DIAGNOSTIC AND PROGNOSTIC TOOLS IN HAEMATOLOGICAL MALIGNANCIES IN 2010 (PART II) Flow cytometry

Immunophenotyping

• Identification of molecular protein characteristics of abnormal cells • Labelling with fluorescent monospecific monoclonal antibodies • Multiparametric flow cytometry

The objectives of immunophenotyping in hematological disorders • Lineage assignment – Lymphoid acute/chronic – Myeloid acute/chronic – Erythroid – Megakaryocytic • Classification /Scoring • Detection of MPAL • Maturation anomalies • Identification of Minimal Residual Disease patterns

Before....

Now .....

Harrogate 7th HLDA : 250 CD Adelaide 2004 8th HLDA : 339 CD Barcelona 2010 : >360 CD

Immunophenotype of lymphoid cells

• Well established maturation sequence • Staged and controlled to prepare long lived antigen-specific cells • Identified by studying leukemias • Validated on normal cells • Numerous similarities between B and T cells: – Lineage committment – Gene rearrangements for antigen recetpors – Selection

B lineage associated markers

CD22

or

CD19 BCR CD79 ITAM ITAM ITAM ITAM CD21 ITAM ITIM ITIM ITAM ITIM ITIM CD23

COOH

CD20 CD24

P NH2

BONE MARROW Stem Cell Pro-B Cell Early-B Cell Pre-B Cell Naive B cell TISSUES ACTIVATION & CLONAL PROLIFERATION Immunocyte Immunoblast H0L0 H0L0 HxL0 HxL0 HRL0 HRLR CD34 DR cCD79 cCD22 CD19 cCD79 cCD79 cCD79 sCD79 CD22 CD19 CD22 CD19 CD22 CD19 CD22 CD19 CD21 CD10 CD20 CD21 CD10 CD20 CD21 cµ CD20 CD21 sµ/s

d

Memory B cells Plasma cells

Classification of B- ALL (EGIL)

B-I (pro-B) B-II (common) B-III (pre-B) B-IV (mature) cCD79/CD 19/c or sCD22 + + + + CD10 + + + c µ + + sIg +

CD3

g d e

T lineage associated markers

TCR CD5 CD7 CD2

e zz/zh

ITAMITAMITAM ITAM ITAM ITAM ITAM ITAM ITAM ITAM CD4 CD8 CD1

BONE MARROW Stem cell THYMUS Cortico thymocyte Pro-T cell Medullary Thymocyte sCD3 CD7 CD2 CD5 CD8 TISSUES ACTIVATION & CLONAL PROLIFERATION

g0d0 a0b0

CD34 DR

g0d0 a0b0

cCD3 CD7 CD2 CD5

g

X

d

X

a0b0

cCD3 CD7 CD2 CD5 CD1

g

X

d

X

a

X

b

X cCD3 CD7 CD2 CD5 CD4 CD8 Naïve T cell Immunocyte Immunoblast

g a

X R

d b

sCD3 CD7 CD2 CD5 CD4 X R cells Memory T cells Effector T

Classification of T-ALL (EGIL)

T-I (pro-T) T-II (pre-T) T-III (cortical T) T-IV (mature T) cCD3 + + + + CD7 + + + + CD2 CD5 CD8 + + + CD1a+ sCD3+/ CD1a + + -

Lymphoproliferative disorders

MARGINAL ZONE NODAL MALT

HCL

T LYMPHOMAS

SEZARY)

LARGE CELLS ANAPLASTIC PERIPHERAL ANGIOCENTRIC INTESTINAL MANTLE ZONE BURKITT INTESTINAL OR LUNG MALT Follicular, mantle zone, CENTROBLASTIC or marginal zone IMMUNOBLASTIC SLVL Large cell Lymphomas Circulating marginal zone cells FOLLICULAR WALDENSTROM MYELOMA

Matutes’ scoring of CLL

CD5 CD23 sIg FMC7 CD22/CD79b POSITIVE 1 1 0 0 0 NEGATIVE OR WEAK 1 1 0 0 1

Comparative immunophenotypes

SMZL/SLVL HCL HCLv CLL MCL CD19 CD22 CD5 CD23 sIg CD43 FMC7 CD10 CD103 CD11c CD25 CD79b CD20 IgM IgM, IgG..

