Immunophenotyping of Leukemia and Lymphoma. Douaa Moh. Sayed

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1 Immunophenotyping of Leukemia and Lymphoma By Douaa Moh. Sayed

2 Immunological Classification of Acute leukemia ALL Hybrid AML T-ALL Intermediat T ALL early T-ALL mature T-ALL B-ALL pro-b-all C-ALL pre-b-all B-ALL M1 M2 M3 M4 M5 M6 M7

3 Immunological Classification of ALL Morphology Cytochemistry Surface markers Gene rearrangement

4 Monoclonal Antibodies CD = Cluster of Differentation Classification system of all mabs against human leukocytes antigens Defined in workshops (HLDA, VI, Kobe 97) A CD number comprises all antibody clones detecting the same human antigen - not epitope specific

antigens Defined in workshops (HLDA, VI, Kobe 97) A CD number

5 Bone marrow CFU-GM CD13, CD34, CD33, CD38, CD117 pluripotent Stem Cell CD34, CDw90, Thymus CFU-M CD13 CD33 CDw65 CD117 Monoblast CD13 CD15 CD33 CDw65 unreifer Monozyt CD13 CD33 CD64 CDw65 Monozyt CD13 CD15 CD64 CDw65 Blood CFU-G CD13 CD33 CDw65 CD117 cmpo Myeloblast CD13 CD33 CDw65 CD117 cmpo Myelozyt CD13 CD33 CD15 CDw65 cmpo Metamyelozyt CD13 CD33 CD15 CD65s cympo Granulozyt CD15 CD16 CD65s cympo CFU-Meg CD34 CD33 Megakoryblast CD34 CD61 Megakoryzyt CD41 CD42 CD61 Thrombozyt CD41 CD42 CD61 CFU-E CD34 CD33 Erythroblast CD34 CD36 Retikulozyt CD36 Glyko A Erythrozyt Glyko A? NK-Zelle +/-CD2 CD7 CD8 CD16 CD56 +/-CD57 prä-prä-b-zelle CD19 ccd22 CD34 ccd79a TdT prä-b-zelle CD19 CD22 CD24 ccd79a cm unreife B-Zelle CD10 CD19 CD20 CD22 CD24 CD79a IgM B-Lymphozyt CD19 CD20 CD22 CD24 CD79a IgM/IgD CD34, TdT subkortikaler Thymozyt ccd3 CD5 CD7 CD34 TdT cortiklaer Thymozyt CD1 +/-CD2 ccd3 CD5 CD7 medullärer Thymozyt CD2 CD3 CD5 CD7 +/-CD4 +/-CD8 T-Lymphozyt CD2 CD3 CD5 CD7 +/-CD4 +/-CD8

cympo CFU-Meg CD34 CD33 Megakoryblast CD34 CD61 Megakoryzyt CD41 CD42 CD61 Thrombozyt CD41 CD42 CD61 CFU-E CD34 CD33 Erythroblast CD34 CD36 Retikulozyt CD36 Glyko A Erythrozyt Glyko A?

6 T cell development

8 B cell development

9 Immunophenotype of Leukemia vs Normal cells Leukemia cells do not obey rules of normal differentiation: Some antigens expected to be expressed may be lacking Some differentiation antigens may appear in aberrant combinations

antigens expected to be expressed may be lacking Some

10 B Cell Leukemia Pro-B-ALL Common-ALL Pre-B-ALL B-ALL CD79a CD19 CD20 CD22 CD24 CD34 TdT CD10 IgM Legende: Expression no Expression variable Expression exclusive Expression cytoplasmatic Expression

11 Immunological classification of ALL (Catovsky and Hoffbrand, 1999) Marker Pro-B call Pre-B B-ALL Pre-T T-ALL TdT CD19 (m) CD22 (cyt) CD79a (cyt) CD10 (m) /+ -/+ - µ chain (cyt) Smlg (m) CD7 (m) CD3 (cyt) CD2 (m) CD5 (m) /+ + CD1a (m) /+

+ + + - - CD10 (m) - + + -/+ -/+ - µ chain (cyt) - - + + - - Smlg (m) - - - + - - CD7 (m) -

12 B Cell Leukemia Pro B-ALL (BI) Common B-ALL Pre B-ALL (BII) (BIII) B-ALL (BIV) TdT CD22 CD79a CD19 CD19 CD10 CD22 CD79a µ CD19 CD10 CD22 CD79a IgM Y CD19 CD20 CD22 CD79a CD65/15 CD24 CD34

