantitation • Multiple myeloma 99% – M protein (+) in serum, urine – Dx: M protein, bone study, BM study • Protein electrophoresis(단백전기영동) • Immunofixation electrophoresis(면역고정 전기영동)…확진 • Quantitation of immunoglobulin • Free light chain quantitation

Photo - PEP & densitometry PEP Immunofixation

Photo -osteolytic lesion

Photo - BM plasma cell

MPD Myeloproliferative disorders • Polycythemia Vera – Jak 2 V 617 F or JAK 2 • Chronic myelocytic leukemia – t(9; 22)(q 34; q 11. 2) • Essential thrombocythemia • Idiopathic myelofibrosis

Myelodysplastic syndrome(MDS) • • Ineffective Hemopoiesis Apoptosis 증가 Peripheral cytopenia BM blast <20% BM morphology : Daysplasia Old age Chromosomal abnormality 동반