IgG FL

Immunophenotype of myeloid cells

• Known maturation sequence • Massive production of cells with no specificity • Early and late differentiation markers • Important lineage promiscuity

MPO CD14 L L L L L L L L L L P

Myeloid lineage markers

CD11b CD117 Mg Mg Mg CD35 CD13 COOH E E CD33 NH2 CD36 K CD15 CD65 3-fucosyl-N-acetyl-lactosamine céramide dodecasaccharide

BONE MARRROW CD34 DR Stem cell CD117 CD13 CD33 MPO CD7 PERIPHERAL BLOOD TISSUES Immature precursors DR CD4 CD19 CD11B CD14 CD36 CD15 CD65 CD16 CD32 CD64 Differentiated cells

Evolution immunohénotypique au cours de la maturation granuleuse (B Husson) CFU-GM Myeloblast HLA-DR Promyelocyte Myelocyte Metamyelocyte Band CD15 Granulocyte CD66 CD11b CD16 CD117 CD34 CD33 CD13 CD13 CD11c CD11c CD33 CD15 CD11b CD66 CD16

Detection of BAL : EGIL’scoring system

• NOT to be used for lineage assignment • Scoring based on the lineage specificity of critical differentiation antigens • Calculation of each lineage “score” • In BAL, at least two lineages have scores HIGHER than 2 • In “variant” AL coexpression with a score <2 is not rare

Detection of BAL : EGIL ’scoring system

2 points 1 point 0.5 point B LINEAGE CD79 cµ cCD22 CD19 CD10 CD20 TdT CD24 T-LINEAGE CD3 TCR MYELOID LINEAGE MPO (lysozyme) CD2 CD5 CD8 CD10 TdT CD7 CD1a CD13 CD33 CDw65 CD117 CD14 CD15 CD64

WHO’s new classification

Extensive immunophenotype AUL Acute undifferentiated leukemia MPAL Mixed Phenotype Acute leukemia With t(9;22) With t(n;11q23) NOS Others Incl NK

WHO’s new criteria of MPAL

• Myeloid lineage – MPO – Or strong monocytic engagement • NSE • CD14, CD11c, CD36, CD64, Lysozyme • B-lineage – Bright CD19 + another B marker – Low CD19 + two other B markers • T-lineage : cCD3 (strong, PE or APC)

One word on MDS

0 1 2 3

Proposition de score Wells, 2003

Working conference 2008

M Loken, A van de Loosdrecht, K Ogata, A Orfao, D Wells • Classical blasts



enumeration – DR+/11b – CD34 – CD117 • Anomalies of the CD13/CD16 pathway • Abnormal expression anormale of DR on monocytes

Stachurski, 2008

•

• • Blasts – CD34, CD117 – Abnormal CD2, CD5, CD7, CD56 – Increased CD117 Granulocytes – Deganulation – Abnormal expression of CD33, CD13, CD11b, CD16, CD15, CD64, CD10, CD14, DR Monocytes – Anomalies of CD33, CD13, CD11b, CD15, CD64, CD14, DR

In practice, for hematological malignancies

• Depending on – Previous clinical and morphological information – Sample volume – Monoclonal antibodies availability

Consensual European Panel, European LeukemiaNet (2005) For quick orientation or paucicellular samples • cCD3, MPO, cCD79a, TdT • CD7, CD2, CD10, CD19, CD22 (s or c), sIg, CD13, CD33, CD34 • CD45 for gating purposes Sublineage classification and definition of clinical entities (also with adapted gating strategy) • DR, CD1a, CD4, CD5, CD8, CD3 (m), IgM (c), CD14, CD117, CD56, CD65, CD41 or CD61, RBC marker such as glycophorin A Complementary panel of useful referenced markers

