Leukemia (1997) 11, 408–415  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

Adhesion receptors on peripheral blood leukemic B cells. A comparative study on chronic lymphocytic and related / G Csanaky1, E Matutes2, JA Vass1, R Morilla2 and D Catovsky2

1Department of Pathology, University Medical School of Pe´cs, Hungary; and 2Academic Department of Haematology and Cytogenetics, The Royal Marsden Hospital, London, UK

The expression of a series of adhesion receptors: L- the same disease, or change during the clinical course of the (CD62L): Leu-8, several integrins (LFA-1: CD11a/CD18, VLA-4: disease. For example, in chronic lymphocytic leukemia (CLL), CD49d/CD29 and VLA-5: CD49e/CD29), ICAM-1(CD54) and the the most common of the B lymphoid disorders, clinical staging ‘homing ’ (CD44) were investigated by a dual color flow 4,5 cytometry in 56 cases of B cell disorders namely, 39 chronic systems not only consider the degree of anemia and lymphocytic leukemias (CLL), four (HCL), thrombocytopenia, but also splenic and nodal infiltration as seven splenic lymphoma with villous (SLVL) and well as the number of nodal regions involved. Similarly, six other non-Hodgkin’s lymphoma (NHL). The functional dissemination of B-NHL may correlate with bulky disease activity of L-selectins was assessed with L- ligand ana- and resistance to standard therapy. logs (polyphosphomonester core polysaccharide: PPME and fucoidin). Leukemic B cells were identified with phycoerythrin- Since these patterns have proven to be of clinical relevance, conjugated monoclonal (McAbs) anti-CD19, anti- it seems important to investigate the factors which may play kappa/lambda investigated simultaneously for the expression a role in the dissemination and tissue distribution in these of adhesion receptors estimated with fluorescein-isothiocyan- malignancies. In this context, analysis of interactions between ate (FITC) conjugated McAbs. The percentage of leukemic cells certain receptor and ligand molecules,6,7 which result in the expressing L-selectins (Leu-8) was high in CLL (52% of positive binding of lymphocytes to the endothelial cells and give them cases) and integrin expression (LFA-1, VLA-4, 5) was low (19 and 33%, respectively), while a reverse pattern, low Leu-8 the opportunity to leave the blood via the high endothelial (17%), and a high VLA-4 (77%), was observed in non-CLL cases. venules (HEVs) and enter into the secondary lymphoid organs, The expression of LFA-1 ␣-chain was variable in non-CLL may give some clues in the patterns of dissemination. Thus, cases, and the LFA-1 heterodimer was expressed on most the attachment of leukemic cells to the endothelium rep- clonal B cell in NHLs (92%). LFA-1 ␣-chain was detected on resents a crucial step in the spreading of such malignancies. ␤ cells from only one HCL case, while 2 integrin was regularly Over the last few years, there have been a number of reports expressed on hairy cells. VLA-5 integrin was found on a rela- tively small number (26%) of mature B cell leukemias. A remark- on the expression and function of cell-adhesion molecules on able finding was the detection of ICAM-1 in all CLL cases albeit leukemic B cells, however, only few studies have focused on the number of positive cells was significantly lower (P Ͻ 0.05) the correlation between the expression of these molecules and compared to non-CLL cases. CD44 was expressed on a high the clinical and laboratory findings.8–16 number of neoplastic cells in all the investigated categories. In this study, we have analyzed circulating neoplastic cells There was no correlation between the expression of the from a variety of B cell diseases, namely CLL and hairy cell adhesion molecules and clinical and laboratory parameters except for CD18 which was expressed on a significantly leukemia (HCL) and B-NHL in leukemic phase for the (P Ͻ 0.05) higher number of leukemic cells in CLL with more expression of a number of adhesion molecules which advanced stages. This study demonstrates that even closely included: (1) L-selectins (CD62L) (their functional activity was related B cell leukemia/ have a certain well defined assessed by biotinylated ligand analogs); (2) some key dimeric and strictly variable adhesion profile which is characteristic of integrins such as LFA-1 (leucocyte function associated the disease entity and therefore, the