UvA-DARE (Digital Academic Repository)
Cell adhesion receptors in lymphoma dissemination
Drillenburg ook genaamd Lelijveld, P.
Publication date 1999 Document Version Final published version
Link to publication
Citation for published version (APA): Drillenburg ook genaamd Lelijveld, P. (1999). Cell adhesion receptors in lymphoma dissemination.
General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.
UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
Download date:04 Oct 2021 JÈ^PE "- - • : im -"* Cell Adhesion Receptors iii Lymphoma Dissemination Paul Drillenburg UBA003000065 Cell Adhesion Receptors in Lymphoma Dissemination Financial support for the publication of this thesis is greatly acknowledged from: Compex Nederland BV ISBN 90-9012497-7 This thesis was prepared at the Department of Pathology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands Cover: low grade lymphoma of MALT. a4ß7 positive tumor cells (blue) invade epithelial structures (brown) to form lympho-epithelial lesions (by J. Mulder). Cell Adhesion Receptors in Lymphoma Dissemination ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus Prof.dr. J.J.M. Franse ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Aula der Universiteit op donderdag 1 april 1999, te 13.00 uur door Paul Drillenburg ook genaamd Lelijveld geboren te Voorschoten Promotiecommissie Promotor: Prof. dr. S.T. Pais Leden: Prof. dr. S.J.H, van Deventer Prof. dr. Ph.M. Kluin Prof. dr. G. Kraal Prof. dr. C.J.L.M. Meijer Dr. L.A. Noorduyn Dr. M.H.J. van Oers Table of Contents Abbreviations Chapter 1 Introduction Chapter 2 Expression of the mucosal homing receptor a4ß7 in malignant 27 lymphomatous polyposis of the intestine. Gastroenterology 1994; 107:1519-1523 Chapter 3 Preferential expression of the mucosal homing receptor integrin 33 a4ß7 in gastrointestinal non-Hodgkin's lymphomas. American Journal of Pathology 1997; 150:919-927 Chapter 4 Expression of adhesion molecules in pagetoid reticulosis 43 (Woringer-Kolopp disease) British Journal of Dermatology 1997; 136:613-616 Chapter 5 CD44 expression predicts disease outcome in localized large 49 B-cell lymphoma. submitted Chapter 6 Local therapy alone is effective for stage I, CD44' 67 diffuse large B-cell lymphomas. Chapter 7 Cell adhesion receptors in lymphoma dissemination 75 submitted Summary /Samenvatting 101 Nawoord 108 Curriculum vitae 109 Abbreviations AIDS acquired immunodeficiency syndrome APC antigen presenting cell CLA cutaneous lymphocyte antigen DLCL diffuse large B-cell lymphoma DC dendritic cell EAE experimental allergic encephalitis ECM extracellular matrix FDC follicular dendritic cell HA hyaluronic acid HEV high endothelial venule HSPG heparan sulfate proteoglycan ICAM-1 intercellular adhesion molecule-1 IPI International prognostic index LFA-1 lymphocyte function associated antigen-1 mAb monoclonal antibody MAdCAM-1 mucosal addressin cell adhesion molecule-1 MALT mucosa associated lymphoid tissue MLP malignant lymphomatous polyposis NHL non-Hodgkin's lymphoma PLN peripheral lymph node PNAd peripheral lymph node addressin REAL revised European-American lymphoma classification VCAM-1 vascular cell adhesion molecule-1 VLA very late antigen Chapter 1 Introduction Chapter 1 1. Introduction In the human immune system three distinct compartments can be recognized. The primary lymphoid organs, bone marrow and thymus, produce naive lymphocytes which disseminate through the body to search for their cognate antigen. In the secondary lymphoid organs (lymph nodes and spleen) the anatomy is optimal for efficient lymphocyte-antigen interaction. After antigen-specific lymphocyte activation, the effector and memory cells migrate to the extralymphoid or tertiary lymphoid organs to be functionally active [1,2]. The functioning of the immune system depends on the pool of lymphocytes travelling between the different lymphoid compartments. These lymphocytes can be divided into two groups based on activation stage. The naive, non-activated lymphocytes leaving the bone marrow and thymus, freely move from the bloodstream through the secondary lymphoid organs and back to the blood. This process of lymphocyte recirculation comprises binding of lymphocytes to the specialized vascular endothelium of high endothelial venules (HEV), extravasation, migration through the lymphoid tissue and, via the efferent lymph vessels back to the systemic circulation [1-4]. After antigen-specific stimulation in the secondary lymphoid organs, the activated lymphocytes alter their recirculation pattern. These activated, effector or memory cells no longer freely move through the secondary lymphoid organs but migrate specifically to the sites that mimiek those where they originally were activated. The recirculation patterns of naive and effector or memory cells depend on the presence of adhesion molecules on the surface of lymphocytes called homing receptors. Binding of these adhesion molecules to tissue specific counterparts on the vascular endothelium (addressins) is essential in the first step of lymphocyte recirculation. After antigen stimulation of a naive lymphocyte, altered expression of adhesion molecules restricts recirculation of the resultant effector or memory cell to the site and related organs of initial antigen presentation [1,2,5]. The adhesion molecules involved in lymphocyte interaction with endothelium belong to 4 families: the selectins, the immunoglobulin super family, the integrins and CD44 (fig.l). 2. Adhesion molecules 2.1 Selectins The selectin family, comprising L-selectin(CD62L), E-selectin (CD62E) and P-selectin (CD62P), is involved in leukocyte-endothelium interaction. These molecules are characterized by an N-terminal lectin domain, an epidermal growth factor domain, a variable number of tandem consensus repeats similar to domains of complement-bindingproteins, a transmembrane segment and a short cytoplasmic domain [6-10]. The N-terminal lectin domain is, in the presence of calcium ions, the binding site of these molecules with their carbohydrate ligands [10]. L-selectin, which is also known as peripheral lymph node homing receptor, is only present on leukocytes. It is expressed on all neutrophils, monocytes and on essentially all 8 Introduction Selectins Ig -family Integrins CD44 L-selectin ICAM-1 a4ß1 multiple E-selectin ICAM-2 aLß2 splice- P-selectin VCAM-1 a4ß7 variants MAdCAM-1 aEß7 Figure 1. Adhesion molecules involved in lymphocyte homing. virgin/naive T and B lymphocytes. On CD4+ memory T cells expression of L-selectin is restricted to a subset with homing properties to the peripheral lymph nodes [11]. In mice, functional inactivation of L-selectin by gene targeting results in diminished lymphocyte binding capacity to HEV, and a reduction in the number of lymphocytes localized to peripheral lymph nodes [12]. Cytokines can modulate the expression of L-selectin, but only interferon-a increases the synthesis [13]. After activation L-selectin is rapidly shed from the surface of leukocytes as a mechanism of downregulating its function [14]. Expression of E-selectin is limited to endothelium. Inflammatory stimuli, such as interleukin-1 and tumor necrosis factor-a upregulate E-selectin within a few hours. It is postulated that E-selectin functions as a skin-selective endothelial addressin that preferentially binds skin-homing lymphocytes [15]. However, in case of inflammation, expression of E-selectin is not restricted to the skin [6]. P-selectin is stored preformed in small cytoplasmic granules and is only expressed on platelets and endothelium [10,16]. Activation stimuli, i.e. thrombin, histamine, activators of protein kinase C and complement fragments, mediate rapid expression of P-selectin at the cell surface as a result of fusion of the cytoplasmic granules with the plasma membrane. 2.2 Immunoglobulin family The molecules of the immunoglobin family share the presence of one or more Chapter 1 immunoglobulin domains composed of 90-100 amino acids arranged in a loop that is stabilized by a disulphidebond. Members of this family, ICAM-1, ICAM-2, VCAM-1 and MAdCAM-1, are expressed on endothelium and are involved in lymphocyte adhesion and extravasation. The intercellular adhesion molecule-1 (ICAM-1), the counter receptor of the integrins LFA-1 and Mac-1, mediates stable adhesion of lymphocytes to endothelium in the process of leukocyte extravasation [1,17-19]. LFA-1/ICAM-1 interaction has been implicated in T- lymphocyte mediated killing and T cell interaction with antigen presenting cells [20,21]. ICAM- 2 is constitutively expressed on endothelium and lymphocytes and can also bind to LFA-1 [22], VCAM-1 is the ligand for the integrin a4ß, [23,24]. In vivo VCAM-1 is expressed on endothelium at very low levels. However, cytokine activation can induce high expression of VCAM-1 [25]. a4ßi/VCAM-l interaction is important in adhesion of lymphocytes to cytokine activated endothelium [26]. MAdCAM-1 is a member of the immunoglobulin family with expression on high endothelium in Peyer's patches and mesenterial lymph nodes and on flat-walled vessels in the lamina propria of the gut [27,28]. The main receptor for MAdCAM-1 is the integrin a4ß7, an adhesion molecule on lymphocytes involved in selective homing to mucosal sites [29-31 ]. Other leukocyte surface molecules, such as L-selectin and a4ß,, can also mediate initial binding to MAdCAM-1 in the process of leukocyte-endothelium interaction [32-35]. In the spleen, MAdCAM-1 is expressed on sinus-lining cells closest to the lymphoid white pulp, but so far no functional role has been demonstrated [36]. 2.3 Integrins Integrins are heterodimeric transmembrane glycoproteins composed of noncovalently associated a and ß chains [23,37]. Most integrins are expressed on a wide variety of cells, and most cells express several integrins. According to their ß chain, the integrins have been divided into different subfamilies. In leukocyte-endothelium interactions the ß1; ß2 and ß7 integrins are important. The ß, integrins, synonymous with 'very late activation' antigens (VLA), can form a heterodimer with 8 different a chains (VLA-1 - VLA-8). They are expressed in a range of different cell types and are predominantly involved in cell binding to the extracellulair matrix molecules collagen, fibronectin and laminin [23,37,38]. The specificity of the binding is determined by the a chain. On peripheral blood T and B lymphocytes a4ß, (VLA-4) is the most abundant ß, integrin and is upregulated after activation. It is not only involved in cell-matrix but also in cell-cell interactions [39,40]. Besides binding to the CS-1 domain of fibronectin, it can bind to the vascular-cell adhesion molecule-1 (VCAM-1), expressed on activated endothelium. a4ß, is an important molecule in the process of lymphocyte migration to inflammatory sites [41- 43]. Furthermore, it can mediate cell-cell interactions in a homotypic manner via the a4 chain 10 Introduction [44]. The family of ß2 integrins consists of LFA-1, Mac-1, p 150,95 and the recently described aDß2, which are expressed on lymphoid and myeloid cells [37,45]. The ß2 integrins are involved in the regulation of cell-cell interactions. LFA-1 is expressed on nearly all lymphocytes and is one of the most important integrins of the immune system[46]. LFA-1 mediates lymphocyte adhesion to activated endothelium and HEV [19,37,47,48], binding to antigen presenting cells [49,50], follicular dendritic cells [51] and epithelium [52]. Particularly after lymphocyte activation, LFA-1 can bind to the endothelial intercellular adhesion molecules ICAM-1 and ICAM-2, creating a stable lymphocyte-endothelialinteraction [1,17,21,22,34,53]. Lymphocytes express only low levels of Mac-1 and pi 50,95 and it is likely that these ß2 integrins do not play a major role in lymphocyte-endothelial interaction. Although expressed at low levels on other leukocytes, aDß2 is mainly involved in macrophage functions [54]. At present the ß7-integrin subfamily consist of two members: a4ß7 and aEß7. a4ß7 is present at low levels on all naive peripheral blood lymphocytes and on a subset of memory T cells with gut tropism [55,56]. It is involved in lymphocyte migration to the intestinal mucosa by binding to mucosal vascular addressin (MAdCAM-1 ), an addressin selectively expressed on mucosal endothelium [28,29,57,58]. The integrin aEß7 is present on all intra-epithelial lymphocytes in the gut, and on approximately 50% of the T-lymphocytes in the lamina propria. Via binding to E-cadherin on epithelial cells, 2.4 CD44 CD44 is a family of transmembrane glycoproteins, encoded by a single gene on chromosome 11 q 13. (fig.2) [61]. Members of the CD44 family have been implicated in a number of important physiological and pathological processes, including lymphocyte functioning, hematopoiesis, tumor progression and metastasis [62-73]. The CD44 gene consists of 19 exons; as the result of alternative RNA-splicing of at least 10 exons, by which additional domains can be inserted in the extracellular part of the molecule, a large number of CD44 isoforms can be generated [74]. Furthermore, CD44 can posttranslationally be modified by N&O linked sugars and glycosaminoglycan side chains [71]. On lymphocytes and hematopoietic cells the 85-95 kD 'standard' or 'hematopoietic' isoform of CD44 is widely expressed, while larger isoforms are present on activated lymphocytes and malignant lymphomas [61,70,71,75-78]. Expression of CD44 on lymphocytes is important in lymphopoiesis, lymphocyte activation, trafficking and homing [62,66,79-83]. In the process of lymphocyte-endotheliuminteraction, CD44 can bind to high endothelial venules, mediates rolling on post-capillair venules, and migration of lymphocytes to inflammatory sites [71,82,84-88]. Most of these functions involve interaction of CD44 with hyaluronic acid. 11 Chapter I 166 • 2 134 H 3 69 • 4 Cartilage link protein homology domain 230 • 5 (223 aa) Growth factors: HGF/SF 126 \vß è-„ k. FGF 126 |~v3| 7 114 [v4] 8 117 {yë\ 9 129 |~v6l 10 132 |~v7| 11 102 fv8l 12 (0-381 aa 90 fv9|l3 204 [yjö] 14 (45 aa 63 • 15 72 Bi 16 nnnnfinnê^nrinnnnnnnnnnnnnnnnnn 79 ^17TM (23 aa) ^A Plasma membrane 4 i=p f 8~ " ~ -uuuuuuüBwuuuyuuuuuuuuuuuuuuuu 205 rn 19 Cytoplasmic domain (3 or 70 aa) Figure 2. Schematical representation of the CD44 gene and its encoded proteins. The extracellular domain and cytoplasmic tail of CD44 isoforms vary in size as the result of alternative splicing. The alternatively spliced exons are indicated by open boxes. The human vl exon contains a stop codon. In the model of the protein, all putative glycosylation sites are indicated: O-glycosylation (open circles); N-glycosylation (closed circles); chondroitin sulfate (open squares); heparan sulfate (rod). As indicated, the heparan sulfate binding site in exon v3 has the ability to bind growth factors. In addition, the HA-binding sites (double line); the disulfide bonds (S-S); the ankyrin binding site ( ); the Ezrin binding site (—-); the phosphorylation sites (P); and the putative interaction sites for SRC-family kinases, are indicated. 12 Introduction Recently, CD44 splice variants decorated with heparan sulfate side chains have been shown to bind and present chemokines and growth factors [89-91]. 3. Regulation of lymphocyte homing Lymphocyte-endothelium interactions leading to stable adhesion and diapedesis, involve a number of sequential adhesive interactions. Besides adhesion per se, these interactions are also important in regulating other cell functions e.g. cell growth, and cell survival [92,93]. Selective expression and regulation of adhesion molecules on lymphocytes and endothelium forms the basis for tissue-specific lymphocyte homing. The interaction of lymphocytes with endothelium is a multistep process [ 1,19,34,94]. The first step is a loose "tethering" engagement between the lymphocyte and the endothelium, leading to a characteristic rolling movement over the vascular endothelium of the post-capillary venule. In this step the long molecules of the selectin family localized on the tips of the cell membrane's microvilli are involved [95,96]. However, other molecules such as the integrins ß„ ß7 and CD44 can also mediate lymphocyte rolling [34,86,87,97,98]. This rolling movement is transient if it is not followed by activationof adhesion molecules of the integrin family. These molecules, i.e. a4ß,, LFA-1 and a4ßy, mediate stable adhesion and the possibility for diapedesis. Extremely rapid integrin activation is regulated by chemokines which can be presented to lymphocytes by binding to proteoglycans on the endothelium [99-104]. Expression of adhesion molecules on lymphocytes is influenced by antigen and/or chemokine mediated activation signals. Initial lymphocyte-antigen interaction will alter the cell surface profile drastically, resulting in a set of adhesion molecules involved in tissue specific homing to the sites of primary antigenic stimulation. The ligands for these lymphocyte 'homing' receptors are site-specific endothelial surface molecules ('vascular addressins') crucial in tissue specific recirculation of lymphocytes. Combinations of lymphocyte adhesion molecules and vascular addressins involved in tissue-specific homing are: a4ß7/MAdCAM-l for MALT; cutaneous lymphocyte antigen (CLA) /E-selectin for skin and L-selectin/ PNAd for peripheral lymph node. Binding of aEß7 to E-cadherin expressed on epithelial cells, is important in positioning of lymphocytes in epithelium. 4. Non-Hodgkin's lymphomas Malignant lymphomas form a diverse group of tumors, heterogeneous in clinical behaviour and morphologic appearance. Based on the morphology of Reed-Sternberg cells in an appropriate environmentofreactiveleukocytes,Hodgkin'sdisease is distinctive within this broad group of neoplasms. All other forms of the malignant lymphomas constitute the non-Hodgkin's lymphomas (NHL). In 1995 in the USA 22700 people died from NHL, which represent 4% of all cancer 13 Chapter 1 deaths [105]. NHL incidence rates increase exponentially with age: 8.5 annual cases per 100 000 under 65 years of age versus 68.8 per 100 000 in persons 65 and over. There is a male predominance, which is more outspoken in younger than older patients. Compared to other neoplasms, the incidence of NHL has increased rapidly over the last decades. In the USA, in the period between 1947-1988 the annual NHL incidence increased with 150% [105]. The etiology of NHLs is mostly unknown. However, two viruses, Epstein-Barr virus (EBV) and human T cell leukemia/lymphomavirus (HTLV-1) are etiologically associated with respectively, endemic Burkitt's lymphoma in central equatorial Africa and T cell leukemia/lymphoma in areas of Japan. The risk of NHL is greatly increased in patients with a congenital or acquired immunodeficiency. Particularly in patients with AIDS or in transplant recipients treated with immunosuppressiva,there is an increased risk for aggressive B-cell NHLs. In approximately 40% of AIDS-related lymphomas EBV DNA can be detected, however the precise role of EBV in the malignant transformation of these lymphomas is not understood. For a number of occupational and environmental factors (i.e. herbicide, hair dyes and benzene) a role in the pathogenesis of NHL has been suggested, however, at the moment no unequivocal evidence is present. At present a number of genetic changes, important in the formation of non-Hodgkin's lymphomas have been identified. For B-cell NHLs, the most frequent alterations are chromosomal translocations involving the immunoglobulin heavy (chr. 14) or light chain gene loci (lambda, chr. 22; kappa, chr. 2) and oncogenes encoding proteins involved in cell proliferation (c-myc) and differentiation(bcl-6), cell cycle control (cyclinDl/bcl-1) or apoptosis inhibition (bcl-2) [106-109]. In approximately one third of the anaplastic large-cell NHLs the t(2,5)(p23;q35) has been observed [110]. This translocation results in the fusion of the amino terminus of the nucleophosmin gene on chromosome 5 with the anaplastic lymphoma kinase (ALK) gene on chromosome 2 [111]. Rearrangements involving the T-cell a and ô receptor gene loci, localized on 14q 11, are often observed in T-cell NHLs. The most common of these are the reciprocal translocations t(l 1;14) and t(8;14) [112,113]. The development of lymphocytes from precursor cell to effector cell, is characterized by phenotypical changes. Since maturation arrest and clonal expansion are important in the formation of non-Hodgkin' s lymphomas, the phenotype of the tumor cells is reminiscent to that of their normal counterparts. Small lymphocytic lymphomas/ chronic lymphocytic leukemia (CLL) and mantle cell lymphomas are thought to be derived from 'naive' pre-germinal center B-cells. By contrast, follicle center lymphomas, diffuse large B-cell lymphomas, Burkitt's lymphomas and the myelomas are related to stages of antigen driven B-cell development. These lymphomas are related to germinal center and post-germinal center cells, or in the case of myelomas to plasma cells [114,115]. In 1966 Rappaport proposed a classificationof NHLs based on morphology [116]. In this 14 Introduction system, growth pattern (nodular or diffuse) and the shape of the tumor cells (lymphocytic or histiocytic) were used to predict the clinical grade of malignancy. Although applied for many years in clinicopathological investigations, it became clear that the classification of NHLs had to be improved. Based on the physiology of lymphocyte development, new classifications were proposed, i.e. the Kiel classification and the Lukes-Collins classification [117,118]. However, none of them gained worldwide acceptance and in a study of the National Cancer Institute in the US, none of them proved to be superior in reproducibility and predictive value for disease outcome [119]. This led to the so called Working Formulation for clinical usage, which divides malignant lymphomas into a low, intermediate and high grade of malignancy based on strictly morphological criteria. In an attempt to categorize NHL in well described subgroups relevant for treatment and research, recently, the Revised European-American Lymphoma (REAL) classification was proposed. This classification is not only based on morphology but also on modern insights in the tumorbiology of lymphomas [114]. Since in the REAL classification the reproducibility of criteria for categorization is emphasized, it is accepted that some subgroups of NHL comprise a diverse group of neoplasms. For instance the diffuse large B-cell lymphomas (DLCL) are heterogeneous in genetic background, morphology and treatment response, but form a single entity since criteria for a clinically relevant subdivision are currently lacking [115,120-123]. New, prognostic markers (morphologically, functionally or genetically) are needed to solve this problem in the diagnosis of NHL. 5. Lymphoma dissemination Dissemination of malignant lymphomas depends on the type of NHL and the site of origin. While B-cell chronic lymphocytic leukemia/small lymphocytic lymphomas are extensively disseminated in the early phase of the disease, diffuse large B-cell lymphomas remain often localized initially and tend to disseminate primarily to adjacent lymph nodes. The extranodal lymphomas, low grade B-cell lymphoma of MALT and mycosis fungoides, show often at diagnosis dissemination limited to mucosal sites and skin, respectively. These observations indicate that NHLs follow, in part, the lymphocyte migration pathways of their presumptive non-neoplastic counterparts. Since adhesion molecules are important in the physiology of lymphocyte migration and recirculation, these molecules may also be involved in lymphoma dissemination. Expression of adhesion molecules might hence characterize specific subgroups of NHLs, and might be a marker for dissemination propensity. 6. Outline of this study In the process of normal lymphocyte migration and recirculation,cell-adhesionmolecules are important in lymphocyte-endotheliuminteractions. Subsets of adhesion molecules expressed 15 Chapter 1 on lymphocytes ('homing receptors'), are involved in tissue-specificmigration, by binding to site specific endothelial ligands (addressins). Since the dissemination patterns of malignant lymphomas, at least partly, resemble the normal lymphocyte migration pathways, adhesion molecules may also be involved in the spread of NHLs. In this study the role of adhesion molecules in the dissemination of non-Hodgkin's lymphomas was investigated. Malignant lymphomatouspolyposis (MLP) is a malignant lymphoma of mantle cell type, located in the gastro-intestinal tract, with the endoscopic picture of multiple polyps. This multifocal distribution throughout the intestinal tract suggest a role for tissue-specific homing mechanisms in the dissemination. In chapter 2 the expression of adhesion molecules in MLP is compared to that in nodal mantle cell lymphomas. The role of a4ß7 in lymphoma dissemination was further investigated in chapter 3. In a large panel of B- and T-cell NHLs, a4ß7 expression was related to the primary site of origin of the lymphoma. Furthermore, expression of a4ß7 was investigated on normal lymphocytes from the tonsil, mucosa and peripheral blood. Pagetoid reticulosis or Woringer-Kolopp disease is a cutaneous T-cell lymphoma with a characteristic distribution of tumor cells within the epidermis. The selective dissemination to the skin, and the extreme epitheliotropism of the tumor cells suggest the involvement of a specific set of adhesion molecules in the pathogenesis of pagetoid reticulosis. In chapter 4 the expression of adhesion molecules in a case of pagetoid reticulosis is described. At the moment, NHLs are categorized according to the Revised European-American Lymphoma classification. In this classification diffuse large B-cell lymphomas form a diverse group of neoplasms, heterogeneous in genetic background and prognosis. The presence of adhesion molecules might be of value as a marker for predicting disease outcome, since these molecules play a role in tumor dissemination. In chapter 5 and 6 the value of the cell adhesion molecule CD44 in predicting disease outcome in diffuse large B-cell lymphomas was investigated in a multi-center study material. Chapter 7 comprises a review on the role of adhesion molecules in the dissemination of non-Hodgkin's lymphomas. References 1. Butcher EC, Picker LJ. Lymphocyte homing. Adv Immunol 1997; 64:139 homing and homeostasis. Science 1996; 3. Gowans JL, Knight EJ. The route of 272:60 lymphocyte recirculation in the rat. Proc 2. Salmi M, Jalkanen S. How do R Soc London Ser B 1964; 159:257 lymphocytes know where to go: current 4. Kraal G, Mebius RE. High endothelial concepts and enigmas of lymphocyte venules: lymphocyte traffic control and 16 Introduction controlled traffic. Adv Immunol 1997; 12. Arbonés ML, Ord DC, Ley K, Ratech 65:347 H, Maynard-Curry C, Otten G, Capon Mackay CR Marston WL Dudler L DJ, Tedder TF. Lymphocyte homing Spertini O Tedder TF Hein WR. Tissue- and leukocyte rolling and migration are specific migration pathways by impaired in L-selectin-deficient mice. phenotypically distinct subpopulations Immunity 1994; 1:247 of memory T cells. Eur J Immunol 13. Evans SS, Collea RP, Appenheimer 1992; 22:887 MM, Gollnick SO. Interferon-oc induces 6. Bevilacqua P, Stengelin S, Gimbrone the expression of the L-selectin homing MA, Seed B. Endothelial leukocyte receptor in human B lymphoid cells. J adhesion molecule 1: an inducible Cell Biol 1993; 123:1889 receptor for neutrophils related to 14. Jung TM, Dailey MO. Rapid complement regulatory proteins and modulation of homing receptors lectins. Science 1989; 243:1160 (gp90mel"14) induced by activators of Lasky LA, Singer MS, Yednock TA, protein kinase C. Receptor shedding Dovvbenko D, Fennie C, Rodriguez H, due to accelerated proteolytic cleavage Nguyen T, Stachel S, Rosen SD. at the cell surface. J Immunol 1990; Cloning of a lymphocyte homing 144:3130 receptor reveals a lectin domain. Cell 15. Picker LJ, Kishimoto TK, Smith CW, 1989; 56:1045 Warnock RA, Butcher EC. ELAM-1 is Siegelman MH, van de Rijn M, an adhesion molecule for skin-homing Weissman IL. Mouse lymph node T cells. Nature 1991; 349:796 homing receptor cDNA clone encodes a 16. McEver RP, Beckstead JH, Moore KL, glycoprotein revealing tandem Marshall-Carlson L, Bainton DF. GMP- interaction domains. Science 1989: 140, a platelet cc-granule membrane 243:1165 protein, is also sythesized by vascular Vestweber D. The selectins and their endothelial cells and is localized in ligands. Curr Topics Microbiol Weibel-Palade bodies. J Clin Invest Immunol 1993; 184:65 1989; 84:92 10. Kansas GS. Selectins and their ligands: 17. Dustin ML, Springer TA. Lymphocyte current concepts and controversies. function-associated antigen-1 (LFA-1) Blood 1996; 88:3259 interaction with intercellular adhesion 11. Picker LJ, Treer JR, Ferguson-Darnell molecule-1 (ICAM-1) is one of at least B, Collins PA, Buck D, Terstappen three mechanisms for lymphocyte LWMM. Control of lymphocyte adhesion to cultured endothelial cells. J recirculation in Man I. Differential Cell Biol 1988; 107:321 regulation of the peripheral lymph node 18. Diamond MS, Staunton DE, de homing receptor L-selectin on T cells Fougerolles AR, Stacker SA, Garcia- during the virgin to memory transition. Aguilar J, Hibbs ML, Springer TA. J Immunol 1993; 150:1105 ICAM-1 (CD54): a counter-receptor for 17 Chapter 1 Mac-1 (CDllb/CD18). J Cell Biol endothelial cells that is induced by 1990; 111:3129 tumor necrosis factor, IL-1, or 19. Springer TA. Traffic signals for lipopolysaccharide. J Immunol 1990; lymphcyte recirculation and leukocyte 144:2558 emigration: the multistep paradigm. 