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© 1999 American Society for Clinical Oncology Expression of Beta-Integrin Adhesion Molecules in Non-Hodgkin's Lymphoma: Correlation With Clinical and Evolutive FeaturesFrom the Hematopathology Unit, Departments of Hematology and Internal Medicine, Instituto de Investigaciones Biomédicas "August Pi i Sunyer," Hospital Clínic, Barcelona, Spain. Address reprint requests to Emilio Montserrat, MD, Department of Hematology, Hospital Clínic de Barcelona, Villarroel 170, 08036 Barcelona, Spain; email emili{at}medicina.ub.es
PURPOSE: To analyze beta-integrin expression in non-Hodgkin's lymphomas (NHLs) in order to assess its distribution among histologic subtypes and correlate with clinical features and outcome.
PATIENTS AND METHODS: The expression of
RESULTS: MCL cells did not show CONCLUSION: In NHL, beta-integrin expression is related to histologic subtype. The expression pattern of these molecules probably influences disease dissemination and patients' prognoses.
ADHESION MOLECULES ARE cell-surface receptors that mediate cell–cell and cell–extracellular matrix interactions. They are required for the development of several physiologic processes, such as hematopoiesis, immune response, lymphocyte migration, and homing.1 Lymphocyte adherence is largely mediated by three families of specific cell-surface adhesion molecules: integrins, immunoglobulin-related molecules, and selectins. Integrins are large heterodimeric glycoproteins that recognize extracellular matrix components such as collagen, fibronectin, laminin, and vitronectin, as well as cell-surface molecules (ie, intercellular adhesion molecule [ICAM] 1, ICAM-2, ICAM-3, and vascular cell adhesion molecule 1). Integrins are usually classified into seven subfamilies according to the common beta subunit.2-6 The ß1 (CD49/CD29 molecules or very late activation antigen [VLA] proteins) and the ß3 through ß7 subfamilies are widely expressed, whereas the ß2 subfamily (CD11a/CD18 or leukocyte function-associated antigen 1 [LFA-1] molecule, CD11b/CD18, CD11c/CD18, and CD11d/CD18) is expressed only in leukocytes.7,8 The migration and extravasation of lymphoid cells into different lymphoid tissues (homing) is a complex process mediated by interactions between lymphocyte cell-surface molecules, the so-called homing receptors, and the high endothelial venules present at secondary lymphoid tissues. This occurs through several sequential steps mediated by different adhesion molecules.9 Such a process is strictly regulated to selectively address "appropriate" lymphoid subsets to specific microenvironments. Non-Hodgkin's lymphoma (NHL) represents neoplastic counterparts of lymphoid cells arrested at different stages of activation and differentiation.10 There is increasing evidence that some of the mechanisms that control the traffic of normal lymphoid cells also regulate the circulation and dissemination of neoplastic lymphoid cells.11 In several B-cell neoplasias, VLA expression has been correlated with the profile of normal B-cell development, with the expression pattern being closely related to the degree of lymphoma differentiation. In this regard, it has been suggested that the characteristic pattern of dissemination of each subtype of NHL could be explained, in part, by VLA expression.12 Furthermore, the expression of CD44 in lymphoma and leukemia parallels the expression shown during T- and B-cell ontogeny, with a high expression during the earliest and mature differentiation phases and a reduction in the expression during subsequent stages of differentiation.13-15 Moderate to strong expression of CD44 has been associated with advanced disease and worse prognosis in several studies.16-18 Moreover, CD44V6 expression has proved to be an independent adverse prognostic factor in NHL.19 Leukocyte integrins play an important role in the immune response and lymphocyte migration into tissues. Through the binding to its counterreceptor (ICAM-1), LFA-1 sends a costimulatory signal for T-cell activation triggered by specific antigen recognition.20 It has been proposed that downregulation of LFA-1 may impair T-cell recognition, resulting in an escape from immunosurveillance.21 In fact, cytokine upregulation of LFA-1 and ICAM-1 restores the susceptibility of hairy cell leukemia cells to lysis by cytotoxic lymphocytes.22 Experimental studies have shown that LFA-1/ICAM-1 interactions mediate invasion of fresh lymphoma cells into cultures of hepatocytes.23 Moreover, antibodies to LFA-1 block the invasive ability of lymphoma cells, and LFA-1–deficient lymphoma cell lines show a reduced invasive potential in vitro as well as an attenuated metastatic capacity in vivo.24 We recently demonstrated a relationship between ICAM-1 expression and tumor dissemination.25 Finally, a prognostic role of LFA-1 expression in NHL has been suggested, but not confirmed, in human series.26,27 The aim of this study was to analyze the distribution of the main beta-integrin adhesion molecules in a large series of patients with NHL to determine the pattern of expression among the different subtypes of lymphoma, as well as the correlation with clinicobiologic characteristics and the outcome of the patients.
