—  SYMPOSIUM #47  —

The Role of Infectious Agents in B-cell Lymphomas
Moderators: Dr. Miguel A. Piris and Dr. Steven H. Swerdlow

Section 4 - Hepatitis C Virus and Lymphoproliferative Disorders
The role of lymphoma microenvironment in Splenic Marginal Zone Lymphoma

B Herreros, A Sanchez-Aguilera, M Mollejo , MA Piris
Lymphoma group, Spanish Nacional Cancer Centre (CNIO)
elchor Fernandez Almagro
3. Madrid , Spain


Introduction
The growth of lymphoma cells may depend not only on the accumulation of genetic alterations but also on signals derived from the milieu that drives cell proliferation or inhibits apoptosis. The relevance of the microenvironment to lymphoma pathogenesis has been highlighted by the observations linking specific microbial and viral pathogens to specific types of lymphoproliferative syndromes. However, it has also become apparent that lymphoma development requires a particular stromal microenvironment that conditions the nature of the developed neoplasms. This evidence has come from a range of conditions such as inflammation, bacterial and viral infection, endemic diseases and autoantigen stimulation of the immune system.

The Normal Marginal Zone
Presence of a discernible marginal zone was initially noticed in the spleen. It is usually a well-developed functional and topographical area consisting of medium-sized lymphoid elements with regular nuclei and a variable amount of clear cytoplasm [1] . Marginal zone cells in the spleen are CD20+, CD79a+, IgM+, CD21+, alkaline phosphatase+, but CD23-, Bcl6-, and IgD-negative or weakly IgD-positive. The size and immunophenotype of the splenic marginal zone may vary with age and specific pathological conditions . Marginal zone B cells have also been described in normal lymph nodes, mainly in those draining mucosae, and have a similar immunophenotype and topographical distribution to their splenic counterparts. By analysis of the mutated IgVH genes, marginal zone cells have been demonstrated to be mostly memory B cells, with a minor component of naive elements, the latter probably being responsible for thymus-independent type 2 antigen responses, such as bacterial capsular polysaccharide. Splenic marginal zone B cells play an important role in transporting circulating immune complexes into the splenic follicles and depositing them on the surface of follicular dendritic cells [2]. This functional assignation seems to be consistent with the regular distribution of MZ B cells around lymphoid follicles.

Splenic Marginal Zone Lymphoma
The term splenic marginal zone lymphoma (SMZL) was introduced by Schmid et al. in 1992, in relation to a study performed on a series of 4 cases [3] . Subsequently, larger series have been published, reediting some diagnostic criteria and increasing the knowledge of their molecular and clinical characteristics [4] . Splenic marginal zone lymphoma is a relatively rare type of lymphoma, which accounts for around 1-2% all non-Hodgkin's lymphomas.

The tumour in the spleen is characterized by a micronodular lymphoid infiltrate located in white pulp, with variable red pulp infiltration, marginal zone differentiation and follicular replacement by neoplastic cells. The white pulp tumoral nodules are composed of an inner central zone of small lymphocytes, located in the mantle zone and replacing the germinal centre, and a peripheral zone of medium-sized cells with clear cytoplasm and scattered blasts, the marginal zone component. Lymphoma cells may have variable degree of plasmacytic differentiation, including as a characteristic feature the presence of monoclonal plasma cells in the germinal centre. The tumour includes numerous admixed T cells.

The bone marrow infiltration is characterised by intertrabecular polymorphic nodules and interstitial and intrasinusoidal infiltration of small lymphocytes, with a pattern highly characteristic. The morphology of the tumoral cells in the peripheral blood includes villous cells, small cells, plasmacytic cells, centrocytoid cells and cells with monocytoid appearance.

Splenic hilar lymph node involvement shows a micronodular pattern, centred on pre-existing replaced follicles. As in the bone marrow, marginal differentiation is generally absent. These morphological variations suggest that SMZL cells, depending on their background, show morphological and immunophenotypic changes that reflect the tumoral-growth dependence of the microenvironment. Thus, it seems that marginal zone differentiation is mainly restricted to the spleen, while at other locations, the lack of the appropriate architecture or signalling mechanisms does not induce marginal zone differentiation. At the same time, the cell composition of the tumoral aggregates somehow reflects the now-recognised capacity of marginal zone B cells to induce germinal centre development through the transport of immune complexes to the follicular dendritic cells .

