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Appropriate staging and evaluation of residual disease is critical to improving the treatment of patients with lymphoma. The specific expression of homing receptors may determine the preferential dissemination pattern of tumoral cells. We investigated the expression of the mucosal homing receptor α4β7 on tumoral cells from peripheral lymph node in patients with newly diagnosed mantle cell lymphoma (MCL) to check whether it is associated with gastrointestinal involvement. Expression of the α4β1 integrin and the peripheral lymph node addressin CD62L were also examined. Thirteen MCL patients presenting with peripheral lymphadenopathy were studied. Expression of the mucosal homing receptor integrin α4β7 by peripheral lymph node lymphoma cells was found to be frequent (5/13) and associated with gastrointestinal involvement (5/7). In contrast, lymphoma cells from patients without gastrointestinal involvement did not express α4β7 (6/6) (P = 0.03). These data suggest that α4β7 integrin is expressed by a subset of MCLs and that its expression may predict digestive tract involvement in MCL, furnishing a basis for recognizing two distinct clinical and phenotypic forms, ie, “digestive homing (or digestive primitive)” versus “peripheral” MCL. Further studies on more patients will be needed to understand the impact of biological differences on the prognosis of these two clinical forms.
Recent studies focused mainly on T-lymphocytes have led to the concept that finely tuned mechanisms involving adhesion molecules regulate leukocyte migration and organ targeting in the human body.
Discrete subpopulations of leukocytes appear to be able to migrate specifically to certain tissues due to the restricted expression of receptor-ligand pairs.
The α4β7/MAdCAM-1 pair is one of the best characterized receptor-ligand pairs shown to play a role in the control of leukocyte circulation. Integrin α4β7 (LPAM-1) mediates murine and human B and T memory lymphocyte migration into the intestinal mucosa by binding to MAdCAM-1, a vascular recognition molecule selectively expressed on digestive tract lamina propria, Peyer's patch endothelium, and the spleen.
Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1) demonstrates structural and functional similarities to the α4β7-integrin binding domain of murine MAdCAM-1, but extreme divergence of mucin-like sequences.
Indeed, in mice with targeted disruption of the β7 (β7−/−) or the α4 integrin, the formation of the gut-associated lymphoid tissue (GALT) is severely impaired, whereas β7−/− mice exhibit otherwise normal immune system development and function.
Human MAdCAM-1 was recently cloned and MAdCAM-1 mRNA and protein were found to be expressed mainly in the small bowel and to a lesser extent in the colon and spleen.
Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1) demonstrates structural and functional similarities to the α4β7-integrin binding domain of murine MAdCAM-1, but extreme divergence of mucin-like sequences.
α4 integrin may also be expressed on leukocytes as a heterodimeric integrin with the β1 integrin chain (CD18). α4β1 is a ligand for VCAM-1 (CD106) and is involved in adhesion to cytokine-activated endothelial cells, germinal centers within lymph nodes,
Follicular non-Hodgkin's lymphoma cell adhesion to normal germinal centers and neoplastic follicles involves very late antigen-4 and vascular cell adhesion molecule-1.
We previously proposed that expression of α4β7 on peripheral tumoral cells was associated with digestive tract involvement in Langerhans cell histiocytosis, a clonal proliferative disease of dendritic cell origin.
Mantle-cell lymphoma (MCL) is a recently reappraised entity among B-cell non-Hodgkin's lymphoma, on the basis of its clinical course and morphological, immunophenotypic, cytogenetic, and molecular features.
Although no prospective study is available, digestive tract involvement appears to be more frequent in MCL at diagnosis compared to other peripheral lymphomas; it was diagnosed at presentation in 11.5% to 15% of MCL patients.
In the present study, we investigated whether the “homing model” may be of clinical relevance in MCL digestive tract involvement and whether the α4β7 adhesion molecule may, if it is expressed on peripheral tumoral cells, help to predict the existence of gastrointestinal (GI) tract involvement.
Materials and Methods
MCL Patients
Thirteen consecutive patients with nodal peripheral MCL for whom material from frozen lymph node biopsy was available and who underwent digestive tract endoscopic examination and biopsies were studied.
