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Prepublished online as a Blood First Edition Paper on May 31, 2002; DOI 10.1182/blood-2002-01-0169. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-01-0169v1 100/7/2659    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Zhu, D. Articles by Stevenson, F. K. Search for Related Content PubMed PubMed Citation Articles by Zhu, D. Articles by Stevenson, F. K. Related Collections Immunobiology Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 October 2002, Vol. 100, No. 7, pp. 2659-2661 BRIEF REPORT VH gene analysis of splenic marginal zone lymphomas reveals diversity in mutational status and initiation of somatic mutation in vivo Delin Zhu, Jennifer Orchard, David G. Oscier, Dennis H. Wright, and Freda K. Stevenson From the Molecular Immunology Group, Tenovus Laboratory, Southampton University Hospitals, United Kingdom; and the Department of Haematology, Royal Bournemouth Hospital, United Kingdom.     Abstract Top Abstract Introduction Study design Results and discussion References Tumors of the splenic marginal zone can present in spleen or blood. The maturational status of the neoplastic B cells from each site appears heterogeneous, with either unmutated or mutated variable-region heavy chain (VH) genes. To determine an influence of tissue location, we assessed matched blood and splenic tumor cells from 4 patients and found them identical. However, one patient with unmutated VH genes in blood and spleen developed a clonally related diffuse large B-cell lymphoma in the chest wall. Strikingly, this subclone had undergone significant somatic mutation, with clear intraclonal heterogeneity. To our knowledge, this is the first case of a B-cell tumor showing initiation of somatic mutation in vivo. The finding emphasizes that the tissue microenvironment can influence tumor cell behavior and possibly affect disease progression. Importantly, because several replacement mutations were located within or close to the complementarity-determining regions (CDRs), it raises the question of a role for antigen in driving tumor growth. (Blood. 2002;100:2659-2661) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Splenic marginal zone lymphoma (SMZL) is a chronic B-cell disorder in which the splenic white pulp is infiltrated by an inner zone of small lymphocytes, frequently surrounding a residual germinal center, and an outer zone comprising larger cells with abundant pale cytoplasm and occasional blasts. Cases with blood involvement often include a proportion of cells with a characteristic villous appearance, and are diagnosed as splenic lymphoma with villous lymphocytes (SLVL). The tumor cells are positive for surface immunoglobulin M (IgM) and IgD and express CD20, CD22, and CD79a, but not CD5, CD10, CD23, CD43, or cyclin D1. SMZL usually pursues an indolent clinical course, but transformation to a large-cell lymphoma may occur.1 The distinctive lymphocyte morphology, immunophenotype, and cytogenetic abnormalities distinguish SMZL from extranodal MZL and from the majority of cases of nodal MZL. Analysis of mutated variable-region heavy chain (VH) genes from a small number of cases with SMZL or SLVL has shown that most have undergone somatic mutation with or without intraclonal heterogeneity.2-4 Because tissue location may influence mutational activity, we analyzed matched splenic and blood tumor cells in 4 patients. One patient who subsequently developed a clonally related transformed tumor site in the chest wall allowed the comparison of tumor cells in 3 sites. The findings indicate no difference in mutational patterns between spleen and blood, but reveal the potential for initiating mutation within the tumor clone in the appropriate environment in vivo.     