26. Shimizu Y, Newman W, Gopal TV, Cell 1994; 76:301 Horgan KJ, Graber N, Beall LD, van 20. Inaba K, Steinman RM. Monoclonal Seventer GA, Shaw S. Four molecular antibodies to LFA-1 and to CD4 inhibit pathways of T cell adhesion to the mixed leukocyte reaction after the endothelial cells: roles of LFA-1, antigen-dependent clustering of VCAM-1, and ELAM-1 and changes in dendritic cells and T lymphocytes. J pathway hierarchy under different Exp Med 1987; 165;1403 activation conditions. J Cell Biol 1991; 21. Makgoba MW, Sanders ME, Ginther 113:1203 Luce GE, Gugel EA, Dustin ML, 27. Streeter PR, Lakey Berg E, Rouse BTN, Springer TA, Shaw S. Functional Bargatze RF, Butcher EC. A tissue- evidence that intercellular adhesion specific endothelial cell molecule molecule-1 (ICAM-1) is a ligand for involved in lymphocyte homing. Nature LFA-1-dependent adhesion in T cell- 1988; 331:41 mediated cytotoxicity. Eur J Immunol 28. Briskin M, Winsor-Hines D, Shyjan A, 1988; 18:637 Cochran N, Bloom S, Wilson J, 22. de Fougerolles AR, Stacker SA, McEvoy LM, Butcher EC, Kassam N, Schwarting R, Springer TA. Mackay CR, Newman W, Ringler DJ. Characterization of ICAM-2 and Human mucosal addressin cell adhesion evidence for a third counter-receptor for molecule-1 is preferentially expressed LFA-1. J Exp Med 1991 ; 174:253 in intestinal tract and associated 23. Hynes RO. Integrins: versatility, lymphoid tissue. Am J Path 1997; modulation, and signaling in cell 151:97 adhesion. Cell 1992; 69:11 29. Berlin C, Berg EL, Briskin MJ, Andrew 24. Postigo AA, Sânchez-Mateos P, DP, Kilshaw PJ, Holzmann B, Lazarovits AI, Sânchez-Madrid F, Weissman IL, Hamann A, Butcher EC. Landâzuri MO. a4ß7 integrin mediates a4ß7 integrin mediates lymphocyte B cell binding to fibronectin and binding to the mucosal vascular vascular cell adhesion molecule-1. addressin MAdCAM-1. Cell 1993; Expression and function of a4 integrins 74:185 on human B lymphocytes. J Immunol 30. Briskin MJ, Rott L, Butcher EC. 1993; 151:2471 Structural requirements for mucosal 25. Wellicome SM, Thornhill MH, Pitzalis vascular addressin binding to its C, Thomas DS, Lanchbury JSS, Panayi lymphocyte receptor a4ß7. J Immunol GS, Haskard DO. A monoclonal 1996; 156:719 antibody that detects a novel antigen on 31. Rott LS, Briskin MJ, Andrew DP, Berg Introduction EL, Butcher EC. A fundamental Schwarz L, Strominger JL, Clabby ML. subdivision of circulating lymphocytes Characterization of the cell surface defined by adhesion to mucosal heterodimer VLA-4 and related addressin cell adhesion molecule-1. J peptides. J Biol Chem 1987; 262:11478 Immunol 1996; 156:3727 40. Hemler ME, Elices MJ, Parker C, 32. Berg EL, McEvoy M, Berlin C, Takada Y. Structure of the integrin Bargatze RF, Butcher EC. L-selectin- VLA-4 and its cell-cell and cell-matrix mediated lymphocyte rolling on adhesion functions. Immunol Rev 1990; MadCAM-1. Nature 1993; 366:695 114:45 33. Hamann A, Andrew DP, Jablonski- 41. van Dinther-Janssen ACHM, Horst E, Westrich D, Holzman B Butcher EC. Koopman G, Scheeper RJ, Newman W, Role of a4-integrins in lymphocyte Meijer CJLM, Pals ST. The homing to mucosal tissues in vivo. J VLA-4/VCAM-1 pathway is involved Immunol 1994; 152:3282 in lymphocyte adhesion to endothelium 34. Bargatze RF, Jutila MA, Butcher EC. in rheumatoid synovium. J Immunol Distinct roles of L-selectin and integrins 1991; 147;4207 35. Davenpeck KL, Sterbinsky SA, antibodies against a4ß, integrin. Nature Bochner BS. Rat neutrophils express cc4 1992; 356:63 and ß, integrins and bind to vascular 43. Lobb RR, Hemler ME. The cell adhesion molecule-1 (VCAM-1) pathophysiologic role of a4 integrins in and mucosal addressin cell adhesion vivo. J Clin Invest 1994; 94:1722 molecule-1 (MadCAM-1). Blood 1998; 44. Altevogt P, Hubbe M, Ruppert M, Lohr 91:2341 J, von Hoegen P, Sammar M, Andrew 36. Kraal G, Schornagel K, Streeter PR, DP, McEvoy L, Humphries MJ, Butcher Holzmann B, Butcher EC. Expression EC. The a4 integrin chain is a ligand for of the mucosal vascular addressin, a4ß7 and cc4ß, J Exp Med 1995; MAdCAM-1, on sinus-lining cells in 182:345 the spleen. Am J Pathol 1995; 147:763 45. Danilenko DM, Rossitto PV, van der 37. Springer TA. Adhesion receptors of the Vieren M, Trong HL, McDonough SP, immune system. Nature 1990; 346:425 Affolter VK, Moore PF. A novel canine 38. Shimizu Y, van Seventer GA, Horgan leukointegrin, adß2, is expressed by KJ, Shaw S. Regulated expression and specific macrophage subpopulations in binding of three VLA (ß,) integrin tissue and a minor CD8* lymphocyte receptors on T cells. Nature 1990; subpopulation in peripheral blood. J 345:250 Immunol 1995; 155:35 39. Hemler ME, Huang C, Takada Y, 46. Krensky AM, Sânchez-Madrid F, 19 Chapter 1 Robbins E, Nagy JA, Springer TA, Dermatol 1989; 92:746 Burakoff SJ. The functional 53. de Fougerolles AR, Springer TA. significance, distribution, and structure Intercellular adhesion molecule 3, a of LFA-1, LFA-2, and LFA-3: cell third adhesion counter-receptor for surface antigens associated with CTL- lymphocyte function-associated target interactions. J Immunol 1983; molecule 1 on resting lymphocytes. J 131:611 Exp Med 1992; 175:185 47. Haskard D, Cavender D, Beatty P, 54. van der Vieren M, Le Trong H, Wood Springer TA, Ziff M. T lymphocyte CL, Moore PH, St John T, Staunton DE, adhesion to endothelial cells: Gallatin WM. A novel leukointegrin, mechanisms demonstrated by anti-LFA- alpha d beta 2, binds preferentially to 1 monoclonal antibodies. J Immunol ICAM-3. Immunity 1995; 3:683 1986; 137:2901 55. Schweighoffer T, Tanaka Y, Tidswell 48. Hamann A, Jablonski-Westrich D, M, Erle DJ, Horgan KJ, Ginther Luce Duijvestijn A, Butcher EC, Baisch H, GE, Lazarovits AI, Buck D, Shaw S. Harder R, Thiele HG. Evidence for an Selective expression of integrin a4ß7 on accessory role of LFA-1 in lymphocyte- a subset of human CD4T memory T high endothelium interaction during cells with hallmarks of gut-trophism. J homing. J Immunol 1988; 140:693 Immunol 1993; 151:717 49. Scheeren RA, Koopman F, van der 56. Erle DJ, Briskin MJ, Butcher EC, Baan S, Meijer CJLM, Pals ST. Garcia-Pardo A, Lazarovits AI, Adhesion receptors involved in Tidswell M. Expression and function of clustering of blood dendritic cells and T the MAdCAM-1 receptor, integrin o,ß7, lymphocytes. Eur J Immunol 1991; on human leukocytes. J Immunol 1994; 21:1101 153:517 50. Steinman RM. The dendritic cell system 57. Parker CM, Cepek KL, Russell GJ, and its role in immunogenicity. Annu Shaw SK, Prosnett DN, Schwarting RL, Rev Immunology 1991; 9:271 Brenner MB. A family of ß7 integrins 51. Koopman G, Parmentier HK, on human mucosal lymphocytes. Proc Schuurman H-J, Newman W, Meijer Nati Acad Sei USA 1992; 89; 1924 CJLM, Pals ST. Adhesion of human B 58. Farstad IN, Halstensen TS, Lazarovits cells to follicular dendritic cells AI, Norstein J, Fausa O, Brandtzaeg P. involves both the LFA-1/ICAM-1 and Human intestinal B-cell blasts and VLA-4/VCAM-1 pathways. J Exp Med plasma cells express the mucosal 1991; 173:1297 homing receptor integrin a4ß7 Scan J 52. Singer KH, Tuck DT, Sampson HA, Immunol 1995; 42:662 Hall RP. Epidermal keratinocytes 59. Cerf-Bensussan N, Jarry A, Brousse N, express the adhesion molecule Lisowska-Grospierre B, Guy-Grand D, intercellular adhesion molecule-1 in Griscelli C. A monoclonal antibody inflammatory dermatoses. J Invest (HML-1) defining a novel membrane 20 Introduction molecule present on human intestinal block lympho-hemopoiesis in long-term lymphocytes. Eur J Immunol 1987; bone marrow cultures. J Exp Med 1990; 17:1279 171;477 60. Cepek KL, Shaw SK, Parker CM, 67. Jalkanen S, Joensuu H, Soderstrom KO, Russell GJ, Morrow JS, Rimm DL, Klemi P. Lymphocyte homing and the Brenner MB. Adhesion between clinical behavior of non-Hodgkin's epithelial cells and T lymphocytes lymphomas. J Clin Invest 1991; mediated by E-cadherin and the aEß7 87;1835 integrin. Nature 1994, 372;190 68. Sy MS, Guo YJ, Stamenkovic I. 61. Stamenkovic I, Amiot M, Pesando JM, Distinct effects of two CD44 isoforms Seed B. A lymphocyte molecule on tumor growth in vivo. J Exp Med implicated in lymphocyte homing is a ' 1991; 174:859 member of the cartilage link protein 69. Herrlich P, Zöller M, Pals ST, Ponta H. family. Cell 1989; 56;1057 CD44 splice variants: metastases meet 62. Jalkanen ST, Bargatze RF, Herron LR, lymphocytes. Immunol Today 1993; Butcher EC. A lymphoid cell surface 14;395 glycoprotein involved in endothelial 70. Koopman G, Heider KH, Horst E, cell recognition and lymphocyte Adolf GR, van den Berg F, Ponta H, homing in man. Eur J Immunol 1986; Herrlich P, Pals ST. Activated human 16:1195 lymphocytes and non-Hodgkin's 63. Günthert U, Hofmann M, Rudy W, express a homoloque of the rat Reber S, Zoller M, Haussmann I, metastasis-associated variant of CD44. Matzku S, Wenzel A, Ponta H, Herrlich J Exp Med 1993; 177;897 P. A new variant of CD44 confers 71. Lesley J, Hyman R, Kincade PW. CD44 metastatic potential to rat carcinoma and its interaction with extracellular cells. Cell 1991; 65;13 matrix. Adv Immunol 1993; 54;271 64. Haynes BF, Telen MJ, Hale PL, 72. Wielenga VJM, Heider KH, Offerhaus Denning SM. CD44 - A molecule GJA, Adolf GR, van den Berg FM, involved in leucocyte adherence and T Ponta H, Pals ST. Expression of CD44 cell activation. Immunol Today 1989; variant proteins in human colorectal 10;423 cancer is related to tumor progression. 65. Pals ST, Horst E, Ossekoppele GJ, Cancer Res 1993; 53:4754 Figdor CG, Scheper RJ, Meijer CJLM. 73. Naor D, Vogt Sionov R, Ish-Shalom D. Expression of lymphocyte homing CD44: structure, function and receptor as a mechanism of association with the malignant process. disseminiation in non-Hodgkin's Adv Cancer Res 1997; 71:241 lymphoma. Blood 1989; 73:885 74. Screaton GR, Bell MV, Jackson DG, 66. Miyake K, Medina KL, Hayashi S, Ono Cornelius FB, Gerth U, Bell JI. S, Hamaoka T, Kincade PW. Genomic structure of DNA encoding Monoclonal antibodies to Pgp-1/CD44 the lymphocyte homing receptor CD44 21 Chapter 1 reveals at least 12 alternatively spliced blood T cell activation. J Immunol exons. Proc Natl Acad Sei USA 1992; 1990; 144;7 89;12160 82. Koopman G, van Kooyk Y, de Graaff 75. Arch R, Wirth K, Hofman M, Ponta H, M, Meyer CJLM, Figdor CG, Pals ST. Matzku S, Herrlich P, Zoller M. Triggering of the CD44 antigen on T Participation in normal immune lymphocytes promotes T cell adhesion responses of a metastasis-inducing through the LFA-1 pathway. J Immunol splice variant of CD44. Science 1992; 1990; 145;3589 257;682 83. Pierres A, Lipcey C, Mawas C, Olive D. 76. Heider KH, Hofmann M, Horst E., van A unique CD44 monoclonal antibody den Berg F, Ponta P, Herrlich P, Pals identifies a new T cell activation ST. A human homologue of the rat pathway. Eur J Immunol 1992; 22;413 metastasis-associated variant of CD44 84. Jalkanen S, Bargatze RF, de los Toyos is expressed in colorectal carcinomas J, Butcher EC. Lymphocyte recognition and adenomatous polyps. J Cell Biol. of high endothelium: antibodies to 1993; 120;227 distinct epitopes of an 85-95-kD 77. Fox SB, Fawcett J, Jackson DG, Collins glycoprotein antigen differentially I, Gatter KC, Harris AL, Gearing A, inhibit lymphocyte binding to lymph Simmons DL. Normal human tissues, in node, mucosal, or synovial endothelial addition to some tumors, express cells. J Cell Biol 1987; 105:983 multiple different CD44 isoforms. 85. Camp RL, Scheynius A, Johansson C, Cancer Res 1994; 54;4539 Pure E. CD44 is necessary for optimal 78. Mackay CR, Terpe HJ, Stauder R, contact allergic responses but is not Marston WL, Stark H, Giinthert U. required for normal leucocyte Expression and modulation of CD44 extravasation. J Exp Med 1993; variant isoforms in humans. J Cell Biol 178:497 1994; 124:71 86. Clark RA, Alon F, Springer TA. CD44 79. Huet S, Groux H, Caillou B, Valentin and hyaluronan-dependent rolling H, Prieur AM, Bernard A. CD44 interactions of lymphocytes on tonsillar contributes to T cell activation. J stroma. J Cell Biol 1996; 134:1075 Immunol 1989; 143;798 87. DeGrendele HC, Estess P, Picker LJ, 80. Shimizu Y, van Seventer GA, Siegelman MH. CD44 and its ligand Siraganian R, Wahl SL, Shaw S. Dual hyaluronate mediate rolling under role of the CD44 molecule in T cell physiologic flow: a novel lymphocyte- adhesion and activation. J Immunol endothelial cell primary adhesion 1989; 143;2457 pathway. J Exp Med 1996; 183 :1119 81. Denning SM, Le PT, Singer KU, 88. DeGrendele HC, Estess P, Siegelman Haynes BF. Antibodies against the MH. Requirement for CD44 in CD44 p80, lymphocyte homing receptor activated T cell extravasation into an molecule augment human peripheral inflammatory site. Science 1997; 22 Introduction 278:672 Shaw S. Lymphocyte interaction with 89. Bennett KL, Jackson DG, Simon JC, endothelial cells. Immunol Today 1992; Tanczos E, Peach R, Modreli B, 13:106 Stamenkovic I, Plowman G, Aruffo A. 95. von Andrian UH, Hasslen SR, Nelson CD44 isoforms containing exon v3 are RD, Erlandsen SL, Butcher EC. A responsible for the presentation of central role for microvillous receptor heparin-binding growth factor. J Cell presentation in leukocyte adhesion. Cell Biol 1995; 128:687 1995; 82:989 90. Tanaka Y, Kimata K, Adams DH, Eto 96. Warnock RA, Askari S, Butcher EC, S. Modulation of cytokine function by von Andrian UH. Molecular heparansulfate proteoglycans: mechanisms of lymphocyte homing to sophisticated models for the regulation peripheral lymph nodes. J Exp Med of cellular responses to cytokines. 1998; 187:205 Proceedings of the Association of 97. Alon R, Kassner PD, Woldemar Carr American Physicians. 1998; 110:118 M, Finger EB, Hemler ME, Springer 91. van der Voort, Taher TEI, Wielenga TA. The integrin VLA-4 supports VJM, Prevo R, Smit L, David G, tethering and rolling in flow on VCAM- Groenink M, van Krimpen C, Hartman 1. J Cell Biol 1995; 128:1243 G, Gherardi E, Pals ST. Heparan 98. Berlin C, Bargatze RF, Campbell JJ, sulfate-modified CD44 promotes von Andrian UH, Szabo MC, Hasslen hepatocyte growth factor/scatter factor- SR, Nelson RD, Berg EL, Erlandsen induced signal transduction through the SL, Butcher EC. aA integrins mediate receptor tyrosine kinase c-Met. J Biol lymphocyte attachment and rolling Chem, 1999 in press under physiologic flow. Cell 1995; 92. van Noesel C, Miedema F, Brouwer M, 80:413 De Rie MA, Aarden LA, van Lier 99. Lloyd AR, Oppenheim JJ, Kelvin DJ, RAW. Regulatory properties of LFA-1 Taub DD. Chemokines regulate T cell alpha and beta chains in human T- adherence to recombinant adhesion lymphocyte activation. Nature 1988; molecules and extracellular matrix 333:850 proteins. J Immunol 1996; 156:932 93. Koopman G, Keehnen RM, Lindhout E, 100. Rollins BJ. Chemokines. Blood 1997; Newman W, Shimizu Y, van Seventer 90:909 GA, de Groot C, Pals ST. Adhesion 101. Baggiolini M. Chemokines and through the LFA-1 leukocyte traffic. Nature 1998; 392:565 (CDlla/CD18)-ICAM-l (CD54) and 102. Campbell JJ, Hedrick J, Zlotnik A, the VLA-4 (CD49d)-VCAM-l (CD 106) Siani MA, Thompson DA, Butcher EC. pathways prevents apoptosis of Chemokines and the arrest of germinal center B cells. J Immunol lymphocytes rolling under flow 1994; 152:3760 conditions. Science 1998; 279:381 94. Shimizu Y, Newman W, Tanaka Y, 103. Luster AD. Chemokines - Chemotactic 23 Chapter 1 cytokines that mediate inflammation. N 1994; 263:1281 Eng J Med 1998; 338:436 112. Offit K, Wong G, Filippa DA, Tao Y, 104. Piali L, Weber C, LaRosa G, Mackay Chaganti RSK. Cytogenetic analysis of CR, Springer TA, Clark-Lewis I, Moser 434 consecutively ascertained B. The chemokine receptor CXCR3 specimens of non-Hodgkin's mediates rapid and shear-resistant lymphoma: clinical correlations. Blood adhesion-induction of effector T 1991; 77:1508 lymphocytes by the chemokines IP 10 113. Hammond DW, Hancock BW, Goyns and Mig. Eur J Immunol 1998; 28:961 MH. Clinical implications of molecular 105. Rabkin CS, Ward MH, Manns A, and cytogenetic studies of non- Blattner WA. Epidemiology of non- Hodgkin's lymphomas. Cancer Hodgkin's lymphomas. In Magrath I treatment rev 1998; 24:157 (ed): The non-Hodgkin's lymphomas. 114. Harris NL, Jaffe ES, Stein H, Banks London: Arnold, 1997, p 171-186 PM, Chan JKC, Cleary ML, Delsol G, 106. Della-Favera R, Martinotti S, Gallo RC, De Wolf-Peeters C, Falini B, Gatter Erikson J, Croce CM. Translocations KC, Grogan TM, Isaacson PG, Knowles and rearrangements of the c-myc DM, Mason DY, Muller-Hermelink oncogene locus in human HK, Pileri SA, Piris MA, Ralfkiaer E, undifferentiated B-cell lymphomas. Warnke RA. Perspective: a revised Science 1983; 219:963 European-American classification of 107. Croce CM, Nowell PC. Molecular lymphoid neoplasms: a proposal from genetics of human B cell neoplasia. the International Lymphoma Study Adv Immunol 1986; 38:245 Group. Blood 1994; 84:1361 108. Gaidano G, Dalla-Favera R. Proto- 115. Klein U, Goossens T, Fischer M, oncogenes and tumor suppressor genes. Kanzler H, Braeuninger A, Rajewsky, In: Neoplastic Hematology, edited by Kuppers R. Somatic hypermutation in Knowles, DM Baltimore: Williams and normal and transformed B cells. Wilkins, 1992, p. 245-261. Immunol Rev 1998; 162:261 109. Korsmeyer SJ. Bcl-2 initiates a new 116. Rappaport H. Tumors of the category of oncogenes: regulators of haematopoietic system. Atlas of tumour cell death. Blood 1992; 80:879 pathology. Washington DC: AFIP, 110. Franssila K. t(2;5)(p23;q35) a specific 1966; section 3, fascicle 8. chromosome abnormality in large cell 117. Gerard-Marchant R, Hamlin I, Lennert anaplastic (Ki-1) lymphoma. Leukemia K, Rilke F, Stansfeld AG, Van Unnik Lymphoma 1990; 3:53 JAM. Classification of non-Hodgkins 111. Morris SW, Kirstein MN, Valentine lymphomas. Lancet 1974; ii:406 MB, Dittmer KG, Shapiro DN, Satlman 118. Lukes RJ, Collins RD. A functional DL, Look AT. Fusion of a kinase gene, approach to the classification of ALK, to a nucleolar protein gene, NPM, malignant lymphoma. Resent Results in non-Hodgkin's lymphoma. Science Cancer Res 1974; 46:18 24 Introduction 119. The non-Hodgkin's lymphoma pathologic classification project. National Cancer Institute sponsored study of classifications of non- Hodgkin's lymphomas. Summary and description of a working formulation for clinical usage. Cancer 1982; 49:2112 120. Stein H, Dallenbach F. Diffuse large cell lymphomas of B and T cell type, in Knowles DM (ed): Neoplastic Hematopathology. Baltimore: Williams andWilkins, 1992, p 675. 121. Della-Favera R. BCL-6 in diffuse large- cell lymphomas, in DeVita VT, Hellman S, Rosenberg SA (eds): Important advances in oncology 1996. Philadelphia, Lippencott, 1996, p 139. 122. Kramer MHH, Hermans J, Parker J, Krol ADO, Kluin-Nelemans JC, Haak HL, van Groningen K, van Krieken JHJM, de Jong D, Kluin PhM. The clinical significance of BCL2 and p53 protein expression in diffuse large B- cell lymphoma: a population based study. J Clin Oncol 1996; 14:2131 123. Kramer MHH, Hermans J, Wijburg E, Philippo K, Geelen E, van Krieken JHJM, de Jong D, Maartense E, Schuuring E, Kluin PM. Clinical Relevance of BCL2, BCL6, and MYC Rearrangements in Diffuse Large B-Cell Lymphoma. Blood 1998; 92:3152 25 26 Chapter 2 Expression of the mucosal homing receptor a4ß7 in malignant lymphomatous polyposis of the intestine Steven T. Pals1, Paul Drillenburg', Brigitte Dragosics2, Andrew I. Lazarovits3, and Thaddäus Radaszkiewicz2 'Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; : Department of Pathology, University of Vienna, Vienna, Austria; and1 University Hospital, Robarts Research Institute, University of Western Ontario, London, Ontario, Canada Gastroenterology 1994; 107:1519-1523 27 Chapter 2 Expression of the Mucosal Homing Receptor cc4ß7 in Malignant Lymphomatous Polyposis of the Intestine STEVEN T. PALS,* PAUL DRILLENBURG,* BRIGITTE DRAGOSICS,f ANDREW I. LAZAROVITS,8 and THADDÄUS RADASZKIEWICZ1, »Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 'Department of Pathology, University of Vienna, Vienna, Austria; and sUniversity Hospital, Robarts Research Institute, University of Western Ontario, London, Ontario, Canada Recent studies have identified the integrin 28 a4ß- in MLP Table 1. Clinical Findings and Course of Disease From Eight Patients With MLP Age (yO/Sex Presenting Time (M/F) symptoms Gastrointestinal tract site Stage' Therapy Course In») Outcome i. 67/M Bloody stools Colon, stomach, duodenum, III CT CR 36 Death without lymphoma epipharynx 2. 66/F Diarrhea Colon, duodenum, jejunum II CT PR 14 Death of lymphoma 3. 78/F Dysphagia Tonsil, colon IV CT PR 14 Alive with disease 4. 65/M Diarrhea Stomach, duodenum II CT Progression 14 Death of lymphoma 5. 87/M Ileus lleocecum II Resec/RT PR 2 Death of lymphoma 6. 58/M Abdominal pain Stomach, colon IV" CT PR 62 Alive with disease 7. 78/M Weight loss Colon IV" CT Progression 12 Death of lymphoma 8. 60/M Dysphagia Tonsil, duodenum, jejunum, IV' CT/RT Progression 38 Death of lymphoma colon CR, complete remission; CT, chemotherapy; PR, partial remission; RT, radiotherapy. aAnn Arbor system. "Stage IV because of bone marrow involvement. up to 1.5-cm diameter in the cecum and the right-sided colon. Case 3 Biopsy specimens showed an MC lymphoma. A partial remis A 78-year-old women presented with dysphagia caused sion was obtained after seven terms of chemotherapy, including by enlargement of the left tonsil. Biopsy specimen showed an endoxan and Oncovin. The patient died of a small bowel perfo MC lymphoma. At staging, a cervical and hilary lymphadenop- ration 14 months after diagnosis. No autopsy was performed. athy and bone marrow involvement were found. The patient received six terms of chemotherapy at reduced dosage, re sulting in a partial remission. At 14 months, routine colonos copy showed multiple 6-8-mm polyps in a lawnlike pattern in the left-sided colon (Figure Iß). The patient refused further diagnostic procedures and therapy. Methods Tissue specimens of the eight patients with MLP were from the files of the Department of Pathology, University of Vienna, Vienna, Austria. They were histologically classified as MC lymphoma.2 Frozen tissue specimens were studied for expression of the mucosal homing receptor Ct4ß7 by using monoclonal antibody Act-1 (immunoglobulin Gl).8'9 Mono clonal antibodies against other adhesion receptors were Leu-8 (Becton Dickinson, Sunnyvale, CA) against L-selectin; HECA- 452 against CLA1"; TB133 against LFA-la (CDlla)"; RR/1 Table 2. Expression of Adhesion Molecules in Primary Multiple MC Lymphoma of the Gastrointestinal Tract (MLP) and in Primary Nodal MC Lymphoma Mucosal site (MLP) Nodal site (no. (MC) (no. Adhesion positive/ Staining positive/ Staining receptor no. tested) intensity no. tested) intensity a4/?7 8/8 1-2+ 0/5 0 L-selectin 8/8 1-2+ 5/5 1-2+ CLA 0/8 0 0/5 0 LFA-1 (CDlla) 6/8 1+ 3/5 1-2+ ICAM-1 (CD54) 4/8 1-2+ 2/5 1+ a4 (CD49d) 8/8 2+ 5/5 1-2+ CD44 8/8 2 + 5/5 2 + Figure 1. MLP. (A) Barium enema. Multiple small polyps throughout the LFA, lymphocyte function-associated antigen; ICAM, intracellular ad entire colon. (S) Colonoscopy. Numerous polyps in the left-sided colon. hesion molecule. 29 Chapter 2 .,:,;.- o*a» •"» \ © @ Figure 2. MLP. (A) Dense tumor infiltration of the mucosa and submucosa of the jejunum. The surface epithelium is intact (arrows). Giemsa stain (original magnification 15x). (B) Higher magnification of A. Mucosal infiltration by mantle cells. No lymphoepithelial lesions are formed. * Lumen of a crypt (original magnification 160x). (C) Immunostainingwith monoclonal antibody Act-1 directed against ct4ß7 integrin. (D) Negative control stained with monoclonal antibody OKT-6 against CD1. (£) Positive control stained with monoclonal antibody B4 against CD19. against intracellular adhesion molecule (CD54) "; HP2/1 For comparison, 6ve cases of nodal MC lymphoma were also against Ct4 (CD49d)n; and NKI-P1 against CD44.11 Mono studied. clonal antibodies OKT-6 immunoglobulin Gl (Ortho Diag nostic Systems, Raritan, NY) against CD1 and B4 {immuno Discussion globulin Gl; Coulter Clone, Hialeah, FL) against CD19 were The results of our immunohistochemical studies used as positive and negative controls, respectively. Immuno- using monoclonal antibody Act-1 against Cx4ß7 show peroxidase staining was performed on acetone-fixed cryostat expression of this homing receptor in all cases of MLP sections using the streptavidin-biotin-peroxidase complex examined (Table 2 and Figure 2A—C). In each case, method. Sections were counterstained with hematoxylin. Stain expression of Ct4ß7 was detectable on a majority of the ing intensity was scored semiquantitative^ on a scale of 0-2 tumor cells. The expression level on individual tumor (0, no staining; 1, weak staining; 2, moderate/strong staining). For a lymphoma to be scored positive, a minimum of 20% of cells was heterogeneous and ranged from moderate to the cell has to be stained. weak. By contrast, Ot4ß7 was absent from nodal MC 30 aß- in MLP lymphomas (Table 2). This difference between MC References lymphoma of the gastrointestinal tract (i.e., MLP) and 1. Isaacson PG. Gastrointestinal lymphomas and lymphoid hyper nodal MC lymphoma did not reflect a general differ plasias. In: Knowles DM, ed. Neoplastic hematopathology. Bal ence in the regulation of adhesion molecule expression timore: Williams & Wilkins, 1992:953-978. but was specific for a4ß7 (Table 2). Expression of a 2. Weisenburger D. Mantle cell lymphoma. In: Knowles DM, ed. number of other adhesion receptors involved in lym- Neoplastic hematopathology. Baltimore: Williams & Wilkins, 1992:617-628. phocyte-endothelial cell interaction and lymphocyte 3. Streeter PR, Berg E, Rouse BTN. Bargatze RF. Butcher EC. A homing, including L-selectin, cutaneous lymphocyte tissue-specific endothelial cell molecule involved in lympho antigen, leukocyte function —associated antigen 1 cyte homing. Nature 1988;331:41-46. (LFA-lCC, CDlla), intercellular adhesion molecule 1 4. Briskin MJ, McEvoy, Butcher EC. MAdCAM-1 has homology to immunoglobulin and mucin-like adhesion receptors and to (ICAM-1, CD54), VLA-4 (CD49d), and CD44 in nodal IgAl. Nature 1993;363:461-464. MC and MLP, was not different (Table 2). Hence, 5. Holzmann B, Mclntyre BW, Weisman IL. Identification of a mu (X4ß7 is specifically expressed in MLP and might medi rine Peyer's patch-specific lymphocyte homing receptor as an ate the mucosa-specific tumor dissemination that is integrin molecule with an a chain homologous to human VLA- 4a. Cell 1989;56:37-46. chatacteristic of this disease. 