When is morphology mandatory in hematological practice?

François MULLIER Bernard CHATELAIN BHS Belgian Society for Analytical Cytology La Hulpe, Brussels, 30 January 2015 Hematopoietic Maturation

• Morphological vision – Differentiated steps – Nomenclature • Immunophenotypic vision – Maturation continuum – Different nomenclature Multipotential stem cell

Lymphoid progenitor Myeloid progenitor

Lymphoblast Megacaryoblast

Eosinophilic Neutrophilic Basophilic Prolymphocyte Promegacaryocyte promyelocyte promyelocyte Basophilic erythroblast Bone Eosinophilic Neutrophilic Basoophilic myelocyte myelocyte Polychromatic erythroblast

Eosinophilic Neutrophilic Basophilic Megacaryocyte metamyelocyte metamyelocyte metamyelocyte Acidophil erythroblast Eosinophilic Neutrophilic Basophilic Band cell Band cell

Reticulocyte Reticulated Thrombocytes

B Lymphocyte T Lymphocyte Basophile Erythrocyte Thrombocyte

Plasma cell NK cell

Dendritic cell Tissue Lymphoid progenitors Myeloid progenitors CD2 CD7 CD34 DR T lineage NK B lineage Monocytic CD117 lineage DR CD34 CD117 Proerythroblast Megacaryoblast CD33 CD13 CD34 DR CD13 CD19 CD33 CD10 CD38 Bone Marrow Bone

CD33 CD36 CD33 CD13 CD71 CD13 CD235 CD36

CD64 CD41 CD42 CD61

T lymphocytes NK Blymphocytes Erythrocytes Platetets

CD294 CD2 CD2 CD19 DR CD13 CD294 Blood CD41 CD5 CD16 CD22 CD13 CD10 CD123 groups CD2 CD7 CD56 CD24 CD33 CD24 CD61 CD3 CD20 CD38 CD11b CD21 CD36 CD16 Blood CD4:CD8 CD11b CD11b CD15 DR CD11c CD65 sIg CD14 CD64

Dendritic cells Macrophages Llin- DR CD33 CD68 CD1 CD11c Tissues CD123 CD14 Bone marrow and flow in the literature Numerous and non systematic patterns (pattern recognition VS operator independent analysis software)

B.Wood Fluorescence activated Cell Sorting

+ -

JF Classen, ESCCA, luxembourg 2013 Myeloid maturation CD16

CD11B Myeloid maturation CD16

CD11B Myeloid maturation CD16

CD11B CD64+dim CD14-

CD64+ dim /CD14-/+

JF Classen, ESCCA, luxembourg 2013 A.M.L. (mut NPM1) Morphology, IHC, FCM, Imaging Flow Cytometry

NPM (AP)

Mut

NPM-DRAQ5

WT

Kathy Heel, Blood Reviews 27 (2013) 193-207 A.M.L. t(15;17)

Kathy Heel, Blood Reviews 27 (2013) 193-207 Sample preparation: recommendations from the European Leukemia Net

• Morphology should be checked on the sample that will be immunophenotyped (integrity of the cells and absence of selection) Acute Leukemia

• Usefullness of phase microscopy • Cytochemistry is mandatory – Peroxidase – Esterases – (Acid phosphatase, Perls, PAS) Phase Contrast microscopy CYTOCHEMISTRY MPO: assignment of myeloid lineage

Threshold: 28% (residual lymphocytes)

Sens: 97% Spec: 96%

Julien Guy, British Journal of Haematology, 2013, 161, 551–555 MPO ALL / AML M0

ALL

AML M0 MPO

AML MO (AML M1 cytochem.)

AML M3

AML M3

AML M2 Complex Caryotype MDS MDS DIAGNOSIS European LeukemiaNet MDS DIAGNOSIS European LeukemiaNet Is flow cytometry needed in the diagnosis of MDS?

NO

Morphology is still the cornerstone of diagnosis !

Jan Philippé, ABCA Luxembourg, 2010 WHO 2008 • Immunophenotyping has focused on: 1. Determining the size of the blast population 2. Immunophenotype of the blast population 3. Assessment of the maturation pattern of the lineages

• Discordancies have been noticed: 1. Enumeration of blasts, due to hemodilution or presence of myelofibrosis 2. Aberrant immunophenotypes of blasts is of limited value

Jan Philippé, ABCA Luxembourg, 2010 Peripheral Blood contamination

Morpho FCM % CD34+ % % immature cells immature %

Bone Marrow purity (%) Bone Marrow purity (%)

R. A. Brooimans, Cytometry Part B (Clinical Cytometry) 76B:18–26 (2009) WHO 2008 3. Aberrant maturation patterns – (FC) erytroid abnormalities can predict morphological erythroid dysplasia in 98% – (FC) granulocytic abnormalities can predict morphological dysplasia in 90% – FC is of limited value ~

– However, in cases with borderline dysplasia by morphology w/o cytogenetic abnormalities, FC results are highly suggestive for MDS only if 3 or more aberrant features are found in erythroid, granulocytic or monocytic maturation (single aberrant features by FC are NOT significant).

