Lymphocytes from patients with B-chronic lymphocytic leukemia (B-CLL) express large numbers of P2X7 receptors for extracellular adenosine triphosphate (ATP). Activation of P2X7 receptors induces multiple downstream effects, of which the best documented is the opening of an ionic channel that is selective for divalent cations. Another effect of ATP is to induce the shedding of L-selectin (CD62L), a molecule which is involved in the adhesive interactions of lymphocytes on endothelial cells. High levels of soluble L-selectin and CD23 are found in the serum of patients with B-CLL, although the mechanisms involved in their production are poorly characterized. Because extracellular ATP causes shedding of L-selectin, we studied the effect of ATP on shedding of CD23, an adhesion molecule expressed on the surface of B-CLL lymphocytes. ATP induced the shedding of CD23 at an initial rate of 12% of that for L-selectin, while the EC50 of ATP (35 uM) and BzATP (10 uM) was identical for shedding of both molecules. Inactivation of the P2X7 receptor by pre-incubation with OxATP, an irreversible inhibitor of P2X7 purinoceptor, abolished ATP-induced shedding of both molecules. Moreover, KN-62, the most potent inhibitor for the P2X7 receptor inhibited ATP-induced shedding of both CD23 and L-selectin with the same IC50 (12 nM). Ro 31-9790, a membrane permeant zinc chelator which inhibits the phorbol-ester stimulated shedding of L-selectin also inhibited shedding of CD23 from B-CLL lymphocytes, but the IC50 was different for the two shed molecules (25 versus 1 ug/ml respectively). Although L-selectin was completely shed by incubation of cells with phorbol-ester no CD23 was lost under these conditions. Also, Ca2+ inhibits ATP-induced CD23 shedding but not L-selectin shedding. Since soluble CD23 and L-selectin are found in the serum of normal subjects and B-CLL patients, the expression of these two adhesion molecules on lymphocytes before and after transendothelial migration was studied in an in vitro model of this process. In normal and B-CLL subjects, 71�b5% of L-selectin from both T and B cells and 90% of CD23 from B cells was lost following transmigration, while the expression of a range of other adhesion molecules such as VLA-4, ICAM-1, LFA-1 and CD44 was unchanged. Lymphocytes incubated with OxATP retained their capacity for transendothelial migration and showed the same loss of L-selectin as control leukaemic lymphocytes. Ro 31-9790, which can protect ATP-induced both L-selectin and CD23 shedding, had no effect on inhibiting L-selectin and CD23 lost during transmigration. These data show the presence of a second pathway for the downregulation of L-selectin and CD23 from the lymphocyte surface. Data in vivo from 'knock-out' mice show that L-selectin is essential for the emigration of lymphocytes through high endothelial venules into lymph nodes. The migration of normal and B-CLL lymphocytes across confluent human umbilical vein endothelial monolayers was studied in an in vitro model of this process. Lymphocytes treated with ATP or BzATP showed 56�b25% or 67�b16% loss of L-selectin on the surface and 36�b24% or 64�b19% decrease of transmigration, respectively, while OxATP, which does not alter the L-selectin level, had no effect on lymphocyte transmigration. Further experiments examined this correlation between L-selectin expression and lymphocyte transendothelial migration in this model system. A quantitative assay for cell surface L-selectin showed that expression of L-selectin was lower on B-CLL lymphocytes (8,880�b5,700 molecules/cell) than on normal lymphocytes (29,500�b7,500 molecules/cell, p less than 0.001). Also the rate of transmigration of B-CLL lymphocytes (1.5�b0.9 migrated cells/HUVEC) was lower than normal peripheral lymphocytes (2.4�b0.9 migrated cells/HUVEC, p=0.04). Incubation of lymphocytes in complete medium for 24 hrs increased the expression of L-selectin on B-CLL lymphocytes by 1.5 to 2 fold while the normal lymphocyte L-selectin remained at the initial level. This upregulation of B-CLL L-selectin correlated with a 2 fold increased rate of transendothelial migration. A correlation was found between L-selectin expression on lymphocytes and their ability for transendothelial migration (r^2=0.6). This study shows that the adhesion molecules L-selectin and CD23 can be lost from lymphocytes by two different physiological pathways. One is by P2X7 receptor activation by extracellular ATP while the second is activated by transendothelial migration of these cells. A second finding is that B-CLL lymphocytes have lower level of L-selectin expression and an impaired ability for transendothelial migration compared with normal peripheral blood lymphocytes. Do these results explain the high serum levels of soluble L-selectin and CD23 observed in B-CLL? Although B-CLL lymphocytes do not recirculate as rapidly as normal peripheral blood lymphocytes, the greatly increased number of leukaemic cells in B-CLL ensures that much more soluble L-selectin and CD23 is generated during the recirculation of these cells through the body.