6. Berlin C, Berg EL, Briskin M, Andrew DP, Kilshaw PJ, Holzman Maintenance of the integrity of distinct lymphoid B, Weissman IL, Hamann A, Butcher EC. a4ß7 integrin medi ates lymphocyte binding to the mucosal vascular addressin compartments, such as mucosa-associated lymphoid MAdCAM-1. Cell 1993;74:185-195. tissues, is critically dependent on selective recircula 7. Parker CM, Cepek KL, Russell GJ, Shaw SK, Prosnett DN, tion and homing of lymphocytes. Results from a large Schwarting RL, Brenner MB. A family of ß7 integrins on human mucosal lymphocytes. Proc Natl Acad Sei USA 1992; body of studies show that this homing process is 89:1924-1928. carefully regulated through specialization of both en 8. Schweighoffer T. Tanaka Y, Tidswell M, Erle DJ, Horgan KJ. dothelial cells and lymphocyte subsets in both their Luce GEG, Lazarovits AI, Buck D, Shaw S. Selective expression expression and regulation of adhesion teceptors and of integrin a4ß7 on a subset of human CD4+ memory T cells with hallmarks of gut-tropism. J Immunol 1993:151:717- counterreceptors." In non-Hodgkin's lymphomas, the 729. normal expression programs of these receptors seem to 9. Lazarovits AI, Mosciki RA, Kurnick JT, Camerini D, Bhan AK, be at least partly preserved.16 Like in normal lympho Baird LG, Erikson M, Colvin RB. Lymphocyte activation anti gens: a monoclonal antibody, anti-Act-1, defines a new late cytes, adhesion receptor expression in lymphomas is lymphocyte activation antigen. J Immunol 1984:133:1857- related to their stage of maturation and anatomic local 1862. ization. This selective receptor expression seems to 10. Duijvestijn AM, Horst E, Pals ST, Rouse BN, Steere AC, Picker U, Meijer CJLM, Butcher EC. High endothelial differentiation contribute importantly to the specific patterns of dis in human lymphoid and inflammatory tissues defined by mono semination of non-Hodgkin's lymphomas. This notion clonal antibody HECA-452. Am J Pathol 1988;130:147-155. is not only supported by the selective expression of 11. Noesel C, van Miedema F, Brouwer M, Rie MA, de Aarden LA, Ct4ß7 in MLP as found in our present study. Previous van Lier RAW. Regulatory properties of LFA-la and ß chains in human T lymphocyte activation. Nature 1988;333:850-853. studies from our own and other laboratories have 12. Rothlein R, Dustin ML, Marlin SD, Springer TA. A human inter shown that the skin homing receptor cutaneous lym cellular adhesion molecule ICAM-1 distinct from LFA-1. J Immu phocyte antigen is selectively expressed on cutaneous nol 1986;137:1270-1274. T-cell lymphomas. Furthermore, expression of 13. Sanchez-Madrid F, de Landazuri MO, Morago G, Cebrian M, Acevedo A, Bernabeu c. VLA-3: a novel polypeptide associated Cc4ß7 mucosal homing recepror is not unique to MLP, within the VLA molecule complex: cell distribution and bio but this molecule is also expressed on other lymphomas chemical characterization. Eur J Immunol 1986; 16:1343- of mucosa-associated lymphoid tissue (unpublished 1349. 14. Pals ST, Hogervorst F, Keizer GD, Thepen T, Horst E, Figdor observation). The close relationship between the ex CG. Identification of a widely distributed 90-kD glycoprotein pression of particular tissue-specific homing receptors that is homologous to the hermes-1 human lymphocyte homing and the anatomic site of lymphoma involvement sug receptor. J Immunol 1989;143:851-857. 15. Picker U, Butcher EC. Physiology and molecular mechanisms gests that lymphomas at different sites may represent of lymphocyte homing. Ann Rev Immunol 1992;10:561-591. biologically distinctive groups of tumors, a notion that 16. Pals ST, Horst E, Scheper RJ, Meijer CJLM. Mechanisms of is also emerging from recent clinical, morphological, human lymphocyte migration and their role in the pathogenesis and molecular genetic studies.1'"'20 Assessment of of disease. Immunol Rev 1989;108:111-133. 17. Picker LJ, Michie SA, Rott LS, Butcher EC. A unique phenotype homing receptor expression in non-Hodgkin's lym of skin-associated lymphocytes in humans: preferential ex phomas, which can be performed by routine immuno- pression of the HECA-452 epitope by benign and malignant T histochemistry, may contribute to the classification of cells at cutaneous sites. Am J Pathol 1990; 136:1053-1068. 18. Noorduyn LA, Beljaards RC, Pals ST, Heerde P van, Radaszkie- non-Hodgkin's lymphomas and may help to predict wicz T, Wtllemze R, Meijer CJLM. Differential expression of the lymphoma dissemination. HECA-452 (cutaneous lymphocyte associated antigen, CLA) in 31 Chapter 2 cutaneous and non-cutaneous T-cell lymphomas. Histopathol- Received February 3, 1994. Accepted July 4, 1994. ogy 1992; 21:59-64. Address requests for reprints to: Steven T. Pals, M.D., Ph.D., De- Isaacson PG, Spencer J. Malignant lymphoma of the mucosa- partaient of Pathology, Academic Medical Center, University of Am- associated lymphoid tissue. Histopathology 1987:11:445- sterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands. 462. Fax: (31) 20-696-0389. Van Krieken JHJM, Raffeld M, Raghoebier S, Jaffe ES, van Supported by grant IKA-91/9 of the Dutch Cancer Foundation and Ommen GJB, Kluin PM. Molecular genetics of gastrointestinal the Crohn's and Colitis Foundation of Canada. non-Hodgkin's lymphomas: unusual prevalence and pattern of The authors thank Drs. R. A. W. van Lier, E. C. Butcher, T. A. Springer, c-myc rearrangements in aggressive lymphomas. Blood F. Sanchez-Madrid, and C. G. Figdorfor providing monoclonal antibodies, 1990; 76:797-800. and M. Burghuber and I. Mosberger for technical assistance. 32 Chapter 3 Preferential expression of the mucosal homing receptor integrin a4ß7 in gastrointestinal non-Hodgkin's lymphomas Paul Drillenburg1, Robbert van der Voort', Gerrit Koopman', Brigitte Dragosics2, Johan H. J. M. van Krieken3, Philip Kluin3, John Meenan4, Andrew I. Lazarovits5, Thaddäus Radaszkiewicz2, and Steven T. Pals' Departments of Pathology' and Gastroenterology4, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Department of Pathology2, University Hospital Vienna, Vienna, Austria; Department of Pathology3, Leiden University Hospital, Leiden, The Netherlands; and University Hospital, Robarts Research Institute5, University of Western Ontario, London, Ontario, Canada American Journal of Pathology 1997; 150:919-927 33 Chapter 3 Preferential Expression of the Mucosal Homing Receptor Integrin a4ß7 in Gastrointestinal Non- Hodgkin's Lymphomas Paul Drillenburg,* Robbert van der Voort,* primary localization; in mucosal, nodal, and cu Gerrit Koopman,* Brigitte Dragosics,1" taneous T cell lymphomas the percentage of pos Johan H. J. M. van Krieken,* Philip Kluin,* itive cases was 56%, 17%, and 0%, respectively. 5 John Meenan, Andrew I. Lazarovits," All cases of precursor T-cell lymphoma were ct4ß7 1 Thaddaus Radaszkiewicz, " and negative. High expression of a4ß7 was found on Steven T. Pals* a subset of peripheral blood memory T cells as From the Departments of Pathology and Gastmenterology}* well as on lymphocytes in the intestinal mucosa. Academic Medical Center, University of Amsterdam, We conclude that non-Hodgkin's lymphomas that Amsterdam, The Netherlands: the Department of Pathology} are related to mucosa-associated B- and T-lym- University Hospital Vienna, Vienna, Austria; the phocyte populations selectively express the mu Department of Pathology} Leiden University Hospital, cosal homing receptor ot4ß7. Tl>e presence of this Leiden, the Netherlands; and University Hospital, Robarls receptor underscores their distinctive character Research Institute} University of Western Ontario, London. and may play an important role in determining Ontario, Canada their characteristic mucosal dissemination pat tern. (Am f Pathol 1997, 150:919-927) Recent studies have identified the integrin ot4ß7 as a mucosal homing receptor that mediates lym Maintenance of the integrity of distinct lymphoid phocyte migration to the intestinal mucosa by compartments, such as mucosa or skin-associated binding to MAdCAM-1, a vascular recognition lymphoid tissues, is critically dependent on selective molecule (addressin) selectively expressed on recirculation and homing of lymphocytes. This hom mucosal endothelium. In the present study, we ing process is carefully regulated through special have assessed the expression of ot4ß7 on B- and ization of both endothelial cells and lymphocyte sub T-cell non-Hodgkin's lymphomas of different pri sets in their expression and regulation of adhesion mary localization and on related normal lympho receptors and counter-receptors.' Evidence from cytes. Among B-lineage lymphomas, expression several sources indicates that malignant lympho of a4ß7 was present in the majority of cases of cytes may use these physiological homing pathways malignant lymphomatous polyposis of the intes as a mechanism of dissemination2 For example, tine and low-grade lymphoma of the mucosa- non-Hodgkin's lymphomas (NHLs) of the mucosa- associated lymphoid tissue/monocytoid B-cell associated lymphoid tissues (MALTs) and the skin lymphoma and in some cases of precursor B-cell tend to spread to mucosal sites and skin, respec lymphoma. CLL/small lymphocytic lymphoma, tively. In the latter tumors, this dissemination presum (nodal) mantle cell lymphoma, follicular center ably is mediated by cutaneous lymphocyte antigen cell lymphoma, Burkitt's lymphoma, and diffuse (CLA), a skin homing receptor, which is selectively large B-cell lymphoma were virtually always expressed on cutaneous T-cell lymphomas3,4 and 5 ot4ß7 negative, as was the case when localized in interacts with E-selectin on skin endothelium. the mucosa-associated lymphoid tissue. The nor mal B cells of the follicle mantles and part of the B cells of the extrafollicular B-cell compartment Supported by grant IKA 91-9 from the Dutch Cancer Foundation and by the Crohn's and Colitis Foundation of Canada. of lymphoid tissues expressed moderate levels of Accepted for publication October 24, 1996. a4ß7. By contrast, follicular center cells were Address reprint requests to Dr. Steven T. Pals, Department of ct^ßy negative. Among T-lineage lymphomas, ex Pathology, Academic Medical Center, University of Amsterdam, pression of a4ß7 was also strongly related to the Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands 34 a4ß- expression in non-Hodgkin 's lymphomas Recently, important new insights have been ob was blocked by 0.3% H202 in methanol. As an en tained in the molecular basis of lymphocyte homing zyme for color development, horseradish peroxidase to the MALT. In mice, high endothelial venules of was used, which was coupled to the biotin via a Peyer's patches and lamina propria venules selec streptavidin-biotin-peroxidase complex (Dako). After tively express a glycoprotein called mucosal vascu incubation for 30 minutes, the sections were incu lar addressin (MAdCAM-1 ).6 MAdCAM-1 is an immu bated with 3,3-amino-9-ethylcarbazole (Sigma noglobulin family member with domains that display Chemical Co., St. Louis, MO) for 5 to 10 minutes. homologies to the vascular adhesion receptors for Sections were counterstained with hematoxylin. leukocytes ICAM-1 (CD54) and VCAM-1 (CD106) as Double staining was performed with a two-step well as to another mucosa-associated Ig family indirect immunotechnique using subclass-specific 7 17 member, lgA1. The integrin a4/37, which is strongly second-step antibodies, as described previously. expresssed on mucosal lymphocytes, appears to The second step consisted of a cocktail of goat represent the dominant lymphocyte receptor for anti-mouse IgG-, (Southern Biotechnology Associ MAdCAM-1 and for regulating lymphocyte homing to ates, Birmingham, AL) labeled with alkaline phos 89 mucosal sites. In man, aAß7 may have a similar phatase and goat anti-mouse lgG2a (Southern Bio function; it was shown to be expressed on mucosal technology Associates) labeled with horseradish 10 T-cell lines and on a subset of peripheral blood peroxidase. For color development, 3,3-amino-9-eth- 11,12 memory T cells with gut homing properties. Fur ylcarbazole (Sigma) and naphthol-AS-MX-P/fast blue thermore, we have recently shown that a4ß7 is ex BB was used (Sigma). pressed in malignant lymphomatous polyposis of the Staining intensity was scored semiquantitatively intestine (MLP), suggesting that it may play a role in on a scale of 0 to 2 (0, no staining; 1, weak staining; the multifocal intestinal dissemination characteristic 2, moderate/strong staining) by two independent ob 13 of this lymphoma. servers (T. Radaszkiewicz and S. T. Pals). Discrep To further explore the relationship between a4ß7 ancies were solved by consensus. For a lymphoma expression and lymphoma localization/dissemina to be scored positive, a minimum of 20% of the cells tion, we have now studied the expression of a4ß7 on had to be stained. The antibody used for the detec 18 a panel of NHLs and on related normal lymphocytes. tion of a4ß7 was Act-1 (IgG,), which has been 11,12 shown to be specific for a4ß7 The antibody E against a ß7 was Bez-Act8 (Dako). The anti-cyto- Materials and Methods keratin antibody was CAM 5.2 (lgG2a; Becton Dick inson, San Jose, CA). Case Selection and Classification A panel of NHLs of different subcategories was Cell Isolation selected from the files of the Departments of Pa thology, Academic Medical Center, University of Peripheral blood mononuclear cells (PBMCs) from Amsterdam, the Netherlands; the University Hos normal donors were isolated by Ficoll-lsopaque den pital Leiden, the Netherlands, and the University sity gradient centrifugation. For isolation of tonsil lym Hospital Vienna, Austria. Histological subclassifi phocytes, tonsillar tissue was dissected free from cation of NHLs was based on the Revised Euro surface epithelium and finely minced into a cell sus pean American Lymphoma Classification.14 Lym pension. Mononuclear cells were isolated by Ficoll- phomas of the MALTs were classified according to lsopaque density gradient centrifugation. Monocytes the criteria described by Isaacson.15 Normal lym were removed by plastic adherence (1-hour incuba phoid tissues were retrieved from the files of the tion at 37°C in 10-cm petri dishes (Costar, Cam Department of Pathology, Academic Medical Cen bridge, MA). T cells were depleted using 2-amino- ter, Amsterdam. ethyl-isothiouronium-bromide-modified sheep red blood cells. Immunohistochemistry Normal duodenal biopsies were obtained from pa tients undergoing evaluation for peptic ulcer dis Immunoperoxidase staining was performed on ace ease. For isolation of mononuclear cells, four endo tone-fixed cryostat sections using the streptavidin- scopic biopsies were taken into RPMI 1640 biotin-peroxidase complex method as described supplemented with fetal calf serum and 10% genta- previously.16 Before incubating with the secondary mycin. Biopsies were teased apart, added to a 14-ml biotinylated anti-mouse F(ab')2 antibody (Dako tube (Falcon, Cambridge, MA) containing RPMI Corp., Glostrup, Denmark), endogenous peroxidase 1640/10% fetal calf serum and gentamycin with 50 Chapter 3 CD3 CD19 Figure 1. Expression of a ,ß- (Act-1) on periph eral blood T and B cells. PBMCs were triple stained for either CD3. CD45RO, and a ,ß- or CDJ9. IgD. and aß-. Left: Expression of CD45RO versus a,ß- on T cells ( CD3 gated). Right: E\pression of IgD versus a ß- on B cells (CD19 gated). The black dot in each histogram IQ* 10' 10' 10' 10' ID indicates Ibe median fluorescence obtained when irrelevant MAbs itère used as commis CD45RO (representative of four samples). IU/ml collagenase type IV (Sigma), and placed on a performed in PBS/bovine serum albumin with 10% mixing table (multi-purpose rotor, Scientific Indus human serum for 30 minutes at 0°C. tries, New York, NY) at 37°C. After 1 hour, the super natant was pelleted and washed, and cells were resuspended in FACS buffer to a concentration of 2 x 106/ml. Cell viability was >90%, Differential iso Results lation of intra-epitheliai lymphocytes was performed Expression of the Mucosal Homing by an initial incubation with 2 mmol/L EDTA and Receptor a ß on Normal Lymphocytes dithiothreitol (Sigma) before collagen digestion. 4 7 Peripheral blood lymphocytes from healthy volun teers were analyzed for the simultaneous expression FACS Analysis of CD3, CD45RO, and a4ß7 or CD19, IgD, and a4ß7 1112 For determining the expression of a4ß7 on lympho (Figure 1). In accordance with previous reports, cyte subpopulations, triple-staining experiments the CD45RO" (naive) T cells homogeneously ex were performed. Cells were preincubated with 10% pressed a4ß7, whereas the expression of a4ß7 on the + human serum in phosphate-buffered saline (PBS) memory T-cell subset (CD45RO ) was heteroge containing 1% bovine serum albumin and then se neous with a a4ß7 low/negative subset and a sub- quentially incubated with appropriate dilutions of population expressing high levels of a4ß7 represent Act-1 and phyooerythrin-conjugated goat anti- ing gut homing T lymphocytes. The vast majority of mouse Ig (Southern Biotechnology Associates) for B-cell peripheral blood lymphocytes showed a mod 30 minutes at 0°C. Free binding sites of the goat erately strong homogeneous expression of a4ß7. anti-mouse antibody were then blocked by incuba In histological sections of lymph nodes, tonsils, tion with 5% normal mouse serum. For detection of and small intestine, a4ß7 was weakly expressed on a4ß7 expression in peripheral blood T and B lympho the cells in the mantle zones of B-cel! follicles (Figure cytes, the staining procedure was continued by in 2A) and on approximately 30% of the cells in the cubating the cells with either fluorescein isothiocya- extrafollicular compartments of lymph nodes and nate (FITC)-labeled monoclonal antibody directed tonsils. Germinal center cells were consistently e*4ß7 against CD45RO (UCHL-1; Dako) followed by biotin- negative (Figure 2A). In the mucosa of the small labeled anti-CD3 (leu-4; Becton-Dickinson, Mountain intestine, approximately 50% of the cells in the lam View, CA) and RED613-labeled streptavidin (Gibco, ina propria showed expression of a4ß7 whereas in Grand Island, NY) or with FITC-labeled rabbit anti- tra-epitheliai T lymphocytes were not stained (Figure IgD (Dako) followed by biotin-labeled anti-CD19 2B-D). (HD37; Dako) and RED613-labeled streptavidin. For Restriction of a4ß7 expression to specific lympho + detection of a4ß7 subpopulations in tonsil B cells, cyte populations was also demonstrated on isolated the 5% normal mouse serum incubation step was tonsillar B lymphocytes (Figure 3) and duodenal T followed by FITC-labeled rabbit anti-lgD (Dako), bi lymphocytes (Figure 4). Of the B lymphocytes, the otin-labeled anti-CD38 (CALTAG Laboratories, San lgD+ B-cell subset, which represent naive B-cells Francisco, CA) and RED613-labeled streptavidin. largely derived from follicle mantle zones, were a4ß7 The incubations with the conjugated antibodies were positive, whereas (lgD~/CD38+) germinal center B 36 a_,ß- expression in non-Hodgkin 's lymphomas .' a .. 't i V i i- V K Figure 2. Expression of a^ßy in normal lymph node (A) anclMAlTiB and C).D: Expression of a ß7 on the intm-epithelial lymphocytes in the normal mucosa. Magnification. x O' 10' 10! 10" 10' Act-1 Act-1 Figure 3. Expression of aß- ( Act-1) on tonsil B cell subpopulations Fünf led tonsil B cells ( CD19 > 98%) were triple stained for IgD, CDJS. and a ,/3-. Upper left: Expression oflgD versus CD38 showing three distinct subpopulations, ie. IgD". CD38+ (germinal center B cells): lgD+. CD33~~ (naive B cells). and IgD". CD38" ( memory B cells). Expression of a.ß7 is shown in histogram plotsfor IgD', CD38* (upper right), IgD*, CD38" (lower rightl, and IgD". CD38" (lower left) B cell subpopulations , background. , aß- expression, representative of four samples 2) and with the observation in the mouse that aAß7 is monocytoid B-cell lymphoma) and MLP (Table 1 and down-regulated upon T-lymphocyte migration to the Figure 5, A, C, and D). Positive cases of marginal- epithelium.'9 zone lymphoma were located in the gastrointestinal tract (n = 7), tonsil plus regional lymph nodes (n = Expression of a ß on B-Cell 1 ), and salivary gland (n = 1 ). A detailed description 4 7 of the clinical and pathological findings in the cases Non-Hodgkin's Lymphomas of MLP included in this study, which all had multiple To assess ctAß7 expression on B-NHLs, a panel of intestinal tract lesions, has been published else 13 tumors representing different pathological subtypes where. Unlike low-grade B-cell lymphoma of MALT and with primary localization in either lymph nodes or and MLP, cases of mucosa-associated diffuse large MALT was analyzed (Table 1). Interestingly, Figure 4. Expression ufaß-, (Act-1) on muco sal T lymphocytes Left: Intra-epitbelial T cells. Right: Lamina propria T cells. , back 10' 10< 10' 10' 10' 10' 10 ground. , aß- expression; representative of four samples. Act-1 Act-1 Of the primary nodal peripheral (mature) B-cell primary localization of the tumor. a4ß7 was found in 5 neoplasms (n = 41), which included cases of CLL/ of 9 cases of primary mucosal T-cell lymphoma, in 1 small lymphocytic lymphoma, nodal mantle cell lym of 6 cases of nodal T-cell lymphoma, and in 0 of 7 phoma, follicular center lymphoma, Burkitt's lym cases of primary cutaneous T-cell lymphoma (myco phoma, and diffuse large B-cell lymphoma, only 2 sis fungoides). Furthermore, a4ß7 was expressed in expressed aAßT. 2 of 17 cases of anaplastic large-cell lymphoma. Hence, like in lymphomas of the B lineage, a4ß7 expression in T-lineage lymphomas is a characteris Expression of a4ß7 on T-Cell Non-Hodgkin's tic of primary mucosal T-cell lymphomas. Lymphomas A panel of T-NHLs representing different pathologi cal subtypes with primary localizations in lymph Discussion node, skin, and mucosa was analyzed for the ex The key observation of this study is that the mucosal pression of ct4ßT (Table 2). Neoplasms of precursor T homing receptor a4ß7 is expressed on the vast ma cells, ie, T-iymphoblastic lymphomas, were invari jority of cases of low-grade B-cell lymphoma of ably a ß negative. In peripheral T-cell lymphomas, 4 7 MALT, MLP, and intestinal T-cell lymphoma but that expression of a ß was strongly correlated with the 4 7 expression of this molecule is uncommon in B- and T-cell lymphomas that are not MALT related (Tables Table 1. Expression of the Mucosal Homing Receptor 1 and 2). This selective expression of the mucosal a4ß7 on B-Lineage Non-Hodgkin's Lymphomas homing receptor a4ß7 on MALT lymphomas strongly Num oer a4ß7*l Number tested Subtype (%) Intensity Table 2. Expression of the Mucosal Homing Receptor a4ß7 on T-lineage Non-Hodgkin s Lymphomas B-cell lymphoma + Precursor B Number a4ß7 l Lymphoblastic 2/4 (50) 1 + Number tested Peripheral B Subtype (%) Intensity CLL/lymphocytic 0/6 (0) 0 Mantle eel 1/6 (17) 1 + T-cell lymphoma MLP 7/8 (87) 2 + Precursor T Follicle center 0/11 (0) 0 Lymphoblastic 0/8 (0) Marginal zone Peripheral T Low-grade B MALT 9/10(90) 1-2 + Unspecified (nodal) 1/6 (17) 2 + Monocytoid B 2/2(100) 1-2 + Enteropathy associated 5/9 (56) 1-2 Diffuse large B 1/24 (4) 2 + Mycosis fungoides/CTCL 0/7 (0) 0 Burkitt's 0/5 (0) 0 Anaplastic large cell 2/12(17) 1-2 Lymphomas were classified according to the Revised Lymphomas were classified according to the Revised European American Lymphoma Classification. Scoring for European American Lymphoma Classification Scoring for intensity was as follows: 0, no staining; 1+ weak staining; 2 + . intensity was as follows: 0, no staining: 1+, weak staining, 2+, moderate to strong staining. moderate to strong staining CTCL, cutaneous T-cell lymphoma. 39 Chapter 3 a o %&£ / '^S* $Lè<, Figure 5. Expression ofa.ß7 on mucosal NHLs. A: Low-grade B-cell lymphoma of MALT localized in the stomach with positive staining for a ß- B: Diffuse large B-cell NHL localized in the stomach, with no aß7 expression. C and D: Low-grade B-cell lymphoma of MALT localized in the stomach, with double staining for a ß^ (blue) and cytokeratin {red), showing extensive destniction of epithelium by a ß- tumor cells with lymphoepithelial lesions (arrows). Magnification. X460(A anclB). X2J0(C). and X 345(D), 40 aß- expression in non-Hodgkin 's lymphomas supports the concept that these lymphomas repre a4ß7 was not only expressed on intestinal tumors but sent a biologically distinctive group of tumors.15,20 also on lymphomas localized in the tonsil and the Furthermore, the selective expression of a4ß7 may salivary gland favors their relation to a common mu explain the propensity of mucosal lymphomas to cosal immune system involving lymphocytes com disseminate to and relapse at distant mucosal mitted to mucosal sites.25 In this context, the recent sites2'15'20-22 report by Diss et al21 of a single neoplastic B-cell The existence of distinctive recirculation pathways clone in sequential biopsy specimens from a patient for different lymphocyte subpopulations1,23,24 is one with primary gastric-mucosa-associated lymphoma of the striking features of lymphocyte homing. and Sjogren's syndrome is of interest. Whereas naive T cells recirculate preferentially We observed that a4ß7 is expressed at relatively through secondary lymphoid tissues such as lymph high levels in MLP. The expression of this mucosal nodes, memory (and activated) T cells preferentially homing receptor on the tumor cells in this uncommon leave the blood in peripheral vascular beds of, eg, but dramatic disease, characterized by multifocal the skin and the mucosa.23 Among memory T cells, gastrointestinal involvement, might be an important there is yet further specialization; distinct subsets of factor in its dissemination. MLP has been proposed memory T cells home to the skin or gut lamina pro to be related to follicle mantle cells,14 which repre pria, respectively.1,11,24 In the human peripheral sent naive B cells. By analogy with naive T cells, they blood, a4ß7 is expressed on a subset of gut-trophic express a4ß7 (Figures 2A and 3) in concert with memory T lymphocytes11,12 (Figure 1). Moreover, several other adhesion receptors including L-selec- a4ß7 is expressed at high levels on T cells in the tin, thus presumably providing them with a relatively lamina propria of the intestine but is down-regulated broad homing specificity.26 Interestingly, most cases on intra-epithelial lymphocytes (Figures 2C and 4). of nodal mantle cell lymphoma did not express ct4ß7, This a4ß7 memory T-cell subset is phenotypically although they were L-selectin positive, and hence and functionally distinct from other subsets of mem may have lost their potential to home to mucosal ory T cells11 and, for example, is nonoverlapping sites13. with a memory T-cell subset defined by expression Taken together, our data indicate that NHLs that 11 of CLA, a skin homing receptor. The presence of are related to mucosa-associated B- and T-lympho- aAß7 on intestinal T-cell lymphomas strongly sug cyte populations selectively express the mucosa! gests that these tumors (which all expressed homing receptor a4ß7. The presence of this receptor CD45RO) are directly derived from gut homing a4ß7- underscores their distinctive character and may play positive memory T cells. Like in the normal memory an important role in determining their characteristic T-cell subsets, a4ß7 and CLA expression on T cell mucosal dissemination pattern. lymphomas was also mutually exclusive; the intesti nal T-cell lymphomas in our series did not express the skin homing receptor CLA (our own unpublished Acknowledgments observation), and vice-versa, we did not observe a4ß7 expression in any of the cutaneous T-cell lym We thank M. Burghuber, I. Mosberger, and J. B. G. phomas examined (Table 2). Mulder for technical assistance. Interestingly, most cases of low-grade B-cell lym phoma of MALT and monocytoid B-cell lymphoma References were found to express a4ß7 (Figure 5, A, C, and D). These tumors represent closely related lymphoma 1. Picker LJ, Butcher EC: Physiology and molecular subtypes, are believed to originate from memory B mechanisms of lymphocyte homing. Annu Rev Immu cells residing in the