Aggiornamento in Ematologia Clinica Diagnosi differenziale dei processi

Aggiornamento in Ematologia Clinica Diagnosi differenziale dei processi linfoproliferativi leucemizzati Catania, 6 -7 novembre 2008

1. FCM studies -> diagnosis of lymphoid neoplasms through the identification of phenotypically abnormal cells belonging to the lymphoid lineage and recognition of phenotypes characteristic of separate disease entities. 2. FCM can be used to identify expression of targets for potential antibody-directed therapy. 3. FCM can provide prognostic information such as CD 38, ZAP-70 and CD 49 d expression in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). 4. Following therapy, flow cytometry is becoming an established method for the evaluation of minimal residual disease.

BLOOD FCM LYMPHOMA TYPING BONE MARROW LYMPH NODES

Natural Abs and Ig. A B 1 cell Marginal zone B cell Germinal centre Ig. M Proliferation and mutation memory B cell apoptotic cell Follicular B cell Selection and CSR Ig. M week 1 Ig. G Ig. A Ig. E week 2 Survival in homing tissues week 3

The wrong approach to FCM characterization of NHL CD 5+ CD 23+ CD 10 CD 5+ CD 23 - CD 10 CD 5 CD 10+ CD 5 - CD 10 - CLL ? MCL ? FL ? MZL ? This simplistic (and misleading) view of NHL typing is one of the main reasons for the unsufficient scientific communication between histo-pathologists and FCMetrists

CD 5+CD 10 CLL MCL CD 5 -CD 10+ PLL MZL DLBCL LPL CD 11 c+ CD 103± CD 25+ CD 138+ FL BL DLBCL CD 11 c+ CD 103+ CD 25+ CD 22++ HCL c. Ig+ s. Ig. CD 138+ CD 5 -CD 10 FL MZL DLBCL HCL MCL CD 23 CD 43+ Bcl-1 CD 5+CD 10+ FL BL DLBCL MCL CD 23 CD 43+ Bcl-1

Reagents of clinical utility in mature B-lymphoid neoplasms (from Craig & Foon, 2008, modified) CD 5 CD 10 CD 19 CD 20 CD 45 k and l CD 9 CD 11 c CD 15 � � CD 22 CD 23 CD 25 CD 13 CD 34 CD 38 CD 43 CD 58 � CD 79 a/b CD 103 FMC-7 CD 138 � bcl-2 k and l (cy) Zap-70 Td. T c. Ig. M

NOVEL RISK STRATIFICATION: NON-MUTATED VERSUS MUTATED CLL LDT<12 LDT>12 CD 38 high Zap-70 high CD 38 low NM M CD 49 dhigh CD 69 p 53 defects poor risk FISH (17 p-; 11 q-) Zap-70 low CD 49 dlow CD 71 good risk FISH (13 q-) normal p 53

1. A type of phenotypic aberrancy is abnormal expression of antigens (eg, bcl-2 expression on CD 10 B cells). 2. Normal germinal center B cells and hematogones are both CD 10+ and bcl-2 -, whereas bcl-2 is expressed by most other B-cell subsets. 3. Abnormally increased bcl-2 expression can be found in most FL, some diffuse large B-cell lymphoma (DLBCL), and some B-lineage ALL. 4. In contrast, Burkitt lymphoma (BL) is usually CD 10+ and bcl-2 -.

FL (bone marrow) bcl-2/CD 3/CD 45/CD 19 B-CELLS T-CELLS PMN 10 0 BCL 2 -> 10 1 10 2 10 3 10 4

Differential diagnosis between FL and regenerative BM

Identification of composite lymphomas

Structure and expression of CD 200 Rosenblum M, Yancey K, Olasz E, Truitt R. J Dermatol Sci 2006; 41: 165 -174

Function of anti-CD 200 Treg CD 200+ Tumor Cell TH 2 TH 1

CD 200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell chronic lymphocytic leukemia • CD 200 is OX 2 • Membrane gp, Ig superfamily, T and B • Immunosuppressive/ tolerogenic + - CLL 84 0 MCL 3 5 • Myeloma and B-CLL • Cancer stem cells Palumbo GA, Catania

Expression of CD 200 in LPD and MM L. Brunetti, ASH 2008

Expression of CD 200 in HCL hairy Consistently expressed with high fluorescence intensity

Significance of small populations of phenotypically abnormal B cells. 1. In the staging of patients with NHL, FCM identification of a small population of abnormal cells can be used to determine the presence of involvement by the neoplasm (particularly if the phenotype matches that of the original diagnostic specimen). 2. In patients without a previous diagnosis of NHL, the significance of a small population of abnormal B cells (less than 5% of the total cells analyzed) is less clear. 3. Small populations of B cells with abnormal phenotypes have been reported in peripheral blood and bone marrow specimens, and are not necessarily associated with a diagnosable neoplasm. 4. Therefore, if a small population of phenotypically abnormal B cells is identified in a patient with no previous diagnosis of a lymphoid neoplasm, it should not be used to establish a new diagnosis of malignancy, but correlated with the morphology, clinical information, and other findings.

A case of MZL

Clinical role of flow cytometry in redefining bone marrow involvement in diffuse large B-cell lymphoma – a new perspective p=0. 173 p=0. 026 Talaulikar D, Histopathology 2008

% pos cells 22 Hyper. CVAD M 18 M 0. 20 M 0. 10 M ABMT 0 Jan 9 Apr 3 Apr 28 Jun 1 Sep 1

Flow cytometry and morphology on the same cell Abnormal localization of selected David Basiji Antigens

Centro di Citometria Clinica e Sperimentale M. Fiammetta Romano Giulia Scalia Rita Bisogni Giovanna Abate Marisa Gorrese Maddalena Raia Ceterina Pascariello Marica Gemei Angela Gravetti Annalisa Di Santi Lorenzo Brunetti