Splenic Marginal Zone Lymphoma with Villous Lymphocytes Shows On-Going Immunoglobulin Gene Mutations

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1 American Journal of Pathology, Vol. 162, No. 2, February 2003 Copyright American Society for Investigative Pathology Splenic Marginal Zone Lymphoma with Villous Lymphocytes Shows On-Going Immunoglobulin Gene Mutations Anne Tierens,* Jan Delabie,* Agnieszka Malecka,* Junbai Wang, Alicja Gruszka-Westwood, Daniel Catovsky, and Estella Matutes From the Departments of Pathology* and Tumor Biology, The Norwegian Cancer Institute and Radiumhospital, University of Oslo, Oslo, Norway; and Academic Haematology and Cytogenetics, The Royal Marsden Hospital, London, United Kingdom Splenic marginal zone lymphoma (also splenic lymphoma with villous lymphocytes) is a B-cell non- Hodgkin s lymphoma with a characteristic morphology and phenotype. We studied the pattern of somatic hypermutation of the rearranged immunoglobulin heavy chain genes on 23 cases and have correlated these data with survival as well as immunophenotypic and genetic characteristics of the cases. Twothirds of the cases show immunoglobulin gene mutations, half of which show evidence of antigen selection, whereas one-third of the cases show no significant mutations. On-going mutation, a feature characteristic of follicular lymphoma, was demonstrated in all six cases randomly selected for this analysis, including one case with a low number of mutations (<2%). No statistical significant correlation was found between immunoglobulin mutation status and clinical, immunophenotypic, or genetic characteristics. Our results demonstrate that on-going somatic hypermutation is a prominent feature of splenic marginal zone lymphoma with circulating villous lymphocytes. On-going somatic hypermutation has previously been demonstrated in extra-nodal and nodal marginal zone lymphoma. Our results indicate that marginal zone lymphomas at different anatomical localizations may derive from a similar B-cell subset. (Am J Pathol 2003, 162: ) Splenic marginal zone lymphoma (SMZL), a lymphoma previously also described as splenic lymphoma with villous lymphocytes, is usually characterized by variable numbers of circulating lymphoma cells with fine cytoplasmic projections, intrasinusoidal bone marrow involvement, and monoclonal gammopathy in one-third of the patients. 1 4 The lymphoma infiltrates the white pulp of the spleen. 5 SMZL expresses pan B-cell markers such as CD19, CD20, and CD22 as well as surface IgM and IgD. One-third of the cases are positive for CD10, CD11c, CD23, or CD38. 6 Although no specific cytogenetic abnormalities have been identified, loss of chromosome 7q21-32 and trisomy 3 have, respectively, been documented in 40% and 17% of the cases SMZL is a low-grade lymphoma, but a more aggressive behavior is observed in a minority of cases. The molecular changes leading to the differences in clinical behavior are not yet elucidated, although p53 mutations as well as 7q deletions have been associated with a worse survival. 12,13 Mutation analysis of the rearranged immunoglobulin (Ig) genes provides information on the differentiation stage of normal and neoplastic B cells. 14,15 As such, three levels of peripheral B-cell maturation can be recognized. Before antigen exposure, mature but naive B cells display unmutated rearranged Ig genes. On encounter with antigen, affinity maturation of the B cells takes place in the microenvironment of the germinal center. There, B cells proliferate, acquire somatic mutations, and undergo isotype switching. Those B cells with the best fit for the antigen are selected and differentiate into plasma cells or memory B cells on leaving the germinal center. Accordingly, B-cell neoplasms can be classified in three categories: pregerminal center cell, germinal center cell, and of postgerminal center cell origin. Thus, follicular lymphoma displays highly mutated Ig genes and on-going mutations consistent with its germinal center cell origin. In contrast, mantle cell lymphomas typically, but not always, have rearranged Ig genes without or few somatic mutations, which is consistent with its derivation from naive B cells. Finally, lymphomas with Ig gene somatic mutations but lack of on-going mutations such as approximately half of chronic lymphocytic leukemia (CLL) cases, are thought to have a postgerminal center cell origin. The mutation status of the rearranged Ig genes provides insight into the origin of B-cell neoplasms and also may be a prognostic indicator as has been shown in CLL and most recently in a series of SMZLs. 