adhesion profile may offer antigen-1: CD11a/CD18), VLA-4,5 (very late activated: additional information useful for differential diagnosis and study of disease pathogenesis. CD49d/CD29, CD49e/CD29); (3) the intercellular adhesion Keywords: B cell lymphoproliferative disorders; immunopheno- molecule-1 (ICAM-1: CD54) which belongs to the immuno- type; adhesion receptors globulin superfamily, as well as (4) the hyaluron binding pro- tein (homing receptor: CD44). Findings have been correlated to the various disease categories and clinical and laboratory Introduction features such as nodal status, pattern of bone marrow infil- tration, count and progressive vs stable disease. B cell lymphoproliferative disorders encompass a variety of disease entities which can be distinguished on clinical and laboratory features.1,2 Peripheral blood involvement is, as a Materials and methods rule, present in cases of primary leukemias and may be present in B cell non-Hodgkin’s lymphoma (B-NHL), parti- Patients and cell samples cularly in some subtypes such as splenic lymphoma of villous lymphocytes (SLVL) which almost always constantly evolve Peripheral blood cells from 56 patients with a B lymphopro- with leukemia.3 liferative disorder which included: 39 CLL; eight of which had In addition, the dissemination pattern may also differ within more than 10% circulating (CLL/PL); four HCL, three typical and one HCL-variant and 13 cases of B cell NHL in leukemic phase (Ͼ5 × 109/l lymphoma cells) were analyzed. The diagnosis was based on clinical, morphological Correspondence: G Csanaky, Department of Pathology, Marku- sovszky Teaching Hospital, Szombathely, Markusovszky u.3, Hun- and immunological features according to the French–Amer- gary, H-9700 ican–British (FAB) Cooperative Group proposals.1 Received 18 October 1996; accepted 27 November 1996 The clinical data were obtained from the patient files of the Adhesion receptors in mature B cell leukemia/lymphomas G Csanaky et al 409 Royal Marsden Hospital, and a questionnaire was sent to Preparation of biotinylated L-selectin ligand analogs physicians of outside hospitals to collect the relevant clinical (b-PPME and b-fucoidin) data. CLL patients were staged according to Binet and Rai.4,5 The 39 CLL patients included 18 males and 21 females (M/F Amino-derivatives of L-selectin ligand17 analogs, polyphos- ratio: 18/21) with a median age of 64 years (range: 41–91); phomonester (PMME)18,19 and fucoidin20 were first produced the four HCLs were all males with a median age: 50 years as described elsewhere,21 then these derivatives were biotinyl- (range: 42–56); and the B-NHL included seven SLVL (M/F: 4/3, ated with the use of biotinyl-N-hydroxysuccinimide ester median age: 66 years, range: 40–74) and six patients with (BNHS) similarly to some biologically active , such as 22 B-NHL which included four , one follicular and antibodies, lectins or polypeptide hormones. one lymphoplasmocytic lymphoma (M/F: 1, median age: 63 Briefly, commercial fucoidin (Sigma, St Louis, MO, USA) years, range: 53–81). was treated with a protease, then dialyzed against running and Nodal involvement (number and site of the involved lymph distilled water. The final product of the dialysis was run to node areas) was graded as follows: G1: no lymph node Sepharose 4B gel filtration, and a high molecular-weight enlargement; G2: one node, or one group of lymph nodes in carbohydrate peak was collected. a localized area; G3: involvement of two or more sites con- The PPME preparation started by a mild acid hydrolysis of H. holstii phosphomannan, then saturated by barium hydrox- tiguous along pathways of lymphatic drainage and limited to ide, and the barium salt of high molecular weight PPME was one side of diaphragm (eg neck and axillae); G4: two or more precipitated by ethanol. After the removal of barium by a cat- noncontiguous sites of involvement on both sides of dia- ion exchange resin, a sodium salt was obtained. The acid phragm; and G5: generalized dissemination (bone marrow, hydrolysis resulted in a pentasaccharide phosphonoester spleen and liver involvement were not considered). (PENT) which was also isolated from the supernatant of bar- The histological patterns of bone marrow infiltration were ium precipitation. Prior reduction with sodium borohydride recorded as nodular, interstitial, mixed (nodular and