Histology and Immunohistochemistry
Deparaffinized lymph node sections were stained with hematoxylin-eosin-safran, Giemsa stain, and silver stain. Immunohistochemistry was performed on deparaffinized formalin-fixed sections with an avidin-biotin-peroxidase protocol
revealed by 3–3′ diaminobenzidine as chromogen (Vectasin ABC Kit, Vector, CA), using antibodies against CD20 (clone L26, IgG2a, Dako, Glostrup, Denmark), and CD3 (clone PS1, IgG2a, Immunotech, Marseille, France). Immunohistochemistry was then performed on frozen lymph node biopsies using 5-micrometer-thick cryostat sections with the same avidin-biotin-peroxidase protocol revealed by 3–3′ diaminobenzidine as chromogen. Antibodies used on frozen sections were CD22 (clone To15, IgG2b, Dako), CD3 (clone PSI, IgG2a, Immunotech), CD5 (clone L17F12, IgG2a, Becton Dickinson, Mountain View, CA), CD10 (clone ALB1, IgG1, Immunotech), CD23 (clone MHM6, IgG1, Dako), CD62L (L-selectin, clone DREG-56, IgG1, R&D Systems, Minneapolis, MN), anti-α4β7 (clone Act-1, IgG1, kindly provided by Dr. A.I. Lazarovits, Robarts Research Institute, University of Western Ontario, London, Ontario, Canada), CD49d (VLA4, α4 integrin chain, clone 44H6, IgG1, T-Cell Diagnostics, Woburn, MA), and CD29 (β1 integrin chain, clone K20, IgG2a, Immunotech). Cases were considered positive for α4β7 staining when most tumoral cells stained positive (>70%). The topography of α4β7 positivity was compared to B-cell antigen staining to distinguish between T cells and tumoral B-cell nodules. When only minor populations showed positive α4β7 staining, this positivity was shown to correspond to T cells' areas.
Molecular Analysis
Cytogenetic and molecular studies of the t (11;14) translocation and of the cyclin D1 mRNA could be performed as previously described
Mauvieux L, Canioni D, Hermine O, Valensi F, Radford-Weiss I, Azagury M, Magen M, Flandrin G, Brousse N, and BV, Macintyre EA: Quantitative RNA slot-blot analysis of CCND1/cyclin D1 expression in suspected mantle cell lymphoma. Leukemia 1998 (in press)
Troussard X, Mauvieux L, Radford-Weiss I, Rack K, Valensi F, Garand R, Vekemans M, G F, Macintyre E: Genetic analysis of splenic lymphoma with villous lymphocytes (SLVL): A Groupe Français d'Hématologie Cellulaire (GFHC) study. Br J Haematol 1998, (in press)
Mauvieux L, Canioni D, Hermine O, Valensi F, Radford-Weiss I, Azagury M, Magen M, Flandrin G, Brousse N, and BV, Macintyre EA: Quantitative RNA slot-blot analysis of CCND1/cyclin D1 expression in suspected mantle cell lymphoma. Leukemia 1998 (in press)
(Table 1). To further confirm the diagnosis, cytogenetic and molecular studies were informative in 10 of 13 cases (Table 1). Patients were staged according to the Ann Arbor classification on the basis of physical examination, routine laboratory tests, chest X-ray and/or CT scan, abdominal ultrasound and/or CT scan, and bone marrow trephine biopsy.
Table 1Clinical and Biological Data of 13 Patients with MCL
Patients were staged according to the Ann Arbor system. NS, normal or nonspecific lesion; P, features of lymphomatous polyposis (abnormal thick folds in stomach and/or polyps in small bowel); G, gastric; GI, gastrointestinal; 0, no digestive tract involvement; SM, splenomegaly; CS, circulating lymphoma cells; BM, bone marrow involvement; LDH, lactate dehydrogenase; nd, not done.
* Detection of the t(11;14) by karyotype and/or polymerase chain reaction.
Because of the frequency of GI involvement in MCL, upper and lower digestive tract endoscopic examination was done in the absence of prominent digestive symptoms in all 13 cases, 11 at diagnosis and 2 at relapse. Seven patients displayed histological features of GI involvement by lymphoma cells (six at diagnosis, one at relapse); six patients did not (five at diagnosis, one at relapse).
Characteristics of Digestive Tract Involvement
Digestive tract involvement was not associated with specific digestive clinical symptoms. In the seven patients with histologically proven digestive tract involvement, one patient had diarrhea (patient 2), three had a history of abdominal pain (patients 1, 4, and 5), and three patients had no symptoms. Endoscopy revealed features of lymphomatous polyposis consisting of abnormal thick folds in the stomach and/or polyps in the small bowel in four cases (patients 2, 4, 5, and 6), and either a normal aspect or nonspecific lesions (ie, gastritis) in three cases (patients 1, 3, and 7). Histological examination revealed multifocal involvement in six out of the seven positive cases (Table 1).