13,16,17 CLL can be segregated into two prognostic subgroups based on Supported by a grant from the Norwegian Cancer Society. Accepted for publication November 6, Address reprint requests to Jan Delabie, M.D., Ph.D., The Norwegian Cancer Institute and Radiumhospital, Dept. of Pathology, University of Oslo, Montebello N-0310, Oslo, Norway. jan.delabie@labmed.uio.no. 681

2 682 Tierens et al the mutation status of the rearranged IgV genes. CLL cases with unmutated Ig variable (V) genes have a worse prognosis compared to those with mutated IgV genes. Earlier reports of the IgV gene mutation analysis on SMZL indicated that this lymphoma derives from a postgerminal center B cell However, studying a limited series of marginal zone lymphomas we have previously found heterogeneity with respect to the presence of somatic mutations, which has been most recently confirmed. 13,21,22 Here we report on the mutation analysis of rearranged IgV genes on a large number of SMZL cases and have studied on-going somatic mutation, which has not yet extensively been studied in this disease. The data were correlated with immunophenotype, genotype, and survival. Materials and Methods Patients A total of 29 cases with a diagnosis of SMZL were selected for the Ig mutation analysis and of these, 23 were informative for analysis. All cases were well characterized by morphology and immunophenotype. The bone marrow or spleen histology was reviewed on the majority of cases in which tissue was available. Flow Cytometry Flow cytometry analysis was performed on isolated blood mononuclear cells by indirect immunofluorescence using a panel of monoclonal antibodies (mabs) against B- and T-lymphocyte antigens: CD2, CD5, CD23, CD22, CD79b, FMC7, and anti-ig light chains and as previously described. 23 Control preparations included substitution of the relevant mab by a mouse Ig of a matched isotype. A marker was considered positive when 30% of B cells stained with the mab. Fluorescent in Situ Hybridization Fluorescent in situ hybridization analyses were performed using standard methods. Mononuclear cells were fixed in methanol:glacial acetic acid (3:1) and stored at 20 C until needed. The slides were incubated with RNase A (0.1 mg/ml, Sigma, St. Louis, MO) at 37 C for 1 hour, and washed in 2 standard saline citrate (SSC) at room temperature for 5 minutes. The cells were then digested with pepsin (0.1 mg/ml, Sigma) at 37 C for 10 minutes, rinsed in phosphate-buffered saline at room temperature for 5 minutes, and in 2 SSC at 37 C for 30 minutes. Afterward, the slides were dehydrated in 70%, 90%, and 100% ethanol and dried. Subsequently, denaturation was performed on a dry-heat block at 72 C using a denaturation solution (70% formamide, 2 SSC, 0.05 mol/l sodium phosphate buffer, ph 7.0). The slides were then quickly rinsed in 2 SCC, dehydrated, and dried before hybridization. A biotinylated centromeric probe for chromosome 3 was obtained from Dr. Janet Shipley (ICR, Sutton, Surrey, UK). A p53 locus-specific probe (LSI p53 Spectrum Orange; Vysis, Richmond, UK) was used in combination with a probe specific for the chromosome 17 centromere (CEP17 Spectrum Green, Vysis). The probes were prepared and denatured according to the manufacturer s instructions. The hybridization was performed overnight at 37 C. The posthybridization procedure consisted of three washes in 1 SCC at 45 C, followed by three washes in 0.1 SSC at 60 C and one wash in 4 SSC/Triton X-100 at room temperature. The slides were then dehydrated in 70%, 90%, and 100% ethanol; dried; and mounted with Vectashield mounting medium with 4,6-diamidino-2-phenylindole (Vector Labs, Peterborough, UK). To detect the biotinylated probes, the slides were additionally incubated with avidin-fluorescein isothiocyanate (Vector Labs), washed in 4 SSC/Triton X-100, dehydrated, and mounted as above. Analysis of the Ig