Study design Top Abstract Introduction Study design Results and discussion References Patient and clinical data In all 4 cases, SMZL was diagnosed on splenic tissue with the use of World Health Organization criteria.5 Outer zone cells exhibited clear cytoplasm, and small satellite nodules of similar cells were present in the red pulp. Immunohistochemistry showed tumor cells to be positive for CD20 and CD79a, and negative for CD5, CD10, and CD23. Peripheral blood morphology was consistent with SLVLs in all cases. Patient 4 presented in 1988 with splenomegaly, a lymphocyte count of 5.2 × 109/L, and lymphocyte morphology consistent with SLVL. She underwent splenectomy in 1991, and histology indicated SMZL. The white blood cell (WBC) count rose to 68 × 109/L in 1994, and blood cells from this period were available for analysis. Following 3 cycles of fludarabine, circulating tumor cells were cleared. Further administration of fludarabine in 1996 for recurrent lymphocytosis resulted in prolonged pancytopenia. In 1997, a firm mobile lump was removed from the left anterior chest wall, and histology revealed a diffuse large B-cell lymphoma (DLBCL). Chromosomal analysis of blood and splenic mononuclear cells showed an identical complex karyotype, including deletion of chromosome 7q, the most common cytogenetic abnormality in SMZL. The DLBCL revealed karyotypic evolution with additional genetic material on chromosomes 1p, 6q, and 16q, and deletion of Xq and 3q. Analysis of VH genes Tumor-related VH genes were amplified from cDNA, and predominant sequences with clonally related complementarity-determining region 3 (CDR3) were identified.6 Sequence alignment was to the Entrez database (National Center for Biotechnology Information [NCBI], Bethesda, MD) and to the V Base (MRC Centre for Protein Engineering, Cambridge, United Kingdom).7     Results and discussion Top Abstract Introduction Study design Results and discussion References Tumor VH sequences were obtained from all 4 cases, with clear clonal identity between tumor cells from different tissues of each individual (Table 1). In 3 of 4 patients, homology to the germ line sequence exceeded 98% and they were classified as unmutated.8 In these patients, VH sequences from blood and splenic tumor cells were identical. Patient 3 showed a low but significant level of somatic mutation, with intraclonal heterogeneity, in tumor cells from both blood and spleen. Mutational patterns were similar in both tissues. Data from these patients confirm the mixed origins of tumor cells in SMZL.4 They are consistent with derivation from a heterogeneous population of normal SMZ B cells, including naive B cells poised for T-cell-independent type-2 responses against bacterial polysaccharide antigens,9 and memory B cells.10 Patient 3 represents those cases of SMZL with intraclonal heterogeneity, similar to that commonly observed in follicular lymphoma (FL).11 It illustrates that cells of SMZL may be responsive to factors known to be involved in inducing somatic mutation.12 These include stromal cells, CD4+ T cells, cytokines, and ligation of the B-cell receptor (BCR). A role for antigen may exist, although analysis of mutation distributions in V-genes is no longer considered adequate to reveal this role.13 The identity of mutation pattern between blood and splenic tumor cells is consistent with the movement of tumor cells between the 2 sites.                                View this table: [in this window] [in a new window]   Table 1. Patient data and summary of tumor VH gene sequences For patient 4, tumor samples were obtained from spleen, blood, and chest wall. The only phenotypic difference between the sites was the acquisition of bcl-6 expression in the chest wall biopsy. Immunohistochemical staining of this site showed no expression of CD10, but antibodies against CD21 and CD23 did reveal residual follicular structures among the diffuse infiltrate. The VH gene amplified from the spleen, and from the blood 3 years later, contained no somatic mutations. The VH sequences obtained from the DLBCL in the chest wall biopsy, obtained after a further 3 years, were clearly derived from the same tumor clone with an almost identical CDR3 sequence (Figure 1). However, there had been an accumulation of somatic mutations, with continuing mutational activity indicated by intraclonal heterogeneity (Figure 1). Some mutations were common to all sequences, but others existed only in subclones. The fact that repeated mutations were present in different clones, and that no mutations were detected in multiple clones from blood or splenic tissue, confirms that these changes were derived from mutation initiated within the tumor