differential expression of cell adhesion molecules in b-zone small lymphocytic lymphoma and other...

a94

Differential Expression of Cell Adhesion Molecules in B-Zone Small Lymphocytic Lymphoma and Other Well-Differentiated Lymphocytic Disorders Antonio Pinto, M.D.,*,t Antonino Carbone, M.D.,*,$ Annunziata Gloghini, B.D.,$ Giuseppe Marotta, M.D.,* Rachele Volpe, M.D.,$ and Vittorina Zagonel, M.D.*-t

Background. Cell adhesion molecules (CAM) may de- termine the patterns of growth and dissemination of lymphoproliferative disorders.

Methods. The authors have studied, by flow cytometric and immunohistochemical examination, the expression of several CAM, mediating cell-cell and cell-microenvironment interactions, on B-zone small lymphocytic lymphoma (BZSLL) and other B-cell low-grade non-Hodgkin lymphomas (NHL), including intermediate lymphocytic/mantle zone lymphoma (ILL/MZL), small lymphocytic lymphoma (SLL), and chronic lymphocytic leukemia (CLL).

Results. Relevant differences in the “adhesion phenotype” of BZSLL compared with other low-grade NHL examined were evidenced. Cells from BZSLL displayed a higher rate of expression and/or a stronger intensity of LFA-1, LFA-3, ICAM-1, and BL-CAM and a lower density of H-CAM and LAM-1 homing receptors, as opposed to SLL or CLL. A lower intensity of H-CAM along with a stronger expression of LFA-1, LFA-3, ICAM-1, and BL- CAM were also detected by comparing BZSLL with ILL/ MZL. Malignant cells from BZSLL expressed Leu-CAMb determinants in three cases. BZSLL cells lacked VLA-a5- integrins as opposed to CLL lymphocytes and displayed a stronger reactivity with anti-VLA-a4 antibodies with respect to ILL/MZL and CLL. PI-integrins were consis-

From *The Leukemia Unit, Divisions of tMedical Oncology, and $Pathology, Centro di Riferimento Oncologico, Instituto Nazionale di Ricovero e Cura a Carattere Scientific0 (I.N.R.C.C.S.), Aviano, Italy.

Supported in part by the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy, by the Minister0 della Saniti, Ricerca Finalizzata I.N.R.C.C.S., 1990-1991, Rome, Italy, and by the Consig- lio Nazionale delle Ricerche (CNR), PF-ACRO (grant no. 92.02347.PF39), Italy.

Address for reprints: Antonio Pinto, M.D., The Leukemia Unit, Centro di Riferimento Oncologico, INRCCS, Via Pedemontana Occi- dentale, Aviano 1-33081, Italy.

Accepted for publication March 17, 1993.

tently detected on BZSLL lymphocytes as opposed to ILL/ MZL.

Conclusions. These data suggest that the adhesion phenotype of BZSLL, by favoring homotypic and heterotypic adhesive interactions of tumor cells, might account at least in part for the peculiar intranodal compartmentalization leading to a deceptively benign (reactive) histologic appearance, and for the smoldering clinical course of this lymphoma. The pattern of CAM expression detected by the authors on malignant lymphocytes also is suggestive for a cellular origin of BZSLL from a rare subset of interfollicular or external mantle B-lymphocytes. Cancer 1993; 72894-904.

Key words: cell adhesion molecules, homing receptors, lymph node architecture, malignant lymphoma, B-zone lymphoma.

Non-Hodgkin lymphomas (NHL) reflect the phenotypes of normal lymphocytes subpopulations and the architecture of the various compartments of the lymph node from which they are thought to arise.’,’ It is, therefore, conceivable that the patterns of intranodal growth and spread of NHL are mediated by the same cell adhesion molecules (CAM) that control normal lymphocyte traffi~king.~-~ These include ”homing receptors” (H- CAM/CD44, Leu8/LAM-1) mediating lymphocyte migration through endothelial ~ e n u l e s , ~ , ~ , ~ lymph node h ~ m i n g ~ , ~ - ~ and possibly matrix interaction^,^ pl (a’ to a6),l0 and p2 leukocyte integrins (CDlS/CDl la,b,c,)’* implicated in cell-cell and cell-matrix interactions, and CAM related to the immunoglobulin (Ig) supergene family (ICAM-l/CD54, LFA-3/CD58)’ important for cell-cell and cell-stroma adhesion processes or mediating homotypic adhesion among B-cells (BL-CAM/ CD2 2). *