and mild periodate oxidation, PPME and fucoidin were interstitial) and diffuse. The degree of was derivatized with hexanediamine. × 9 regarded as high when there were over 50 10 /l circulating The b-PPME and b-fucoidin were produced as follows: lymphocytes; lymphocyte doubling time (LDT) less than 12 20 mg of the amino-derivatives of PPME/fucoidin were dis- months was considered a bad prognostic indicator; anemia solved in 550 ␮l phosphate-buffered saline (PBS), and 10 mg Ͻ Ͻ × 9 (Hb 11 g/dl) and thrombocytopenia (platelets 100 10 /l) of BNHS in 200 ␮l dimethyl-sulfoxide (DMSO). Both solutions were also recorded. The progressive or non-progressive nature were mixed at room temperature, and the mixture was incu- of the disease was determined, considering if the patient bated overnight, then precipitated with three volumes of 95% required treatment for active disease. ethanol, and incubated again for 20 min at −20°C, decanted Peripheral blood mononuclear cells (PBMCs) were isolated and the precipitate was repeatedly washed with ethanol, cen- by Ficoll–Hypaque gradient centrifugation from samples trifuged and dried. referred for immunophenotyping to The Royal Marsden Hos- pital. Cryopreserved cells were used in eight cases (five SLVL, two HCL and one B-NHL). Cell morphology was reviewed in Specificity of L-selectin ligand analogs all cases and all were investigated with a panel of B and markers on a FACScan flow cytometer (Becton Dickinson, The specificity of L-selectin ligand analogs was tested by pre- Mountain View, CA, USA). Assays were performed using the incubation of CLL cells with LECAM-1, and by saturating the following McAbs: RFT11 (Prof G Ja´nossy, Royal Free Hospital, binding sites with unconjugated analogs (250 ␮g/ml PPME London, UK) for CD2, Leu-3a for CD4, Leu-2a for CD8, Leu- and fucoidin, 30 min, 4°C). Binding of b-PPME was reduced M5 for CD11c, Leu-12 (PE: phycoerythrin conjugated) for with −79 ± 8% (LECAM-1) and −51 ± 21% (PPME). Preincu- CD19, anti-IL-2 for CD25 (all supplied by Becton Dickinson, bation with LECAM-1 and with non-conjugated fucoidin also Mountain View, CA, USA), UCTH-2 for CD5 (Prof P Beverley, resulted in a decrease of b-fucoidin binding (−24 ± 30% and − ± University College, London, UK), J5 for CD10 (Coulter, Hia- 90 6%, respectively). leah, FL, USA), OKB22 for CD22 (Ortho, Raritan, NJ, USA), MHM6 for CD23 (Prof D Crawford, ICR, London, UK), L30 for CD24 (Dr K Kikuchi, Sapporo Medical University School Immunofluorescence analysis of Medicine, Sapporo, Japan), WR17 for CD37 (Prof JL Smith, Southampton University Hospitals, Southampton, UK), GRB1 In order to detect the adhesion profile specifically on clonal B for HLA-DR (Prof F Garrido, General Hospital of Baza, Gran- cells a double labelling with phycoerythrin conjugated CD19 McAb (Becton Dickinson) was applied in CLL cases, while in ada, Spain), FMC7 (Sera-Lab, Crawley Down, Sussex, UK), B- HCL and B-NHL, phycoerythrin-conjugated anti-kappa or ly-7 for CD103 (Dr S Poppema, Cross Cancer Institute, anti-lambda McAb (DAKO) were used. Some of the McAbs Edmonton, Alberta, Canada), HC2 (non-clustered; Dr D Pos- against adhesion molecules such as Leu-8 for CD62L, anti- nett, Cornell University Medical College, New York, NY, CD11a for LFA-1␣, anti-CD18 for integrin ␤2 chain (Becton USA), SN8 for CD79b (Dr BK Seon, Roswell Park Cancer Insti- Dickinson) and anti-CD29 for integrin ␤1 chain (Immunotech, tute, Buffalo, NY, USA), anti-IgM, anti-IgA, anti-IgD and anti- Marseille, France) were fluorescein-isothiocyanate (FITC) con- IgG (DAKO, Copenhagen, Denmark). In a few cases, the jugated, and a double direct immunofluorescence technique immunoalkaline phosphatase (APAAP) technique with anti- was performed. In the case of unconjugated McAbs, such as kappa and anti-lambda reagents (DAKO) was applied on cyto- anti-CD49d for VLA ␣-4, anti-CD49e for VLA ␣-5, anti-CD54 spin slides when clonality could not be established by mem- for ICAM-1 and anti-CD44 (Immunotech), before the incu- brane staining. bation with the PE-conjugated McAb (anti-CD19 and kappa/lambda) a mouse-serum (1:2000) was applied to block the free binding sites of the primary McAb layer. All the incu- Adhesion receptors in mature B cell leukemia/lymphomas G Csanaky et al 410 Table 1 Expression of adhesion molecules and b-PPME/fucoidin binding of leukemic B-cells