Acute leukemia Diagnosis Panel Other useful markers (>20) • • • • • • • • • • • • • • • • MPO/LF (lactoferrin) (c): (i) Identification of late neutrophil granulocyte compartment (Lactoferrin positive) (25,52,54); (ii) for refined detection/quantification of MPO+ early myeloid cells in combination with CD14 (25,49) LZ (lysozyme) (c): (i) for myeloblastic leukaemia; (ii) to discriminate pDCs and myeloid cells (53); (iii) to positively identify early monocytic cells (48) K/L : (i) on surface for clonality, (ii) in the cytoplasm for rare B-IV cases CD11b, CD11c : negative in APL (14) CD15 : for myeloblastic leukemia (42) CD16 : to discriminate mature PMNs (9) CD35/36 : for GEIL´s AML classification (11), for RBC after excluding monocytes (10) CD58 : to distinguish between normal regenerating B cells and B-cell blasts (59) CD64 : for AML (9) CD68 (c): (i) for AML (bright) and subset of B-ALL (weak) (53); (ii) for positive identification of normal pDCs (bright) (55) CD71 : for cell proliferation/activation and/or RBC (22,41) CD86 : prognostic factor in AML (34) CD99 : to differentiate between blasts and non blastic T-cells (19) CD123 : IL-3 R, for pDC and AML, some NK (24) TCR chains for T-ALL, c and/or s (50) Therapeutic targets: CD20 (40), CD52 (40) , (46), CD44 (20), uPAR(CD87)/uPACD116 (1) CD45 (39) , CD33 (31), CD123 (3), CD87

Chronic lymphoproliferative diseases.

Mandatory panel (20 Abs) • Samples : peripheral blood, bone marrow, LN suspensions… (Fine needle aspiration for primary screeening to avoid unnecessary biopsies) • Gating markers CD19, CD3, CD56* • B oriented panel gated on CD19 CD5, CD20, CD23, CD103, CD10, K, L, Ig, CD25, CD79b, CD38 • T oriented panel gated on CD3 or other T-lineage marker CD2, CD3, CD4, CD5, CD8, CD7,

Lymphoproliferative diseases: additional useful markers (< 15) • • • B lineage – CD2, CD7, CD123, FMC7, CD138, DR, CD24*, CD43 – (G, A, M, D), CD81* – Cytoplasmic : Bcl2, Zap70 (relative to internal control) T lineage – TCRs*, CD30, CD10 NK panel excluding CD19 and CD3 cells/ CD56 – CD57, CD16, CD94, perforin, granzyme B • *CD81/CD22 useful for CLLfollow-up based on dim co-expression level *V-beta panel and imunophenotype *Absence of CD24 on marginal zone and HCL Note : CD52 if alemtuzumab considered

Chemosensitivity

• Minimal residual disease • Quick assessment of response to therapy

Why look for minimal residual disease?

The founders

San Miguel, 1997 Kern, 2004 Campana, 1999

MRD in ALL

MRD B-II ALL

1.5 x 10-2 CD19

MRD in AML

MRD - AML CD34

2.1 10-3

AML peripheral blast cells decrease

A D0 c CD14 B a FSC C b CD45 d CD16 g CD11b D e CD45 h 5.3% Blasts 100 CD11b f CD45 E

a CD11b D1 c CD11b D2 e CD11b D3 b 3.9% Blasts 73.7

CD11b d 1.1% Blasts 21.2

CD11b f 0.5% Blasts 9.4

CD11b g CD11b D4 h 0.2% Blasts 3.6

CD11b

Blast slope 100 90 80 70 60 50 40 30 20 10 0 0 Slope < -25 Slope -25 < > -15 Slope > -15 200 400 600 800 Time (Days) 1000 1200 1400

Conclusions

• Consensual approach • Rapid and informative diagnostic tool • Precise definition of the disease at diagnosis • Therapeutic indications • Aberrant immunophenotypes useful for follow up • Search for new markers: – New monoclonals – Microarrays