marginal zones of mucosal lym nol 1992, 10:561-591 phoid tissues,14 and hence might be related to the 2. Pals ST, Horst E, Scheper RJ, Meijer CJLM: Mecha a4^7+/lgD"i CD38~ subset of tonsil lymphocytes nisms of human lymphocyte migration and their role in identified in the present study (Figure 3). They typi the pathogenesis of disease. Immunol Rev 1989, 108. cally arise at mucosal sites where they give rise to 111-133 lympho-epithelial lesions. Although data on the mo 3. Picker LJ, Michie SA, Rott LS. Butcher EC: A unique phenotype of skin-associated lymphocytes in humans, lecular basis of normal B cell homing are scarce, we preferential expression of the HECA-452 epitope by envision that expression of the mucosal homing re benign and malignant T cells at cutaneous sites. Am J ceptor a ß in these tumors might be instrumental in 4 7 Pathol 1990, 136:1053-1068 their often very typical dissemination to distant mu 4. Noorduyn LA, Beljaards RC, Pals ST, Heerde P van, 2,13,15,20 22 cosal sites. ~ Also, the observation that Radaszkiewicz T, Willemze R, Meijer CJLM: Differential 41 Chapter 3 expression of the HECA-452 (cutaneous lymphocyte as 15. Isaacson PG: Gastrointestinal lymphomas and lym sociated antigen, CLA) in cutaneous and non-cutaneous phoid hyperplasias. Neoplastic Hematopathology. Ed T-cell lymphomas. Histopathology 1992, 21:59-64 ited by DM Knowles. Baltimore, Williams and Wilkins, 5. Berg EL, Yoshino T, Rott LS, Robinson MK, Warnock RA, 1992, pp 953-978 Kishimoto TK, Picker LJ, Butcher EC: The cutaneous lym 16. Pals ST, Hogervorst F, Keizer GD, Thepen T, Horst E, phocyte antigen is a skin lymphocyte homing receptor for Figdor CG: Identification of a widely distributed 90-kD the vascular lectin endothelial cell-leukocyte adhesion glycoprotein that is homologous to the hermes-1 hu molecule 1, J Exp Med 1991, 174:1461-1466 man lymphocyte homing receptor, J Immunol 1989, 6. Streeter PR, Berg E, Rouse BTN, Bargatze RF, Butcher 143:851-857 EC: A tissue-specific endothelial cell molecule involved 17. van der Loos CM, Becker AE, van den Oord JJ: in lymphocyte homing. Nature 1988, 331:41-46 Practical suggestions for successful immunoenzyme 7. Briskin MJ, McEvoy, Butcher EC: MAdCAM-1 has ho double-staining experiments. Histochem J 1993, 25: mology to immunoglobulin and mucin-like adhesion 1-13 receptors and to lgA1. Nature 1993, 363.461-464 18. Lazarovits AI, Mosciki RA, Kurnick JT, Camerini D, 8. Holzmann B, Mclntyre BW, Welsman IL: Identification Bhan AK, Baird LG, Erikson M, Colvin RB: Lymphocyte of a murine Peyer's patch-specific lymphocyte homing activation antigens: a monoclonal antibody, anti-Act-1, receptor as an integrin molecule with an a chain ho defines a new late lymphocyte activation antigen. J Im mologous to human VLA-4«. Cell 1989, 56'37-46 munol 1984, 133:1857-1862 9. Berlin C, Berg EL, Briskin M, Andrew DP, Kilshaw PJ, 19 Kilshaw PJ, Murant SJ: Expression and regulation of j37 Holzmann B, Weissman IL, Hamann A, Butcher EC: integrins on mouse lymphocytes: relevance to the mu a4ß7 integrin mediates lymphocyte binding to the mu cosal immune system. Eur J Immunol 1991, 21:2591- cosal vascular addressin MAdCAM-1. Cell 1993, 74 2597 185-195 20. Radaszkiewicz T, Dragosics B, Bauer P: Gastrointesti 10. Parker CM, Cepek KL, Russell GJ, Shaw SK, Prosnett nal malignant lymphomas of the mucosa-associated DN, Schwarting RL, Brenner MB: A family of (37 inte- lymphoid tissues: factors relevant for prognosis. Gas grins on human mucosal lymphocytes. Proc Natl Acad troenterology 1992, 102:1628-1638 Sei USA 1992, 89:1924-1928 11 Schweighoffer T. Tanaka Y, Tidswell M, Erle DJ, Hor- 21 Diss TC, Peng H, Wotherspoon AC, Pan L, Speight PM, gan KJ, Luce GEG, Lazarovits AI, Buck D, Shaw S. Isaacson PG: A single neoplastic clone in sequential biopsy specimens from a patient with primary gastric- Selective expression of integrin aAß7 on a subset of human CD4* memory T cells with hallmarks of gut- mucosa-associated lymphoid-tissue lymphoma and tropism. J Immunol 1993, 151:717-729 Sjogren's syndrome. N Engl J Med 1993, 329:172-175 12 Erle DJ, Briskin MJ, Butcher EC, Garcia-Pardo A, Laz 22. Ree HJ, Rege VB, Knisley, Thayer WR, D'Amigo RP, arovits AI, Tidswell M: Expression and function of the Song JY, Crowley JP: Malignant lymphoma of Waldey- MAdCAM-1 receptor integrin a4ß7, on human leuko er's ring following gastrointestinal lymphoma. Cancer cytes. J Immunol 1994, 153:517-528 1980, 46:1528-1535 13. Pals ST, Drillenburg P, Dragosics B, Lazarovits AI, 23. Mackay CR, Marston WL, Dudler L: Naive and memory Radaszkiewicz T: Expression of the mucosal homing T cells show distinct pathways of lymphocyte recircu receptor a4ß7 in malignant lymphomatous polyposis of lation. J Exp Med 1990, 171:801-817 the intestine. Gastroenterology 1994, 107:1519-1523 24. Mackay CR, Marston WL, Dudler L, Spertini O, Tedder 14. Lee Harris N.Jaffe ES, Stein H, Banks PM, Chan JKC, TF, Hein WR: Tissue-specific migration pathways by Cleary ML, Delsol G, De Wolf-Peeters C, Falini B, Gatter phenotypically distinct subpopulations of memory T KC, Grogan TM, Isaacson PG, Knowles DM, Mason cells. Eur J Immunol 1992, 22:887-895 DY, Muller-Hermelink H-K, Pileri SA, Piris MA, Ralfkiaer 25. Bienenstock J, Befus AD: Mucosal immunology. Immu E, Warnke RA: A revised European-American classifi nology 1980, 41:249-270 cation of lymphoid neoplasms: a proposal from the 26. Girard JP, Springer T: High endothelial venules (HEVs): international lymphoma study group. Blood 1994, 84: specialized endothelium for lymphocyte migration. Im 1361-1392 munol Today 1995, 16:449-457 42 Chapter 4 Expression of adhesion molecules in pagetoid reticulosis (Woringer-Kolopp disease) P. Drillenburg1, CM. Bronkhorst', A.C. van der Wal', L.A. Noorduyn', R. Hoekzema2, and S.T. Pals1 Departments of Pathology' and Dermatology2, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands British Journal of Dermatology 1997; 136:613-616 43 Chapter 4 Expression of adhesion molecules in pagetoid reticulosis (Woringer-Kolopp disease) P.DRILLENBURG, C.M.BRONKHORST, A.C.VAN DER WAL. L.A.NOORDUYN. R.HOEKZEMA* AND S.T.PALS Departments of Pathology and •Dermatology. Academic Medical Centre. University of Amsterdam. 1105 AZ Amsterdam, the Xetherlands Accepted for publication 24 September 1996 Summary Cell adhesion molecules play a critical role in lymphocyte migration and homing. They convey tissue-specific homing properties to lymphocyte subsets and regulate the positioning of these subsets in the body. In a patient with pagetoid reticulosis, a rare form of cutaneous T-cell lymphoma characterized by extreme epitheliotropism. we examined the expression of adhesion molecules. The neoplastic T lymphocytes showed a strong expression of cutaneous lymphocyte antigen, a skin- E homing receptor which interacts with E-selectin on skin endothelium. a ß7, an adhesion molecule interacting with E-cadherin on epithelial cells, was also expressed on tumour cells. These findings suggest that adhesion molecules are responsible for the unique growth pattern in pagetoid reticulosis, and for the clinical behaviour of the disorder. The formation of distinct lymphoid compartments related monoclonal.12 Clinically, the disease can be subdivided to specific organs, such as mucosa- or skin-associated into an indolent localized type and a generalized type that lymphoid tissues, depends on selective recirculation may run an unfavourable course.1314 The most charac and homing of lymphocytes. This homing process is care teristic morphological feature is the strict cutaneous loca fully regulated through specialization of both endothelial lization and the extreme epitheliotropism of the tumour cells and lymphocyte subsets in their expression and cells, which suggests the involvement of a highly specific regulation of adhesion receptors and counter-recep set of adhesion molecules in the pathogenesis of PR. tors.1 By mediating interactions with the extracellular In the present study, we have examined the expression of matrix (ECM)2 and with a variety of cells, including adhesion receptors in a patient with PR. antigen presenting* and epithelial cells,4'5 adhesion recep tors also play a pivotal role in the subsequent migration and positioning of lymphocytes in the tissues. Case report Lymphomas are the malignant counterparts of normal lymphocytes. Adhesion receptors that direct normal A 74-year-old-man was referred because of a lesion on lymphocyte homing (homing receptors) may hence the medial part of the left lower leg. It consisted of a play an important role in lymphoma dissemination.6 flat-surfaced, scaly plaque which varied in colour (from In cutaneous non-Hodgkin's lymphomas this dissemi pinkish pale to reddish brown). The diameter was 6 cm nation is presumably mediated by cutaneous lymphocyte and it had a sharp irregular border (Fig. 1). Induration antigen (CLA),7'8 a skin homing receptor which interacts was minimal and the lesion was not painful. Although with E-selectin on skin endothelium.9 the colour variation of the lesion was unusual, a clinical diagnosis of Bowen's disease or superficial basal cell Pagetoid reticulosis (PR), or Woringer-Kolopp dis carcinoma was suspected and punch biopsies were taken. ease,10 is a rare lymphoproliferative disorder which is A biopsy taken from the affected skin was fixed in histologically characterized by an infiltrate consisting 10% buffered formalin and paraffin-embedded. A second of atypical T lymphocytes localized in the epidermis.11 biopsy was directly snap-frozen in liquid nitrogen. Consistent with a neoplastic character, these T cells are Immunohistochemical stainings were performed on frozen and/or paraffin sections using the streptavidin- Correspondence: Dr Steven T.Pais. Department of Pathology. Academic- biotin-peroxidase complex method (DAKO, Glostrup, Medical Centre. University of Amsterdam. Meibergdreef 9. 1105 AZ Amsterdam, the Netherlands. Denmark). For tumour typing, a panel of commercially 44 adhesion molecules in pagetoid reticulosis Table 1. Expression of adhesion molecules Adhesion Positivilv Intensity* molecule tumour ceils (%i a J-chain 0% U aj-chaln 0% 0 a4-chaln 10%-50% 1 + a,07 0% (1 a,,-chain 0% 0 a% a 90% 2+ ai -chain 2 90% 2+ pVchain a 90% 2+ 0i-chain 50%-90% 2+ L-selectln 1 ()"«,- 50%, 2 + Figure 1. The variably coloured and scaly plaque, diameter b cm. on ICAM-1 10%-50% 2 + the medial part of the left lower leg. V'CAM-1 5()%-90% 1 + CLA a 90'», 2+ available monoclonal antibodies (mAbs) against CD1. CD2. CD3. CD4. CD5. CDS. CD10. CD20, CD25. CD30. * U. no staining: 1+. moderate staining: 2+. strong staining. CD45, CDhX. TcRa/3. ÏCR7Ô, HLA-DR. vimentin and keratin was used IDAKO). The mAbs against adhesion molecules used were as follows: TS 2/7 against a, U.S.A.); BB1G-I1 against ICAM-1 (CD54) (British Biotech nology. Abingdon. Oxon. U.K.I: 4B9 against VCAM-1 [CD49a) (ATCC Rockville. Maryland. U.S.A.): J143 against 15 a, (CD490 (ATCC): Ber-ActS against a% (CD103I (CD106): and HECA452 against CLA."' HP2/1 against (DAKO): 4B4 against /3, (CD29) (Coulter. Hialeah. FL. 0:4 (CD49d): Ml 7 against «,. (CDlla): and MUS against ß2 (CD1S) were kindly provided by Dr F.Sanchez-Madrid (Universidad Autonoma de Madrid. Spain). 1A10 against ah (CD490 and Act-1 against a+ß7 (CD49d/ ß7) were a gift from Dr A. Sonnenberg (N'KI. Amster dam, the Netherlands) and Dr A.I. Lazarovits (Univer sity of Western. London. Ontario. Canada), respectively. The skin biopsy showed an intraepithelial lesion con sisting of atypical cells with a moderate amount of cytoplasm and large irregular, hyperchromatic nuclei with inconspicuous nucleoli (Fig. 2). The atypical cells were arranged as individual cells or clusters between the keratinocytes. There was no evident hyper- and/or para mBmmBBBS keratosis. In the dermis underneath the lesion, a reactive mm infiltrate consisting of plasma cells and small lymphocytes '.V was present. Immunohistochemistry on paraffin and/or frozen sections showed uniform expression of CD2. CD5. CD8. CD25. CD30. CD45. HLA-DR and TCR-a/3 on the tumour cells, and hence identified them as activated cytotoxic T-lymphocytes. The tumour cells showed an adhesion phenotype as described in Table 1. .«f- After PR was diagnosed based on the histopathological features, staging, which included a thorough physical examination, blood and bone marrow smears and a chest X-ray were performed. No evidence of internal J* » involvement was found. The lesion was treated by Ml radiation with 6 MeV electron beam therapy using 20 Figure 2. Pagetoid reticulosis: (a) extreme epitheliotropism. staining doses of 2Gy in 25 days. This resulted in a complete for CD30 1x35): (b) atypical cells with irregularly, hyperchroma tic nuclei arranged individual or in clusters (x350). clinical remission. 45 Chapter 4 a •*<•"!: . Ï . ' . Figure ï. Emmunohistochemical stainings * V ;•;••' i of the skin lesion: (al cutaneous E l\ mphocyte antigen: (bl a^ßr'. (c) a ß7; (d) CD30 1x175). jgfi* Discussion mycosis fungoides (unpublished observation). The expres sion of CLA in PR reflects its ontogenetic relation to the The atypical cells in the present case of PR strongly skin-homing T-cell subset and may contribute to the expressed cutaneous lymphocyte antigen (CLA) (Fig. 3a). specific pattern of dissemination of the malignant lym Cutaneous lymphocyte antigen is a skin-homing receptor phocytes. '"s which directs T lymphocytes to the skin through inter Interestingly, all tumour cells were found to strongly 1,9 action with E-selectin on dermal vessels. In the periph express a ß7 (Fig. 3c). Under physiological conditions. eral blood. CLA is expressed on a subset of memory T a 'ß- is expressed on nearly all intestinal intraepithelial lymphocytes.'~' Cutaneous lymphocyte antigen expres lymphocytes and on approximately 50% of the T cells sion is presumably acquired during virgin to memory in the lamina propria. In the normal skin expression transition of T cells in skin-associated peripheral lymph of 46 adhesion molecules in pagetoid reticulosis E LFA-1 may also contribute to this interaction since the cells and T lymphocytes mediated by E-cadherin and the a ß7 epidermal keratinocytes in the lesions expressed ICAM-1 integrin. Nature 1994: 372: 190-3. 5 Cerf-Bensussan N, (arry A. Brousse N et al. A monoclonal antibody (not shown). In this context, the recent finding of a (H.ML-1) defining a novel membrane molecule present on human correlation in mycosis fungoides between loss of epithe- intestinal lymphocytes. Eur ƒ Immunol 1987: 17: 1279-85. E liotropism and loss of ct ß7 expression is of interest since 6 Pals ST. Horst E, Scheper Rf. Meijer CJLM. Mechanisms of human it also supports a role for this integrin in tumour cell lymphocyte migration and their role in the pathogenesis of interaction with the epidermis.19 disease. Immunol Rev 1989: 108: 111-33. 7 Noorduyn LA, Beljaards RC. Pals ST et al. Differential expression of To enter the epithelium, lymphocytes have to cross the HECA-452 antigen (cutaneous lymphocyte associated antigen. the epithelial basement membrane (EBM). Recently, inter CLA) in cutaneous and non-cutaneous T-cell lymphomas. Bisto- action of the integrin a^ßi to laminin-5 in the EBM. was pathology 1992: 21: 59-64. suggested to present the first step in this process.20 In the 8 Picker LI. Michie SA, Rott LS. Butcher EC. A unique phenotype of skin-associated lymphocytes: preferential expression of the HECA- present case, the tumour cells did not express a3. In PR 452 epitope by benign and malignant Tcells at cutaneous sites. the tumour cells are localized in the epithelium: down- Am I Pathol 1990: 136: 1053-68. regulation of aj might occur after transition of the EBM. 9 Berg EL. Yoshino T. Rott LS et al. The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular The present tumour showed strong expression of CD30 lectin endothelial cell-leucocyte adhesion molecule 1. ƒ Exp Med (Fig. 3d). Among primary cutaneous T-cell lymphomas, 1991: 174: 1461-6. the classical cases of mycosis fungoides/Sézary syndrome 10 Woringer F. Kolopp P. Lesion erythemato-squameuse polycyclique can be separated from a group of large cell lymphomas de l'avant-bras évoluant depuis 6 ans chez un garçonnet de 13 with a distinctive clinical and histological picture. ans: histologiquement infiltrat intra-epidermique d'apparence tumorale. Ann Dermatol Venereal 1939: 10: 945-8. Expression of CD30 on the tumour cells further subdivides 11 Willemze R. Beljaards RC. Meijer CJLM. Classification of primary this latter group since, irrespective of the morphology, cutaneous T-cell lymphomas. Histopathology 1994: 24: 405-1 5. expression of CD 30 is related to a favourable prognosis.11 12 Wood GS. Weiss LM. Hu C-H et al. T-cell antigen deficiencies and These CD 30* lymphomas have a tendency to remit clonal rearrangements of T-cetl receptor genes in pagetoid spontaneously and are highly responsive to radiother reticulosis (Woringer-Kolopp disease). .\' Engl ƒ Med 1988: 318: 1 164-7. apy. ' CD30. a member of the TNF/NGF family, might 13 Wood GS. Benign and malignant cutaneous lympho-proliferative play a direct role in causing this favourable response to disorders including mycosis fungoides. In: Neoplastic Hemato- therapy since triggering can induce apoptosis.2' palhology (Knovvles DM, ed.), Baltimore: Williams and Wilkins, 1992: 932-3. In conclusion, this case shows that the atypical 14 Yagi H. Hagiwara T. Shirahama S et al. Disseminated pagetoid lymphocytes in PR are equipped with a set of adhesion reticulosis: need for long-term follow-up. ] Am Acad Dermatol molecules that allow specific skin-homing as well as 1994; 30: 345-9. highly effective interaction with epidermal keratinocytes. 15 Carlos TM. Schwartz BR. Kovach NL et al. Vascular cell adhesion molecule-1 (VCAM-1) mediates lymphocyte adherence to cytokine- This suggests that these adhesion molecules are an activated cultured human endothelial cells. Blood 1990: 76: important factor in determining the unique growth 965-70. pattern of PR and its characteristic clinical behaviour. 16 Duljvestijn AM. Horst E. Pals ST et al. High endothelial cell differentiation in human lymphoid and inflammatory tissues defined by monoclonal antibody HECA-452. Am ƒ Pathol 1988: 130: 147-55. Acknowledgment 17 Picker LJ. Treer JR. Ferguson-Darnell B et al. Control of lymphocyte recirculation in man. II. Dilferential regulation of the cutaneous This research was supported by Grant IKA 91/9 of the lymphocyte-associated antigen, a tissue-selective homing receptor Dutch Cancer Society. for skin-homing T cells. / Immunol 1993; 150: 1122-36. 18 Schweighoffer T. Tanaka Y. Tidswell M et al. Selective expression of integrin a^ßj on a subset of human CD4 + memory T cells with References hallmarks of gut-tropism. ƒ Immunol 1993; 151: 717-29. 19 Simonitsch I. Volc-Platzer B. Mosberger I. Radaszkiewicz T. 1 Butcher EC. Picker LJ. Lymphocyte homing and homeostasis. E Expression of monoclonal antibody HML-1 defined a ß7 Science 1996; 272: 60-6. integrin in cutaneous T-cell lymphoma. Am J Pathol 1994: 145: 2 Reynolds PJ. Shimizu Y. Mobiey JL. Lymphocytes and extracellular 1148-58. matrix. In: Lymphocyte Adhesion Molecules (Shimizu Y. ed.). Austin: 20 Wayner EA. Gil SG. Murphy GF et al. Epiligrin. a component of RG Landes Company. 1993: 192-220. epithelial basement membranes, is an adhesive ligand for aißi 3 Koopman G. Parmentier HK. Schuurman H-J et al. Adhesion of positive T lymphocytes, ƒ Cell Biol 1993; 121: 1141-52. human B-ce[ls to follicular dendritic cells involves both the LFA-1/ 21 Gruss HJ, Boiani N. Williams DE et al. Pleiotropic effects of the ICAM-1 and VLA-4/VCAM-1 pathways, ƒ Exp Med 1991: 173: CD30 ligand on CD30-expressing cells and lymphoma cell lines. 1297-304. Blood 1994: 83: 2045-56. 4 Cepek KL. Shaw SK, Parker CM et al. Adhesion between epithelial 47 48 Chapter 5 CD44 expression predicts disease outcome in localized large B-cell lymphomas Paul Drillenburg', Vera J. M. Wielenga1, Mark H. H. Kramer, Johan H. J. M. van Krieken2, Hanneke C. Kluin-Nelemans3-7, Jo Hermans4-7, Siem Heisterkamp5, Evert M. Noordijk6,7, Philip M. Kluin2-7, and Steven T. Pals' Departments of Pathology' and Clinical Epidemiology and Biostatistics3 Academic Medical Center, Amsterdam, The Netherlands; Departments of Pathology1, Hematology3, Medical Statistics'1 and Clinical Oncology6 Leiden University Medical Center, Leiden, The Netherlands, and Comprehensive Cancer Center West (CCCWf, Leiden, The Netherlands submitted 49 Chapter 5 Abstract Diffuse large B-cell non-Hodgkin's lymphomas (DLCL) form a heterogeneous group of tumors with diverse morphology, clinical features, treatment response and prognosis. The biological variables underlying this heterogeneity are unkown. In the present study, we explored the value of the lymphocyte homing receptor CD44, a putative determinant of lymphoma dissemination, in predicting prognosis in DLCL. Expression of the standard form of CD44 (CD44s) and of CD44 isoforms containing exon v6 (CD44v6) on tumor cells was assessed by immunohistochemistry in a cohort of 276 DLCL patients from a population based lymphoma registry. We observed that CD44s as well as CD44v6 expression correlated with tumor dissemination in patients with primary nodal DLCL. Importantly, in patients with localized nodal disease, CD44s was a strong prognosticator predicting tumor related death independent of the other parameters of the International Prognostic Index (IPI). Incorporation of CD44s in the IPI parameter "stage", increased the prognostic value of this parameter in nodal DLCL. Our data identify CD44 as a biological prognosticator, which can be used to "fine-tune" the IPI for nodal DLCL. 50 CD44 in large B-cell lymphoma Introduction Diffuse large B-cell lymphomas (DLCL) constitute 30 to 40% of adult non-Hodgkin's lymphomas and are biologically related to (activated) mature B cells '"3. In the recently proposed Revised European American Lymphoma (REAL) classification these tumors are categorized as one single group, since previous attempts to subclassify them on morphological grounds, were largely irreproducible 2. However, there is consensus that DLCL represent a diverse group of neoplasms, with heterogeneous genetic background, clinical features, treatment response, and prognosis 1"6. Although prognosis in DLCL can be predicted on the basis of clinical characteristics 6, the genetic and molecular basis underlying the heterogeneity in disease aggressiveness and tumor progression, as well as in response to therapy remain to be elucidated. Normal recirculating lymphocytes express cell-surface glycoproteins of the CD44 family. These molecules have a wide tissue distribution and function in several important biological processes including lymphocyte homing and activation710, hematopoiesis7", and tumor progression and metastasis712'16. Due to extensive alternative splicing and posttranslational modification by N- and O-linked sugars as well as by glycosaminoglycan (GAG) side chains1317-19, CD44 is highly diverse. On lymphocytes, the short 80-90 kDa standard (hematopoietic) form of CD44 (CD44s) is most abundant, while larger variants predominate on some normal and neoplastic epithelial cells and are also expressed on activated lymphocytes and on aggressive lymphomas 17-20-23. Clinical studies in non-Hodgkin's lymphomas have demonstrated that expression of CD44s epitopes is associated with disseminated disease and tumor related death1214-24-25. A causal role of CD44s in determining this unfavorable disease outcome was suggested by experiments in nude mice, showing an enhancing effect of CD44s on lymphoma growth and dissemination15. More recently, CD44 splice variants containing exon v6 were shown to be expressed in a subgroup of high-grade lymphomas with poor prognosis 21. In rodents, homologues of these variants confer metastatic potential on carcinoma and lymphoma cell lines13-26. Together, these data indicate that CD44s and CD44v6 are important mediators of lymphoma dissemination that could be of use as molecular markers to identify subgroups of high-risk DLCL patients. Here, we have explored this hypothesis by studying CD44s and CD44v6 expression in tumor tissue of patients with DLCL from a population-based NHL registry; the results indicate that CD44 can be considered as a major prognosticator in patients with localized DLCL. 51 Chapter 5 Materials and methods Patients and material. Between 1981 and 1989 1168 patients with a histologically proven diagnosis of NHL were included in the population based non-Hodgkin's lymphoma registry program of the Comprehensive Cancer Center West (CCCW) in The Netherlands4,27,28'29'30. The region of the CCCW comprises 1.6 million inhabitants and 15 hospitals. Only newly diagnosed NHL were included. All tumors were reviewed by a panel of hematopathologists. Patients were staged according to the Ann Arbor classification31 with modifications for extranodal lymphoma32. For adequate staging at least a chest radiograph and/or CT scan, a CT scan of the abdomen or lymphangiogram supplemented by isotope spleen and liver scan in the early years of registration, and a bone marrow biopsy had to be performed. In addition to clinical stage, the presence or absence of lymphoma at various anatomical sites was recorded. Patients were treated according to the preference of the local specialist in internal medicine, and this treatment was considered 'adequate' for large cell NHL, when doxorubicin- containing polychemotherapy was administered with or without radiotherapy, whereas for stage I patients, radiotherapy alone was also considered as adequate. All tissue specimens were obtained prior to treatment. Clinical parameters as well as treatment modalities and outcome were recorded. The follow-up was annually updated up to 1996. In the CCCW records 494 B-cell lymphomas were diagnosed according to the Kiel classification33 as diffuse centroblastic-centrocytic (diff CbCc), diffuse centroblastic (diff Cb), immunoblastic (lb) and mucosa-associated lymphoid tissue lymphomas of high malignancy (MALT high), compatible with large B-cell NHL as recently defined by the REAL- classification2. From 310 patients, tissue blocks containing tumor were available and slides of paraffin embedded tumor tissue were made. From 276 patients, interprétable staining of tumor tissue for CD44s and CD44v6 was obtained. For 236 of these patients, all prognostic parameters of the International Prognostic Index (IPI) were available (table 1); demographics, and stage distribution of these 236 patients was not different than those of the total group of 494 B-cell lymphomas in the CCCW registry (data not shown). According to our previous reports4,27,28'29 at presentation three patterns of lymphoma localization were defined: (1) "primary nodal" NHL with presentation in lymph node, Waldeyer's ring or spleen (n=155); (2) "primary extranodal" NHL with presentation at other sites without or with regional lymph node involvement (n=91); (3) other NHL with disseminated disease at presentation and no possibility to determine the primary site (n=30); the latter were called "extensive". Both in primary nodal and extranodal disease, bone marrow involvement was accepted in cases with a positive staging biopsy. All parameters of the IPI were available in these three subgroups for 137, 75 and 24 patients respectively. 