α4β7 Expression on Peripheral Lymph Node Lymphoma Cells Is Associated with GI Tract Involvement in Patients with Mantle Cell Lymphoma
α4β7 heterodimeric integrin expression on peripheral lymph node lymphoma cells at diagnosis was absent in all patients (6/6) without GI tract involvement. In contrast, expression of α4β7 on peripheral lymphoma cells was detected in five of the seven patients with histologically proven digestive involvement (P = 0.03) (Figure 1, Table 1, Table 2). Patients 3 and 7, who displayed microscopic digestive tract involvement without α4β7 positivity of tumoral cells from peripheral lymph node, did not present macroscopic GI involvement at endoscopy. Lymphoma cells infiltrating the GI tract expressed α4β7 in 3/3 tested patients with α4β7 positivity of tumoral cells from peripheral lymph node, whereas in patient 7, tumoral cells from the GI tract were not recognized by the anti-α4β7 antibody. Thus, in this patient tumoral cells from both the GI tract and the peripheral lymph node did not express α4β7.
Figure 1A: Peripheral lymph node in MCL patient without digestive tract involvement. Lymphoma cells are not stained with the anti-α4β7 ACT-1 antibody, although scattered infiltrating lymphocytes express α4β7 (brown peroxidase staining). B: In contrast, in a case with digestive tract involvement, peripheral lymph node lymphoma cells are positive for α4β7. Endothelial cells are negative.
In contrast, peripheral tumoral cells from all tested patients (11/11) co-expressed α4 and β1 integrin chains. Lymphoma cells in almost all MCL patients (11/12) did not express the lymph node homing receptor CD62L.
Interestingly, splenomegaly was present in all cases with digestive involvement, but in only two of six cases without digestive involvement (P = 0.04) (Table 2). However, when comparing α4β7-positive patients to α4β7-negative patients, the correlation with splenomegaly was not statistically significant (5/5vs. 4/8).
Discussion
The present study shows evidence that a large proportion of peripheral nodal B-cell MCLs express the mucosal homing receptor α4β7 and are associated with multifocal lymphomatous involvement of the GI tract. In contrast, MCL without digestive tract involvement did not express α4β7. α4β7 integrin expression may thus represent a specific marker of GI tract involvement in MCL. Moreover, splenomegaly appears to be more frequent in α4β7-positive MCL. These data on lymphoma cells are consistent with the physiological pattern of expression of α4β7 ligand MAdCAM-1 at the mRNA and protein levels in the human bowel and spleen.
Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1) demonstrates structural and functional similarities to the α4β7-integrin binding domain of murine MAdCAM-1, but extreme divergence of mucin-like sequences.
However, not all patients with digestive tract involvement were found to express α4β7 in our study. Two patients displayed microscopic digestive tract involvement without α4β7 positivity of tumoral cells in the peripheral lymph node. Both patients had circulating lymphoma cells, as did three of the five patients with α4β7-positive lymph node. Both patients also displayed unremarkable macroscopic features at endoscopy, in contrast to four of the five α4β7-positive patients. Interestingly, in one of these two patients (patient 7) α4β7 immunostaining could be performed on digestive tract tumoral cells and was negative. Thus, in these two cases digestive tract involvement may be due to a nonspecific leukemic spreading, as suggested by the lack of macroscopic lesions and the negativity of the α4β7 staining of digestive tract tumoral cells, or to an unknown adhesion molecule. Alternatively, we cannot exclude the possibility that the α4β7 antigen might be present but not recognized by the ACT-1 antibody in these two patients.
While the present study was in progress, others reported that T-cell leukemia/lymphoma dissemination to the digestive tract may also be related to α4β7 expression detected at the peripheral level.
these data strongly suggest that GI tract involvement is associated with α4β7 expression in T-cell, B-cell, and dendritic cell neoplasms.
Although data on the molecular basis of normal B-cell homing are scarce, we may hypothesize that expression of the mucosal receptor α4β7 on a subset of MCL might be instrumental in their dissemination to the digestive tract. MCL cells have been proposed to originate from follicle mantle cells,
α4β7-positive MCL may thus represent the neoplastic counterpart of follicle mantle cells originating from digestive tract-associated lymphoid tissue, having thus acquired α4β7,
whereas α4β7-negative MCL may originate from the peripheral lymph node's follicle mantle cells.