Adhesion Molecules in B-Cell Lymphoma/Pinto et al . 895

Recently, we have described an unusual histologic variant of low-grade NHL13,14 phenotypically distinct from other well-differentiated low-grade B-cell neo- plasms including small lymphocytic lymphoma (SLL), intermediate lymphocytic lymphoma (ILL) and its mantle zone variant (MZL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL) and monocytoid B-cell lymphoma (MBCL). In this SLL variant, opera- tionally termed B-zone lymphoma (BZSLL), neoplastic cells showed a nondestructive intranodal growth pattern with a selective replacement of the B-areas of lymph nodes, and respect for sites such as the T-zone and sin use^'^^'^ leading to a deceptively benign (reactive) histologic pattern at a low magnifi~ation.'~,'~ The peculiar intranodal compartmentalization, along with the maintenance of a pseudonormal lymph node architecture, the strong association of neoplastic cell clusters with dendritic reticulum cells (DRC), and the smoldering clinical c o u r ~ e , ' ~ , ~ ~ suggest that BZSLL might pos- sess specific adhesive properties allowing a strong co- hesion among tumor cells or their preferential interaction with microenvironmental components of the lymph node (extracellular matrix, accessory cells) as opposed to other low-grade lymphoma^.'^

The current study investigates whether adhesion receptors profile of BZSLL might account, at least in part, for the unusual intranodal compartmentalization of this lymphoma variant and for its peculiar clinicopathologic features.13,14 To achieve this goal, we inves- tigated the expression on BZSLL of different groups of adhesion molecules expected to influence the patterns of growth and spread of NHL.3-6 The results were compared with those obtained with other well-differentiated B-cell lymphomas/leukemias including SLL, ILL/MZL, and CLL, characterized by a different pattern of intranodal spread and dissemination. Finally, since adhesion receptors are differentially expressed by defined B-cell subsets within the normal lymph node cornpartrnent~,'~-'~ we determined whether the eluci- dation of adhesion profile of BZSLL provided a further insight into the cellular origin of this putative new entity among low-grade lymphomas.

Materials and Methods

Case Selection

Eight cases of SLL showing the B-zone att tern'^,'^ (BZSLL) on lymph node specimens were identified according to previously described riter ria.'^,'^ Nine patients with SLL (absolute lymphocyte count less than 5 X 109/1) and 10 patients with ILL/MZL were selected

according to the International Working Forrnulati~n'~ and to Weisenburger et a1.,20,21 respectively. Eighteen additional patients with an absolute lymphocytosis in excess of 5 X 109/1 and fulfilling the conventional crite- ria for a diagnosis of CLLZ2 were also included in the study. The clinicopathologic features of BZSLL cases have been previously rep~rted. '~ , '~ All patients displayed bone marrow involvement by neoplastic B-cells.

Histologic Study and In Situ Immunophenotype

For light microscopic examination, tissue fragments of lymph nodes, bone marrow biopsy material, as well as marrow and peripheral blood smears were available for all patients. Representative fresh samples were obtained from lymph nodes. Immunohistochemical staining was performed on cryostat tissue sections with the use of avidin-biotin-peroxidase complex immunoperoxidase method as previously de~cribed.'~, '~ Monoclo- nal antibodies recognizing different types of adhesion molecules are listed in Table 1. The antibody panel used for the immunophenotypic studies also included the fol- lowing antibodies: BA-2/FMC8-CD9, Leu12/B4-

OKB2-CD24, FMC7, Hle- 1 -CD45, IL2R/OKT26a- CD25,0KTlO-CD38, Leul-CD5, J5-CD10, Leu4-CD3, M02-CD14, anti-K, anti-A, anti-surface immunoglobulins (SIg), and DRC-1.

CD19, Bl-CD20, OKB7/B2-CD21, B6-CD23, BA-1/

Cell Isolation and Flow Cytometric Study

Mononuclear cells were obtained by Ficoll-Hypaque centrifugation of bone marrow aspirates and peripheral blood samples. Before staining with the antibodies listed in Table 1, cell samples showing more than 30% of residual CD3+ T-cells, were depleted of T-lymphocytes by immunomagnetic sorting with anti-CD2- coated magnetic beads (Dynabeads, Dynal, Oslo, Nor- way) as previously de~cribed.'~ T-cell-depleted populations contained more than 95% of CD19+ and surface Ig+ cells. Such a procedure allowed a direct comparison of fluorescence data among neoplastic B-cells which were evidenced by discrete peaks when analyzed on a flow cytometer. Indirect and two-color immunofluorescence was performed as previously d e s ~ r i b e d . ' ~ , ' ~ * ~ ~ Nonspecific binding of monoclonal antibodies was assessed by labeling cells with unconjugated or phycoer- ythrinated and fluoresceinated isotype-matched control mouse Ig. Viable, antibody-labeled lymphocytes were identified according to their forward and right- angle scattering, electronically gated, and analyzed for