52 CD44 in large B-cell lymphoma Table 1. Patient characteristics total nodal extranodal extensive n=276 n=155 n=91 n=30 (%) (%) (%) (%) male/female 131/145 82/73 38/53 11/19 age (range) 20-93 21-91 20-93 29-86 age (mean) 65 64 67 65 Ann Arbor stage I 88 (32) 39 (25) 49 (54) 0 stage II 73 (26) 53 (34) 20 (22) 0 stage III 38(14) 38 (25) 0 0 stage IV 77 (28) 25(16) 22 (24) 30(100) IPI low 97 (35) 57(37) 40 (44) 0 low/intermediate 62 (23) 46 (30) 14(15) 2(7) high/intermediate 39(14) 23(15) 10(11) 6(20) high 38(14) 11(7) 11(12) 16(53) not évaluable 40(14) 18(11) 16(18) 6(20) no therapy and surgery only 36(13) 11(7) 23 (25) 2(7) radiotherapy only 67 (25) 38 (25) 28(31) 1(3) chemotherapy and +/- radiotherapy without doxorubicin 48(17) 24(15) 11(12) 13(43) containing doxorubicin 125 (45) 82(53) 29 (32) 14(47) Immunoltistochemistry. Tissues were fixed in buffered formalin and routinely embedded in paraffin. Sections of 5um were cut and put on APES-coated slides. After deparaffinization in xylene and rehydration in alcohol endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxide in methanol for 20 minutes. Prior to staining with monoclonal antibody (mAb) VFF18 (see below), the tissue sections were incubated in a microwave oven at 100° C for 10 minutes in citrate buffer (0.01 M, pH=6). The slides were washed in PBS, pre-incubated for 15 minutes with 10% normal goat serum, and then incubated for 1 hour with the primary mAb. The primary monoclonal antibodies used were Hermes-334 against an epitope on the constant part of CD44 (kindly provided by Dr S. Jalkanen, Turku, Finland), and VFF 18 (Bender Co. Vienna, Austria) against the epitope QWFGNRWHEGYRQT on exon CD44v6 35. After incubation with the primary mAb, the sections were incubated with biotinylated rabbit anti-mouse antibodies (DAKO, Glostrup, Denmark) in PBS containing 10% normal human serum, and with streptavidin-biotin peroxydase complex (DAKO) each for 30 minutes. HRP activity was detected by incubation in 1 mg/ml 3,3-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, U.S.A.) and 0.015% H,02 in 50mM Tris-HCL (pH= 7.6). Slides were counterstained with Mayer's hematoxylin. Normal tonsil tissue was used as a 53 Chapter 5 positive control in each staining procedure. Furthermore, within the tumors, non-neoplastic lymphocytes and macrophages expressing high levels of CD44s were always present and served as internal standard and control. To facilitate identification of the neoplastic lymphoid cells, serial sections were routinely stained for CD20 (CD20, DAKO) and CD3 (CD3, DAKO). On the basis of the estimated percentage of positively staining tumor cells, CD44s and CD44v6 expression was scored as follows: 0 (negative/low) = less than 10% positive tumor cells; 1 (intermediate) 10-50% tumor cells positive; 2 (high) more than 50% positive tumor cells. This scoring system is based on our experience from prior studies24-36 . Intensity of the stainings was also estimated, but subdivision of the groups based on staining intensity proved of no additional prognostic value (data not shown). All slides were read by two independent observers (P. Drillenburg and S.T. Pals); discrepancies were solved by consensus. During the whole procedure, the observers were blinded for clinical data/disease outcome. Statistical analysis. The correlation of CD44s and CD44v6 with biological and clinical parameters was estimated with the Spearman correlation coefficient. Survival curves were plotted following the Kaplan-Meier method and tested for statistical significance using the log-rank test. Overall survival (OS) was calculated from the date of diagnosis until death (all causes) or last follow-up. For patients who reached complete remission (CR), disease free survival was estimated from the date of CR until first relapse or last contact, if disease free. For the multivariate analysis and calculation of the hazard ratios and its confidence intervals Cox proportional hazard model was used. In Cox regression, the forward stepwise selection method was used, the results were controlled with backward stepwise selection. Results CD44s and CD44v6 expression in diffuse large B-cell lymphoma (DLCL). CD44s expression in DLCL was highly variable, ranging from negative to strongly positive, which confirms a previous study from our laboratory24. Intermediate or high expression of CD44s was present in 13% and 63% of the tumors, respectively; 24% of the tumors were CD44s negative. Unlike CD44s, CD44v6 was only expressed in a minority of the tumors: whereas 84% showed no detectable staining for CD44v6, intermediate or high expression was present in 10% and 6% of the tumors, respectively. Subclassification of DLCL on the basis of morphology (Kiel-classification) or primary tumor localization did not reveal significant differences in distribution of CD44s or CD44v6 between the groups (data not shown). 54 CD44 in large B-cell lymphoma 100 100 +/++. o 90 E 3 80 70 70 60 60 Q U 50 50 A B p=0.003 p=0.01î f 1 1 i ~T ~I I II III IV 12 3 4 >5 (n=39) (n=53) (n=38) (n=25) (11=50) (n=56) (n=23) (n=13) (n=13) stage number of anatomical sites Figure 1. CD44s expression in nodal DLCL is related to tumor dissemination. A) Relation between CD44s expression and Ann Arbor stage; B) Relation between CD44s expression and the number of anatomical sites involved by tumor. Since intermediate (1+) and high (2+) expression of CD44s were both positively correlated with tumor stage/dissemination, and since the survival curves for these patient groups were identical, both groups have been combined (+/++). Correlation ofCD44s and CD44v6 with clinical risk factors. In view of the strong evidence for a role of CD44 in lymphocyte homing and lymphoma dissemination, we analyzed the relation between CD44 expression and tumor dissemination in our study group. In the total group (n=276), no correlation between CD44s expression and tumor dissemination was found (p=0.20). However, in the tumors with a primary nodal localization (n=155), CD44s expression significantly correlated to clinical stage (Fig.lA) (p=0.003) and, as an alternative measure of dissemination, to the total number of anatomical sites involved by tumor (Fig. IB) (p=0.018). In extranodal lymphomas (n=91), CD44s did not correlate with tumor dissemination (p=0.21). Also, CD44v6 expression showed a significant correlation to tumor stage in nodal, but not in extranodal tumors (p=0.04). Bone marrow involvement did not correlate with expression of CD44s or CD44v6 (p=0.28 and p=0.72 respectively). Table 2 shows the relation between CD44 expression and the clinical parameters of the International Prognostic Index (IPI). With the exception of the parameter "stage", neither of the IPI parameters correlated to CD44 expression. CD44 predicts prognosis in localized DLCL. We next explored the prognostic value of CD44s and CD44v6. For the whole cohort 55 Chapter 5 Table 2. Expression of CD44 related to the clinical parameters of the International Prognostic Index (n=236). N=2 36 N= 137 (nodal) CD44s(%) CD44v6(%) CD44s(%) CD44v6(%) n _* +/++* P - +/++ P n - +/++ p - +/++ p age classes <60 years 80 24 76 NS 81 19 NS 48 29 71 NS 81 19 NS >60 156 22 78 85 15 89 19 81 84 16 LDH Normal 114 21 79 NS 83 17 NS 60 30 70 NS 87 13 NS Abnormal 122 24 76 84 16 77 17 83 81 19 Karnofsky >80% 174 23 77 NS 85 15 NS 113 23 77 NS 85 15 NS <80% 62 21 79 79 21 24 21 79 75 25 Ann Arbor Stage I+II 140 26 74 NS 86 14 NS 81 31 69 .005 88 12 NS III+IV 96 18 82 80 20 56 11 89 77 23 Extranodal sites <1 197 22 78 NS 83 17 NS 137 23 77 83 17 >1 39 26 74 87 13 0 * -(negative/low)= less than 10% positive tumor cells; +/++(intermediate/high)= more than 10% positive of patients (n=276), CD44s and CD44v6 did not predict overall survival (p=0.17 and 0.96, respectively). Interestingly, however, in patients with localized (stage I) disease (n=88), CD44s proved to be a highly significant unfavorable prognosticator (p=0.0076) (Fig. 2A). No statistically significant prognostic value was present in stage II or IV disease (Fig. 2B,C) although there was a similar trend in stage II patients. The stage III patient group was too small to be analyzed. Although a strong prognostic effect of CD44s was evident in the whole stage I group, this was largely accounted for by the effect of CD44s in the patient subgroup (n=39) with a primary nodal lymphoma (p=0.0014) (Fig. 2D). If stage I extranodal lymphomas were analyzed separately, CD44s had no significant prognostic value (p=0.79). Hence, CD44s was a strong prognosticator in patients with localized nodal DLCL. In a multivariate comparison with the clinical parameters of the International Prognostic Index (IPI), CD44s was the major prognosticator in this patient group (RR= 5,07; 95% CI: 1,12-22,90). These data were also found for the 26 patients with nodal stage I disease who were treated with doxorubicin- containing polychemotherapy with of without radiotherapy, or radiotherapy alone (Fig.3A). 56 CD44 in large B-cell lymphoma 100 75 •" s,i+.^a«^.^ n—25 50 •- 25 25 •" S3 > 3 "I- 10 15 10 100 a.. D ..+..+ _ y""""" n=15 75 - \ 50 • - \ stage I n=24 25 • - nodal n=39 +/++ p=0.0014 0 1 1 1— 10 15 Time (years) Figure 2. CD44s predicts outcome in patients with localized DLCL. Overall survival for patients with CD44s positive (1+ and 2+) and negative (-) DLCL subdivided according to Ann Arbor stage. A) stage I, whole study group; B) stage II, whole study group; C) stage IV, whole study group: D) stage I; primarily nodal lymphomas. The stage III group was too small to be analyzed. It is important to note that the proportion of patients who received doxorubicin- containing chemotherapy and/or radiotherapy did not differ significantly between the CD44s positive and negative groups (table 3). Also the percentage of patients who entered complete 57 Chapter 5 remission was similar (table 3). However, the relapse rate for CD44s positive lymphomas (58%) was significantly increased in comparison to the negative lymphomas (10%, p=0.018) (table 3). Of the relapses 86% took place outside the region originally involved by tumor. In line with the high relapse rate in CD44s positive patients, CD44s expression was also an unfavorable prognosticator for disease free survival (p=0.03) (Fig. 3B). The number of patients with a relapse was too small to analyse the impact of treatment on relapse rates in CD44s positive and negative cases. Table 3. CD44s expression in stage I, nodal DLCL in relation to therapy, complete remission and relapse in 'adequately' treated patients. chemotherapy- radiotherapy complete relapse +/- radiotherapy only remission rate n=9 n=17 n % P % P % P % P - 11 45 55 91 10 CD44s* NS NS NS 0.018 +/++ 15 27 73 80 58 - doxorubicin-containing polychemotherapy * -(negative/low)= less than 10% positive tumor cells; +/++(intennediate/high)= more than 10% positive. Incorporation ofCD44 in the IPIparameter "stage" improves its prognostic power in nodal lymphoma. In view of the evidence that CD44s is instrumental in mediating lymphoma dissemination, we adopted the working hypothesis that the adverse prognostic effect of CD44s in clinically localized tumors might result from occult dissemination. If so, CD44s might be used as a tool to predict outcome. As the International Prognostic Index is currently widely used to predict prognosis in DLCL, we addressed this hypothesis by exploring whether the prognostic power of the IPI parameter "stage" might be improved by including CD44s as an additional parameter in primary nodal lymphomas. Hence, we replaced the parameter Ann Arbor stage >II disease by Ann Arbor stage >II disease or positivity for CD44s. This redefined parameter ("stage/CD44s") was compared in multivariate analysis with the conventional parameters of the IPI, i.e., age >60; elevated LDH; Karnofsky performance status <80 and Ann Arbor stage >II. Upon regression analysis (table 4), the parameter "stage/CD44s" (and not the conventional IPI parameter "stage") was selected, indicating that inclusion of CD44s increases the prognostic power of the IPI parameter "stage". As is shown in figure 4, incorporation of CD44s into the IPI parameter "stage"' clearly improves the separation between the risk groups (Fig. 4A versus 4B). 58 CD44 in large B-cell lymphoma 100 1UU B n=ll K"""~ n=10 >cd 75 75 --{ 50 - 50 -.!_! u DFS +/++ M n=12 "3 25 25 - - stage I n=15 +/++ nodal s n=22 3 p=0.03 u 0 -1 1 1—' 10 15 10 15 Time (years) Figure J. Cumulative survival for adequately treated patients with CD44s positive (1+ and 2+) and negative (-) stage I, nodal DLCL. A) overall survival (OS); B) disease free survival (DFS). Discussion The heterogeneity in outcomes in aggressive non-Hodgkin's lymphoma has prompted the development of the IPI6, a clinical prognostic factor model, which identifies patients with different likelihoods of being cured of their disease. However, there is consensus that the clinical prognostic features incorporated in the IPI are, in large part, surrogate variables, with a remote relation to the biological variables determining disease heterogeneity 6-37. Identification of these unknown biological variables is of great importance since they may improve prognostic factor models and, in addition, serve as targets for therapy in specific subsets of patients. In our present study, we demonstrate that CD44s, a biological determinant of lymphoma dissemination, predicts outcome in patients with localized DLCL and that the IPI parameter "stage" can be improved by incorporating CD44s as an additional parameter. Previous studies from our own and other laboratories have related CD44 expression to tumor dissemination in non-Hodgkin's lymphoma u-]4-24-25. This association is confirmed in our present population based study group: In patients presenting with a primary nodal DLCL lymphoma, but not in those with extranodal disease, both CD44s and CD44v6 were correlated 59 Chapter 5 Table 4. Factors Independently Prognostic of Overall Survival in nodal DLCL (n=1371 Factor Relative Risk 95% CI p value step of selection Karnofsky >80% 1.0 <80% 1.87 1.15-3.06 0.012 1 "stage/CD44s" * negative 1.0 positive 2.62 1.31-5.27 0.007 2 Age <60 years 1.0 >60 years 2.08 1.32-3.28 0.002 3 LDH normal 1.0 abnormal 1.55 1.02-2.37 0.042 4 * see text for definition with tumor spread (Fig. 1). We currently have no explanation for the differential effect of CD44 in nodal versus extranodal lymphoma. It should be noted, however, that nodal and extranodal lymphomas show important genetic, functional and phenotypic differences suggesting that they represent distinct biological entities 2-27-38-39. These differences include differential expression of adhesion/homing receptors other than CD44, for example of L- selectin, which may co-determine tumor spread40. The key finding of our present study is that CD44s is a strong independent prognosticator in localized nodal DLCL (Fig. 2). Whereas patients with localized (stage I) disease and a CD44s negative tumor have a favorable prognosis, the prognosis of those with CD44s positive tumors is poor, even at stage I. The observation that CD44s expression is both correlated with lymphoma dissemination in the entire group and predictive of relapse of disease in patients with localized disease, matches with studies indicating an important physiological role of CD44s in lymphocyte trafficking. In this trafficking process, CD44s mediates lymphocyte binding to high endothelial venules 810-34, lymphocyte rolling 41,42, and migration to inflammatory sites 4344. Presumably, CD44s expression in localized DLCL reflects a high propensity to disseminate and/or the presence of occult dissemination, a scenario which is supported by the fact that CD44s promotes the dissemination of human lymphoma cells xenografted into nude mice '5. A similar scenario has also been proposed to explain the prognostic value of CD44 splice variants containing exon v6 in colorectal and breast cancer 3 60 CD44 in large B-cell lymphoma specifically its recently described role in counteracting apoptosis 46, may also contribute to the less aggressive biological behavior of CD44 negative tumors. In view of the strong evidence that CD44 is instrumental in mediating lymphoma dissemination, we argued that CD44s might be used as an additional prognostic parameter in patients with localized disease. As the IPI is currently widely accepted as a major tool for the identification of "high" or "low" risk in DLCL, this was analyzed by including CD44s in the IPI parameter "stage". Indeed, we observed that the IPI parameter "stage" can be improved by including all CD44s positive patients in the 'high risk for stage' group: In a multivariate comparison with the other IPI parameters, the modified parameter "stage/CD44s" incorporating CD44s, and not the conventional IPI parameter "stage", was selected (table 4). Furthermore, by using this modified staging parameter a superior separation of the distinct IPI risk groups was obtained (Fig. 4A versus 4B). We considered the possibility that more effective treatment of CD44s negative lymphomas might explain the favorable outcome, however, there were no differences in therapy between the CD44s negative and positive groups (table 3). The percentage of patients entering a complete remission was also identical in the CD44s positive and negative groups, by contrast, relapses were much more common in patients with CD44s positive tumors (table 3). As the vast majority of these relapses occurred outside the anatomical area of initial tumor loo x: 75 50- 25 Time (years) Figure 4. Incorporation of CD44s into the IPI (see text) improves the separation between the risk groups for nodal DLCL A) conventional IPI risk groups; B) IPI risk groups in which the staging parameter has been modified by incorporating CD44s. L denotes low risk, LI low intermediate risk, HI high intermediate risk and H high risk. 61 Chapter 5 involvement, this high relapse rate in CD44s positive tumors is consistant with a role of CD44s in (occult) lymphoma dissemination. Analysis of these patients with a relapse for the impact of therapy was not possible due to the small number of patients. Since recent data indicate that the most adequate therapy for stage I patients is a combination of 3 cycles of polychemotherapy and radiotherapy, instead of radio- or chemotherapy alone 47, it will be very interesting to see whether the impact of CD44s holds in a prospective study including this novel therapeutic modalities. Meanwhile, our current study clearly shows that stage I DLCL patients with a CD44 negative tumor have an excellent prognosis. Our current findings extend earlier studies from our own and Jalkanen's laboratory 1424 We previously reported that B-DLCL with low CD44s expression have a favorable prognosis. In the current much larger study, this finding is confirmed and, moreover, the prognostic effect of CD44s is shown to be independent of other IPI parameters24. Jalkanen and collègues reported CD44s to have independent prognostic value14, however, their study and our present study differ at important points. Whereas our present study focusses on a single clinicopathological entity, the study group of Jalkanen was heterogeneous, containing lymphomas of different subtype and grade. Furthermore, in their study from the pre-IPI era, not all clinical parameters of IPI were included in the analysis and, instead of overall survival rates, survival rates corrected for intercurrent death and death of unkown cause, were used as end point. Although these differences make a direct comparison between the two studies difficult, both studies underscore the prognostic value of CD44s. In our present study CD44v6 expression was not a significant prognosticator. In contrast to our data, Stauder and colleagues reported CD44v6 to be a better prognosticator than CD44s 2I. This discrepancy might be due to differences in the composition of the study groups. Unlike in our study group, which contains only B-DLCL, the 'high-grade' B-NHL group analyzed by Stauder et al. consists of a number of distinctive, unrelated, clinicopathological entities including Burkitt's lymphoma, precursor B-cell lymphoma, and DLCL. In view of their distinctive character, Burkitt's lymphoma and precursor B-cell lymphoma were not included in the international non-Hodgkin's lymphoma prognostic factor project. As CD44 is known to be differentially expressed at various stages of B-cell differentiation and in different lymphoma subtypes 48, lumping of different entities may greatly influence, and potentially confound, the results. In conclusion, this population based study identifies CD44s as a strong risk factor in B-DLCL patients with localized nodal disease and shows that incorporation of CD44s into the IPI improves its prognostic value. Unlike the other parameters of the IPI, which have a remote relation to the biological variable determining disease heterogeneity, CD44s most likely represents a true biological prognosticator (co)determining lymphoma dissemination. 62 CD44 in large B-cell lymphoma Acknowledgements We thank Mr. J.B.G. Mulder for expert technical assistance; Drs G. Adolf, E. Patzelt and K.H. Heider, Bender Medsystems, Vienna, Austria for mAb VFF18; Dr S. Jalkanen, Turku University, Turku, Finland, for mAb Hermes-3; and Dr. G.J.A. Offerhaus for critical reading of the manuscript. References Stein H, Dallenbach F: Diffuse large Hellman S, Rosenberg SA (eds): cell lymphomas of B and T cell type, Important advances in oncology 1996. in Knowles DM"(ed): Neoplastic Philadelphia, Lippencott, 1996, p 139 Hematopathology. Baltimore: 6. The International Non-Hodgkin's Williams and Wilkins, 1992, p 675 Lymphoma Prognostic Factors Harris NL, Jaffe ES, Stein H, Banks Project: A predictive model of PM, Chan JKC, Cleary ME Delsol G, aggressive non-Hodgkin's lymphoma. De Wolf-Peeters C, Falini B, Gatter N Engl J Med 329:987, 1993 KC, Grogan TM, Isaacson PG, 7. Lesley J, Hyman R, Kincade PW: Knowles DM, Mason DY, Muller- CD44 and its interactions with the Hermelink HK, Pileri SA, Piris MA, extracellular matrix. Adv. Immunol Ralfkiaer E, Warnke RA: Perspective: 54:271, 1993 a revised European-American 8. Jalkanen S, Bargatze RF, Herron LR, classification of lymphoid neoplasms: Butcher EC: A lymphoid cell surface a proposal from the International glycoprotein involved in endothelial Lymphoma Study Group. Blood cell recognition and lymphocyte 84:1361, 1994 homing in man. Eur J Immunol Klein U, Goossens T, Fischer M, 16:1195, 1986 Kanzler H, Braeuninger A, Rajewsky, 9. Huet S, Groux H, Caillou B, Valentin Kuppers R: Somatic hypermutation in H, Prieur AM, Bernard A: CD44 normal and 'transformed B cells. contributes to T cell activation. J Immunol Rev (in press) Immunol 143:798, 1989 Kramer MHH, Hermans J, Parker J, 10. Koopman G, van Kooyk Y, de Graaf Krol ADG, Kluin-Nelemans JC, Haak M, Meijer CJLM, Figdor CG, Pals ST: HL, van Groningen K, van Krieken Triggering of the CD44 antigen on T JHJM, de Jong D, Kluin PhM: The lymphocytes promotes T cell adhesion clinical significance of BCL2 and p53 through the LFA-1 pathway. J protein expression in diffuse large B- Immunol 145:3589, 1990 cell lymphoma: a population based 11. Miyake K, Medina KL, Hayashi S, study. J Clin Oncol 14:2131, 1996 Ono S, Hamaoka T, Kincade PW: 5. Della-Favera R: BCL-6 in diffuse Monoclonal antbodies to Pgp-1/CD44 large-cell lymphomas, in DeVita VT, block lympho-hemopoiesis in long- 63 Chapter 5 term bone marrow cultures. J Exp spliced exons. Proc Natl Acad Sei Med 171:477, 1990 USA 89:12160, 1992 12. Pals ST, Horst E, Ossekoppele GJ, 19. Jackson D, Bell JI, Dickinson R, Figdor CG, Scheper RJ, Meijer CJLM: Timans J, Shields J, Whittle N: Expression of lymphocyte homing Proteoglycan forms of the lymphocyte receptor as a mechanism of homing receptor CD44 are dissemination in non-Hodgkin's alternatively spliced variants lymphoma. Blood 73:885, 1989 containing the v3 exon. J Cell Biol 13. Gunthert U, Hofman M, Rudy W, 128:673, 1995 . Reber S, Zoller M, Haussmann I, 20. Koopman G, Heider KH, Horst E, Matzku S, Wenzel A, Ponta H, Adolf GR, van den Berg F, Ponta H, Herrlich P: A new variant of Herrlich P, Pals ST: Activated human glycoprotein CD44 confers metastatic lymphocytes and aggressive non- potential to rat carcinoma cell lines. Hodgkin's lymphomas express a Cell 65:13, 1991 homologue of the rat metastasis- 14. Jalkanen S, Joensuu H, Soderstrom associated variant of CD44. J Exp KO, Klemi P: Lymphocyte homing Med 177:897, 1993 and clinical behaviour of non- 21. Stauder R, Eisterer W, Thaler J, Hodgkin's lymphoma. J Clin Invest Gunthert U: CD44 variant isoforms in 87:1835, 1991 non-Hodgkin's lymphomas: a new 15. Sy MS, Guo YJ, Stamenkovic I: independent prognostic factor. Blood Distinct effects of two CD44 isoforms 85:2885, 1995 on tumor growth in vivo. J Exp Med 22. Terpe HJ, Koopmann R, Imhof BA, 174:859, 1991 Gunthert U: Expession of integrins 16. Wielenga VJM, Heider KH, Offerhaus and CD44 isoforms in non-Hodgkin's GJA, Adolf GR, van den Berg F, lymphomas: CD44 variant isoforms Ponta H, Herrlich P, Pals ST: are preferentially expressed in high- Expression of CD44 splice variant grade malignant lymphomas. J proteins in human colorectal cancer is Pathology 174:89, 1994 related to tumor progression. Cancer 23. Salles G, Zain M, Jiang W-M, Res 53:4754, 1993 Boussiotis VA, Shipp MA: 17. Stamenkovic I, Amiot M, Pesando JM, Alternatively spliced CD44 transcripts Seed B: A lymphocyte molecule in diffuse large-cell lymphomas: implicated in lymph node homing is a characterization and comparison with member of the cartilage link protein normal activated B cells. Blood family. Cell 56:1057, 1989 82:3539, 1993. 18. Screaton GR, Bell MV, Jackson DG, 24. Horst E, Meijer CJLM, Radaszkiewicz Cornells FB, Gerth K, Bell Jl: T, Ossekoppele GJ, van Krieken Genomic structure of DNA encoding JHJM, Pals ST: Adhesion molecules the lymphocyte homing receptor in the prognosis of diffuse large-cell CD44 reveals at least 12 alternatively lymphoma: expression of a 64 CD44 in large B-cell lymphoma lymphocyte homing receptor (CD44), Cancer (in press) LFA-1 (CD1 la/18), and ICAM-1 30. Hermans J, Krol ADG, van Groningen (CD54). Leukemia 4:595, 1990 K, Kluin PhM, Kluin-Nelemans JC, 25. Jalkanen S, Joensuu H, Klemi P: Kramer MHH, Noordijk EM, Ong F, Prognostic value of lymphocyte Wijermans PW: International homing receptor and S phase fraction prognostic index for aggressive non- in non-Hodgkin's lymphoma. Blood Hodgkin's lymphoma is valid for all 75:1549, 1990 malignancy grades. Blood 86:1460, 26. Yakushijin Y, Steckel J, Kharbanda S, 1995 Hasserjian R, Neuberg D, Jiang W-M, 31. Carbone PP, Kaplan HS, Musshoff K, Anderson I, Shipp MA: A directly Smithers DW, Tubiana M: Report of spliced exon 10-containing CD44 the committee on Hodgkin's disease variant promotes the metastasis and staging classification. Cancer Res homotypic aggregation of aggressive 31:1860, 1971 non-Hodgkin's lymphoma. Blood 91: 32. Musshoff K: Klinische 4282, 1998 Stadieneinteilung der nicht-Hodgkin 27. Kramer MHH, Hermans J, Wijburg E, lymphome. Strahlentherapie 153:218, Philippo K, Geelen E, van Krieken 1977 JHJM, de Jong D, Maartense E, 33. Stansfeld AG, Diebold J, Kapanci Y, Schuuring E, Kluin PhM: Clinical Kelenyi G, Lennert K, Mioduszewska relevance of BCL2, BCL6 and myc O, Noel H, Rilke F, Sundstrom C, van rearrangements in diffuse large B-cell Unnik JAM, Wright DH: Updated lymphoma. Blood 92:3152, 1998 Kiel classification for lymphomas. 28. Krol AD, Hermans J, Kramer MHH, Lancet 1:292, 1988 Kluin PhM, Kluin-Nelemans HC, 34. Jalkanen S, Bargatze RF, De Los Blok P, Heering KJ, Noordijk EM, van Toyos J, Butcher EC: Lymphocyte Krieken JHJM: Gastric lymphomas recognition of high endothelium: compared with lymph node antibodies to distinct epitopes of an lymphomas in a population-based 85-90Kd glycoprotein differentially registry differ in stage distribution and inhibit binding to lymph node, dissemination patterns but not in mucosal and synovial endothelial patient survival. Cancer 79:390, 1997 cells. J Cell Biol 105:983, 1987 29. Krol AD, Hermans J, Dawson L, 35. Heider KH, Mulder JWR, Ostermann Snijder S, Wijermans PW, Kluin- E, Susani E, Patzelt E, Pals ST, Adolf Nelemans HC, Kluin PhM, van GR: Splice variants of the cell surface Krieken JHJM, Noordijk EM: glycoprotein CD44 associated with Treatment, patterns of failure and metastatic tumor cells are expressed in survival of stage I nodal and normal tissues of humans and extranodal non-Hodgkin's lymphomas cynomolgus monkeys. Eur J Cancer in the Comprehensive Cancer Centre 31A:2385, 1995 West population based registry. 