Patients: Clinical Data and Treatment A total of 137 patients with a diagnosis of NHL in a single institution were included in the present study. The only inclusion criterion was the availability of frozen lymph node samples to analyze the integrin expression. The histologic classification according to the revised European-American classification of lymphoid neoplasms10 was as follows: immunocytoma (IM), 10 cases; follicular lymphoma (FL), 31 cases; mantle-cell lymphoma (MCL), seven cases; diffuse large-cell lymphoma (DLCL), 64 cases; T-cell lymphoblastic lymphoma (LL), 11 cases; anaplastic large-cell Ki-1–positive T-cell lymphoma, three cases; and other peripheral T-cell lymphomas, 11 cases.
Eighty of 137 patients (median age, 58 years; 54% male) were consecutively diagnosed between 1986 and 1994 and had the initial slides available to assess beta-integrin expression. The histologic distribution of the patients included in the study was as follows: IM, five cases; FL, 19 cases; MCL, four cases; DLCL, 40 cases; LL, six cases; anaplastic large-cell Ki-1–positive T-cell lymphoma, one case; and other peripheral T-cell lymphomas, five cases. Advanced Ann Arbor stage (III to IV) was observed in 56 patients (70%), extranodal involvement in 54 (67%), and bone marrow infiltration in 30 (40%). Forty patients (50%) presented with high serum lactate dehydrogenase (LDH) levels, and 28 (38%) had high or high/intermediate risk according to the International Prognostic Index (IPI).28 The median follow-up duration was 6.5 years (range, 2.4 to 12.5 years). The correlation between beta-integrin expression and both the main clinicobiologic data at diagnosis and the outcome of the patients was analyzed. For this purpose, the following information from each patient was recorded and analyzed: age, sex, performance status (according to the Eastern Cooperative Oncology Group scale), presence of "B" symptoms, bulky disease ( All patients were treated with combination chemotherapy, in most cases cyclophosphamide, doxorubicin, vincristine, and prednisone. Posttherapy restaging consisted of repetition of tests and/or biopsies that previously had abnormal results. Complete response (CR) was defined as the total disappearance of tumor masses and disease-related symptoms and normalization of abnormal test results for at least 1 month. Partial response was defined as a decrease in tumor masses or organ infiltration of at least 50%, along with the disappearance of disease-related symptoms. Patients not included in these categories and early deaths were considered nonresponders.
Determination of Integrin Expression Staining intensity was scored semiquantitatively on a scale from 0 to 3 (0, negative; 1+, weak; 2++, moderate; 3+++, strong). For a lymphoma to be scored, a minimum of 15% of the lymphoma cells had to stain. All the slides were reviewed twice by the same observer in a random manner. Reactive nonneoplastic lymph nodes were used as normal controls to determine the physiologic expression of beta-integrins. Finally, in most patients, tumor ICAM-1 expression had previously been determined25; therefore, its correlation with the beta-integrin expression could be determined.
Statistical Analysis
Beta-Integrin Expression The expression of  chains and the ß2 common chain of VLA integrins by tumor cells according to the histologic subtype is listed in Table 1. In DLCL, such expression, when present, was weak in most cases (only 13% and 16% of these patients showed moderate 4 and 5 expression, respectively). No patient with MCL expressed 2 on tumor cells, whereas most showed weak to moderate levels of 3, 4, and 5. ß1 integrins were present in 64% to 100% of LLs. Of note, 4 and 5 were expressed in LL at moderate to strong intensity in nearly all cases (91%). 6 was positive (weak) in seven of 11 patients with LL (64%), a significantly higher proportion (P < .01) than that in any other histologic subgroup. Expression of 2 through 6 was observed in the interfollicular vessel endothelium in all the cases. Figure 1 shows examples of 4 chain expression in patients with FL, DLCL, and LL.
Overall,
The expression of ß2 integrins on tumor cells according to the histologic subtype is listed in Table 2
No significant relationship was found between ICAM-1 expression in tumor cells, as previously published,25 and LFA-1 expression (CD11a plus ß2 common chain).