896 CANCER August 1, 2993, Volume 72, No. 3

Table 1. Monoclonal Antibodies

Cluster of differentiation Antibody Specificity Source* Method

l l a MHM24 LFA-1 (a chain) Dakopatts A/S A 1 l a R3/1 III/IV Workshop B l l b MO 1 Leu-CAMb (a chain) Coulter A 1 l c p150.95 Leu-CAMc ( a chain) Dakopatts A/S B l l c LeuM5 Becton-Dickinson A 18 MHM23 LFA-1 (6 chain) Dakopatts A/S A 18 TS1/18 III/IV Workshop B 22 Leu 14 BL-CAM Becton-Dickinson A, B 54 84H10 ICAM-1 Immunotech A, B 58 TS2/9 LFA-3 IV Workshop A, B 44 F10-44-2 H-CAM Serotec A, B

Leu8 LAM-1 Becton-Dickinson A, B - TS2/3 VLA-a' T cell Science A

w49b IOP49b VLA-a' Immunotech A - P1B5 VLA-a3 Telios A

w49d IOP49d VLA-a4 Immunotech A w49e IOP49e VLA-tu5 Immunotech A w49f IOP49f V L A - ~ ~ lmmunotech A 29 484 VLA-#I (common chain) Coulter A

-

LFA: leukocyte function antigen; Leu-CAM: leukocyte cell adhesion molecule; BL-CAM: B-lymphocyte cell adhesion molecule; ICAM-I: intercellular adhesion molecule-1; H-CAM: homing-associated cell adhesion molecule; LAM-1; leukocyte adhesion molecule-I; VLA: very late antigen; A: bone marrow and peripheral blood, flow cytometric study; B: lymph nodes, cryostat sections, immunoperoxidase. * Dakopatts A/S, Glostrup, Denmark; Coulter Co., Hialeah, FL; Becton-Dickinson, Mountain View, CA; Immunotech, Marseille, France; Serotec, Oxford, UK; T cell Science Inc., Cambridge, MA; Telios Pharmaceutical AS, San Diego, CA.

surface fluorescence on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA). The presence of residual T-lymphocytes and monocytes in the analysis gate was assessed by staining cells with anti-CD3 and anti-CD14 antibodies. Antibody staining was considered to have positive results if more than 25% of gated lymphocytes exhibited fluorescence intensity greater than 95% of cells stained with negative control antibodies.

Fluorescence was collected by using a four-decade logarithmic amplifier. Mean fluorescence intensity was converted to a linear scale by utilizing a 9153C Hew- lett-Packard microcomputer (Mountain View, CA). Laser output was maintained constant and the fluorescence gain settings were adjusted daily to obtain the same fluorescence signals from calibrating micro- spheres (Polyscience, Inc., Niles, PA) to allow fluorescence intensity comparisons. In all experiments, the percentage of stained cells and the mean fluorescence intensity were considered. Mean fluorescence intensity of neoplastic cells was evaluated by setting-appropriate markers around the discrete fluorescence histograms of positive cell populations and expressed as arbitrary units.

Results

His t o p a t hol ogic Fin dings

In all cases of BZSLL, the normal architectural compartments of the lymph nodes were readily recognizable: the trabecular and medullary sinuses were patent and frequently dilated. Some reactive follicles were seen in three cases. The neoplastic tissue, consisting of a mono- morphous lymphoid population of small-sized and me- dium-sized cells with round or slightly indented nu- clear contours (Fig. l), consistently replaced the outer cortical zone, spared mildly expanded paracortical areas, and completely involved the medullary cords (Fig. 1). Thus, lymphoma cells showed a complete and selective replacement of the B-zones without local aggressiveness in relation to the paracortex, the majority of the marginal and medullary sinuses, and the cap- ~ u l e . ' ~ ~ ' ~ This type of growth resulted in a deceptively benign microscopic pattern at a low magnifi~ation.'~

Immunophenotypic Findings

The immunophenotypic characterization of BZSLL cases has been described in detail and compared with

Adhesion Molecules in B-Cell Lymphoma/Pinto et 01. a97

Figure 1. In a lymph node involved by BZSLL, lymphoma cells replace the outer cortical zone (CZ) (top left), surround a spared paracortical (PC) area showing defined borders (asterisks) (top right), and show no local aggressiveness in relation to the sinuses (S) (top left, bottom) and the capsule (C) (top left). The BZSLL cells are small cells with round or slightly irregular nucleus (top right, bottom) (H & E; top, X220; top right, X510; bottom, X510).