17 Immunophenotyping of AML Cells Marker CD13 CD33 CD34 HLA-DR CD11b CD14 CD41 CD42a CD42b CD61 Glycophorin A TdT Lineage Myeloid Myeloid Early precursor Positive in most AML, negative in APL Mature monocytes Monocytes Platelet glycoprotein IIb/IIIa complex Platelet glycoprotein IX Platelet glycoprotein Ib Platelet glycoprotein IIIa Erythroid Usually indicates acute lymphocytic leukemia, however, may be positive in M0 or M1

Monocytes Platelet glycoprotein IIb/IIIa complex Platelet glycoprotein IX Platelet glycoprotein Ib

18 Immunophenotyping of AML (Campana and Behm, 2000): <10

22 Panel of antibodies recommended by the European Group for the Immunological Characterization of Leukemias (EGIL) for the diagnosis and classification of acute leukemia (1995) First Panel B lymphoid T lymphoid Myeloid Non-lineage related Second panel If B lineage IF T lineage If myeloid CD19, ccd22, CD10 ccd3, CD2, CD7 Anti-MPO, CD13, CD33, CD65, CD117 TdT, CD34, HLA-DR Cyµ, κ, λ, CD20, CD24 CD1a, mcd3, CD4, CD5, CD8, anti- TCR αβ, anti-tcr γδ Anti-lysozyme, CD14, CD15, CD41, CD61, CD64, anti-glycophorin A

23 Leukemia with 2 lineage markers expression Biphenotypic : Synchronous Billineal : Synchronous Metachronous Biclonal : Two discrete transforming events

24 Aim of Immunophenotyping of Leukemias To establish the line of origin and stage of differentiation To identify various prognostic subgroups To tailor treatment protocols To define peculiar phenotypes that may help to detect MRD

25 Methods of Detection of MRD Immune receptor gene rearrangement RT-PCR for chromosomal translocations immunophenotyping

26 MRD Detection by Immunophentyping Sensitivity Limit: -Morphologically -Phenotyping 1 : malignant cells Practical Applications: -Treatment monitoring and identification of patients with persistent residual malignant cells - Prediction of clinical relapse early enough -Verifying morphologically suspicious cells - Detection of residual malignant cells in the harvest of autologous BM and test efficacy of purging

27 Immunological Detection of MRD - Markers not usually present in PB e.g. TDT, CD10, CD1 - Aberrant coexpression on malignant cells in BM : - different lineage combinations - asynchronous expression of markers - Measuring the intensity of the antigen - - Phenotyping on morphlogically defined cell populations (light scattering properties)

28 Antigen Pattern I aberrant TdT CD22 CD79a CD19 Definition Cells are clearly defined by several pan-lineage antigens and co-express an antigen of a different lineage e.g. CD65 - myeloid antigen - expressed on a immature B-cell (CD19 +, CD34 +, cycd22 +, cycd79a + ) CD65 CD34

29 Antigen Pattern II asynchronous Definition The differentiation stage of the cells are clearly defined by several antigens e.g. antigens of mature B-cells, but co-express an antigen of immature B-cells like CD34. IgM Y CD19 CD20 CD22 CD79a CD24 CD34

30 Aberrant Antigen Patterns Coexpression CD15/CD65 CD7 CD2 CD19/CD56 Subtyp Pro-B-ALL AML M0/M1/M2 AML FAB 4eo AML FAB M2 Detection of minimal residual disease

31 Screening for Lymphoma Flow Cytometric Analysis by monoclonal antibodies can then add additional information B-, T- or LGL-cells clonality of B-Cell Lymphomas subtyping

35 Immunophenotyping of B chronic lymphoid leukemia Discrimination between CLL, CLL/PLL, PLL, HCL & leukemic phase of NHL: FL, MCL, SLVL Phenotypic changes have clinical & biological significance MRD detection - use of combination of reagents - Quantification of cellular antigens

36 Diagnosis of NHL s B-cell Y < 20% CD19 CD19 Y Κ and λ B-NHL Y > 20 % CD19 CD19 Y Κ or λ CD5 CD23 Proliferation Light chain restriction Co-expression of immature antigens activation antigens NHL

38 B-ALL

39 THANK YOU FOR YOUR ATTENTION

Pathology No: SHS-CASE No. Date of Procedure: Client Name Address

Pathology No: SHS-CASE No. Date of Procedure: Client Name Address TEL #: (650) 725-5604 FAX #: (650) 725-7409 Med. Rec. No.: Date of Procedure: Sex: A ge: Date Received: Date of Birth: Account No.: Physician(s): Client Name Address SPECIMEN SUBMITTED: LEFT PIC BONE MARROW,