Malignant lymphoproliferative disorders include highly heterogeneous entities, i.e. lymphomas (Non-Hodgkin - NHL, as well Hodgkin's lymphoma), lymphoid leukemias, multiple myeloma and others. As currently many chromosomal aberrations with diagnostic and prognostic significance are known, molecular cytogenetic analyses of tumor cell genome has become a substantial examination also in lymphoproliferative disorders. This thesis focuses primarily on chronic lymphocytic leukemia (CLL), which is one of the mature B-cell neoplasms and represents the most common type of leukemia. We analyzed four most frequently found aberrations (13q14 deletion, ATM and TP53 gene deletion, and trisomy 12) by fluorescence in situ hybridization (FISH) and also IgH gene aberrations in some patients. We compared the findings with other factors and clinical characteristics. This work shows that the conventional G-banding is analysis relatively little relevant. FISH was more effective in detecting aberrations in CLL. Although none of the four aforementioned changes is specific to CLL, the prognostic impact is significant, particularly that of TP53 deletion. Next, detection of some IgH gene translocations is essential in differential diagnosis of CLL and other NHL (follicular, mantle cell, diffuse large B cell, Burkitt's...

Malignant lymphoproliferative disorders include highly heterogeneous entities, i.e. lymphomas (Non-Hodgkin - NHL, as well Hodgkin's lymphoma), lymphoid leukemias, multiple myeloma and others. As currently many chromosomal aberrations with diagnostic and prognostic significance are known, molecular cytogenetic analyses of tumor cell genome has become a substantial examination also in lymphoproliferative disorders. This thesis focuses primarily on chronic lymphocytic leukemia (CLL), which is one of the mature B-cell neoplasms and represents the most common type of leukemia. We analyzed four most frequently found aberrations (13q14 deletion, ATM and TP53 gene deletion, and trisomy 12) by fluorescence in situ hybridization (FISH) and also IgH gene aberrations in some patients. We compared the findings with other factors and clinical characteristics. This work shows that the conventional G-banding is analysis relatively little relevant. FISH was more effective in detecting aberrations in CLL. Although none of the four aforementioned changes is specific to CLL, the prognostic impact is significant, particularly that of TP53 deletion. Next, detection of some IgH gene translocations is essential in differential diagnosis of CLL and other NHL (follicular, mantle cell, diffuse large B cell, Burkitt's...

Diagnosis of two distinct malignant entities existing concurrently and at the same location (synchronous malignancy) by fine-needle aspiration (FNA) is unusual but may occur. Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) in particular is associated with an increased incidence of secondary tumor, likely due to associated immunodeficiency. Co-occurrence of some carcinomas such as squamous cell carcinoma (SCC), may show especially aggressive behavior. A 57-year-old Caucasian male presented with recurrent upper extremity lymphedema and diffuse lymphadenopathy ofthe axillary and cervical regions. FNA ofa large cervical lymph node was diagnostic for both atypical lymphocytic proliferation and SCC. Flow cytometric analysis showed the atypical lymphocytic proliferation to be positive for CD5, CD23, CD19, CD20, HLA-DR, CD38, and the population was kappa light chain restricted. These cells were negative for CD-10 and FMC-7 antigens, suggesting a phenotype of B-cell SLL/CLL. We report a rare occurrence of metastatic SCC to a lymph node infiltrated by SLL/CLL. The diagnosis was achieved by a combination of cytomorphologic examination of FNA smears, immunohistochemical staining of cell block material, and flow cytometry on the sample obtained by FNA. To the best of our knowledge, only three cases of SCC metastasis to SLL/CLL diagnosed by FNA have been reported in the English literature. Though rare, awareness of such a possibility and careful cytological examination under the appropriate clinical conditions is warranted.

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