both SLL/CLL and ILL/MZL.'3#'4 Neoplastic cells from all BZSLL cases displayed monoclonal SIg (kappa light chains in six cases and lambda light chains in two cases) as detected by in situ staining of lymph node tissue sections and flow cytometric study of bone marrow

(Pinto et al. Unpublished data, 1992). The immunophenotypic profile of BZSLL allowed its distinc- tion from SLL, CLL, ILL/MZL, and other B-cell lymphoproliferative disorders including prolymphocytic leukemia, HCL and its variants, MBCL, and splenic villous lymph~ma. '~ , '~ The composite final phenotype of BZSLL cells, (CD5-, SIg+ strong, CD19+, CD20+, CD2l+, CD22+, CD24+, CD9+, Leu 8+, CD35+, CD23-, FMC7-, CD38-, CD25-, CDIO-) was con- firmed by two-color flow cytometric testing on diagnostic cryopreserved bone marrow or peripheral blood samples, or both, and during patients' follow-up. In lymph node sections, DRC that appeared to be intimately associated with the lymphoma cells were visual- ized by DRC-1, and OKB7-CD21 antibodies in seven BZSLL and in seven MZL/ILL cases (Carbone et al. Un- published data, 1992). DRC were not evidenced in the neoplastic tissue of SLL and CLL cases.14

Expression of CAM

The expression of several surface molecules involved in lymphocyte adhesion and migration was examined on lymphoma cells by indirect immunofluorescence analysis (Tables 2 and 3). Cells from BZSLL cases expressed LFA-la, LFA-1P, LFA-3, ICAM-1 (seven of eight), and BL-CAM antigens which were conversely inconsistently detected on SLL and CLL samples (Table 2). Cells from ILL/MZL specimens were always LFA-1 positive, expressed LFA-3 and BL-CAM in most of the cases, but were rarely ICAM-1 positive (Table 2). Consistent differences in the expression of LFA-1, a and 0, LFA-3, ICAM-I, and BL-CAM were detected when comparing BZSLL to SLL or CLL cases (Table 2). The rate of expression of ICAM-1 on BZSLL cases was also different with respect to MZL/ILL (Table 2). Leu-CAMb determinants were detected on neoplastic cells from three BZSLL cases whereas an inconsistent expression of Leu-CAMc was observed in all lymphoma samples (Ta- ble 2). In general, the fluorescence intensity level of antibody staining correlated with the frequency of antigen-positive cells, allowing the detection of relevant

898 CANCER August 1, 1993, Volume 7 2 , No. 3

Table 2. Expression of Cell Adhesion Molecules in B-Zone Small Lymphocytic Lymphoma and Other Well-Differentiated Lymphocytic Disorders*