CLLa HCLb SLVL NHL

Leu-8 20/38 (23–81)c 1/4 (58) 1/7 (50) 1/6 (38) b-PPME 36/39 (23–80) 3/4 (41–91) 4/7 (27–61) 3/6 (28–67) b-fucoidin 35/38 (26–72) 4/4 (32–80) 5/7 (33–63) 5/6 (21–79) CD11a 9/39 (37–68) 1/4 (47) 7/7 (34–64) 5/6 (21–93) CD18 11/34 (23–84)d 4/4 (80–94) 6/6 (71–81) 5/6 (36–94) CD49d 17/39 (21–85) 3/4 (25–60) 6/7 (31–79) 5/6 (45–78) CD49e 10/36 (23–67) 1/4 (84) 1/7 (22) 2/6 (21–25) CD29 17/38 (21–92) 3/4 (37–96) 6/7 (35–71) 5/6 (37–85) CD54 39/39 (25–97)d 4/4 (25–98) 7/7 (44–88) 6/6 (68–90) CD44 38/38 (76–100) 3/3 (90–98) 7/7 (70–99) 5/5 (68–100)

Results are shown as proportion of positive cases. aIncludes eight cases of CLL/PL. bIncludes one case of HCL-V. cRange of clonal B cells expressing the antigen. dCD18 was expressed on a significantly higher number of leukemic cells in more advanced disease stages, the number of CD54 positive cells was significantly lower in CLL than in non CLL cases (P Ͻ 0.05).

bations were performed on ice. Controls were set up by but often in the other B cell diseases including HCL, SLVL and replacing the McAb with a mouse Ig of the same isotype. In NHL (15/16, 93% and 14/17, 82%, respectively) (Figures 1 case of b-PPME and b-fucoidin, an extravidin-FITC conjugate and 3). These differences were statistically significant (Sigma, St Louis, MO, USA) was applied to detect the binding (P Ͻ 0.001 for CD18 and CDw49d, P Ͻ 0.05 for CD11a and sites (control set up: no biotinylated analogs added). CD29). LFA-1 (CD11a/CD18) was highly expressed in SLVL The cut-off point to consider a positive case was when the (Figure 1), the differences also being statistically significant for adhesion molecule was expressed on over 20% of clonal B both the ␣ and ␤ chains when compared to CLL and to HCL cells. (P Ͻ 0.05) (Table 1). The VLA-5 ␣ chain (CD49e) expression was usually low in the majority of the cases regardless of the disease subtypes Statistics (10/36, 27% in CLL and 4/17, 23% in the other cases) (Table 1, Figure 3). Data were analyzed by a two-tailed t test and by the A positive correlation was found between the expression of calculation of the Kendall correlation coefficients. ␣ and ␤ chains of the LFA-1, VLA-4 and VLA-5 integrins (P Ͻ 0.05) in CLL cases.

