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

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. FCM can be used to identify expression of targets for potential antibody-directed therapy. FCM can provide prognostic information such as CD38, ZAP-70 and CD49d expression in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Following therapy, flow cytometry is becoming an established method for the evaluation of minimal residual disease.

FCM LYMPHOMA TYPING BLOOD BONE MARROW LYMPH NODES

Survival in homing tissues Natural Abs and IgA B1 cell Germinal centre Marginal zone B cell IgM Proliferation and mutation memory B cell apoptotic cell IgG IgA IgE Follicular B cell Selection and CSR Survival in homing tissues IgM week1 week2 week3

The wrong approach to FCM characterization of NHL CD5+ CD23+ CD10- CLL ? CD5+ CD23- CD10- MCL CD5- CD10+ FL CD5- CD10- 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

CD5+CD10- CD5-CD10+ CLL MCL PLL MZL LPL FL BL HCL CD5-CD10- CD5+CD10+ DLBCL LPL FL DLBCL BL HCL CD11c+ CD103+ CD25+ CD22++ CD11c+ CD103± CD25+ CD138+ cIg+ sIg- CD138+ CD5-CD10- CD5+CD10+ FL DLBCL HCL MCL MZL FL DLBCL BL MCL CD23- CD43+ Bcl-1 CD23- CD43+ Bcl-1

CD5 CD22 CD79a/b CD10 CD23 CD103 CD19 CD25 FMC-7 CD20 CD13 CD138 CD45 Reagents of clinical utility in mature B-lymphoid neoplasms (from Craig & Foon, 2008, modified) CD5 CD22 CD79a/b CD10 CD23 CD103 CD19 CD25 FMC-7 CD20 CD13 CD138 CD45 CD33 bcl-2 k and l CD34 k and l (cy) CD9 CD38 Zap-70 CD11c CD43 TdT CD15 CD58 cIgM    

NOVEL RISK STRATIFICATION: NON-MUTATED VERSUS MUTATED CLL LDT<12 LDT>12 CD38high CD38low Zap-70 high Zap-70 low NM M CD49dhigh CD49dlow CD69 CD71 p53 defects normal p53 poor risk FISH (17p-;11q-) good risk FISH (13q-)

A type of phenotypic aberrancy is abnormal expression of antigens (eg, bcl-2 expression on CD10 B cells). Normal germinal center B cells and hematogones are both CD10+ and bcl-2-, whereas bcl-2 is expressed by most other B-cell subsets. Abnormally increased bcl-2 expression can be found in most FL, some diffuse large B-cell lymphoma (DLBCL), and some B-lineage ALL. In contrast, Burkitt lymphoma (BL) is usually CD10+ and bcl-2-.

FL (bone marrow) bcl-2/CD3/CD45/CD19 BCL2 -> B-CELLS T-CELLS PMN 10 1 2 3 4 B-CELLS T-CELLS PMN

Differential diagnosis between FL and regenerative BM

Identification of composite lymphomas

Structure and expression of CD200 Rosenblum M, Yancey K, Olasz E, Truitt R. J Dermatol Sci 2006;41:165-174

Function of anti-CD200 Treg TH2 CD200+ Tumor Cell TH1

CD200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell chronic lymphocytic leukemia CD200 is OX2 Membrane gp, Ig superfamily, T and B Immunosuppressive/ tolerogenic Myeloma and B-CLL Cancer stem cells + - CLL 84 MCL 3 5 Palumbo GA, Catania

Expression of CD200 in LPD and MM L. Brunetti, ASH 2008

Consistently expressed with high fluorescence intensity Expression of CD200 in HCL hairy Consistently expressed with high fluorescence intensity

Significance of small populations of phenotypically abnormal B cells. 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). 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. 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. 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 HyperCVAD M Jan 9 Apr 28 Jun 1 Sep 1 ABMT Apr 3 22 18 0.20 0.10 0.20 18 22

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

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