In agreement with this hypothesis, a primary GI lymphoma known as multiple lymphomatous polyposis (MLP), which represents about 8% of the primary GI lymphomas,
However, these studies did not investigate the correlation between α4β7 expression by peripheral tumoral cells and digestive tract involvement in presenting peripheral MCL. The two clinical entities, MLP and MCL, clearly overlap. Recent studies showed that 29% to 42% of lymphomas diagnosed as MLP are associated at diagnosis with enlarged peripheral lymph nodes.
Multiple lymphomatous polyposis of the gastrointestinal tract. A clinicopathologically distinctive form of non-Hodgkin's lymphoma of B-cell centrocytic type.
Therefore, MCL appears to be a heterogeneous disease with two distinct clinical and phenotypic forms, ie, α4β7-positive “digestive homing,” which may originate from the mucosal associated lymphoid tissue and be identical to MLP, and α4β7-negative peripheral MCL, usually without preferential tropism for the digestive tract.
The homing of lymphocytes to peripheral lymph nodes is thought to depend on L-selectin (CD62L, peripheral lymph node homing receptor)
Surprisingly, although lymph nodes were involved, CD62L was not expressed by either α4β7-positive or α4β7-negative MCL nodal tumoral cells in this study, in contrast to small lymphocytic lymphoma (unpublished data), suggesting that L-selectin is not responsible for lymph node involvement in MCL. However, all tested MCL expressed both α4 and β1 integrins. The α4β1 integrin is involved in adhesion to cytokine-activated endothelial cells, to germinal centers within lymph nodes,
Follicular non-Hodgkin's lymphoma cell adhesion to normal germinal centers and neoplastic follicles involves very late antigen-4 and vascular cell adhesion molecule-1.
However, GI tract involvement was not systematically explored in patients with MCL until the present work, so a definitive conclusion cannot be drawn, and no prospective study is available. More advanced disease in MCL patients with digestive tract involvement might reflect the late discovery (ie, when peripheral lymph nodes are involved) of the primary intestinal disease known as MLP. Alternatively, it might result from more aggressive behavior of α4β7-positive lymphoma cells, because it was recently suggested that α4β7-positive lymphoblastic lymphomas share an aggressive clinical course.
The present study shows that α4β7 expression is strongly associated with and may predict GI tract involvement in MCL. This finding may support the recognition of two distinct clinical and phenotypic forms of MCL, ie, “digestive homing” vs. “peripheral,” and help to identify patients for whom GI tract endoscopic examination is suitable. Further studies addressing the prognostic significance of α4β7 expression should be considered. Moreover, therapeutic strategies currently under development should include exploration of the digestive tract to ensure the quality of response to therapy.
Acknowledgements
We thank Dr. A.I. Lazarovits, who kindly provided the ACT-1 antibody, the nurses who took care of the patients, and Ms. Michelle Leborgne for excellent technical assistance.
Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1) demonstrates structural and functional similarities to the α4β7-integrin binding domain of murine MAdCAM-1, but extreme divergence of mucin-like sequences.
Follicular non-Hodgkin's lymphoma cell adhesion to normal germinal centers and neoplastic follicles involves very late antigen-4 and vascular cell adhesion molecule-1.
Mauvieux L, Canioni D, Hermine O, Valensi F, Radford-Weiss I, Azagury M, Magen M, Flandrin G, Brousse N, and BV, Macintyre EA: Quantitative RNA slot-blot analysis of CCND1/cyclin D1 expression in suspected mantle cell lymphoma. Leukemia 1998 (in press)
Troussard X, Mauvieux L, Radford-Weiss I, Rack K, Valensi F, Garand R, Vekemans M, G F, Macintyre E: Genetic analysis of splenic lymphoma with villous lymphocytes (SLVL): A Groupe Français d'Hématologie Cellulaire (GFHC) study. Br J Haematol 1998, (in press)
Multiple lymphomatous polyposis of the gastrointestinal tract. A clinicopathologically distinctive form of non-Hodgkin's lymphoma of B-cell centrocytic type.
Supported by grants from the Association pour la Recherche sur le Cancer (no. 4023) and the Comité de Paris/Ligue Nationale contre le Cancer no. (97/RS-RC/52). FG is a recipient of a Prix de l'Internat fellowship from the Assistance Publique-Hôpitaux de Paris.
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