Results Expression of the ICAM-1 (CD54) molecule and Expression and function of L-selectins on leukemic B hyaluron binding (CD44) cells All cases regardless of the disease category expressed CD54. The number of cases positive with Leu-8 was significantly However, the proportion of CD54-positive cells was very vari- higher in CLL which includes CLL/PL (20/38, 52% of cases) able and significantly lower (P Ͻ 0.01) in CLL (56 ± 17%, (Figure 1) than in other B cell disorders (3/17, 17%) (Table 1). range: 25–97%) compared to non-CLL cases (71 ± 19%, Within positive cases, the proportion of cells staining with range: 25–98%) (Figure 3). Leu-8 was 52 ± 12% (range: 23–81%) in CLL and 48 ± 10% Expression of CD44 molecule was also high in all the types (range: 38–58%) in other cases. The functional activity of L- of diseases, but with no differences among them (Table 1, selectins, the b-PPME binding, correlated well with the Leu-8 Figure 3). expression in CLL and CLL/PL (P Ͻ 0.001 and P Ͻ 0.01, respectively). Among the other B cell diseases, the L-selectin expression and its function was low, particularly, in SLVL (1/7, Adhesion profile of leukemias/lymphomas related to 14%) (Figures 1 and 2, Table 1). clinical stages and lymph node status

In CLL and CLL/PL there was no correlation between the Expression of the LFA-1 (CD11a/CD18) and VLA-4,5 expression of adhesion molecules and clinical stages: con- (CD49d,e/CD29) integrins sidering stages A vs B, A vs B + C, and according to Rai 0 vs 1, 2, 3 and 4, or 0 and 1 vs 2, 3 and 4; 0, 1 and 2 vs 3 and The expression of LFA-1 ␣ and ␤ chains and VLA-4 integrins 4, or the patterns of lymph node involvement. In contrast was variable (Table 1). Overall these integrins were expressed CD18 was expressed on a significantly higher number of CLL on a higher number of non-CLL cases as follows: CD11a was cases in stage B, or stages B and C (2/5, 40%, 39–66%, often positive in NHL and SLVL (12/13, 92%), whereas less 41 ± 19% and 4/10, 40%, 24–66%, 39 ± 19%, respectively) common in patients with primary leukemias: CLL and HCL compared to stage A (2/17, 11%, 29–48%, 38 ± 13%) (10/43, 23%) (Figure 1). CD18 and CD49d were rarely (P Ͻ 0.05). A similar correlation was found when the Rai expressed in CLL (11/34, 32% and 17/39, 43%, respectively), classification was applied comparing stages 1, 2, 3 and 4 Adhesion receptors in mature B cell leukemia/lymphomas G Csanaky et al 411

Figure 1 Adhesion profile of leukemia/lymphomas. Representative cases are depicted on dot plots. B cells are identified with the use of PE- conjugated anti-CD19 McAb, while the adhesion molecules (CD62L; CD11a, CD18 and CD29) are detected directly by FITC-conjugated McAbs. Double-labeled lymphocytes are shown in the right upper quadrants. Percentage of positive cells are given as the ratio of double-labeled lymphocytes (right upper quadrants) and CD19 positive B cells (above the horizontal bars).

(4/12, 33%, 24–66%, 39 ± 19%) vs stage 0 (2/14, 11%, 29– The most important observation of this study is the demon- 48%, 38 ± 13%) (P Ͻ 0.05). stration of a reverse expression of L-selectins (as well as func- tion: b-PPME-binding), VLA-4 integrin, and ICAM-1 in CLL vs non-CLL cases. The LFA-1 integrin showed a variable pattern Adhesion profile of leukemias/lymphomas related to and the CD44 was expressed on an equally high number of some laboratory and clinical parameters cells in all the investigated categories. We have not been able to show a correlation between the expression of these Neither of the laboratory parameters (lymphocyte count, lym- adhesion receptors and the pattern of hematogenous dissemi- phocyte doubling time, pattern of bone marrow infiltration) nation (see below). nor progressive vs non-progressive nature of the disease corre- Our results showed that half of the CLL cases expressed L- lated to expression of any adhesion molecules. selectins, while these were only expressed on small number of cells at a detectable level in the other B cell lymphoprolifer- ative disorders (HCL, SLVL, NHL) (17% of cases). A low Discussion expression of L-selectins (Leu-8) has been reported by Hanson et al,26 in 11 cases of Leu-M5 (CD11c) positive B-lymphopro- The leukemias/lymphomas studied represent neoplasias of B liferative disorder which probably included some SLVL, and cells at different stages of maturation. CLL cells reflect an anti- the authors implied that this related to the reduced capacity gen-independent prefollicular state of differentiation, while of the leukemic cells to involve lymph nodes.26 cells from SLVL, HCL and NHL are thought to be equivalent Both LFA-1 and VLA-4 were expressed on a few cells in a to a subsequent antigen-dependent differentiation.23 Although small number of CLL (19 and 33% of cases, respectively), the diagnostic criteria of mature B cell leukemias are well whereas VLA-4 (CD49d/CD29) was almost uniformly established,23–25 our findings on different pattern of L-selectin expressed on leukemic cells from non-CLL cases (HCL, SLVL and integrin expression offer additional markers for differen- and NHL) (77% of cases). These findings are in agreement tial diagnosis. with Baldini et al,27 who reported a reduced VLA-4 expression Adhesion receptors in mature B cell leukemia/lymphomas G Csanaky et al 412