Blood 2009, Vardiman et al.

Minimal morphological criteria for defining bone marrow dysplasia

MG.Della Porta,Leukemia (2015) 29, 66–75 Minimal morphological criteria for defining bone marrow dysplasia

In myeloid disorders with marrow hypoplasia, granulocytic and/or megakaryocytic dysplasia, increased CD34. progenitors and chromosomal abnormalities are consistent with a diagnosis of MDS.

MG.Della Porta,Leukemia (2015) 29, 66–75 Minimal morphological criteria for defining bone marrow dysplasia

The proposed morphological score may be useful to evaluate the presence of dysplasia in cases without a clearly objective myelodysplastic phenotype. The integration of cytological and histological parameters improves the identification of MDS cases among myeloid disorders with fibrosis and hypocellularity.

MG.Della Porta,Leukemia (2015) 29, 66–75 Partial loss of erythroid cells may influence the blast cell percentage

Monocytes

Basophilic and polychromatic Erythroblasts Percoll

Pyknotic Erythroblast CD36

Poly,Pykno Erythroblasts and Erythrocytes CD36

CD235a

NH 4Cl CD235a 2008 WHO criteria for plasma cell myeloma

• Symptomatic myeloma - M-protein in serum or urine - Bone marrow clonal plasma cells or plasmacytoma - Related end-organ damage (hypercalcemia, renal insufficiency, anemia, bone lesions)

• Asymptomatic myeloma - M-protein in serum >=3g/dl and/or - >10% clonal plasma cells in bone marrow - No related end-organ damage

Flow cytometry

Morphology Other limitation: Quantification: comparison between morphology and flow cytometry

• Quantification: Morphology > FCM (Paiva et al. Haematologica 2009/ Rawtron et al. Haematologica 2008) - Heterogenous distribution of plasmocytes in BM - Aspirate sample sent for flow cytometry: often a second-draw bone marrow aspirate - Contamination by peripheral blood 1ml= pure BM (Batinic BMT 1990) - Selection of area riched in plasmocytes by the cytologist - Adhesion to lipids (Nadav et al. BJH 2006) - Quantification on biopsy: probably more accurate but standardization needed - Weak plasmocyte  * Loss in the needle? * Loss during RBC lysis (Smock et al. APLM 2007): * Lowered CD138 expression by lysis (Smock et al. APLM 2007) * Loss during centrifugations (Smock et al. APLM 2007) C.M.L. CML acceleration and blast crisis

Baccarani et al.,BLOOD, 8 AUGUST 2013 VOLUME 122, NUMBER 6 Systemic Mastocytosis Mastocytosis: Toluidin blue Mastocytes Mastocytosis: FCM

Mastocytes (15%) : CD117+ CD25+ CD2+(fd) CD34+(fd) CD45+ CD13+ CD33- Lymphomas

Diffuse Large B Cell lymphoma (lymph node) Diffuse Large B Cell lymphoma (lymph node) Lymph node dissociation

Personal information from F.Preiers Pitfalls of FCM on Lymph Nodes

• MALIGNANT POPULATIONS THAT LACK THE TYPICAL LYMPHOID SCATTER PATTERN (Diffuse large B-cell lymphoma (DLBCL), anaplastic lymphoma kinase (ALK)-positive and negative ALCL, Classical Hodgkin lymphoma) • Lack of CD45 expression • Loss of B-cell lineage markers • Lack of clonal surface immunoglobulin light chain expression • Lack of TCR Vbeta • REACTIVE POPULATIONS THAT SHOW CLONAL EXPANSIONS

K.Hebeda, F.Preiers, Int. Jnl. Lab. Hem 2014 T Anaplastic (buffy coat)

CD 30 CLL (Matutes 5) ….Richter CLL (Matutes 5) ….Richter Future? Raman Microscopy

Using biophotonics techniquesto retrieve prognostic intracellular signatures IRBM 32 (2011) 72–75 J. Klossa a,, S. Daliphard b, X. Troussard c, P. Vielh d, M. Manfait e, J. Angulo f, G. Flandrin a, A. Beljebbar e, V. Untereiner e, T. Happillon e, C. Gobinet e, S. Velasco-Forero f, S. Roux a, V. Saada d, R. Dagiral g, V. Coomans g, E. Froigneux h, P. Rideau i, A. Vievard a a TRIBVN, 39, rue Louveau, 92320 Châtillon, France Conclusion

• Morphology is still very often mandatory in order to establish correct Hematological diagnosis.

• Link between morphology and other techniques (F.C.M., cytogenetic and molecular biology) will allow to define a more appropriate use of these ones.

• New techniques are under clinical evaluation to improve the cost-effectiveness of the diagnosis, staging and prognosis in hematological malignancies. THANKS – Nicolas BAILLY – Alex DROMELET – Nicolas NEYMAN