Cluster of differentiation

1 l a I l b l l c 18 22 54 58 -

44

~~~~

No. positive/no. testedt Cell adhesion molecule BZSLL ILL/MZL SLL CL L

LFA-1 (a) Leu-CAMb Leu-CAMc LFA-I (la) BL-CAM ICAM-1 LFA-3 LAM-1 H-CAM

10/10 0/10 1/10

10/10 8/10 4/10 7/10 8/10

10/10

4/9 0/9 0/9 4/9 4/9 1/9 3/9 4/9 9 /9

6/18 1/18 1/18 7/18 4/18 6/18 4/18 9/18

18/18 BZSLL B-zone small lymphocytic lymphoma; ILL/MZL intermediate/mantle zone lymphoma; SLL small lymphocytic lymphoma; CLL chronic lymphocytic leukemia. * Malignant cells were analyzed by flow cytometric study after they were stained with appropriate monoclonal antibodies. Data are given for bone marrow cells, except for eight cases of chronic lymphocytic leukemia, in which peripheral blood cells were analyzed. t A sample was classified as positive when more than 25% of the cells were stained by monoclonal antibodies.

differences among the various lymphoma cells analyzed. The expression of LFA-1 and LFA-3 molecules was, in fact, higher in BZSLL as opposed to ILL/MZL, SLL, and CLL in terms of both the percentage of stained cells (Fig. 2, top) and mean fluorescence intensity (Fig. 2, bottom). Anti-BL-CAM antibodies stained a higher percentage of BZSLL cells with a brighter fluorescence compared with SLL and CLL lymphocytes. BZSLL cells also displayed a higher expression of BL-CAM in terms of fluorescence intensity compared with ILL/MZL (Fig.

Table 3. Expression of @1-Integrins in B-Zone Small Lymphocytic Lymphoma and Other Well-Differentiated Lymphocytic Disorders*

Integrin

VLA-a’

VLA-a3 VLA-a4 VLA-a5 VLA-a6

V L A - ~ ~

VLA-B1

No. positive/no. testedt

BZSLL ILL/MZL SLL CL L

0/5 0/6 0/6 0/11 0/5 1/6 (1+) 2/6 (1+) 1/13 (1+) 3/5 (1+) 2/6 (1+) 2/6 (I+) 3/8 (3+) 6/6 (2+) 4/7 (2+) 8/9 (I+) 10/18 (2+) 0/5 2 / 7 (I+) 2/6 (I+) 14/18 (2+) 2/5 (1+) 2/7 (I+) 4/6 (2+) 1/17 (I+) 6 /6 (3+) 3 / 7 ( I+) 6 /6 (3+) 14/14 (2+)

BZSLL: 8-zone small lymphocytic lymphoma; ILL/MZL: intermediate/mantle zone lymphoma; SLL small lymphocytic lymphoma; CLL: chronic lymphocytic leukemia; I+: 25%-40% of cells stained; 2+: 41%-6O% of cells stained; 3+: > 6 0 1 of cells stained. * Malignant cells were analyzed by flow cytometric study after they were stained with appropriate monoclonal antibodies. Data are given for bone marrow cells, except for eight cases of chronic lyrnphocytic leukemia, in which peripheral blood cells were analyzed. t A sample was classified as positive when more than 25% of the cells were stained bv monoclonal antibodies.

2, bottom). No differences in terms of fluorescence brightness and percentage of stained cells were detected for ICAM-I, Leu-CAMb, and Leu-CAMc antigens on positive cases from all lymphomas subtypes analyzed (Pinto et al. Unpublished data, 1992). The LAM-1 homing receptor molecule was expressed by all BZSLL cases, 4 of 9 SLL, 9 of 18 CLL samples, and by 8 of 10 ILL/MZL cases. All lymphoma cells except two BZSLL cases displayed H-CAM determinants (Table 2). The expression of both homing receptors was lower in BZSLL than in ILL/MZL, SLL, and CLL in terms of percentage of stained cells (Fig. 2, top) and mean fluorescence intensity (Fig. 2, bottom). Representative fluorescence histograms showing the differential expression of LFA-la, BL-CAM, LAM-1, and H-CAM among BZSLL, ILL/MZL, and CLL are shown in Figure 3. The patterns of staining of lymphoma cells with anti-LFA-3 antibodies are also shown (Fig. 4).

Expression of p l -Integrins

VLAa’,’r5 were not expressed by lymphoma cells with a consistent pattern the only exception being represented by CLL cells which reacted in up to 78% of cases with anti-VLA-a5 antibodies (Table 3). A heterogeneous expression of VLA-a3-integrin was also observed in the various lymphoma subtypes (Table 3). VLA-a6-integrins were detected in 2 of 5 BZSLL cases, 2 of 7 ILL/ MZL samples, in 4 of 6 SLL specimens, and only in 1 of 17 CLL cases (Table 3). VLA-a4-integrins and pl-chains were conversely expressed by all BZSLL cases and by the great majority (88%) of SLL. ILL/MZL cells lacked

Adhesion Molecules in B-Cell LymphomalPinto et al . 899

WA-1 LPA-3 LAM-1 H-CAM BL-CAM (CDlla) (CD58) (Leu8) (CD44) (CD22)

W BZ-SLL ILL/MZL SLL cu Figure 2. Differential expression of cell adhesion molecules on BZSLL, ILL/MZL, SLL, and CLL. Flow cytometric data are expressed as mean percentage of lymphoid cells stained by appropriate monoclonal antibodies in positive samples (top) and as mean fluorescence intensity of positive cell populations (bottom).