Immunophenotype : The tumoral cells express surface IgM and IgD and are CD20+, CD43-, IgD+, bcl2+, CD5-, CD23-, Cyclin D1-, bcl6-, CD10-, DBA44-/+. Immunostaining with bcl2 highlighted follicular replacement of bcl2- cells germinal centre by bcl2+ tumoral cells. The proliferative index is low, and MIB1 staining shows a distinctive annular pattern, outlining the presence of an increased growth fraction in the germinal centre and marginal zone.

Cytogenetic and molecular findings. Cytogenetic and molecular studies have demonstrated that around 45% of SMZL cases have allelic loss in the 7q22-36 chromosomal region. This alteration is found more frequently in SMZL than in other small B-cell lymphomas. Cases with 7q loss behave more aggressively and have more frequent tumoral progression.

Somatic mutations of IgVH genes have been observed in about half of the cases and, in those treated with splenectomy, were associated with longer overall survival . This finding, similar to that observed in CLL, reveals a striking parallel between these two conditions. Immunoglobulin gene studies have additionally revealed that roughly half of the SMZL cases make selective use of the V(H)1-2 segment, suggesting that this tumour derives from a highly selected B-cell population, which in turn implies that either foreign or preserved autoantigens play a role in the genesis of the disease. In addition to IgVH, a smaller proportion (13%) of SMZL cases display somatic mutations in the 5´ non-coding region of the bcl6 gene.

Gene profiling studies, in addition to confirming the relative homogeneity of this entity, indicate potential diagnostic markers and pathogenic pathways involved in the survival of the tumoral cell. Thus, the signature includes upregulated genes involved in apoptosis regulation, BCR and TNF signalling, and NF-κB activation, such as SYK, BTK, BIRC3, TRAF3, TRAF5, CD40 and LTB [5] . Furthermore, genes associated with the splenic microenvironment, like SELL and LPXN, were also overexpressed. Other genes of particular interest are lymphoma oncogenes such as ARHH and TCL1 . The increased expression of TCL1 is linked with the upregulation of genes associated with intracellular signalling via the AKT1 pathway found in SMZL, as described by Thieblemont et al [6] . Upregulation of the AP-1 and Notch 2 transcription factors has also been described by Troen et al [7] . Consistently with previous cytogenetic studies, genes located in the 7q31-7q32 region, such as CAV1, CAV2 and GNG11 are downregulated.

Pathogenesis
The cellular origin of SMZL is still a controversial issue. Although the term seems to indicate a close relationship with marginal zone B cells, the absence of IgVH somatic mutations in half of the cases, the absence of marginal differentiation when the tumour is located outside the spleen and the expression of IgD by the tumoral cells calls into question the marginal zone cell origin of this tumour, and raises the possibility that SMZL tumoral cells could represent the tumoral expansion of hitherto uncharacterised marginal zone precursor splenic B-cell subpopulation. This is supported also by the findings of Troen et al., where Notch 2 [7] , a transcription factor that induces marginal zone B-cell differentiation is overexpressed in SMZL .

Splenic Marginal Zone Lymphoma, Viruses and Bacterias
The role of viral and bacterial antigens in the pathogenesis of splenic marginal zone lymphoma (SMZL) is suggested from the findings of several studies:
  • IgVH-biased selection. IgVH mutational studies have demonstrated that roughly half of the SMZL cases use VH1.2, which contrasts with the use of this IgVH in normal B-cells, since only around 1% of normal peripheral blood B-cells use this gene [8] .

  • Association with malaria and idiopathic splenomegaly [9, 10, 11, 12] . Two different lymphoproliferative disorders, tropical splenic lymphoma, characterized by splenomegaly and circulating naive CD5-negative villous B lymphocytes, and hyper-reactive malarial splenomegaly, have been described in malaria-endemic areas. Although no relation has been found with other B-lymphotropic viruses, patients with tropical splenic lymphoma have been found to exhibit raised EBV antibody levels.

  • A relationship between HCV infection and SLVL has recently been established , which lends support to the hypothesis that the stimulation of marginal zone B cells in the spleen by persistent HCV antigens, particularly the E2 viral antigen, might be involved in the pathogenesis of SMZL. Thus, hepatitis C infection was reported in 10-16% of SMZL patients . Significantly, a higher proportion of HCV positivity (up to 50%) has been found in splenic DLBCL . Even regression of splenic lymphoma with villous lymphocytes (SLVLs) has been described in patients with HCV after antiviral treatment, thereby demonstrating a direct role of HCV in lymphomagenesis [13] . Interestingly, symptomatic mixed cryoglobulinaemia was a common feature in these cases [14] .