Somatic Mutations To be able to study antigen selection, only expressed rearranged immunoglobulin genes have been studied. mrna Extraction and cdna Synthesis Poly A mrna was isolated from either mononuclear cell suspensions of spleen tissues, peripheral blood, or bone marrow using Dynabeads Oligo (dt) 25 following the manufacturer s recommendations (Dynal, Oslo, Norway). Five L of the eluted mrna were reverse-transcribed using Thermoscript reverse transcriptase and Oligo (dt) (Life Technologies, Inc., Grand Island, NY). Ten L of a mastermix [0.01 mol/l dithiothreitol, 40 U of RNase out, 2 mmol/l dntp mix, 15 U of Thermoscript reverse transcriptase in reverse transcriptase-polymerase chain reaction (PCR buffer)] was added to 10 l of diethyl pyrocarbonate H 2 O containing 2 mol/l of Oligo (dt) 25 and 5 l of mrna. The samples were incubated at 60 C for 60 minutes and subsequently at 85 C for 5 minutes to stop the reaction. Finally, 1 l of RNase H was added to the samples that were incubated for 20 minutes at 37 C. PCR Amplification of the Rearranged IgV Genes Five L of cdna was used to amplify the rearranged Ig heavy (H) chain genes using a mixture of six framework (FR) 1 IgH variable gene segment (VH) family-specific and three joining (JH) primers. 21,24 Forty L of a mastermix (200 mol/l dntps, 2.5 mmol/l MgCL 2, 100 nmol/l of each primer in PCR buffer, 5 l of cdna) was heated at 94 C for 10 minutes before adding 1.5 U Taq polymerase diluted in 10 lofdh 2 O. The PCR conditions were as follows: 1 cycle at 95 C for 2 minutes, 59 C for 4 minutes, 72 C for 80 seconds, followed by 34 cycles at 95 C for 90 seconds, 59 C for 30 seconds, 72 C for 80 seconds, and a final cycle at 72 C for 5 minutes. An aliquot of 10 l of PCR product was size fractionated on a agarose gel 1.5% in 1 TBE buffer. The gel bands containing the monoclonal IgH products were excised and purified using the Concert PCR purification kit (Life Technologies, Inc.).

3 On-Going Somatic Mutations in SMZL 683 Table 1. Summary of the Clinical Features, Immunophenotype, and Genotype of the SMZL Cases Case Sex/age (year) Immunophenotype / CD5 CD23 Cytogenetics and FISH for chromosome 3, p53, t(11;14) WBC (10 9 /L) SM/LA SPx Dead/alive 1 M/68 FISH: diploid 3, diploid p53 nd SM /LA No follow up 2 M/70 ND FISH: ND nd nd Dead, 12 months 3 F/65 FISH: diploid 3, diploid p53 14 SM /LA Alive, 72 months 4 M/84 / Complex karyotype/fish: diploid 3, diploid p SM /LA Dead, 10 months 5 M/75 t(2;7)(p12; q21, 12; FISH: diploid 3, del p53 46 SM /LA Alive, 24 months 6 M/NK ND FISH: ND 5 SM /LA Alive, 14 months 7 F/66 FISH: ND 13.9 SM /LA Alive, 5 months 8 F/49 FISH: diploid 3, diploid p53 92 SM /LA ND No follow up 9 M/72 FISH: diploid 3, diploid p SM /LA ND No follow up 10 M/56 FISH: diploid 3, diploid p SM /LA Alive, 72 months 11 M/NK FISH: ND 18 SM /LA Dead, 60 months 12 F/74 FISH: diploid 3, diploid p SM /LA ND No follow up 13 M/48 FISH: diploid 3, del p SM /LA Alive, 48 months 14 M/42 FISH: diploid 3, diploid p53 20 SM /LA Alive, 84 months 15 F/73 FISH: diploid 3, diploid p53 42 SM /LA ND No follow up 16 F/48 FISH: 3, diploid p SM /LA Alive, 84 months 17 M/50 FISH: ND 30 SM /LA Alive, 144 months 18 F/70 / FISH: diploid 3, diploid p SM /LA Alive, 48 months 19 F/67 FISH: diploid 3, diploid p53 20 SM /LA Alive, 12 months 20 F/74 FISH: ND 30 SM /LA Dead 21 F/76 FISH: diploid 3, del p53 38 SM /LA Dead, 32 months 22 F/66 FISH: diploid 3, diploid p SM /LA ND No follow up 23 M/66 FISH: ND nd SM /LA No follow up ND, Not determined; NK, not known; SM, splenomegaly; LA, lymphadenopathy; SPx, splenectomy. Sequencing of the Amplified PCR Products The purified PCR products were cloned using pgem-t easy vector system (Promega, Madison, WI). Purified DNA from at least five clones per case were sequenced on both strands using the DNA sequencing kit (PE Applied Biosystems, Foster City, CA) and Universal M13 primers (Life Technologies). Only those cases in which identical sequences were obtained from the majority of clones, were included in the study and further analyzed. For the study of on-going somatic hypermutation, at least 20 clones were sequenced per selected case. Mac Vector 5.0 sequence analysis software (Oxford Molecular group Inc., Campbell, CA) was