Figure 2 Biotinylated polysaccharide (b-PPME, b-fucoidin) binding of leukemic cells. B cells (CD19 positive lymphocytes) bind b-PPME and b-fucoidin as it is detected by extravidin-FITC (right upper quadrants).

in CLL (global expression of VLA-4 was 37.1%), while an CD49e was only seen in a quarter of CLL and non-CLL cases. opposite reactivity was seen in NHLs (97.1%). Still, some con- The lack of detectable integrin ␤1 chains (only 17 positive out tradictory findings do exist, such as findings reported by of 38 cases) shows that integrin ␤ chain negative CLL cases Mo¨ller et al,28 with all the cases from different low grade B exist and have a prognostic relevance (see below). De Rossi cell neoplasms (CLL, HCL, MZL, centroblastic/centrocytic: et al35 were also able to detect this molecule on the leukemic CB/CC and centrocytic: CC) almost unequivocally expressing cells only in 52% of their studied CLL cases. VLA-4 ␣ chain. Notable differences were also found within The variable expression of ICAM-1 in CLL ranging from 17– the group of non-CLL cases regarding the expression of ␤2 44% in different series12,36 is in contrast with the uniform (CD18) integrins. SLVL and other NHL cases expressed LFA- ICAM-1 positivity of our CLL cases. These differences may 1 ␣ and ␤ chain (CD11a/CD18). Whilst CD11a (LFA-1 ␣ relate to the different McAbs37 and detection systems chain) was detectable on hairy cells from one case only, albeit used.12,15 Some of these studies correlated the expression of all HCL cases were CD18 (LFA-1 ␤ chain) positive. It is also ICAM-1 and serum levels of soluble ICAM-1 with the tumor remarkable that the number of positive B cells with integrin mass.14,15 The origin of soluble ICAM-1 is not clear, although ␤2 chain (CD18) correlated with the LFA-1 ␣ chain (CD11a) it is assumed that it derives from the tumor cells.14 However, expression in CLL and NHL (series which were large enough we considered all our CLL cases positive for ICAM-1 (Ͼ20% to evaluate the correlation statistically). In view of our findings of cells expressed the adhesion molecule), being the number and those reported by others,29–32 both the LFA-1 of cells stained with anti-CD54 significantly higher in non- (CD11a/CD18) and Mac-1 (CD11b/CD18) are expressed on a CLL cases than in CLL. This finding is consistent with reported low number of CLL cells, while the complete LFA-1 heterod- data that certain NHLs (follicular lymphomas, Burkitt’s imer is present on a high number of leukemic cells from SLVL lymphoma, multiple myeloma) express ICAM-1 at a high and other NHL. On hairy cells, the ␤2-heterodimer is formed level.10,38 by CD18 and CD11c (p150,95) molecules.33 VLA-5 ␣ chain In our study, CD44 was unequivocally positive in all types expression on mature leukemic B cells has been docu- of B cell diseases. Estimation of the CD44 density may be mented,27 although, according to Pinto et al34 this molecule relevant in CLL as it seems that CLL patients with intermediate is only present on small proportions of cells (25–40%) in a and high CD44 surface density have longer survival than cases low number of cases of non-CLL. In our study, mature B cell with ‘dim’ CD44 pattern.16 An interesting aspect of this issue leukemias did not express VLA-5 integrin as the expression of is the variant isoform expression of CD44. Whilst the standard Adhesion receptors in mature B cell leukemia/lymphomas G Csanaky et al 413