Beta-Integrin Expression and Initial Characteristics of Patients
Beta-Integrin Expression and Outcome of Patients
Multivariate analysis was performed for the whole series. The model included all initial variables with significant value for OS in the univariate study (data not shown), along with the expression of the different beta-integrins. The most important variables for OS were histologic subtype (indolent v aggressive), serum LDH level, and IPI. In the subset of B-cell DLCL patients,
Integrins are adhesion molecules that are broadly expressed on hematopoietic and nonhematopoietic tissues. The pattern of ß1 integrin expression on normal and neoplastic lymphocytes has been reported by several investigators.13-15,32-39 In normal lymphoid tissues, the integrin most often expressed was 4 on mantle-zone cells followed by 5, which was present on some germinal and mantle-zone cells. The virtual absence of 1 and 2 has been reported universally, whereas expression of 3 has been variable on some germinal cells and mantle-zone cells. In our experience, 3 and 5 were also detectable on some germinal and mantle-zone cells, whereas 4 was mainly expressed at higher levels on mantle-zone cells, although lymphocytes from interfollicular and some from germinal center areas were also positive. VLA expression profile has been correlated with stage of activation and differentiation of neoplastic lymphocytes.13 Therefore, low-grade lymphoproliferative disorders with a tendency toward leukemic expression, such as chronic lymphocytic leukemia or hairy cell leukemia, tend to constantly express VLA-4 and VLA-5. Lymphoid neoplasias that originate from the mantle zone are usually VLA-3–positive, whereas marginal-zone lymphoproliferative disorders are VLA-3–negative. FL showed a heterogeneous distribution of VLA-3, -4, and -5. Primary mediastinal (thymic) large-cell lymphoma always lacks VLA-3, -5, and -6 and frequently –4 as it occurs in normal thymic B cells.39 In FL and DLCL, expression of VLA-3 and -4 was more frequent than that reported in other series.13 Similarly to Baldini et al,14 we found a higher number of VLA-5–positive cases (36% of DLCL and 68% of FL) than did previous studies.13 Furthermore, VLA-2 was present on one third of FLs and some DLCLs, in agreement with previously reported data.32 The differences found among the series may reflect differences in sensitivity of the immunohistochemical procedures. MCL cells were consistently VLA-3–, -4–, and -5–positive, in contrast with IM cells, which always lacked VLA-3.
To date, the expression of ß1 integrins in T-cell NHL has not been described. We found a higher frequency and intensity of expression of all subtypes of I chains of VLA proteins in T-cell NHL, especially LL, compared with B-cell neoplasms. These data reproduce the pattern of expression of VLA in normal lymphoid cells: VLA proteins are more often expressed, and at higher levels, in T lymphocytes than in B cells. On the other hand, in B-cell lymphomas, no discordance was found between the expression of ß1 common chain and all of the alpha subunits, including
In lymphoproliferative disorders, the correlation between VLA protein expression and clinical and biologic features of the disease has not been well established. Some investigators have found that the leukemic phenotype of malignant B cells seems to depend on LFA-1 is strongly expressed by most circulating and normal lymph node lymphocytes and is more prominent on some extrafollicular T and germinal B cells. The lack of expression of this molecule in 41% of DLCLs is in agreement with the results of Medeiros et al,26 suggesting that this molecule could be implicated in metastatic dissemination. It has been proposed that the lack of LFA-1 expression on the surface would lead to an aggressive behavior, probably by escaping from the immunosurveillance system.21 Furthermore, Horst et al27 found that LFA-1 expression tended to be higher in disseminated lymphomas compared with localized disease. In our results, although LFA-1 (CD11a and ß2 common chain) expression was related to lymphoma dissemination, it did not reach statistical significance in terms of CR, OS, or FFS rates.
From the clinical standpoint, the most interesting result was the correlation between different beta-integrins and CR achievement, OS, and FFS in patients with B-cell DLCL. Because these integrins have shown a close relationship with tumor dissemination, this is the most likely explanation for the relationship between beta-integrin molecules and prognosis. However, other mechanisms cannot be discarded, especially with regard to In summary, B-cell lymphomas express several adhesion molecules that are also present on normal B cells. The pattern of expression of such adhesion molecules on lymphoma cells probably influences the spread of the disease and the prognosis of the patients.
Supported in part by grant no. FIS 98/0996 from the "Fondo de Investigacion Sanitaria," Spanish Ministry of Health.
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
10 cm in diameter), nodal and extranodal involvement, bone marrow, CNS and liver infiltration, hemoglobin level, WBC and platelet counts, serum LDH and beta2-microglobulin, IPI, type of treatment, response to therapy, relapse, and survival. 