36. Mulder JWR, Kruyt PM, Sewnath M, 65 Chapter 5 Oosting J, Seldenrijk CA, Weidema Pure E: CD44 is necessary for optimal WF, Offerhaus GJA, Pals ST: contact allergic responses but is not Colorectal cancer prognosis and required for normal leukocyte expression of exon-v6 containing extravasation. J Exp Med 178: 497, CD44 proteins. Lancet 344:1470, 1994 1993 37. Ship MA: Can we improve upon the 44. Degrendele HC, Estess P, Siegelman International Index? Annals of MH: Requirement for CD44 in Oncology 8(suppl.l):S43, 1997 activated T cell extravasation into an Offit K, Le Cocco F, Louie DC, Parsa inflammatory site. Science 278: 672, NZ, Leung D Portlock C, Ye BH, 1997 Lista F, Filippa DA, Rosenbaum A, 45. Kaufmann M, Heider KH, Sinn HP, Ladanyi M, Jhanwar S, Dalla-Favera Minkwitz von G, Ponta H, Herrlich P: R, Chaganti RSK: Rearrangement of CD44 variant exon epitopes in the BCL6 gene as a prognostic marker primary breast cancer and length of in diffuse large cell lymphoma. N survival. Lancet 345:615, 1995 Engl J Med 331:74, 1994 46. Yu Q, Toole BP, Stamenkovic I: van Krieken JHJM, Raffeid M, Induction of apoptosis of metastatic Raghoebier S, Jaffe ES, van Ommen mammary carcinoma cells in vivo by GJB, Kiuin PhM: Molecular genetics disruption of tumor cell surface CD44 of gastrointestinal non-Hodgkin's function. J Exp Med 186:1985, 1997 lymphomas: unusual prevalence and 47. Miller TP, Dahlberg S, Cassady JR, pattern of c-myc rearrangements in Adelstein DJ, Spier CM, Grogan TM, aggressive lymphomas. Blood 76:797, LeBlanc M, Carlin S, Chase E, Fisher 1990 RI: Chemotherapy alone compared 40. Pals ST, Drillenburg P, Radaszkiewicz with chemotherapy plus radiotherapy T, Manten-Horst E: Adhesion for localized intermediate- and high- molecules in the dissemination of non- grade non-Hodgkin's lymphoma. N Hodgkin's lymphoma. Acta Haematol Engl J Med 339:21, 1998 97:73-80,1997. 48. Horst E, Meijer CJLM, Radaskiewicz Clark RA, Alon R, Springer TA: Th, Dongen JJM van, Pieters R, CD44 and hyaluronan-dependent Figdor CG, Hooftman A, Pals ST: rolling interactions of lymphocytes on Expression of a human homing tonsillar stroma. J Cell Biol 134:1075, receptor (CD44) in lymphoid 1996 malignancies and related stages of 42. DeGrendele HC, Estess P, Picker LJ, lymphoid development. Leukemia Siegelman MH: CD44 and its ligand 4:383, 1990 hyaluronate mediate rolling under physiologic flow: a novel lymphocyte- endothelial cell primary adhesion pathway. J Exp Med 183:1119, 1996 43. Camp RL, Scheynius A, Johansson C, 66 Chapter 6 Local therapy alone is effective for stage I, CD44 diffuse large B-cell lymphomas Paul Drillenburg1, Vera J. M. Wielenga', Mark H. H. Kramer, Johan H. J. M. van Krieken2, Hanneke C. Kluin-Nelemans3'7, Jo Hermans4-7, Siem Heisterkamp5, Evert M Noordijk6'7, Philip M. Kluin2-7, and Steven T. Pals' Departments of Pathology' and Clinical Epidemiology and Biostatistics5 Academic Medical Center, Amsterdam, The Netherlands; Departments of Pathology-, Hematology3, Medical Statistics'1 and Clinical Oncology6 Leiden University Medical Center, Leiden, The Netherlands, and Comprehensive Cancer Center West (CCCWf, Leiden, The Netherlands 67 Chapter 6 Introduction Diffuse large B-cell lymphomas (DLCL) are a common type of non-Hodgkin's lymphomas (NHL), biologically related to germinal center or post-germinal center B cells. Although currently classified as a single group, DLCL represent a diverse set of neoplasms, with heterogeneous genetic background, clinical features, treatment response, and prognosis 1.2.3,4 whjig prognosis in DLCL can be predicted on the basis of clinical characteristics ', the biological variables underlying the variance in disease aggressiveness and outcome are unknown. Identification of these variables is of great importance since they may allow definition of patient subgroups with distinctive disease course and treatment requirements. Studies from our own and other laboratories have identified lymphocyte homing receptor CD44 as a biological prognosticator in NHL that determines propensity for dissemination 5.6.7.8.9,10.11.12 jn j3L(2L; CD44 expression is associated with tumor dissemination, and, in patients with localized (stage I) disease, it is a major unfavourable prognosticator predicting relapse and tumor related death '3. By analyzing CD44 expression in tumor tissue of patients with localized (stage I) DLCL from a unique historical population based study group, we now demonstrate that patients with CD44 negative tumors have an excellent prognosis even when treated with local (surgery and/or radiation) therapy alone, making local treatment without chemotherapy an option in these patients. 68 Local therapy for CD44' large B-cell lymphoma Methods The study group consists of 79 patients with Ann Arbor stage I DLCL from the population based lymphoma registry of the Comprehensive Cancer Center West (CCCW) in The Netherlands. Details concerning this registry as well as the current study group have been published previously 3M. All tumors were reviewed by a panel of hematopathologists. Treatment was according to the preference of the local hematologist; treatment modalities were recorded and the follow-up was annually updated. Of the patients, 25 were treated with systemic chemotherapy with or without radiotherapy, including doxorubicin containing polychemotherapy in 17 cases. The remaining 54 patients initially received local treatment with radiotherapy and/or surgery (n=44) or surgery alone (n=10). In case of relapse, second line treatment consisted of radio- and/or chemotherapy. In all patients treated with surgery alone the lymphomas were localized at extranodal sites (stomach n=5; cecum n=l; testis n=3 and breast n=l) and the resection was radical. Patients treated with local therapy only, were comparable to the whole study group of 79 patients with stage I disease (table 1); there was no evidence for selection bias towards better disease outcome in patients treated with local Table 1. Clinical characteristics in locally and systemically treated stage I DLCL. whole local systemic studygroup therapy# therapy* (n=79) (n=54) (n=25) % % age < 60 yr 34 32 40 >60yr 66 68 60 serum LDH normal 73 78 64 increased 19 15 28 not évaluable 8 7 8 performance status > 80 % 72 72 72 < 80 % 19 19 20 not évaluable 9 9 8 No. of IPI risk factors Oor 1 63 63 64 2 22 22 20 3 5 4 8 not évaluable 10 11 8 # radiotherapy and/or surgery * chemotherapy with or without radiotherapy 69 Chapter 6 therapy alone. CD44 expression was studied on formalin-fixed paraffin-embedded tumor tissue using mAb Hermes-3 against an epitope on the constant part of CD44 (kindly provided by Prof. S. Jalkanen, Turku, Finland), by established immune histochemical techniques. Based on the estimated percentage of positively staining tumor cells, the tumors were devided into three categories (-) negative/low, under 10% positive; (+) intermediate, 10-50%; and (++) high, over 50% positive. Overall survival was estimated by the Kaplan-Meier method and statistical significance was tested using the log-rank test. For multivariate analysis, the Cox regression model was used. Results and discussion The expression of CD44 in the whole study group (n=79) varied from negative (29%), intermediate (13%) to strongly positive (58%). For the subgroup of patients locally treated with radiotherapy and/or surgery (n=54) the expression of CD44 was similar (negative 22%; intermediate 16% and positive 62%). In survival analysis the effect of CD44 expression was explored (fig.l). Interestingly, the patients locally treated with radiotherapy and/or surgery (n=54) and a CD44 negative tumor had an excellent prognosis (fig.lA); only 1 out of 12 ïoor 1 IUU Ï B „A "•+*--H— \ n== 12 - CO 75 - > 75 "1 ZL n=14 'E +/++ 3 50 -- 50 - , n==4 2 > n=ll O 25 25 - +/++ p==0.00 2 p=0.77 —h- 0 1 10 10 Time (years) Figure 1. Relation between CD44 expression and overall survival in DLCL patients treated with either local therapy (radiotherapy and/or surgery) (A), or systemic chemotherapy (B). Since the survival curves of the patients with intermediate (+) and high (++) expression for CD44 were identical, both groups have been combined (+/++). 70 Local therapy for CD44' large B-cell lymphoma patients (8%) died of disease. By contrast, in the group CD44 positive lymphomas, 28 out of 42 patients (67%) died. This finding was unrelated to the type of local therapy (radiotherapy with or without surgery, or surgery alone). In the locally treated patients the relapse ratio for the CD44 negative and positive lymphomas was respectively 25% and 55% (p=0.07). In a multivariate comparison with the clinical parameters of the International Prognostic Index, CD44 was an independent prognosticator for overall survival in stage I DLCL treated with radiotherapy and/or surgery (table 2). A relation of CD44 expression with overall survival in the systemically treated patients, at present the recommended therapy for localized DLCL, was not observed (fig.IB). However, in view of the small number of patients (n=25), a reliable conclusion about the effect of CD44 expression in this group is not possible. Until the eighties, local therapy alone was considered an option for the treatment of patients with stage I DLCL 15J6. However, the use of combination chemotherapy regimens including doxorubicin and, more recently, chemotherapy plus radiotherapy, clearly yields Table 2. Independent prognosticators for overall survival in stage I, locally treated DLCL Factor Relative risk 95% CI p value step of selection CD44s negative 1.0 positive 8.41 1.13-62.38 0.0 age < 60 years 1.0 > 60 years 3.21 1.10-9.39 o.o: superior cure rates which can be in excess of 75% 17.1s.19.20.21.22_ The presumed reas0n for the failure of local therapy alone to cure a larger fraction of DLCL patients is the propensity of this lymphoma subtype to spread systemically early in the course of the disease, despite negative clinical staging studies. Indeed extensive staging procedures, including staging laparotomy, can improve identification of patients that can be cured by local treatment alone, but are often not appropriate in these patients, who are usually older 23. However, identification at diagnosis of patients with occult tumor dissemination by non-invasive methods would be beneficial, as it might limit the toxic effects of chemotherapy to those patients at risk for relapse outside the original area of tumor involvement. Our current data showing that locally treated stage I patients with CD44 negative lymphomas, unlike those with CD44 positive tumors, have an excellent prognosis and a low relapse rate, are in line with the postulated role of CD44 in (occult) lymphoma dissemination: Whereas CD44 71 Chapter 6 negative tumors tend to remain localized, CD44 positive have a propensity to disseminate at an early stage of the disease rendering local therapy inadequate. Our findings suggest that local therapy alone is adequate for CD44 negative stage I DLCL. Hence CD44 may represent a tool for therapy selection in DLCL patients. References The International Non-Hodgkin's Jalkanen S, Bargatze RF, De Los Lymphoma Prognostic Factors Toyos J, Butcher EC: Lymphocyte Project: A predictive model of recognition of high endothelium: aggressive non-Hodgkin's antibodies to distinct epitopes of an lymphoma. N Engl J Med 329:987, 85-90Kd glycoprotein differentially 1993 inhibit binding to lymph node, Harris NL, Jaffe ES, Stein H, Banks mucosal and synovial endothelial PM, Chan JKC, Cleary ML, Delsol cells. J Cell Biol 105:983, 1987 G, De Wolf-Peeters C, Falini B, Pals ST, Horst E, Ossekoppele GJ, Gatter KC, Grogan TM, Isaacson Figdor CG, Scheper RJ, Meijer PG, Knowles DM, Mason DY, CJLM: Expression of lymphocyte Muller-Hermelink HK, Pileri SA, homing receptor as a mechanism of Piris MA, Ralfkiaer E, Warnke RA: dissemination in non-Hodgkin's Perspective: a revised European- lymphoma. Blood 73:885, 1989 American classification of lymphoid Horst E, Meijer CJLM, neoplasms: a proposal from the Radaszkiewicz T, Ossekoppele GJ, International Lymphoma Study van Krieken JHJM, Pals ST: Group. Blood 84:1361, 1994 Adhesion molecules in the prognosis 3. Kramer MHH, Hermans J, Parker J, of diffuse large-cell lymphoma: Krol ADG, Kluin-Nelemans JC, expression of a lymphocyte homing Haak HL, van Groningen K, van receptor (CD44), LFA-1 Krieken JHJM, de Jong D, Kluin (CD1 la/18), and ICAM-1 (CD54). PhM: The clinical significance of Leukemia 4:595, 1990 BCL2 and p53 protein expression in Jaikanen S, Joensuu H, Soderstrom diffuse large B-cell lymphoma: a KO, Klemi P: Lymphocyte homing population based study. J Clin Oncol and clinical behaviour of non- 14:2131, 1996 Hodgkin's lymphoma. J Clin Invest Klein U, Goossens T, Fischer M, 87:1835, 1991 Kanzler H, Braeuninger A, Lesley J, Hyman R, Kincade PW: Rajewsky, Kuppers R: Somatic CD44 and its interactions with the hypermutation in normal and extracellular matrix. Adv Immunol transformed B cells. Immunol Rev 54:271, 1993 162:261, 1998 10. Clark RA, Alon R, Springer TA: 72 Local therapy for CD-14" large B-cell lymphoma CD44 and hyaluronan-dependent Walbom-Jorgensen S, Pedersen- rolling interactions of lymphocytes Bjergaard J, Hansen MM, Rygärd J. on tonsillar stroma. J Cell Biol A randomized study of radiotherapy 134:1075, 1996 versus radiotherapy plus 11. DeGrendele HC, Estess P, Picker LJ, chemotherapy in stage I—11 non- Siegelman MH: CD44 and its ligand Hodgkin's lymphomas. Cancer 52:1, hyaluronate mediate rolling under 1983 physiologic flow: a novel 18. Cabanillas F. Chemotherapy as lymphocyte-endothelial cell primary definitive treatment of stage I-II adhesion pathway. J Exp Med large cell and diffuse mixed 183:1119, 1996 lymphomas. Hematol Oncol 3:25, 12. Degrendele HC, Estess P, Siegelman 1985 MH: Requirement for CD44 in 19. Jones SE, Miller TP, Connors activated T cell extravasation into an JM.Long-term follow-up and inflammatory site. Science 278: 672, analysis for prognostic factors for 1997 patients with limited-stage diffuse 13. Drillenburg P, Wielenga VJM, large-cell lymphoma treated with Kramer MHH, van Krieken JHJM, initial chemotherapy with or without Kluin-Nelemans HC, Hermans J, adjuvant radiotherapy. J Clin Oncol Heisterkamp S, Noordijk EM, Kluin 7:1186, 1989 PhM, Pals ST. CD44 expression 20. Longo DL, Glatstein E, Duffey PL, predicts disease outcome in localized Ihde DC, Hubbard SM, Fisher RI, large B-cell lymphoma, submitted Jaffe ES, Gilliom M, Young RC, 14. Kramer MHH, Hermans J, Wijburg DeVita VT. Treatment of localized E, Philippo K, Geelen E, van aggressive lymphomas with Krieken JHJM, de Jong D, combination chemotherapy followed Maartense E, Schuuring E, Kluin by involved-field radiation therapy. J PhM: Clinical relevance of BCL2, Clin Oncol 7:1295, 1989 BCL6 and myc rearrangements in 21. Armitage JO. Treatment of non- diffuse large B-cell lymphoma. Hodgkin's lymphoma. N Eng J Med Blood 92:3152, 1998 328:1023, 1993 15. Jones SE, Fuks Z, Kaplan HS, 22. Miller TP, Dahlberg S, Cassady JR, Rosenberg SA. Non-Hodgkin's Adelstein DJ, Spier CM, Grogan lymphomas. V: Results of TM, LeBlanc M, Carlin S, Chase E, radiotherapy. Cancer 32:682, 1973 Fisher RL Chemotherapy alone 16. Sweet DL, Kinzie J, Gaeke ME, compared with chemotherapy plus Golomb HM, Ferguson DL, Ultmann radiotherapy for localized JE. Survival of patients with intermediate- and high-grade non- localized diffuse histiocytic Hodgkin's lymphoma. N Eng J Med lymphoma. Blood 58:1218, 1981 339:21, 1998 17. Nissen NI, Ersboll J, Hansen HS, 23. Goffinet DR, Castellino RA, Kim H. 73 Chapter 6 Staging laparotomy in unselected previously untreated patients with non-Hodgkin's lymphomas. Cancer 32:672, 1973 74 Chapter 7 Cell adhesion receptors in lymphoma dissemination Paul Drillenburg and Steven T. Pals Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam. The Netherlands submitted 75 Chapter 7 Introduction Cell adhesion receptors play a key role in many biological processes including embryogenesis, hemostasis, inflammation, and tumor metastasis. In these processes, they coordinate cell-cell and cell-extracellular matrix (ECM) interactions as well as cell migration, and they act as co-regulators of cell growth and programmed cell death. In the immune system, adhesion molecules promote interactions of lymphocytes with antigen-presenting cells and target-cells required for the induction and regulation of an immune response and for effector cell function. Furthermore, adhesion molecules regulate leucocyte extravasation and lymphocyte homing by mediating adhesion to endothelium and to ECM components '"5. As discussed in this paper, there is now ample evidence that the adhesion receptors, which direct the homing of normal lymphocytes also mediate the dissemination of their malignant counterparts, the non-Hodgkin's lymphomas (NHLs). In this way, these molecules contribute to lymphoma aggressiveness and determine the highly tissue-specific dissemination patterns of certain NHL-subtypes. Lymphocyte Adhesion Receptors in the Regulation of Lymphocyte Homing. Lymphocytes express members of a number of different adhesion receptor families, i.e. the immunoglobulin 6, the integrin '-7'8, the selectin 9'", and the CD44 families l2"15. The role of these adhesion molecules in lymphocyte interactions with other cells and with the ECM is complex and dynamic: Interaction of adhesion receptors with their ligands does not only lead to cell adhesion per se, but transduces signals into the cell regulating cytoskeletal organization, cell cycle progression, and programmed cell death (outside-in signaling). On the other hand, cytoplasmic signals, e.g. generated via the T-cell receptor, regulate the cell surface expression and functional activity of adhesion molecules (inside-out signaling) l-3-16-18. Selective expression and regulation of adhesion molecules on lymphocytes and endothelium forms the basis for tissue-specific lymphocyte homing 45. Table 1 summarizes some of the important features of the adhesion molecules involved in lymphocyte homing discussed in the present paper. 76 Cell Adhesion Receptors in Lymphoma Dissemination Table 1. Lymphocyte adhesion molecules and their role in homing*. Receptor Expression in Ligands Interacting predominant lymphocytes cells/substrates role(s)in homing aLß2 - broad ex ICAM-1 endothelium, broad function in (LFA-1) pression on T ICAM-2 HEV, DC, FDC, lymphocyte ho and B cells ICAM-3 activated epitheli ming um a4ß, - broad ex VCAM-1, activated endothe homing to (VLA-4) pression on T fibronectin lium, inflammatory and B cells (CS-1) DC, FDC sites a4ß7 - naive T and MAdCAM-1, endothelium intes gut-homing B cells (low) (VCAM-1), tinal mucosa, - memory T (fibronectin) Peyer's patch and cell subset mesenteric lymph (high) node HEV - peripheral B cell subset (low) aEß7 - intra-epithe- E-Cadherin epithelium epitheliotropism lial T-cells L-Selectin - resting T and PNAd, Gly- HEV peripheral B cells CAM, lymph node-ho CD34, ming (MAdCAM-1) CLA '- memory T- E-selectin endothelium skin-homing cell subset (skin) CD44 - broad ex hyaluronate, ECM - homing to sites (multiple pression on T collagen IV, endothelium of inflammation isoforms) and B cells fibronectin, - modulation of - splice vari osteopontin, cell growth and ants on activa FGF-2, motility. ted T and B HGF/SF, cells MIP-lß "only molecules discussed in the present paper are listed 77 Chapter 7 Post-capillary venule Chemokine Sialomuciny « O \/ receptor " Chemokine Activate: SelM-tin* "-Q-- integrin 'tf Integrin Selecting jr- w Proteoglycan S fjigand Figure 1. In the post-capillary venules selectin-sialomucin interactions mediate 'rolling' of lymphocytes on endothelium. Chemokines presented by proteoglycans on the endothelium can bind to the G-protein-coupled 7- membrane spanner chemokine receptor, and activate members of the integrin family on the surface of lympho cytes. Interaction of integrins to their ligands, results in a stable lymphocyte-endothelium adhesion and finally diapedesis. Interaction of lymphocytes (and other leucocytes) with endothelium is a multistep process 3.4.19-22 (pjg ]) -j^g initial step consists of a loose "tethering" engagement between the lymphocyte and the endothelium, leading to a rolling movement of the lymphocyte over the vascular endothelium of the post-capillary venule (Fig.l). This step generally is mediated by molecules of the selectin family, which are strategically localized on the tips of the cell membrane's microvilli 23"25, thus allowing for effective interaction of the selectin with its sialomucin ligand. However, other molecules such as the integrins ß, and ß7, and CD44 can also mediate rolling ~-26-29. Lymphocyte rolling is transient and reversible, unless followed by a signal leading to activation of adhesion molecules of the integrin family. These molecules, i.e. aLß2 (LFA-1), a4ß, (VLA-4) and a4ß7, mediate stable adhesion and promote migration of lymphocytes across the vessel wall (Fig. 1). The extremely rapid integrin activation that has to take place in the blood stream in vivo is mediated by chemokines, as blockade of their G- protein-coupled receptors by pertussis toxin efficiently inhibits lymphocyte emigration from the blood 30. Recently, several chemokines like SDF-1, SLC (6-C-kine), BLC/BCA-1, MIP- 3ß, MIP-3a, IP10 and Mig have been identified that are capable of mediating rapid (milliseconds) integrin dependent lymphocyte arrest under flow conditions 3I"33. Consistent 78 Cell Adhesion Receptors in Lymphoma Dissemination with their important regulatory role in homing, these chemokines display site-specific production as well as specificity for distinct lymphocyte subsets 32-34"38. For example, the chemokine SLC is specifically expressed by HEV and selectively recruits naive T cells, expressing the chemokine receptor CCCR7, into the secondary lymphoid organs 36. BLC/BCA, by contrast, is involved in the recruitment of B cell into B cell areas. It is specifically expressed in B-cell follicles, presumably by FDC. Disruption, of CXCR5, the receptor for BLC leads to a disturbed development of primary follicles and germinal centers in the spleen and Peyer's patches 37-39. In addition to chemokines, heparan sulfate proteoglycans (HSPG) expressed on endothelium or ECM may contribute to integrin activation and promote diapedesis by concentrating and presenting chemokines (Fig. 1). As specific modifications of heparan sulfate chains have been shown to determine growth factors binding specificity, chemokine interaction with HSPG may contribute an additional layer of specificity to the lymphocyte-endothelial cell interaction cascade 40'42. In the multi-step lymphocyte-endothelial cell interaction model described above (Fig. 1), specificity is determined by unique combinations of primary and secondary adhesion receptor pairs, as well as by chemokine mediated activation events. Important factors regulating the adhesion receptor profile of lymphocytes are prior antigenic stimulation and/or state of activation 4i5-43. Imprinting of the lymphocyte at the site of antigenic experience leads to expression of a specific set of adhesion receptors on the lymphocyte involved in tissue- specific homing to the sites of primary antigenic stimulation. These 'homing' receptors permit interaction with endothelial area codes ('vascular addressins'), crucial in tissue-specific recirculation of lymphocytes. Combinations of lymphocyte adhesion molecules and vascular addressins involved in tissue-specific homing are: a4ß7/MAdCAM-l for mucosa associated lymphoid tissues (MALT); cutaneous lymphocyte antigen (CLA)/E-selectin for skin; L- selectin/PNAd, for peripheral lymph nodes. Binding of aEß7, to E-cadherin expressed on epithelial cells, is important in positioning lymphocytes in epithelium (Fig.2). Adhesion Receptors in Lymphoma Dissemination. At least three sets of clinical observations indicate that conserved homing programs play an important role in the dissemination of NHLs 44. First, while NHLs related to small resting lymphocytes usually are disseminated to multiple lymphoid sites at presentation, tumors related to activated lymphocytes (e.g. centroblasts and immunoblasts) are often initially localized and disseminate primarily to adjacent lymph nodes. These differences in dissemination propensity presumably reflect the recirculating versus sessile character of the normal counterparts of these NHLs. Second, extranodal NHLs arising in the MALT or the 79 Chapter 7 skin disseminate preferentially to mucosal sites and skin, respectively. This strongly suggests that they make use of specific area codes, similar to those employed during normal lymphocyte homing. Third, several reports have documented specific lymphoma dissemination to sites of (micro)trauma and inflammation. This phenomenon could be explained by interaction of tumor cells with activated endothelium at the site of injury. In the following paragraphs, studies focussing on the role of specific adhesion receptors in lymphoma dissemination will be discussed. HEV Activated endothelium MAdCAM-1 ' PNAd VCAM-1 . HA , E-/P-Selectin CS-1 naive activated T-cell T-cell / I \ Mucosa Peripheral Mucosal lymph node epithelium memory E-Cadherin T-Cell - intestinal T-cell lymphoma a407 Site specific endothelium - low grade B -cell MALT - nodal T-and B-cell - cutaneous T-cell - intestinal T-cell lymphoma lymphomas lymphoma -MLP Figure 2. Lymphocyte migration is strickly regulated by cell surface molecules on lymphocytes (lymphocyte homing receptors) and endothelium (vascular addressins). Random migration of naive T-cells throughout the body is mediated by the dual expression of a4ß7 and L-selectin. CD44 and members of the integrin and selectin family expressed on activated T-cells can form a stable binding to activated endothelium. Migration of memory T-cells is limited to organs of primary antigenic stimulation. Selective expression of the lymphocyte homing receptors a4ß7, L-selectin and CLA mediate, by binding to their vascular addressins, tissue specific homing to the mucosa, peripheral lymph node and skin respectively. Interaction of aEß7 to E-cadherin, expressed on epithelial cells, is involved in positioning of lymphocytes in the epithelium of skin and mucosa. The non- Hodgkin's lymphomas more or less using these cell surface molecules in their dissemination are mentioned in boxes. 