  • Nevertheless, the presence of HCV in lymphoid neoplasms seems to be a phenomenon characteristic of precise geographic areas, such as Italy and Japan, and restricted to some tumor types, singularly SMZL and other Marginal Zone Lymphomas [15, 16] .


References
  1. van den Oord JJ, de Wolf-Peeters C, De Vos R, Desmet VJ. Immature sinus histiocytosis. Light- and electron-microscopic features, immunologic phenotype, and relationship with marginal zone lymphocytes. Am J Pathol. 1985;118:266-277.

  2. Whipple EC, Shanahan RS, Ditto AH, Taylor RP, Lindorfer MA. Analyses of the in vivo trafficking of stoichiometric doses of an anti-complement receptor 1/2 monoclonal antibody infused intravenously in mice. J Immunol. 2004;173:2297-2306.

  3. Schmid C, Kirkham N, Diss T, Isaacson PG. Splenic marginal zone cell lymphoma. Am J Surg Pathol. 1992;16:455-466.

  4. Mollejo M, Menarguez J, Lloret E, et al. Splenic marginal zone lymphoma: a distinctive type of low-grade B-cell lymphoma. A clinicopathological study of 13 cases. Am J Surg Pathol. 1995;19:1146-1157.

  5. Ruiz-Ballesteros E, Mollejo M, Rodriguez A, et al. Splenic marginal zone lymphoma: proposal of new diagnostic and prognostic markers identified after tissue and cDNA microarray analysis. Blood. 2005;106:1831-1838.

  6. Thieblemont C, Nasser V, Felman P, et al. Small lymphocytic lymphoma, marginal zone B-cell lymphoma, and mantle cell lymphoma exhibit distinct gene-expression profiles allowing molecular diagnosis. Blood. 2004;103:2727-2737.

  7. Troen G, Nygaard V, Jenssen TK, et al. Constitutive expression of the AP-1 transcription factors c-jun, junD, junB, and c-fos and the marginal zone B-cell transcription factor Notch2 in splenic marginal zone lymphoma. J Mol Diagn. 2004;6:297-307.

  8. Algara P, Mateo MS, Sanchez-Beato M, et al. Analysis of the IgV(H) somatic mutations in splenic marginal zone lymphoma defines a group of unmutated cases with frequent 7q deletion and adverse clinical course. Blood. 2002;99:1299-1304.

  9. Bidegain F, Berry A, Alvarez M, et al. Acute Plasmodium falciparum malaria following splenectomy for suspected lymphoma in 2 patients. Clin Infect Dis. 2005;40:e97-100.

  10. Bates I, Bedu-Addo G. Chronic malaria and splenic lymphoma: clues to understanding lymphoma evolution. Leukemia. 1997;11:2162-2167.

  11. Wallace S, Bedu-Addo G, Rutherford TR, Bates I. Serological similarities between hyperreactive malarial splenomegaly and splenic lymphoma in west Africa. Trans R Soc Trop Med Hyg. 1998;92:463-467.

  12. Bates I, Bedu-Addo G, Jarrett RF, et al. B-lymphotropic viruses in a novel tropical splenic lymphoma. Br J Haematol. 2001;112:161-166.

  13. Hermine O, Lefrere F, Bronowicki JP, et al. Regression of splenic lymphoma with villous lymphocytes after treatment of hepatitis C virus infection. N Engl J Med. 2002;347:89-94.

  14. Saadoun D, Suarez F, Lefrere F, et al. Splenic lymphoma with villous lymphocytes, associated with type II cryoglobulinemia and HCV infection: a new entity? Blood. 2005;105:74-76.

  15. Ambrosetti A, Zanotti R, Pattaro C, et al. Most cases of primary salivary mucosa-associated lymphoid tissue lymphoma are associated either with Sjoegren syndrome or hepatitis C virus infection. Br J Haematol. 2004;126:43-49.

  16. Seve P, Renaudier P, Sasco AJ, et al. Hepatitis C virus infection and B-cell non-Hodgkin's lymphoma: a cross-sectional study in Lyon, France. Eur J Gastroenterol Hepatol. 2004;16:1361-1365.