VLA-a4 in three of seven cases and 01 -common chains in four of seven samples (Table 3) . CLL lymphocytes displayed p l -chains in all instances whereas VLA-a4 expression was not detected in 8 of 18 specimens (Table 3) . No significant differences in fluorescence intensity were detected by staining the different lymphoma cells with anti-VLA-a'-6 and anti-pl -integrins antibodies, except for VLA-a3 molecules which were expressed by CLL cells at the highest density (Pinto et al. Unpub- lished data, 1992).

In Situ Expression of CAM

Results of CAM immunostaining of lymphoma cells re- siding in involved lymph nodes and of normal lymph nodes frozen sections by immunoperoxidase method

are shown in Table 4. LFA-la, LFA-10, and LFA-3 antigens were found to be strongly expressed by neoplastic cells in all BZSLL cases and in one ILL/MZL sample, albeit with a lower intensity (Table 4, Fig. 5). LFA-la and molecules were not detected on three CLL and two SLL cases. These latter samples also displayed an inconsistent and weak expression of LFA-3 (Table 4). Anti-ICAM- 1 antibody strongly stained malignant cells in the 3 cases of BZSLL examined, being unreactive with lymphoma cells in 2 of 3 cases tested of ILL/MZL, in 2 SLL samples, and in 9 of 11 cases of CLL. Anti-H- CAM antibodies reacted with neoplastic cells in all cases tested albeit with different staining intensities. Malignant lymphocytes in SLL or CLL showed the strongest expression of H-CAM antigens (Table 4) whereas a weaker expression was detected in BZSLL and ILL/MZL. In normal lymph node sections, within the B-cell areas, germinal center cells were clearly stained by anti-ICAM-1 and anti-LFA-3 antibodies while showing a weak reactivity or a negative staining with anti-LFA- 1 and anti-H-CAM antibodies (Table 4). Mantle zone cells conversely exhibited a weak staining or a negative reaction with all anti-CAM antibodies tested. T-lymphocytes in T-cell areas were strongly stained only by anti-LFA-1 and anti-H-CAM monoclo- nals (Table 4). DRC in normal germinal centers consistently showed immunostaining with anti-ICAM- 1 and

CD 22 LEU 8

C E L L

N U M B E R

LFA- 1 CD44

FLUORESCENCE INTENSITY Figure 3. Representative fluorescence histograms showing the differential expression of BL-CAM/CDZZ, LAM-l/Leu8, LFA-I/ CDlla , and H-CAM/CD44 molecules on lymphoma cells; (. - - matched negative control mouse immunoglobulins, BZSLL (-),

- - ) highest background staining obtained with isotype-

ILL/MZL ( * * * *), CLL (---).

900

c E L L

N U M B E R

CANCER August 1, 1993, Volume 72, No. 3

LFA-3

.. . .

FL .UORE SCEN CE INTENSITY Figure 4. Representative fluorescence histograms showing the differential expression of the LFA-3/CD58 molecule on lymphoma cells. Dotted lines in all panels represent background staining obtained with isotype-matched negative control mouse immunoglobulins. The other lines within each panel represent staining obtained with three different samples of the same lymphoma group. (Top panel) BZSLL, (center panel) CLL, (bottom panel) ILL/MZL.

anti-LFA-3 antibodies (Carbone et al. Unpublished data, 1992).

Discussion

The patterns of lymph node involvement by well-differentiated B-lymphocytic neoplasmsz5 such as SLL,

MZL/ILL, and CLL have been described as pseudofol- l i ~ ~ l a r , ~ ~ ~ ~ ~ mantle zone,z1,28 interfollicular,z7 and, more frequently, d i f f ~ s e . ~ " ~ ~ ~ ~ ~ ~ - ~ ~ We have described a low- grade lymphoma variant (BZSLL),13,14 phenotypically distinct from other well-differentiated B-cell dis- o r d e r ~ , ~ ~ which displays a peculiar intranodal compartmentalization of neoplastic cells resulting in a complete and selective replacement of the B-zones with a sub- stantial sparing of T-dependent zones and sinuses. l3,I4

The smoldering clinical course, the low tendency to leukemic conversion, the marked splenomegaly, and the maintenance of a pseudonormal lymph node architecture which characterize BZSLL,13,14 could be ascribed to intrinsic biologic properties of neoplastic cells and possibly those which involve the homotypic adhesion among tumor cells and their interactions with extracellular m a t r i ~ , ~ - ~ , ~ l neighboring' and accessory cell^^-^ of tissue microenvironment. The cell surface receptors mediating these events are, therefore, likely to play a key role in such interactive p r o c e s s e ~ . ~ - ~ , ~ ~ , ~ ~