80 Cell Adhesion Receptors in Lymphoma Dissemination Selectins and their Carbohydrate Ligands L-selectin; peripheral lymph node homing (Fig 2). L-selectin, like the other members of the selectin family, consists of lectin, epidermal growth factor, and short consensus repeat domains '-9"11. L-selectin is expressed by lymphocytes, monocytes and neutrophils and is rapidly down-regulated upon cell activation 45. The first evidence for a selective role of L- selectin in lymphocyte homing to peripheral lymph nodes (PLN) came from experiments demonstrating that mAbs against L-selectin interfere with in vitro binding of lymphocytes to high endothelial venules (HEV) of PLN, but not to HEV of Peyer's patches 46. In accordance with this finding, inactivation in vivo of L-selectin, either by mAbs or by gene knockout, was found to result in a virtually complete inhibition of lymphocyte homing to PLN 25-47-49. The main ligands for L-selectin are the peripheral node addressins (PNAds). PNAds are selectively 50, but not exclusively, expressed on HEV in peripheral nodes. Monoclonal antibody (mAb) MECA-79, against PNAd, decreases lymphocyte adherence to PLN HEV by 60-90% 50. The MECA-79 epitope is a carbohydrate moiety that decorates a number of different protein backbones including CD34 and GlyCAM-1 5-51-52. a (1,3) Fucosyltransferase Fuc-TVIIA knockout mice, which are deficient in selectin ligands, also show a strongly reduced lymphocyte homing to PLN ". In conclusion, both L-selectin and its ligands are needed for lymphocyte homing to PLN (Fig. 2). Studies of L-selectin expression on NHLs have shown that the vast majority of nodal lymphomas express L-selectin (table 2). This holds for low-grade and aggressive B cell lymphomas, as well as for T-cell lymphomas with a primary nodal localization 54. In extranodal lymphomas, however, a more heterogenous expression of L-selectin is found. Among extranodal lymphomas localized in the gastrointestinal tract, low-grade B-cell lymphoma of the MALT and malignant lymphomatous polyposis (MLP) do express L- selectin (table 2). However, in both tumor types, the mucosal homing receptor a4ß7 is also expressed. In MLP, a variant of mantle cell lymphoma, this double 'homing phenotype' is generally accompanied by widespread lymphoma dissemination to both mucosal sites and PLN 55. By contrast, low-grade B-cell lymphomas of the MALT, although also co-expressing PLN (L-selectin) and mucosal (a4ß7) homing receptors, do not usually involve PLN. For PLN dissemination of these lymphomas, other steps in the lymphocyte-endothelial interaction cascade, e.g. involving integrins or chemokine signals, could be rate limiting. Alternatively, the absence of Helicobacter Pylori antigens in the PLN, which may promote tumor cell growth and survival 56, might explain the low incidence of PLN involvement in low-grade MALT lymphomas. In contrast to the above mentioned tumors, aggressive mucosal B-cell and T-cell lymphomas only occasionally express L-selectin 54-57. Similarly, primary cutaneous T cell 81 Chapter 7 Table 2. Expression of adhesion molecules in non-Hodgkin's lymphomas. REAL L-selectin a4ß7 CLA aEß7 classification B-cell CLL - nodal + Mantle cell lymphoma - nodal + - GI-tract (MLP) + Marginal zone B-cell lymphoma - GI-tract (low grade MALT) + -/+ Follicle centre lymphoma - nodal +/- Diffuse large B-cell lymphoma - nodal +/- - GI-tract Peripheral T-cell lymphoma - nodal + - GI-tract +/- +/- - skin (MF) -/+ -/+ Anaplastic large cell lymphoma - nodal -/+ -/+ - skin -/+ + -/+ + reported cases all or almost all positive; +/- variable reports, most cases positive; -/+ varia ble reports, most cases negative; - reported cases negative or sporadically positive. lymphomas express low levels of L-selectin 58 (table 2). The paucity of L-selectin on most extranodal lymphomas may contribute to their low propensity to disseminate to PLN. Cutaneous lymphocyte antigen (CLA); skin homing. Cutaneous lymphocyte antigen (CLA) is a carbohydrate antigen that is closely related to the sialyl Lewis X antigen (sLex) 2,59,60 CLA is present on a minor subset (10-15%) of memory T lymphocytes in the peripheral blood, tonsils and lymph nodes 60,61. However, of the T cells in the normal and inflamed skin, 40% to >90%, respectively, are CLA positive 60-62-63. CLA mediates skin homing via interaction with E-selectin, which is constitutively expressed on skin endothelium 2M (Fig. 2). Interestingly, cutaneous T cell lymphomas, mycosis fungoides as well as other subtypes, 60 65 66 express CLA - ' . By contrast, they do not express the mucosal homing receptor a4ß7 82 Cell Adhesion Receptors in Lymphoma Dissemination (Drillenburg unpublished observation). Vice versa, gastrointestinal T-cell lymphomas 67 consistently lack CLA but express the mucosal homing receptor a4ß7 (table 2). Hence, like on their normal counterparts, i.e. the skin and gut homing memory-T cell subsets, CLA and a4ß7 expression on NHL is mutually exclusive. This highly selective expression of CLA on cutaneous T cell lymphomas strongly suggests that CLA is mediating the skin-specific dissemination of these lymphomas in vivo. (Fig.3). Integrins Integrins are heterodimeric proteins consisting of non-covalently associated a- and ß- subunits '-8. Most of the eight ß-chains can associate several of the fifteen different a-chains that have so far been discovered. Only a limited number of these integrin molecules are expressed on lymphocytes. They mediate interactions of lymphocytes with a variety of cells including endothelium, epithelium, dendritic cells, FDC, and the ECM u-68. Integrins expressed on lymphocytes are not constitutively active but their function is activation dependent1-3'8. LFA-1 (aLßJ. LFA-1 is one of the most important integrins of the immune system. It is involved in a wide variety of lymphocyte interactions with other cells including adhesion to endothelium u-44-69, binding to APC 70-71, FDC 72, and epithelium 73. The ligands for LFA-1 on endothelium are the intercellular adhesion molecules (ICAM)-l, -2 and -3 2,J4-76. LFA-1 interaction with ICAM-1 plays a major role in lymphocyte adhesion and transmigration 3 2 77 78 through HEV - '- - . Defective expression of the ß2-chain leads to impaired emigration of leucocytes from the blood to sites of inflammation, resulting in a lethal immunodeficiency, called leucocyte adhesion deficiency type I (LAD-I)79. LFA-1 has been shown to support in vitro invasiveness of T cell hybridomas and lymphoma cell lines in hepatocyte and fibroblast monolayers and promotes experimental metastasis in nude mice 80"83. In human lymphomas, expression of LFA-1 is closely related to lineage derivation and stage of differentiation 84. However, notwithstanding its important role in lymphocyte adhesion and migration, LFA-1 expression on NHLs does not predict clinical 84 86 behavior " . Conceivably, functional overlap between LFA-1 and a4ß, causes redundancy of LFA-1 in interactions relevant for lymphoma dissemination, including those with endothelium. Furthermore, as the function of LFA-1 is activation dependent, its expression does not directly reflect function. Integrin a4ß, and other ß, integrins. The leucocyte integrin a4ß, (VLA-4) is unique among 83 Chapter 7 ß, integrins since it is not only involved in cell-matrix but also in cell-cell interaction. It can bind the CS-1 domain of fibronectin as well as vascular-cell adhesion molecule-1 (VCAM-1). The latter molecule is constitutively expressed on FDC and is induced on endothelium at sites of inflammation. a4ß, plays an important role in lymphocyte migration to sites of inflammation, including inflamed joints in rheumatoid arthritis, and experimental allergic encephalitis (EAE) lesions in 87 90 the brain " (Fig. 2). During germinal center reactions, a4ß, in concert with LFA-1, mediates the binding of germinal center lymphocytes to FDC 68". Apart from establishing physical contact, outside-in via a4ß, (and LFA-1) presumably contributes to the B-cell selection process in the germinal center by inhibiting apoptosis of germinal center B cells '792. Recently, a4 knock-out mice were reported to have severe defects in T-cell development as 93 well as in mucosal homing . By using chimeric mice, it was shown that a4 null lymphocytes are unable enter Peyer's patches, while homing to lymph nodes and spleen was not disturbed. 93 These findings demonstrate the importance of a4 in gut homing (see below) . In human NHL, a4ß, is widely expressed in a pattern that matches the expression on related stages of normal lymphocyte differentiation 57-94. Like binding of normal germinal center B cells, binding of the tumor cells of follicle center cell lymphomas to FDC is mediated by a4ß, 9S. Progression of this tumor from a follicular to a diffuse phenotype is associated with loss of FDC from the tumor, rather than with down-regulation of a^ 95. Other ß, integrins expressed on lymphocytes are a,ß,, a3ß!, and a6ß,. These integrins mediate interactions with ECM components including collagen, fibronectin, and laminin, and may play an important role in lymphocyte migration through the ECM. Entry of lymphocytes into epithelia has been suggested to require a3ß, interaction with laminin-5 in basement membrane 96. Integrin a4ß7; mucosal homing. The integrin a4ß7 mediates lymphocyte homing to the intestinal mucosa by binding to MAdCAM-1, a vascular addressin selectively expressed on mucosal endothelium (Fig.2 and 3). MAdCAM-1 is an immunoglobulin family member with domains that display homology to the adhesion receptors ICAM-1 and VCAM-1, as well as to 97 another mucosa associated Ig-family member, IgAl . In mice, a4ß7 is the dominant lymphocyte receptor for MAdCAM-1 and for regulating lymphocyte homing to mucosal sites 48 98 - . In man, a4ß7 appears to have a similar function. It is expressed on mucosal lymphocytes 99l0°, and moreover, it is present on a subset of peripheral blood memory T-cells with gut 61 homing properties . In ß7-knockout mice, the formation of mucosa-associated lymphoid tissues is severely impaired. ß7-deficient lymphocytes failed to stably arrest and transmigrate at Peyer's patch HEV, although the initial rolling was intact 101. Recently, the human MAdCAM-1 has been cloned l02; like its murine equivalent it is preferentially expressed in the 84 Cell Adhesion Receptors in Lymphoma Dissemination e> V / \ 9» « -ii •:', •* T • B S"-':'* t 't V ;?,. va*' "•.••. V'* .,» •• • "*-•' '.. ' ,0 D / Figure 3. Cell adhesion receptors and vascular addressins. a4ß7 (A) / MAdCAM-1 (B) expression in a low grade B-cell lymphoma of MALT type (stomach; arrow: epithelial structures), and CLA (C) / E-selectin (D) expression in a cutaneous T-cell lymphoma. 85 Chapter 7 ^^^ mucosa-associated lymphoid tissue 103. 5567 We have studied the expression of the a4ß7 mucosal homing receptor in NHLs . In malignant lymphomatous polyposis (MLP), an unusual presentation of non-Hodgkin's lymphoma of mantle cell type, characterized by multifocal involvement of the gastrointestinal 55 tract, expression of a4ß7 was present in 7 out of 8 cases . By contrast, all cases of nodal mantle cell lymphoma tested were a4ß7 negative. Furthermore, we observed that most cases of low grade B-cell lymphomas of the MALT and intestinal T-cell lymphomas express a4ß7 while expression on lymphomas with a non-mucosal primary localizations was uncommon 67. These findings suggest that a4ß7 plays an important role in determining the characteristic mucosal dissemination often found in lymphomas of the MALT (table 2). Interestingly, Dogan et al 104 recently reported that low grade B-cell lymphomas of the MALT upregulate a4ß7 after Helicobacter pylori induced T-cell dependent proliferation of neoplastic cells. aEß7; interaction with epithelium. In the gut, the integrin aEß7 is found on nearly all intestinal intraepithelial lymphocytes and on approximately 50% of the T-cells in the lamina propria. aEß7 can bind E-cadherin on epithelial cells and in this way mediates the positioning of lymphocytes in the epithelium 105 (Fig. 2). 6 Expression of Furthermore, ocEß7 was found on the intra-epidermal malignant T-lymphocytes in mycosis fungoides '°7. In advanced stages with loss of epitheliotropism, there was a decrease expression of aEß7, suggesting a direct involvement of aEß7 in the process of epitheliotropism. Consistent with this notion, we recently reported a strong expression of aEß7 in Pagetoid reticulosis, a rare form of cutaneous T-cell lymphoma characterized by an extreme epitheliotropism resulting in a pagetoid pattern of lymphocyte infiltration between keratinocytes '°8. The CD44 family CD44 represents a family of glycoproteins encoded by a single gene on the human chromosome 11 (Fig.4) l3109-114. Members of this family show a broad tissue distribution and have been implicated in a number of important biological processes, including lymphocyte homing and activation, hematopoiesis, and tumor progression and metastasis *2,i3,is.no.ii5-i22_ The CD44 gene consists of 19 exons 123. Structural and functional diversity of CD44 is generated by alternative splicing mRNA-splicing involving 10 exons encoding domains of the extracellular portion of the CD44 molecule. In addition to variable exon usage, variations in glycosylation contribute to the diversity of CD44 '3. Most CD44 expressing cells express the 'standard' CD44 (CD44s) isoform translated from 86 Cell Adhesion Receptors in Lymphoma Dissemination 166 • 2 134 M 3 69 • 4 Cartilage link protein homology domain 230 WE 5 (223 aa) Growth factors: HGF/SF 126 {yß\ è>^N k. FGF 126 ra 7 114 [p 8 v5 117 [vÊO 9 r6V> 129 [veilO 132 [vf] 11 (0 - 381 aa) 102 fv8l 12 v8 ^v10 90 [v9ll3 v9 204 [vjö] 14 SS* (45 aa) 63 |15 72 BH 16 fififffiffi mnnrvinmnnnnnrP.P. 79 ^17 TM (23 aa) Plasma membrane 4 1~8 -uuyyyy uuyuuyuyuuuuuuyyuyyu 205 Cytoplasmic domain T (3 or 70 aa) Figure 4. Schematical representation of the CD44 gene and its encoded proteins. The extracellular domain and cytoplasmic tail of CD44 isoforms vary in size as the result of alternative splicing. The alternatively spliced exons are indicated by open boxes. The human vl exon contains a stop codon. In the model of the protein, all putative glycosylation sites are indicated: O-glycosylation (open circles); N-glycosylation (closed circles); chondroitin sulfate (open squares); heparan sulfate (rod). As indicated, the heparan sulfate binding site in exon v3 has the ability to bind growth factors. In addition, the HA-binding sites (double line); the disulfide bonds (S- S); the ankyrin binding site ( ); the Ezrin binding site (—); the phosphorylation sites (P); and the putative interaction sites for SRC-family kinases, are indicated. 87 Chapter 7 an mRNA species containing none of the 'variant' (v) exons l3-112-121. On hematopoietic cells and lymphocytes this 85-95 kD molecule is the principle CD44 isoform 112121.124-126 Larger CD44 isoforms containing various combinations of variant exons are preferentially expressed 2 , |26 l2M27 on epithelial cells ' ' - j but they can also be expressed on activated lymphocytes and aggressive malignant lymphomas '2'. During lymphocyte ontogeny and activation the expression of CD44 is strictly regulated, suggesting an important functional role for CD44 13.115,128 indeed, CD44 has been reported to be involved in a variety of lymphocyte functions 7 129 |33 including lymphopoiesis " , lymphocyte activation - ; and lymphocytes migration and homing "°. In the latter process, CD44 mediates lymphocyte binding to high endothelial 3I32 I34 2S 29 135136 venules ' - ; lymphocyte rolling - , and migration to inflammatory sites . In T-cells, outside-in signaling through CD44 can costimulate T-cell receptor mediated proliferation, cytokine release, and integrin activation ,29-133. Cross-linking of CD44 on T cells leads to protein tyrosine kinase activation. CD44 is physically and functionally associated with the src-family tyrosine kinase p56lck in these cells '3?. A number of experimental and clinical studies suggest an important role of CD44 in the biological behavior of NHLs and indicate that CD44 represents a clinically useful marker predicting disease outcome in NHLs. In a nude mouse model, CD44s (but not CD44v8-10) enhanced the growth and metastatic capacity of Burkitt's lymphoma cell lines I2°. The capacity of the tumor cells to bind hyaluronate (HA) via CD44s presumably plays an important role in these tumor promoting effects of CD44. HA may enhance tumor cell growth by providing a molecular bridge facilitating tumor cell attachment to the extracellular matrix. The matrix could serve as a scaffold for tumor cell growth and as a reservoir for growth and/or motility factors with biological effects on the tumor cells. In this context, it is of interest that CD44 variants containing exon v3 can be decorated with heparan sulfate (HS) side-chains, and by this virtue, can serve as receptors for heparin binding growth factors, including MIP-lß, FGF-2 and HGF/SF 42-138-"t0. Presentation of HGF/SF to its high-affinity receptor c-Met by CD44-HS, was recently shown to promote signaling, and may enhance tumor growth and motility '40. Cinical studies support an important role of CD44 in the biology of human NHLs. In diffuse large cell lymphomas (DLCL) of the B-lineage, we observed a strong correlation between CD44 expression and lymphoma dissemination 86"6. Furthermore, CD44 expression is an unfavorable prognosticator in these tumors 86. Similar findings were reported by the group of Jalkanen "9H1 for high grade B-cell lymphomas. In a recent study, we have explored the prognostic value of CD44 in a group of 276 patients, diagnosed as having DLCL according to the criteria of the REAL-classification l42. In this multicenter population based study group, expression of CD44s was a powerfull prognosticator for overall survival and disease free survival in patients with localized disease l43. In a multivariate comparison with Cell Adhesion Receptors in Lymphoma Dissemination the clinical parameters of the International Prognostic Index (IPI), which are currently used to predict prognosis in DLCL, CD44s expression was the major prognosticator for the subgroup of patients with localized nodal disease. In addition to CD44s, NHLs may express CD44 isoforms containing variant exons 121-144- 149. These larger variants are predominantly expressed on a subgroup of aggressive lymphomas. They often include exon v6, which was reported confers metastatic behavior in rat carcinoma cell lines 1IS. In a study by Stauder and collègues 147 in a mixed group of high- grade NHLs, including patients with precursor B-cell- and Burkitt's-lymphomas, as well as DLCL, CD44v6 was found to be an independent prognosticator predicting tumor related death. In our own study concentrating on a single diagnostic group, i.e. DLCL, CD44v6 had no prognostic value, while CD44s was an important prognosticator '43. Conclusion A large body of evidence indicates that adhesion receptors do not only regulate normal lymphocyte trafficking but also play a pivotal role in the dissemination of non-Hodgkin's lymphomas (NHLs). Cutaneous lymphocyte antigen (CLA), a4ß7, aEß7, and L-selectin, homing receptors mediating the tissue-specific positioning of normal lymphocytes in the skin, mucosa, epithelium, and peripheral lymph nodes, respectively, are selectively expressed on lymphomas evolving at these sites. By interacting with vascular addressins they mediate tissue-specific lymphoma dissemination. CD44, a family of adhesion receptors involved in both lymphocyte migration and tumor metastasis, promotes lymphoma dissemination by interacting with ECM components, particularly with hyaluronate. In addition, binding of growth and motility factors by heparan sulfate proteoglycan forms of CD44 may play a role. Expression of CD44 on localized aggressive NHLs is an independent unfavorable prognosticator. Taken together, these findings offer a framework for the understanding of tumor dissemination in NHL. In view of the similarities between lymphocyte behavior and the metastatic behavior of solid tumors, lymphoma dissemination may serve as a paradigm for tumor metastasis in non-lymphoid tumors. References 1. Springer TA. Adhesion receptors of the 3. Springer TA. Traffic signals for immune system. Nature 1990; 346:425 lymphocyte recirculation and leucocyte 2. Picker LJ, Butcher EC. Physiological and emigration: the multistep paradigm. Cell molecular mechanisms of lymphocyte 1994; 76:301 homing. Annu Rev Immunol 1992; 4. Butcher EC, Picker LJ. Lymphocyte 10:561 homing and homeostasis. Science 1996; 89 Chapter 7 272:60 with the malignant process. Adv Cancer 5. Salmi M, Jalkanen S. How do Res 1997; 71:241 lymphocytes know where to go: current 16. van Noesel C, Miedema F, Brouwer M, concepts and enigmas of lymphocyte De Rie MA, Aarden LA, van Lier RAW. homing. Adv Immunology 1997; 64:139 Regulatory properties of LFA-1 alpha 6. Williams AF, Barclay AN. The and beta chains in human T-lymphocyte immunoglobulin superfamily-domains activation. Nature 1988; 333:850 for cell-surface recognition. Annu Rev 17. Koopman G, Keehnen RM, Lindhout E, Immunol 1988; 6:381 Newman W, Shimizu Y, van Seventer 7. Hemler ME, Elices MJ, Parker C, Takada GA, de Groot C, Pals ST. Adhesion Y. Structure of the integrin VLA-4 and through the LFA-1 its cell-cell and cell-matrix adhesion (CDlla/CD18)-ICAM-l (CD54) and the functions. Immunol Rev 1990; 114:45 VLA-4 (CD49d)-VCAM-l (CD 106) 8. Hynes RO. Integrins: versatility, pathways prevents apoptosis of germinal modulation, and signalling in cell center B cells. J Immunol 1994; 152:3760 adhesion. Cell 1992; 69:11 18. Lub M, Kooyk Y, Figdor CG. Ins and 9. Stoolman LM. Adhesion molecules outs of LFA-1. Immunol Today 1995; controlling lymphocyte migration. Cell 16:479 1989; 56:907 19. Butcher EC. Leucocyte-endothelial cell 10. Vestweber D. The selectins and their recognition: three (or more) steps to ligands. Curr Topics Microbiol Immunol specificity or diversity. Cell 1991; 1993; 184:65 67:1033 11. Kansas GS. Selectins and their ligands: 20. von Andrian UH, Chambers JD, McEvoy current concepts and controversies. LM, Bargatze RF, Arfors K-E, Butcher Blood 1996; 88:3259 EC. Two-step model of leukocyte- 12. Herrlich P, Zöller M, Pals ST, Ponta H. endothelial cell interaction in CD44 splice variants: metastases meet inflammation: distinct roles for LECAM- lymphocytes. Immunol Today 1993; 1 and the leukocyte ß, integrins in vivo. 14:395 Proc Natl Acad Sei 1991; 88:7538 13. Lesley J, Hyman R, Kincade PW. CD44 21. Shimizu Y, Newman W, Tanaka Y, Shaw and its interaction with extracellular S. Lymphocyte interaction with matrix. Adv Immunol 1993; 54:271 endothelial cells. Immunol Today 1992; 14. Pals ST, Koopman G, Griffioen AJ, 13:106 Ponta H, Herrlich P, van der Berg F, 22. Bargatze RF, Jutila MA, Butcher EC. Horst E: CD44: A variety of isoforms Distinct roles of L-selectin and integrins with functions in cell adhesion and a4ß7 and LFA-1 in lymphocyte homing to metastasis; in Shimizu Y (ed): Peyer's patch-HEV in situ: the multistep Lymphocyte adhesion molecules. RG model confirmed and refined. Immunity Landis Co. Austin USA. 1993, pp 135 1995; 3:99 15. Naor D, Vogt Sionov R, Ish-Shalom D. 23. von Andrian UH, Hasslen SR, Nelson CD44: structure, function and association RD, Erlandsen SL, Butcher EC. A central 90 Cell Adhesion Receptors in Lymphoma Dissemination role for microvillous receptor adherence to recombinant adhesion presentation in leukocyte adhesion. Cell molecules and extracellular matrix 1995; 82:989 proteins . J Immunol 1996; 156:932 24. Hasslen SR, von Andrian UH, Butcher 32. Campbell JJ, Hedrick J, Zlotnik A, Siani EC, Nelson RD, Erlandsen SL. Spatial MA, Thompson DA, Butcher EC. distribution of L-selectin (CD62L) on Chemokines and the arrest of human lymphocytes and transfected lymphocytes rolling under flow murine Ll-2 cells. Histochem J 1995; conditions. Science 1998; 279:381 27:547 33. Piali L, Weber C, LaRosa G, Mackay 25. Warnock RA, Askari S, Butcher EC, von CR, Springer TA, Clark-Lewis I, Moser Andrian UH. Molecular mechanisms of B. The chemokine receptor CXCR3 lymphocyte homing to peripheral lymph mediates rapid and shear-resistant nodes. J Exp Med 1998; 187:205 adhesion-induction of effector T 26. Alon R, Kassner PD, Woldemar Carr M, lymphocytes by the chemokines IP 10 and Finger EB, Hemler ME, Springer TA. Mig. Eur J Immunol 1998; 28:961 The integrin VLA-4 supports tethering 34. Schall TJ, Bacon K, Toy KJ, Goeddel and rolling in flow on VCAM-1. J Cell DV. Selective attraction of monocytes Biol 1995; 128:1243 and T lymphocytes of the memory 27. Berlin C, Bargatze RF, Campbell JJ, von phenotype by cytokine RANTES. Nature Andrian UH, Szabo MC, Hasslen SR, 1990; 347:669 Nelson RD, Berg EL, Erlandsen SL, 35. Taub DD, Proost P, Murphy WJ, Anver Butcher EC. cc4 integrins mediate M, Longo DL, Van Damme J, lymphocyte attachment and rolling under Oppenheim JJ. Monocyte chemotactic physiologic flow. Cell 1995; 80:413 protein-1 (MCP-1), -2, and -3 are 28. Clark RA, Alon F, Springer TA. CD44 chemotactic for human T lymphocytes. J and hyaluronan-dependent rolling Clin Invest 1995; 95:1370 interactions of lymphocytes on tonsillar 36. Gunn MD, Tangemann K, Tam C, Cyster stroma. J Cell Biol 1996; 134:1075 JG, Rosen SD, Williams LT. A 29. DeGrendele HC, Estess P, Picker LJ, chemokine expressed in lymphoid high Siegelman MH. CD44 and its ligand endothelial venules promotes the hyaluronate mediate rolling under adhesion and Chemotaxis of naive T physiologic flow: a novel lymphocyte- lymphocytes. Proc Natl Acad Sei USA endothelial cell primary adhesion 1998; 95:258 pathway. J Exp Med 1996; 183:1119 37. Gunn MD, Ngo VN, Ansel KM, Ekland 30. Bargatze RF, Butcher EC. Rapid G EH, Cyster JG, Williams LT. A B-cell- protein-regulated activation event homing chemokine made in lymphoid involved in lymhocyte binding to high follicles activates Burkitt's lymphoma endothelial venules. J Exp Med 1993; receptor-1. Nature 1998; 391:799 178:367 38. Legier DF, Loetscher M, Stuber Roos R, 31. Lloyd AR, Oppenheim JJ, Kelvin DJ, Clark-Lewis I, Baggiolini M, Moser B. B Taub DD. Chemokines regulate T cell cell-attracting chemokine 1, a human 91 Chapter 7 CXC chemokine expressed in lymphoid Rev 1989; 108:111 tissues, selectively attracts B 45. Tedder TF, Penta AC, Levine HB, lymphocytes via BLR1/CXCR5. J Exp Freedman AS. Expression of the human Med 1998; 187:655 leukocyte adhesion molecule, LAM1. 39. Förster R, Mattis AE, Kremmer E, Wolf Identity with the TQ1 and leu-8 E, Brem G, Lipp M. A putative differentiation antigens. J Immunol 1990; chemokine receptor, BLCR1, directs B 144:532 cell migration to defined lymphoid 46. Gallatin WM, Weissman IL, Butcher EC. organs an specific anatomic A cell-surface molecule involved in compartments of the spleen. Cell 1996; organ-specific homing of lymphocytes. 87:1037 Nature 1983; 304:30 40. Tanaka Y, Adams DH, Hubscher S, 47. Arbonés ML, Ord DC, Ley K, Ratech H, Hirano H, Siebenlist U, Shaw S. T-cell Maynard-Curry C, Otten G, Capon DJ, adhesion induced by proteoglycan- Tedder TF. Lymphocyte homing and immobilized cytokine MIP-Iß. Nature leucocyte rolling and migration are 1993; 361:79 impaired in L-selectin-deficient mice. 41. Tanaka Y, Kimata K, Adams DH, Eto S. Immunity 1994; 1:247 Modulation of cytokine function by 48. Hamann A, Andrew DP, Jablonski- heparansulfate proteoglycans: Westrich D, Holzmann B, Butcher EC. sophisticated models for the regulation of Role of alpha 4-integrins in lymphocyte cellular responses to cytokines. homing to mucosal tissues in vivo. J Proceedings of the Association of Immunol 1994; 152:3282 American Physicians. 