clone. This is likely to be associated with transformation of the tumor and location in the chest wall site. Interestingly, the replacement mutations found in all clones were present in, or near to, the CDRs (Figure 1). Replacement mutations accumulating in intraclonal variants, and verified by their presence in more than 1 clone, were also in these sites. The positions involved were as follows: 30 (AGT [Ser] to ACT [Thr]); 56 (AGT [Ser] to AAT [Asn]); 97 (GGA [Gly] to AGA/AAA [Arg/Lys]); 105 (AGT [Ser] to ACT [Thr]), with the last 2 in CDR3. View larger version (52K): [in this window] [in a new window]   Figure 1. Tumor-derived VH gene and deducted amino acid sequences from case 1.  VH sequences from blood (Bl), spleen (Sp), and chest wall (ChestW) are aligned against the germ line gene counterpart (V3-33). Dashes represent identity with the germline sequence. Replacement amino acid changes observed in more than one clone are starred. CDRs are in bold type and underlined. Amino acid numbering is according to Kabat et al.22 Induction of somatic mutation in vitro in normal B cells,14 and in most cases of neoplastic B cells,15,16 requires engagement of the BCR. This presumably reflects events occurring in the normal immune responses in vivo where antigen provides this stimulus. For gastric mucosa-associated lymphoid tissue (MALT) lymphomas, there is a clear role for stimulation of tumor growth, and possibly somatic mutation, by the presence of Helicobacter pyloris.17 In the case of FL, stimulation of the BCR could occur by direct interaction between glycosylated V-regions and stromal elements.18 This may free these tumors from a requirement for conventional antigen to maintain growth. It is unlikely that somatic mutation continues indefinitely in FL.19-21 SMZL is not considered to be a germinal center tumor, and glycosylation sites are not evident in published4 sequences or our own. The question of a role for interaction with antigen for stimulation of somatic mutation or for growth of tumor cells therefore remains open. The finding that a long-term in vivo tumor with unmutated VH genes can apparently initiate somatic mutation and accumulate intraclonal heterogeneity with replacement mutations clustered in the CDRs is dramatic. It suggests that a factor in the environment, possibly able to bind to the BCR, has stimulated the cell. Alternatively, an event has occurred in the clone to activate somatic mutation independent of exogenous stimuli. Either change could be linked to the transformation to a rapidly dividing diffuse lymphoma, with karyotypic evolution. Transformation is an uncommon event in SMZL and we need to investigate whether this route is the exception or the rule.     Footnotes Submitted January 18, 2002; accepted April 4, 2002. Prepublished online as Blood First Edition Paper, May 31, 2002; DOI 10.1182/blood-2002-01-0169. Supported by the Cancer Research Campaign UK and by Tenovus UK. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Freda K. Stevenson, Molecular Immunology Group, Tenovus Laboratory, Southampton University Hospitals, Southampton SO16 6YD, United Kingdom; e-mail: fs{at}soton.ac.uk.     References Top Abstract Introduction Study design Results and discussion References 1. Camacho FI, Mollejo M, Mateo MS, et al. Progression to large B-cell lymphoma in splenic marginal zone lymphoma: a description of a series of 12 cases. Am J Surg Pathol. 2001;25:1268-1276[CrossRef][Medline] [Order article via Infotrieve]. 2. Zhu D, Oscier DG, Stevenson FK. Splenic lymphoma with villous lymphocytes involves B cells with extensively mutated Ig heavy chain variable region genes. Blood. 1995;85:1603-1607[Abstract/Free Full Text]. 3. Dunn-Walters DK, Boursier L, Spencer J, Isaacson PG. Analysis of immunoglobulin genes in splenic marginal zone lymphoma suggests ongoing mutation. Hum Pathol. 1998;29:585-593[CrossRef][Medline] [Order article via Infotrieve]. 4. Tierens A, Delabie J, Pittaluga S, Driessen A, DeWolf-Peeters C. Mutation analysis of the rearranged immunoglobulin heavy chain genes of marginal zone cell lymphomas indicates an origin from different marginal zone B lymphocyte subsets. Blood. 1998;91:2381-2386[Abstract/Free Full Text]. 