The current study reports the first comparative sur- vey of adhesion molecules expression in BZSLL and other phenotypically related disorders such as SLL, ILL/MZL, and CLL. The adhesion phenotype of BZSLL (LFA-lK, ICAM-lhi, CD44'", LAM-l'", BL-CAMhi, LFA- 3hi, VLAU~~', VLAPlhi, VLAa5"'g) appeared distinctive with respect to other low-grade malignancies examined, including disorders like SLL and CLL, both characterized by the complete effacement of the lymph node architecture. BZSLL displayed a stronger expression of CAM related to the P2-integrin family (LFA-1, CDllb) and Ig superfamily (ICAM-1, LFA-3, BL-CAM) and a lower cell density of homing receptor molecules (H-CAM, LAM-1) compared with ILL/MZL and/or SLL and CLL. Regarding p l -integrin expression, BZSLL appeared consistently VLA-a4 positive and VLA-a' negative whereas VLA-a6 and VLA-a3 molecules were expressed by two and three cases, respectively. This pattern differed overall from that of SLL (VLA-a4 positive and VLA-a' variable), CLL (VLA-a4 negative in 50% of cases and VLA-as positive in up to 75% of samples) and ILL/MZL (VLA-a4 positive in four of seven cases and VLA-a5 negative in most of the samples). Pl-common chains (CD29) were expressed at a strong intensity by all lymphoma cells with the only exception of ILL/MZL lacking Pl-molecules in four of seven cases.

The composite adhesion profile of BZSLL might account, at least in part, for the peculiar biologic properties of this lymphoma. The large amount of ICAM-1 and LFA-1 molecules at the surface of BZSLL lymphocytes might favor both homotypic adhesion among neoplastic cells and a tight adhesion of LFA-1+ BZSLL cells and to DRC and to other accessory cells within the

Adhesion Molecules in B-Cell Lymphoma/Pinto et al. 901

Table 4. Expression of Cell Adhesion Molecules on Lymph Node Malignant Cells in B-Zone Small Lymphocytic Lymphoma and Other Well-Differentiated Lymphocytic Disorders and Comparison With Normal Lymph Node Immunostaining Patterns

Normal lymph node*t

Cell No. positive/No. tested* B-cell areas Cluster of adhesion T-cell differ entiation molecule BZSLL I L L / M Z L SLL CLL GCC MZC areas

l l a LFA-la 5/5 l/lS 0/2 0/3 f f +

54 ICAM- 1 3/3 1/3 0/2 2/11 + - 58 LFA-3 4/4 1/1 1/2$ 1/3$ 44 H-CAM 5/5 1/1 2/2§ 3/3§

18 LFA-10 4/4 l / l S 0/2 0/3 NT NT NT - + - + k

f + -

BZSLL B-zone small lymphocytic lymphoma; ILL/MZL intermediate/mantle zone lymphoma; SLL small lymphocytic lymphoma; CLL chronic lymphocytic leukemia; GCC: germinal center cells; MZC: mantle zone cells; NT: not tested; -: negative; +: 0%-50% positive cells; +: 51%-100% positive cells. * Data obtained by avidin-biotin-peroxidase complex immunoperoxidase staining. t Three samples analyzed. $ Weaker staining intensity as compared with B-zone small lymphocytic leukemia. 5 Stronger staining intensity as compared with 8-zone small lymphocytic leukemia.

lymph node (fibroblasts, endothelial cells) which, in BZSLL cases displayed in fact that DRC-I+ DRC were turn, express the LFA-1 ligand ICAM-1 at high den- closely and intimately associated with tumor cells in sity. 16,3345 Im munohistochemical analysis in most pathologic lymph nodes. 13,14 In addition, we have

Figure 5. Frozen sections of lymph node involved by BZSLL showing LFA-3/CD58 (top left) H-CAM/CD44 (top right), and LFA-l/CDl l a (bottom) antigen expression by neoplastic cells. LFA-3/CD58 and H-CAM/CD44 antigens are strongly expressed by most cells. The cells of a residual germinal center (top right, to the left) show no reactivity for anti-H-CAM/CD44 antibody (Avidin-biotin-peroxidase complex with H & E counterstain, X320).

902 CANCER August I , 3993, Volume 72, No. 3

found that BZSLL consistently express the VLA-a4-integrin, which has been shown to mediate B-cell binding to dendritic cells via the vascular cell adhesion molecule- 1 (VCAM- 1)35,36 and to fibronectin molecules of the extracellular matrix." The multiple homotypic and heterotypic interactions mediated by LFA-l/ICAM-1, LeuCAMb/ICAM- 1 , VLA-a4/VCAM- 1, and VLA-a4/ fibronectin pathways might, therefore, result in a limited capability of BZSLL cells to diffusely proliferate within the lymph node microenvironment leading to the peculiar compartmentalized histologic pattern of this lymphoma. On the other hand, the expression of LFA-1 has been correlated with a low tendency to he- matogenous d i ~ s e m i n a t i o n , ~ ' , ~ ~ , ~ ~ and with a nodular pattern of intranodal growth in low-grade NHL,31*39 whereas the strong expression of ICAM-1 in the same diseases was reported to be associated with a nonleuke- mic c o u r ~ e ~ ~ , ~ ~ and with an increased adhesiveness of B-lymphoid tumors.41