1998; 110:118 49. Ley K, Tedder TF. Leukocyte 42. Koopman G, Taher TEI, Mazzucchelli I, interactions with vascular endothelium. Keehnen RMJ, van der Voort R, Manten- New insights into selectin-mediated Horst E, Ricevuti G, Pals ST. CD44 attachment and rolling. J Immunol 1995; isoforms, including the CD44 v3 variant 155:525 are expressed on endothelium, suggesting 50. Streeter PR, Lakey Berg E, Rouse BTN, a role for CD44 in the immobilization of Bargatze RF, Butcher EC. A tissue- growth factors and the regulation of the specific endothelial cell molecule local immune response. Biochemical and involved in lymphocyte homing. Nature Biophysical research communications 1988; 331:41 1998; 245:172 51. Imai Y, Singer MS, Fennie C, Lasky LA, 43. Mackay CR. T-cell memory: the Rosen SD. Identification of a connection between function, phenotype Carbohydrate-based Endothelial ligand and migration pathways. Immunol Today for a lymphocyte homing receptor. J Cell 1991; 12:189 Biol 1991; 113:1213 44. Pals ST, Horst E, Scheper RJ, Meijer 52. Brustein M, Kraal G, Mebius RE, Watson CJLM. Mechanisms of human SR. Identification of a soluble form of a lymphocyte migration and their role in ligand for the lymphocyte homing the pathogenesis of disease. Immunol receptor. J Exp Med 1992; 176:1415 92 Cell Adhesion Receptors in Lymphoma Dissemination 53. Maly P, Thall AD, Petryniak B, Rogers 1985; 85:199 CE, Smith PL, Marks RM, Kelly RJ, 59. Duijvestijn AM, Horst E, Pals ST, Rouse Gersten KM, Cheng G, Saunders TL, BN, Steere AC, Picker LJ, Meijer CJLM, Camper SA, Camphausen RT, Sullivan Butcher EC. High endothelial FX, Isogai Y, Hindsgaul O, van Andrian differentiation in human lymphoid and UH, Lowe JB. The Expression of the mucosal homing expression of integrin a4ß7 on a subset of receptor cxjß, in malignant lymphomatous human CD4+ memory T cells with polyposis of the intestine. hallmarks of gut-tropism. J Immunol Gastroenterology 1994; 107:1519 1993;151:717 56. Hussell T, Isaacson PG, Crabtree JE, 62. Bos JD, de Boer OJ, Tibosch E, Das PK, Spencer J. Helicobacter pylori-specific Pals ST. Skin homing T lymphocytes: tumour-infiltrating T cells provide detection of cutaneous lymphocyte contact dependent help for the growth of associated antigen (CLA) by HECA-452 malignant B cells in low-grade gastric in normal human skin. Arch Dermatol lymphoma of mucosa-associated Res 1993; 285:179 lymphoid tissue. J Pathol 1996; 178:122 63. de Boer OJ, Horst E, Pals ST, Bos JD, 57. Möller P, Eichelmann A, Mechtersheimer Das PK. Functional evidence that the G, Koretz K. Expression of ß-1 integrins, HECA-452 antigen is involved in the H-CAM (CD44), and LECAM-1 in adhesion of human neutrophils and primary gastrointestinal B-cell lymphocytes to tumour necrosis lymphomas as compared to the adhesion factor-alpha-stimulated endothelial cells. receptor profile of the gut associated Immunology 1994; 81:359 lymphoid system, tonsil and peripheral 64. Berg EL, Yoshino T, Rott LS, Robinson lymph node. Int J Cancer 1991; 49:846 MK, Warnock RA, Kishimoto TK, Picker 58. Abel EA, Wood GS, Hoppe RT, Warnke L, Butcher EC. The cutaneous RA. Expression of leu-8 antigen, a lymphocyte antigen is a skin homing majority T-cell marker, is uncommon in receptor for the vascular lectin mycosis fungoides. J Invest Dermatol endothelial cell-leucocyte adhesion 93 Chapter 7 molecule 1. J Exp Med 1991 ; 174:1461 H-J, Newman W, Meijer CJLM, Pals ST. 65. Meijer CJLM, Beljaards F, Horst E, Adhesion of human B cells to follicular Willemze R, van der Valk P, Pals ST. dendritic cells involves both the LFA- Differences in antigen expression 1/ICAM-l andVLA-4/VCAM-l between primary cutaneous- and node pathways. J Exp Med 1991; 173:1297 based T-cell lymphomas. J Invest 73. Singer KH, Tuck DT, Sampson HA, Hall Dermatol 1989; 92:479 RP. Epidermal keratinocytes express the 66. Noorduyn LA, Beljaards RC, Pals ST, adhesion molecule intercellular adhesion van Heerde P, Radaszkiewicz T, molecule-1 in inflammatory dermatoses. Willemze R, Meijer CJLM. Differential J Invest Dermatol 1989; 92:746 expression of the HECA-452 antigen 74. Dustin ML, Springer TA. Lymphocyte (cutaneous lymphocyte associated function-associated antigen-1 (LFA-1) antigen, CLA) in cutaneous and non- interaction with intercellular adhesion cutaneous T-cell lymphomas. molecule-1 (IC AM-1 ) is one of at least Histopathology 1992; 21:59 three mechanisms for lymphocyte 67. Drillenburg P, van der Voort R, adhesion to cultured endothelial cells. J Koopman G, Dragosics B, van Krieken Cell Biol 1988; 107:321 JHJM, Lazarovits AI, Radaszkiewicz T, 75. de Fougerolles AR, Stacker SA, Pals ST. Preferential expression of the Schwarting R, Springer TA. mucosal homing receptor integrin a4ß7 in Characterization of ICAM-2 and gastrointestinal non-Hodgkin's evidence for a third counter-receptor for lymphomas. Am J Pathol 1997; 150:919 LFA-1. J Exp Med 1991; 174:253 68. Koopman G, Pals ST. Cellular 76. de Fougerolles AR, Springer TA. interactions in the germinal center: role Intercellular adhesion molecule 3, a third of adhesion receptors and significance for adhesion counter-receptor for the pathogenesis of AIDS and malignant lymphocyte function-associated molecule lymphoma. Immunol Rev 1992; 126:21 1 on resting lymphocytes. J Exp Med 69. Haskard D, Cavender D, Beatty P, 1992; 175:185 Springer T, Ziff M. T-lymphocyte 77. Hamann A, Jablonski-Westrich D, adhesion to endothelial cells : Duijvestijn A, Butcher EC, Baisch H, mechanisms demonstrated by anti-LFA-1 Harder R, Thiele HG. Evidence for an antibodies. J Immunol 1986; 137:2901 accessory role of LFA-1 in lymphocyte- 70. Scheeren RA, Koopman G, van der Baan high endothelium interaction during S, Meijer CJLM, Pals ST. Adhesion homing. J Immunol 1988; 140:693 receptors involved in early clustering of 78. Pals ST, den Otter A, Miedema F, Kabel blood dendritic cells and T-lymphocytes. P Keizer GD, Scheper RJ, Meijer CJLM. EurJImmunol 1991; 21:1101 Evidence that leukocyte function- 71. Steinman RM. The dendritic cell system associated antigen-1 is involved in and its role in immunogenicity. Annu recirculation and homing of human Rev Immunology 1991; 9:271 lymphocytes via high endothelial 72. Koopman G, Parmentier HK, Schuurman venules. J Immunol 1988; 140:1851 94 Cell Adhesion Receptors in Lymphoma Dissemination 79. Anderson DC, Schmalstieg FC, Shearer Ossekoppele GJ, van Krieken JHJM, Pals W, Becker-Freeman K, Kohl S, Smith ST. Adhesion molecules in the prognosis CW, Tosi MF, Springer TA. Leukocyte of diffuse large-cell lymphoma: LFA-1, OKM1, pi50,95 deficiency expression of a lymphocyte homing syndrome: functional and biosynthetic receptor (CD44), LFA-1 (CD 11 a/CD 18), studies of three kindreds. Federation Proc and ICAM-1 (CD54). Leukemia 1990; 1985; 44:2671 4:595 80. Roossien FF, de Rijk D, Bikker A, Roos 87. van Dinther-Janssen ACHM, Horst E, E. Involvement of LFA-1 in lymphoma Koopman G, Scheeper RJ, Newman W, invasion and metastasis demonstrated Meijer CJLM, Pals ST. The with LFA-1 deficient mutants. J Cell Biol VLA-4/VCAM-1 pathway is involved in 1989; 108:1979 lymphocyte adhesion to endothelium in 81. Roos E. Adhesion molecules in rheumatoid synovium. J Immunol 1991; lymphoma metastasis. Cancer Metastasis 147:4207 Rev 1991; 10:33 88. Yednock TA, Cannon C, Fritz LC, 82. Harning R, Myers C, Merluzzzi VJ. Sanchez-Madrid F, Steinman L, Karin N. Monoclonal antibodies to lymphocyte- Prevention of experimental autoimmune function-associated antigen-1 inhibit encephalomyelitis by antibodies against invasion of human lymphoma and a4p, integrin. Nature 1992; 356:63 metastasis of murine lymphoma. Clin 89. Baron JL, Madri JA, Ruddle NH, Hashim Exp Metastasis 1993; 11:337 G, Janeway jr. CA. Surface expression of 83. Soede RD, Wijnands YM, van Kouteren- ex4 integrin by CD4 T cells is required for Cobzaru I, Roos E. ZAP-70 tyrosine their entry into brain parenchyma. J Exp kinase is required for LFA-1 dependent T Med 1993; 177:57 cell migration. J Cell Biol 1998; 90. Lobb RR, Hemler ME. The 142:1371 pathophysiologic role of 95 Chapter 7 1997; 27:1 Human intestinal B-cell blasts and 93. Arroyo AG, Yang JT, Rayburn H, Hynes plasma cells express the mucosal homing receptor integrin a ß7. Scan J Immunol RO. Differential requirements for a4 4 integrins during fetal and adult 1995; 42:662 hematopoiesis. Cell 1996; 85:997 101. Wagner N, Lohler J, Kunkel EJ, Ley K, 94. Möller P, Eichelmann A, Mechtersheimer Leung E Krissansen G, Rajewsky K, G. Adhesion molecules VLA-1 to VLA-6 Muller W. Critical role for ß7 integrins in define discrete stages of peripheral B formation of the gut-associated lymphoid lymphocyte development and tissue. Nature 1996; 382:366 characterize different types of B cell 102. Shyjan AM, Bertognolli M, Kenney CJ, neoplasia. Leukemia 1992; 6:256 Briskin MJ. Molecular cloning of human 95. Freedman AS, Munro JM, Morimoto C, mucosal cell adhesion molecule-1 Mclntyre BW, Rhynhart K, Lee N, (MAdCAM-1) demonstrates structural Nadler LM. Follicular non-Hodgkin's and functional similarities to the a4ß7 lymphoma cell adhesion to normal integrin binding domains of murine germinalcenters and neoplastic follicles MAdCAM-1 but extreme divergence of involves very late antigen-4 and vascular mucin-like sequences. J Immunol 1996; cell adhesion molecule-1. Blood 1992; 156:2851 79:206 103. Briskin M, Winsor-Hines D, Shyjan A, 96. Jaspars LH, Bonnet P, Willemze R, Cochran N, Bloom S, Wilson J, McEvoy Meijer CJLM. Mycosis fungoides (MF) LM, Butcher EC, Kassam N, Mackay with extracutaneous localization in the CR, Newman W, Ringler DJ. Human breast. Br J Dermatol 1996; 134:1125 mucosal addressin cell adhesion 97. Briskin MJ, McEvoy LM, Butcher EC. molecule-1 is preferentially expressed in MAdCAM-1 has homology to intestinal tract and associated lymphoid immunoglobulin and mucin-like adhesion tissue. Am J Path 1997; 151:97 receptors and to IgAl. Nature 1993; 104. Dogan A, Du M, Koulis A, Briskin MJ, 363:461 Isaacson PG. Expression of lymphocyte 98. Holzmann B, Mclntyre BW, Weissman homing receptors and vascular addressins IL. Identification of a murine Peyer's in low-grade gastric B-cell lymphomas of patch-specific lymphocyte homing mucosa-associated lymphoid tissue. Am J receptor as an integrin molecule with an Pathol 1997; 151:1361 a chain homologous to human VLA-4a. 105. Cepek KL, Shaw SK, Parker CM, Russell Cell 1989; 56:37 GJ, Morrow JS, Rimm DL, Brenner MB. 99. Parker CM, Cepek KL, Russell GJ, Shaw Adhesion between epithelial cells and T SK, Prosnett DN, Schwarting RL, lymphocytes mediated by E-cadherin and the «Eß integrin. Nature 1994, 372;190 Brenner MB. A family of ß7 integrins on 7 human mucosal lymphocytes. Proc Natl 106. Chott A, Dragosics B, Radaszkiewicz Th. Acad Sei USA 1992; 89:1924 Peripheral T-cell lymphomas of the 100. Farstad IN, Halstensen TS, Lazarovits intestine. Am J Pathol 1992; 141:1361 AI, Norstein J, Fausa O, Brandtzaeg P. 107. Simonitsch I, Volc-Platzer B, Mosberger 96 Cell Adhesion Receptors in Lymphoma Dissemination I, Radaszkiewicz T. Expression of forms of CD44 are distinct polypeptides monoclonal antibody HML-1 defined with different adhesion potentials for ccEß7 integrin in cutaneous T-cell hyaluronate-bearing cells. EMBO J 1991; lymphoma. Am J Path 1994; 145:1148 10:343 108. Drillenburg P, Bronkhorst CM, van der 115. Haynes BF, Telen MJ, Hale PL, Denning Wal AC, Noorduyn LA, Hoekzema R, SM. CD44-A molecule involved in Pals ST. Expression of adhesion leucocyte adherence and T cell molecules in pagetoid reticulosis activation. Immunol Today 1989; 10:423 (Woringer-Kolopp disease). Br J 16. Pals ST, Horst E, Ossekoppele GJ, Figdor Dermatol 1997; 136:613 CG, Scheper RJ, Meijer CJLM. 109. Trowbridge IS, Lesley J, Schulte R, Expression of lymphocyte homing Hyman R, Trotter J. Biochemical receptor (CD44) as a mechanism of characterization and cellular distribution dissemination in non-Hodgkin's of a polymorphic, murine cell-surface lymphoma. Blood 1989; 73:885 glycoprotein expressed on lymphoid 117. Miyake K, Medina KL, Hayashi S, Ono tissues. Immunogenetics 1982; 15:299 S, Hamaoka T, Kincade PW. Monoclonal 110. Jalkanen S, Bargatze RF, Herron LR, antibodies to Pgp-1/CD44 block Butcher EC. A lymphoid cell surface lympho-hemopoiesis in long-term bone glycoprotein involved in endothelial cell marrow cultures. J Exp Med 1990; recognition and lymphocyte homing in 171:477 man. Eur J Immunol 1986; 16:1195 11! Giinthert U, Hofmann M, Rudy W, Reber 111. Carter WG, Wayner EA. Characterization S, Zoller M, Haussmann I, Matzku S, of the class III collagen receptor, a Wenzel A, Ponta H, Herrlich P. A new phosphorylated, transmembrane variant of CD44 confers metastatic glycoprotein expressed in nucleated potential to rat carcinoma cells. Cell human cells. J Biol Chem 1988; 1991; 65:13 263:4193 119. Jalkanen S, Joensuu H, Soderstrom KO, 112. Stamenkovic I, Amiot M, Pesando JM, Klemi P. Lymphocyte homing and the Seed B. A lymphocyte molecule clinical behavior of non-Hodgkin's implicated in lymphocyte homing is a lymphomas. J Clin Invest 1991 ; 87:1835 member of the cartilage link protein 120. Sy MS, Guo YJ, Stamenkovic I. Distinct family. Cell 1989; 56:1057 effects of two CD44 isoforms on tumor 113. Pals ST, Hogervorst F, Keizer GD, growth in vivo. J Exp Med 1991; 174:859 Thepen T, Horst E, Figdor CG. 121 Koopman G, Heider KH, Horst E, Adolf Identification of a widely distributed GR, van den Berg F, Ponta H, Herrlich P, 90-kDa glycoprotein that is homologous Pals ST. Activated human lymphocytes to the Hermes-1 human lymphocyte and non-Hodgkin's express a homoloque homing receptor. J Immunol 1989; of the rat metastasis-associated variant of 143:851 CD44. J Exp Med 1993; 177:897 114. Stamenkovic I, Aruffo A, Amiot M, Seed 122. Wielenga VJM, Heider KH, Offerhaus B. The hematopoietic and epithelial GJA, Adolf GR, van den Berg FM, Ponta 97 Chapter 7 H, Pals ST. Expression of CD44 variant 129. Huet S, Groux H, Caillou B, Valentin H, proteins in human colorectal cancer is Prieur AM, Bernard A. CD44 contributes related to tumor progression. Cancer Res to T cell activation. J Immunol 1989; 1993; 53:4754 143:798 123. Screaton GR, Bell MV, Jackson DG, 130. Shimizu Y, van Seventer GA, Siraganian Cornelius FB, Gerth U, Bell JI. Genomic R, Wahl SL, Shaw S. Dual role of the structure of DNA encoding the CD44 molecule in T cell adhesion and lymphocyte homing receptor CD44 activation. J Immunol 1989; 143:2457 reveals at least 12 alternatively spliced 131. Denning SM, Le PT, Singer KH, Haynes exons. ProcNatl Acad Sei USA 1992; BF. Antibodies against the CD44 p80, 89:12160 lymphocyte homing receptor molecule 124. Heider K.H, Hofmann M, Horst E., van augment human peripheral blood T cell den Berg F, Ponta P, Herrlich P, Pals ST. activation. J Immunol 1990; 144:7 A human homologue of the rat 132. Koopman G, van Kooyk Y, de Graaff M, metastasis-associated variant of CD44 is Meyer CJLM, Figdor CG, Pals ST. expressed in colorectal carcinomas and Triggering of the CD44 antigen on T adenomatous polyps. J Cell Biol 1993; lymphocytes promotes T cell adhesion 120:227 through the LFA-1 pathway. J Immunol 125. Fox SB, Fawcett J, Jackson DG, Collins 1990; 145:3589 I, Gatter KC, Harris AL, Gearing A, 133. Pierres A, Lipcey C, Mawas C, Olive D. Simmons DL. Normal human tissues, in A unique CD44 monoclonal antibody addition to some tumors, express identifies a new T cell activation multiple different CD44 isoforms. Cancer pathway. Eur J Immunol 1992; 22:413 Res 1994; 54:4539 134. Jalkanen S, Bargatze RF, de los Toyos J, 126. Mackay CR, Terpe HJ, Stauder R, Butcher EC. Lymphocyte recognition of Marston WL, Stark H, Günthert U. high endothelium: antibodies to distinct Expression and modulation of CD44 epitopes of an 85-95-kD glycoprotein variant isoforms in humans. J Cell Biol antigen differentially inhibit lymphocyte 1994; 124:71 binding to lymph node, mucosal, or 127. Arch R, Wirth K, Hofman M, Ponta H, synovial endothelial cells. J Cell Biol Matzku S, Herrlich P, Zoller M. 1987; 105:983 Participation in normal immune 135. Camp RL, Scheynius A, Johansson C, responses of a metastasis-inducing splice Pure E. CD44 is necessary for optimal variant of CD44. Science 1992; 257:682 contact allergic responses but is not 128. Horst E, Meijer CJLM, Radaszkiewicz T, required for normal leucocyte van Dongen JJM, Pieters R, Figdor CG, extravasation. J Exp Med 1993; 178:497 Hooftman A, Pals ST. Expression of a 136. DeGrendele HC, Estess P, Siegelman human homing receptor (CD44) in MH. Requirement for CD44 in activated lymphoid malignancies and related stages T cell extravasation into an inflammatory of lymphoid development. Leukemia site. Science 1997; 278:672 1990; 4:383 137. Taher TEI, Smit L, Griffioen AW, 98 Cell Adhesion Receptors in Lymphoma Dissemination Schilder-Tol EJM, Borst J, Pals ST. Blood 1994; 84:1361 Signalling through CD44 is mediated by 143. Drillenburg P, Wielenga VJM, Kramer tyrosine kinases. Association with MHH, van Krieken JHJM, Kluin- p561ck in T lymphocytes. J Biol Chem Nelemans HC, Hermans J, Heisterkamp 1996; 271:2863 S, Noordijk EM, Kluin PhM, Pals ST. 138. Bartolazzi A, Jackson D, Bennett K, CD44 Expression predicts disease Aruffo A, Dickinson R, Shields J, Whittle outcome in localized large B-cell N, Stamenkovic I. Regulation of growth lymphoma, submitted and dissemination of a human lymphoma 144. Salles G, Zain M, Jiang W, Boussiotis V, by CD44 splice variants. J Cell Sei. 1995; Shipp M. Alternatively spliced CD44 108:1723 transcripts in diffuse large-cell 139. Bennett KL, Jackson DG, Simon JC, lymphomas characterization and Tanczos E, Peach R, Modrell B. comparison with normal activated B cells Stamenkovic I, Plowman G, Aruffo A. and epithelial malignancies. Blood 1993; CD44 isoforms containing exon v3 are 82:3539 responsible for the presentation of 145. Terpe HJ, Koopmann R, Imhof BA, heparin-binding growth factor. J Cell Gunthert U. Expression of integrins and Biol 1995; 128:687 CD44 isoforms in non-Hodgkin's 140. van der Voort, Taher TEI, Wielenga lymphomas: CD44 variant isoforms are VJM, Prevo R, Smit L, David G, preferentially expressed in high-grade Groenink M, van Krimpen C, Hartman G, malignant lymphomas. J Pathol 1994; Gherardi E, Pals ST. Heparan sulfate- 174:89 modified CD44 promotes hepatocyte 146. Ristamäki R, Joensuu H, Söderström, growth factor/scatter factor-induced Jalkanen S. CD44V6 expression in non- signal transduction through the receptor Hodgkin's lymphoma: an association with tyrosine kinase c-Met. J Biol Chem, in low histological grade and poor press prognosis. J Path 1995; 176:259 141. Jalkanen S, Joensuu H, Klemi P. 147. Stauder R, Eisterer W, Thaler J, Günthert Prognostic value of lymphocyte homing U. CD44 variant isoforms in receptor and S value in non-Hodgkin's non-Hodgkin's lymphoma: a new lymphoma. Blood 1990; 75:1549 independent prognostic factor. Blood 142. Harris NL, Jaffe ES, Stein H, Banks PM, 1995; 85:2885 Chan JKC, Cleary ML, Delsol G, De 148. Legras S, Gunthert U, Stauder R, Curt F, Wolf-PeetersC, Falini B, Gatter KC, Oliferenko S, Kluin-Nelemans HC, Marie Grogan TM, Isaacson PG, Knowles DM, JP, Proctor S, Jasmin C Smadja-Joffe F. Mason DY, Muller-Hermelink HK, Pileri A strong expression of CD44-6v SA, Piris MA, Ralfkiaer E, Warnke RA. correlates with shorter survival of Perspective: a revised European- patients with acute myeloid leukemia. American classification of lymphoid Blood 1998:91:3401 neoplasms: a proposal from the 149. Yakushijin Y, Steckel J, Kharbanda S, International Lymphoma Study Group. Hasserjian R, Neuberg D, Jiang W-M, 99 Chapter 7 Anderson I, Shipp MA. A directly spiced exon 10-containing CD44 variant promotes the metastasis and homotypic aggreration of aggressive non-Hodgkin's lymphoma. Blood 1998; 91:4282 100 Summary / Samenvatting 101 Summary Summary For an adequate immune surveillance, lymphocytes patrol throughout the body to find pathogens. This dynamic migration process is strictly regulated by interactions of adhesion molecules, expressed on the cell surface of lymphocytes and endothelium. After antigenic stimulation of a naive lymphocyte, altered expression of adhesion molecules restricts recirculation of the resultant effector or memory cell to the site and related organs of initial antigen presentation. Selective expression and regulation of adhesion molecules on lymphocytes (lymphocyte homing receptor) and endothelium (vascular addressin) forms the basis for tissue-specific lymphocyte homing. For non-Hodgkin's lymphomas, the malignant counterparts of normal lymphocytes, tissue specific involvement suggests a role for adhesion molecules in the dissemination process. In this thesis the expression of adhesion molecules on non-Hodgkin's lymphomas was investigated. Malignant lymphomatous polyposis (MLP) is a non-Hodgkin's lymphoma of mantle cell type, located in the gastro-intestinal tract with the endoscopic picture of multiple polyps. Since these lymphomas extensively disseminate to mucosal sites, a role for adhesion molecules mediating lymphocyte migration to the mucosa was suggested. In chapter 2 the expression of the integrin MLP expressed a4ß7 suggesting a role in the characteristic mucosal dissemination pattern of this malignant lymphoma. In chapter 3 the expression of the integrin a4ß7 in a panel of B- and T-cell non-Hodgkin's lymphomas of different primary localizations was explored. The mucosal homing receptor a4ß7 was preferentially expressed on non-Hodgkin's lymphomas related to mucosa-associated B- and T-cell populations. Furthermore, unlike intra-epithelial lymphocytes, 102 Summary stage I primary nodal DLCL, CD44s was a strong prognosticator for overall survival, independent of the clinical parameters used in the International Prognostic Index. Incorporation of CD44s expression in the IPI parameter 'stage' improved the separation between the risk groups in nodal DLCL. The results of the study described in chapter 6 indicate that local therapy alone presumably is adequate for patients with stage I DLCL. In chapter 7 the current concepts about the role of adhesion molecules in lymphoma dissemination are described. Like their normal counterparts, non-Hodgkin's lymphomas express specific subsets of adhesion molecules. The results presented in this thesis indicate that these molecules may, at least partly, explain the tissue-specific dissemination of these non-Hodgkin's lymphomas. 103 Samenvatting Samenvatting De gezondheid van de mens wordt bedreigd door een continue aanval van microorganismen en andere schadelijke stoffen (antigenen), die in potentie de mogelijkheid hebben om een ziekte te veroorzaken. Het lichaam verweert zich hiertegen m.b.v. een dynamisch afweersysteem (immuun systeem) bestaande uit makrofagen, lymfocyten en granulocyten. De doelmatigheid van dit afweersysteem wordt vergroot door lymfocyten die continue recirculeren van bloed naar (lymfoide) organen, en via lymfe weer terug naar het bloed. Door deze recirculatie wordt voor lymfocyten de kans vergroot om in contact te komen met antigenen en bestaat er voor lymfocyten de mogelijkheid om zich naar specifieke plaatsen in het lichaam te verspreiden. Dit proces van recirculatie wordt nauwkeurig gereguleerd door moleculen op het oppervlak van lymfocyten (adhesie moleculen), die specifiek kunnen binden aan moleculen aanwezig op het endotheel van bloedvaten. Lymfocyten die na aanmaak in het beenmerg nog niet in contact zijn gekomen met een antigeen ('naieve' lymfocyten) bezitten adhesie moleculen die verspreiding in het hele lichaam mogelijk maakt. Zodra echter de lymfocyt wordt geactiveerd door een antigeen, veranderd de expressie van de adhesie moleculen met als gevolg dat de verspreiding van de lymfocyt wordt beperkt tot dat orgaan waar de antigeen activatie plaats vond. De adhesie moleculen die via dit mechanisme selectief tot expressie worden gebracht ('homing receptoren') zorgen via binding aan gespecialiseerd endotheel in de organen, voor een weefsel-specifieke verspreiding van lymfocyten. Non-Hodgkin's lymfomen vormen een heterogene groep van kwaadaardige tumoren van lymfocyten. Verondersteld wordt dat in de ontwikkeling van een lymfocyt, door bijvoorbeeld genetische schade, een blokkade van verdere differentiatie optreedt, waarna deze cel door ongecontroleerde groei een tumor kan vormen. Deze tumoren kunnen behalve in de lymfoide organen en het perifere bloed, ook voorkomen in niet-lymfoide organen, waaronder de slijmvliezen van het spijsverteringskanaal en de huid. Het verspreidingspatroon van non- Hodgkin's lymfomen is in het begin stadium van de ziekte vaak overeenkomstig het migratie patroon van de veronderstelde niet-kwaadaardige tegenhanger van de tumorcel. Dit suggereert dat de moleculen die normaal de verspreiding van lymfocyten reguleren in deze lymfomen nog steeds functioneel zijn. In dit proefschrift is de rol van adhesie moleculen in de verspreiding van non-Hodgkin's lymfomen onderzocht. Bij migratie van lymfocyten naar de slijmvliezen van het maag-darm kanaal speelt interactie van het integrine a4ß7, aanwezig op lymfocyten, met MAdCAM-1, selectief aanwezig op het endotheel van de bloedvaatjes in de slijmvliezen, een belangrijke rol. In het perifere bloed kan door expressie van a4ß7 een subgroep van lymfocyten worden gekarakteriseerd die selectief naar het maag-darm kanaal migreert. Maligne lymfomatoide 104 Samenvatting polyposis (MLP) is een mantel cel lymfoom gelocaliseerd in het maag-darm kanaal met endoscopisch het beeld van multipele poliepen. De uitbreiding van de tumor in de slijmvliezen suggereert een rol voor adhesie moleculen in de verspreiding. In hoofdstuk 2 is de expressie van het integrine a4ß7 onderzocht in MLP en vergeleken met een groep mantel cel lymfomen gelocaliseerd in lymfklieren. In tegenstelling tot de mantel cel lymfomen gelocaliseerd in de lymfklieren toonden alle MLP's expressie van a4ß7, wat wijst op een rol voor dit integrine in het opvallende verspreidingspatroon van deze tumor. In hoofdstuk 3 is de expressie van a4ß7 onderzocht in een panel van verschillende B- en T- cel non-Hodgkin's lymfomen. In deze studie kwam a4ß7 vooral tot expressie op de maligne lymfomen die geassocieerd zijn aan het lymfoide weefsel van het slijmvlies van het maag darm kanaal. Verder kon a4ß7 worden aangetoond op de lymfocyten afkomstig van de lamina propria, terwijl de intra-epitheliale cellen negatief waren. Deze bevindingen wijzen op een belangrijke rol voor a4ß7 in de verspreiding van aan de slijmvliezen gerelateerde non- Hodgkin's lymfomen. Pagetoide reticulose is een zeldzame vorm van een cutaan T-cel lymfoom, dat wordt gekenmerkt door een opvallende localisatie van tumorcellen in de epidermis. Op de tumorcellen van een patient met pagetoide reticulose, zoals besproken in hoofdstuk 4, vormen de aanwezigheid van de adhesie moleculen 'cutaneous lymphocyte antigen' (CLA) en aEß7 een verklaring voor het opmerkelijke groeipatroon. CLA geeft migratie naar de huid door binding aan E-selectine aanwezig op de dermale vaatjes, en aEß7 speelt een rol bij de localisatie van de tumorcellen in de epidermis door binding aan E-cadherin, aanwezig op de epitheelcellen. In hoofdstuk 5 en 6 wordt de expressie van CD44 beschreven op diffuus grootcellige B-cel lymfomen. Als deze lymfomen primair gelocaliseerd zijn in de lymfklier (primair nodaal) is de expressie van CD44s en CD44v6 gecorreleerd aan tumor verspreiding. In patiënten met stadium I primair nodale grootcellige B-cel lymfomen had CD44s expressie een voorspellende waarde voor overleving, onafhankelijk van de klinische parameters die gebruikt worden in de Internationale Prognostische Index (IPI). Toevoegen van CD44s expressie aan de IPI parameter 'stadium' verbeterd de differentiatie tussen de overlevingscurves van de risico groepen voor nodale grootcellige B-cel lymfomen. Deze bevindingen tonen aan dat CD44s expressie een risicofactor is voor overleving, voor patiënten met een primair nodaal grootcellig B-cel lymfoom. De resultaten van de studie beschreven in hoofdstuk 6 suggereren dat locale therapie voor patiënten met stadium I grootcellige B-cel lymfomen mogelijk voldoende is. In hoofdstuk 7 wordt een overzicht gegeven van de huidige inzichten in de rol van adhesie moleculen in lymfoom verspreiding. De resultaten van deze onderzoeken tonen aan dat het verspreidingspatroon van non- 105 Samenvatting Hodgkin's lymfomen verklaard kan worden door de expressie van specifieke adhesie moleculen. 106 107 Nawoord Promoveren wordt wel eens vergeleken met de inspanning die geleverd wordt bij het lopen van een marathon. Bij de marathon levert de atleet de inspanning echter zelf, terwijl een promotie onderzoek alleen tot een goed einde kan komen door de inspanning van velen. Ook bij het werk voor dit proefschrift zijn veel mensen betrokken geweest. Iedereen die heeft bijgedragen aan het voltooien van deze wetenschappelijke zevenkamp wil ik daarvoor bedanken. 108 Curriculum vitae De schrijver van dit proefschrift werd op 12 maart 1963 geboren in Voorschoten. In juni 1981 werd het VWO diploma behaald op de Rembrandt scholengemeenschap in Leiden. Vervolgens werd begonnen met de studie Geneeskunde aan de Rijksuniversiteit Leiden waarbij in 1984 het kandidaatsexamen en in 1987 het doctoraalexamen werd behaald. De studie werd op 16 juni 1989 afgesloten met het artsexamen. Door de werkzaamheden als assistent-geneeskundige niet in opleiding op de afdeling pathologie van het Rode Kruis Ziekenhuis in Den Haag (Hoofd: P.J. Spaander), ontstond de interesse voor de pathologie. Op 1 augustus 1991 werd de overstap gemaakt naar de afdeling pathologie van het Academisch Medisch Centrum in Amsterdam waar de opleiding tot patholoog werd gestart (Opleider: Prof.dr. J.J. Weening). Tijdens de opleiding werden de eerste stappen gezet die hebben geresulteerd in dit proefschrift. Na het beëindigen van de opleiding op 1 juni 1996 is hij werkzaam als staflid op de afdeling pathologie van het AMC. 109 Financial support for the publication of this thesis is greatly acknowledged from: Compex Nederland BV ISBN 90-9012497-7 This thesis was prepared at the Department of Pathology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands Cover: low grade lymphoma of MALT. a4ß7 positive tumor cells (blue) invade epithelial structures (brown) to form lympho-epithelial lesions (by J. Mulder). "'SB*,* rfH WW n f' *2Î * 'üCé