Figure 3 Adhesion profile of leukemia/lymphomas. This series represents adhesions molecules (CD49d, CD49e, CD54 and CD44) detected by an indirect method (anti-mouse-FITC). CD19 positive B cells expressing the detected adhesion molecules are found in the right upper quadrants. isoform is highly expressed in all lymphoid tissues and low al46 showed a correlation between a high CD18 expression grade malignant lymphomas as it was seen in our study, high and longer survival in both low and high grade B cell lym- grade malignant lymphomas express several CD44 isoforms, phomas. By contrast, De Rossi et al12,16 in a study of 72 CLL and some of them indicate a more aggressive phenotype.39 patients demonstrated that patients whose cells do not express We have also shown that the biotinylated ligand analog integrin ␤ chains (␤1−␤2−␤3−) show the most favorable clinical molecule, b-PPME – similarly to fluoresceinated analogs such features compared to the other groups. as f-PPME11,40,41 – is a suitable tool to investigate the L-selectin The difficulties to reveal the exact role and real prognostic function.42 Some studies have correlated the Leu-8 expression significance of adhesion molecules in leukemia/lymphoma and PPME-binding (L-selectin expression and function) of leu- dissemination can be explained by many facts. The regulatory kemic CLL cells with their avidity to bind lymph node HEVs, processes of adhesion molecule expression of normal lympho- and suggested that L-selectins are responsible for the cytes are probably operative on malignant cells as well. Since migration of leukemic B lymphocytes from the blood to they are under the regulatory effect of different lymphokines the lymph nodes.11,43 Stauder et al13 demonstrated that and variable chemical agents, adhesion molecules change discontinuous lymph node enlargement (hematogenous their expression dynamically on the cell surface and as a dissemination) correlates with the ability of CLL cells to bind consequence their expression and function depend on a state to HEVs in an in vitro HEV-binding assay, and that the HEV- of activation/differentiation. Even simple processes such bind- binding capacity by the leukemic cells was variable at differ- ing of lymphocytes to endothelial cells (the initial step of hem- ent clinical stages. We have not been able to confirm this,44 atogeneous dissemination) depends on a battery of receptor– as the number of CLL cells bound to HEVs and to PPME- and ligand interactions (reversible rolling, activation, activation- fucoidin-derivatized polyacrylamide gels is higher at early dependent adhesion).6,7 Moreover, clinical parameters such stages of the disease than at the later stages.45 In this study, as the number and location of enlarged lymph nodes (pattern except CD18, none of the adhesion molecules showed corre- of hematogeneous vs lymphogeneous dissemination) depend lation with the clinical stages (Binet, Rai), with pattern of dis- on many additional subsequent cell–cell and cell–matrix inter- semination (hematogeneous vs non-hematogeneous), other actions.47 These facts explain the failure to correlate the most laboratory parameters (lymphocyte count, LDT, type of bone important clinical and laboratory parameters even with a rep- marrow infiltration), or progression. resentative series of adhesion molecules involved in B lym- CD18 (␤2 chain) was expressed on a significantly higher phocyte–endothelial interaction (L-selectins, different inte- number of leukemic B cells in advanced stages of CLL com- grins, ICAM-1, CD44). On the other hand, our study has pared to early stages. In a series of 69 patients, Erikstein et shown that closely related B cell leukemia/lymphomas have Adhesion receptors in mature B cell leukemia/lymphomas G Csanaky et al 414 a certain well defined and strictly variable adhesion 15 Kimby E, Rincon J, Patarroyo M, Mellstedt H. Expression of profile which is characteristic of a particular type of adhesion molecules CD11/CD18 (Leu-Cams, beta 2-intergrins), leukemia/lymphoma. CD54 (ICAM-1) and CD58 (LFA-3) in B-chronic lymphocytic leu- kemia. Leuk Lymphoma 1994; 13: 297–306. 16 De Rossi G, Tenca C, Cerruti G, Favre A, Zarcone D, Tabilio A, Mauro FR, Annino L, Grossi CE. Adhesion molecule expression Acknowledgements on B cells from acute and chronic leukemias. Leuk Lymphoma 1994; 16: 31–36. 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