Other adhesive molecules, however, might be involved in the disruption of lymph node architecture by lymphoma cells and their subsequent dissemination. In this regard, the expression of H-CAM and LAM-1 homing receptors has been associated with lymphoma d i s s e m i n a t i ~ n ~ ~ . ~ ~ and to progressive lymphocyt~sis~~ in NHL and B-cell lymphoproliferative disorders. More recently, a low expression or a loss of LAM-1 expression has been reported in NHL confined to gastrointestinal tract as opposed to nodal or disseminated lympho- m a ~ . ' ~ ' ~ ~ In agreement with these results, BZSLL lacked H-CAM in two cases and displayed a lower density of H-CAM and LAM-1 compared with the other B-cell lymphomas analyzed. In our series, CLL cells displayed the highest levels of LAM-1, which is in agreement to what was reported by Spertini et al.46 Conversely, the expression of the BL-CAM antigen, present at a very high density on BZSLL cells, has been associated with prominent lymphadenopathy, splenomegaly, and low lymphocyte counts in B-cell lymphoproliferative dis- o r d e r ~ . ~ ~ Interestingly, a strong correlation between BL- CAM and LFA-1 expression was evident in BZSLL in analogy to what was reported by analyzing human B- cell lines."

BZSLL also showed a definite expression of the LFA-3 molecule which was, however, comparable with that of ILL/MZL in terms of positive cases. Conversely, SLL and CLL cases, with an overt leukemic course, displayed an inconsistent LFA-3 staining. It has been suggested that LFA-1 and LFA-3 down-regulation by malignant cells is an adaptive change allowing the escape from the immune s u r ~ e i l l a n c e . ~ ~ , ~ ~ It could not be ex-

cluded, however, that a decreased adhesiveness of lymphoma cells resulting from the loss of LFA-3 molecules, might itself account for a high risk of leukemic spread and for a diffuse intranodal growth pattern. In this regard, notice that the decreased adhesiveness of myeloid precursors to bone marrow stromal cells in chronic myelogenous leukemia recently has been correlated to the loss of LFA-3 molecules from clonogenic cells.49 Finally, the lack of VLA-a5-integrins on BZSLL as opposed to the consistent expression of this molecule by CLL cells, which conversely lacked VLA-a4 in 50% of cases, seems to confirm the correlation between VLA-a5 expression and the propensity toward a leukemic dissemination suggested by Moller et al."

In addition to previous immunohistologic find- i n g ~ ' ~ , ' ~ suggesting that BZSLL may arise from a rare B-cell subpopulation sharing some features of Leu-8+/ CD9+ mantle zone lymphocytes, the adhesion profile of BZSLL does not exclude an origin from a subset of extrafollicular B-cells for this lymphoma. Interfollicular B-cells in normal lymph nodes, in fact, have been described to be VLA-ck3 negative, inconsistently expressing VLA-a5*6, clearly reactive with anti-VLA-a4 and an t i -p l -~hains , '~ , '~ ,~~ and weakly expressing H-CAM and LAM-1 homing receptors" as opposed to mantle zone B-cells which appear devoid overall of pl-molecules and display a stronger staining with anti-H-CAM and anti-LAM-1 ant ibodie~ . '~ , '~ ,~~ In addition, a scat- tered population of interfollicular B-cells strongly expressing ICAM-1, LFA-3, and BL-CAM have been detected in different and anti-LFA- 1 antibodies showed a consistent staining of interfollicular areas in several reports as opposed to mantle zone cells. 1 6 , 3 1 3

In conclusion, the complex adhesion phenotype of BZSLL, by conferring an increased capability of homotypic and heterotypic adhesive interactions to malignant B-cells, could explain, at least in part, the preserva- tion of lymph node architecture and the low tendency to an overt leukemic dissemination which characterize this lymphoma. l3,I4 The strong expression of adhesion receptors might also render BZSLL lymphocytes more susceptible to endogenous immune effectors as suggested by the high levels of CD3+, CD4+ T-cells and CD16+, CD56+ natural killer cells detected by us in the peripheral blood from BZSLL patients (Pinto et al. Un- published data, 1992).

Further studying on the functional significance of adhesive molecules in reactive lymph nodes and in BZSLL is warranted to better clarify the cellular origin and the peculiar biology of this lymphoma.

Adhesion Molecules in B-Cell Lymphoma/Pinto et al. 903

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