used for sequence analysis. Sequences were aligned with germline sequences derived from V Base database (V Base Sequence Directory, Tomlinson and colleagues, MRC Center for Protein Engineering, Cambridge, UK). Statistical Analysis The binomial distribution model was used to calculate the probability that the number of R and S mutations in the FR and complementarity determining region (CDR) sequences occurred by chance only. 25 This method gives a good estimation of antigen selection for the purpose of this study, although mutational hot-spots such as the targeting of RGYW sequences, characteristic for somatic hypermutation, are not taken into account. 26,27 A P value of 0.05 was considered as statistically significant. The inherent susceptibility of R mutations of the CDRs and FRs have been calculated for each of the identified germline genes and is based on the chances of the occurrence in each codon of an amino acid replacement given any single nucleotide change not resulting in a termination codon. The likelihood of the occurrence of on-going somatic mutations versus Taq polymerase-induced errors in clonally-related sequences was calculated using the chisquare test. For the calculation, only unique on-going mutations in the clonally related sequences were counted. The rate of Taq polymerase errors was calculated by repeatedly amplifying and sequencing a cloned known germline Ig sequence using the same PCR conditions as described above. The error rate was determined as 0.3 per 1000 bp. Kaplan-Meier life tables were used to analyze the survival of the mutated and nonmutated SMZL cases. Two cutoff values, 98% and 99%, respectively, for the percentage of sequence difference with respect to the germline VH gene sequence were tested for their discriminative value. Indeed, sequence differences can be because of as yet uncharacterized polymorphisms and may not necessarily indicate somatic hypermutation. Differences between survival curves were determined according to the log-rank test. The chi-square test was used to analyze differences in clinical, immunophenotypic, and genetic features between mutated and unmutated cases. Results Clinical Features, Immunophenotype, and Genotype A summary of the clinical, immunophenotypic, and genotypic features of the cases is given in Table 1. The median age of the 23 patients that proved informative for this study was 67 years (range, 42 to 84 years) and male:female ratio 1.1. The median lymphocyte count was /L (range, 2.3 to 410) and all had a circulating

4 684 Tierens et al Table 2. Sequence and Mutation Analysis of Ig VH Genes in SMZL Case DH germline JH germline Closest VH germline % Identity R/S observed P(R) P(S) Conclusion 1 D2-2 JH % FR 0/0 NA NA NA 2 D3-10 JH % FR 0/0 NA NA NA 3 NI JH % FR 0/0 NA NA NA 4 D2-15 JH % FR 0/0 NA NA NA 5 D2-2/D6-13 JH % FR 0/0 NA NA NA 6 D3-10 JH % FR 0/1 NA NA NA CDR 1/0 NA NA 7 D3-9 JH % FR 2/0 NA NA NA CDR 2/0 NA NA 8 D6-6 JH % FR 2/ Random CDR 0/ NI JH % FR 1/ Random CDR 1/ D2-21 JH % FR 2/ Random CDR 1/ D3-22 JH % FR 3/ Random CDR 3/ D6-13 JH % FR 4/ Random CDR 5/ D6-13 JH % FR 2/ Ag selection CDR 0/ NI JH % FR 4/ Ag selection CDR 7/ D1-14 JH % FR 2/ Ag selection CDR 7/ D2 JH % FR 2/ Ag selection CDR 1/ D1-7 JH % FR 2/ Ag selection CDR 2/ D3-9 JH % FR 5/ Ag selection CDR 6/ NI JH % FR 4/ Ag selection CDR 6/ NI JH % FR 3/ Ag selection CDR 4/ D3-9 JH % FR 10/ Ag selection CDR 10/ D3-3 JH % FR 5/ Ag selection CDR 7/ D3-3 JH % FR 5/ Ag selection CDR 9/ VH, IgH variable gene segment; DH, IgH diversity gene segment; JH, IgH joining gene segment; % identity, % homology of the rearranged VH gene with the closest germline; NI, not identified; R, replacement mutations; S, silent mutations; FR, framework regions; CDR, complementarity determining regions; p (R), probability that excess or scarcity of the R mutations in the VH gene CDRs or FRs resulted from chance only; p (S), probability that excess or scarcity of the S mutations in the VH gene CDRs or FRs resulted from chance only; NA, not applicable/not analyzed. clonal B-cell population as assessed by immunophenotyping. All patients presented with splenomegaly, but only a minority presented with lymphadenopathy and/or hepatomegaly. All patients had circulating