5. Jaffe E, Harris N, Stein H, Vardiman J. World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyons, France: IARC Press.; 2001:135-137. 6. Hawkins RE, Zhu D, Ovecka M, et al. Idiotypic vaccination against human B-cell lymphoma: rescue of variable region gene sequences from biopsy material for assembly as single-chain Fv personal vaccines. Blood. 1994;83:3279-3288[Abstract/Free Full Text]. 7. Cook GP, Tomlinson IM. The human immunoglobulin VH repertoire. Immunol Today. 1995;16:237-242[CrossRef][Medline] [Order article via Infotrieve]. 8. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. [Comments appear in Blood. 1999;94:1837-1839 and in Blood. 2001;97:1892-1894.] Blood. 1999;94:1848-1854[Abstract/Free Full Text]. 9. Martin F, Kearney JF. B-cell subsets and the mature preimmune repertoire: marginal zone and B1 B cells as part of a "natural immune memory." Immunol Rev. 2000;175:70-79[CrossRef][Medline] [Order article via Infotrieve]. 10. Dunn-Walters DK, Isaacson PG, Spencer J. Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells. J Exp Med. 1995;182:559-566[Abstract/Free Full Text]. 11. Stevenson FK, Sahota SS, Ottensmeier CH, Zhu D, Forconi F, Hamblin TJ. The occurrence and significance of V gene mutations in B cell-derived human malignancy. Adv Cancer Res. 2001;83:81-116[Medline] [Order article via Infotrieve]. 12. Tarlinton D. Germinal centers: form and function. Curr Opin Immunol. 1998;10:245-251[CrossRef][Medline] [Order article via Infotrieve]. 13. Dorner T, Brezinschek HP, Brezinschek RI, Foster SJ, Domiati-Saad R, Lipsky PE. Analysis of the frequency and pattern of somatic mutations within nonproductively rearranged human variable heavy chain genes. J Immunol. 1997;158:2779-2789[Abstract]. 14. Razanajaona D, Denepoux S, Blanchard D, et al. In vitro triggering of somatic mutation in human naive B cells. J Immunol. 1997;159:3347-3353[Abstract]. 15. Denepoux S, Razanajaona D, Blanchard D, et al. Induction of somatic mutation in a human B cell line in vitro. Immunity. 1997;6:35-46[CrossRef][Medline] [Order article via Infotrieve]. 16. Zan H, Cerutti A, Dramitinos P, Schaffer A, Li Z, Casali P. Induction of Ig somatic hypermutation and class switching in a human monoclonal IgM+ IgD+ B cell line in vitro: definition of the requirements and modalities of hypermutation. J Immunol. 1999;162:3437-3447[Abstract/Free Full Text]. 17. Isaacson PG. Gastric MALT lymphoma: from concept to cure. Ann Oncol. 1999;10:637-645[Abstract/Free Full Text]. 18. Zhu D, McCarthy H, Ottensmeier CH, Johnson P, Hamblin TJ, Stevenson FK. Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma. Blood. 2002;99:2562-2568[Abstract/Free Full Text]. 19. Aarts WM, Bende RJ, Bossenbroek JG, Pals ST, van Noesel CJ. Variable heavy-chain gene analysis of follicular lymphomas: subclone selection rather than clonal evolution over time. Blood. 2001;98:238-240[Abstract/Free Full Text]. 20. Zelenetz AD, Chen TT, Levy R. Histologic transformation of follicular lymphoma to diffuse lymphoma represents tumor progression by a single malignant B cell. J Exp Med. 1991;173:197-207[Abstract/Free Full Text]. 21. Zhu D, Hawkins RE, Hamblin TJ, Stevenson FK. Clonal history of a human follicular lymphoma as revealed in the immunoglobulin variable region genes. Br J Haematol. 1994;86:505-512[Medline] [Order article via Infotrieve]. 22. Kabat E. Sequences of Proteins of Immunological Interest. Bethesda, MD: National Institutes of Health; 1991. Publication 91-3242:3v. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: S. A. Pileri, P. L. Zinzani, P. Went, A. Pileri Jr, and M. Bendandi Indolent lymphoma: the pathologist's viewpoint Ann. Onc., January 1, 2004; 15(1): 12 - 18. [Abstract] [Full Text] [PDF] V. Franco, A. M. Florena, and E. Iannitto Splenic marginal zone lymphoma Blood, April 1, 2003; 101(7): 2464 - 2472. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-01-0169v1 100/7/2659    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Zhu, D. Articles by Stevenson, F. K. Search for Related Content PubMed PubMed Citation Articles by Zhu, D. Articles by Stevenson, F. K. Related Collections Immunobiology Brief Reports Social Bookmarking                   What's this?

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