villous lymphocytes as assessed on May-Grünwald-Giemsa-stained peripheral blood smears. Light chain restriction of the B cells was demonstrated in all cases. The majority of the cases expressed FMC7 and were strongly positive for surface CD22 and/or CD79b. A minority of the cases expressed CD23 and less than one-third expressed CD5. According to the scoring system for the diagnosis of CLL 6 all cases had scores ranging from 0 to 2. Trisomy 3 was demonstrated in one of the cases. Fluorescent in situ hybridization analysis showed a p53 gene deletion in three cases, one of which was associated with overexpression of the protein and a mutation in the p53 gene. Conventional karyotyping demonstrated a t(2;7)(p12; q21) in one patient and a complex karyotype in another. Sequence Analysis of Rearranged IgH Genes of Tumor Cells In 23 of 29 cases a definitive monoclonal IgH gene rearrangement could be identified. The lack of sufficient num-

5 On-Going Somatic Mutations in SMZL 685 bers of tumor cells in the samples or the lack of amplification of the neoplastic rearranged IgH sequences because of mutation, is likely the cause of negative results in six cases. The sequence analysis of the expressed rearranged IgH genes is summarized in Table 2. All sequences are available from GenBank under accession numbers AJ to AJ SMZL preferentially rearranged genes of the VH1 and VH3 families (21 of 23 cases). There was a random use of VH3 family genes, but one member of the VH1 family gene, VH1-2, was used in five of the cases. The IgH diversity gene segment (DH) gene could be identified in 18 of 23 cases. In most of the cases, homology of at least 10 consecutive nucleotides with the closest germline was retained for the DH gene assignment. 28 In the remaining cases, a potential DH gene was identified when using a less strict criterium of a minimum of six successive matches or seven successive matches interrupted by one mismatch. No preferential rearrangement involving any of the DH genes was seen. In 50% of the cases JH4 family genes were used, whereas the other 50% rearranged either the JH6 gene and, to a lesser extent, JH3 and JH5 family genes. Nucleotide sequence analysis of the VH genes, illustrated in Table 2, allowed to distinguish two groups of SMZL as follows: those with 99% homology and those with 99% or 100% homology to germline genes in the database. We used the 99% cutoff value because polymorphisms and PCR errors cannot be excluded above this value. As case 7 illustrates (see further), this cutoff level is not too high. In 7 of 23 cases, IgVH genes were in germline or near-germline configuration, whereas in 16 SMZL cases, IgVH genes were somatically mutated. The homology with the closest identified germline VH gene ranged from 88 to 99%. In addition, we studied the mutation pattern of somatic hypermutations to investigate whether the lymphoma cells showed features of antigen selection. This analysis is based on the fact that as a result of antigen-selective pressure, the ratio of amino acid replacement (R) to silent mutations (S) is higher than expected in the CDR regions, which is consistent with the need to provide the best fit for the antigen. In contrast, the R/S ratio is lower in the FR regions to conserve the antibody structure. The distribution of R and S mutations in CDR and FR regions of the tumor-derived VH genes is summarized in Table 2. P values of 0.05 according to the binomial distribution model were considered statistically significant of antigen-selective pressure. A random distribution of somatic hypermutation was observed in 5 of 16 cases suggesting that the tumor cells derived from nonselected memory B cells. In 11 of 16 cases the pattern of R or S mutations was indicative of antigen-selective pressure. To detect whether a particular antigen might have been responsible for the antigen-selective pressure observed in those cases showing features of antigen selection, we evaluated nucleotide and derived amino acid sequence similarities of the CDR3 regions. However, no consensus sequence could be demonstrated between the CDR3 regions of the rearranged Ig genes (results not shown). Intraclonal Heterogeneity Because two-thirds of the cases apparently derived from a mutated B cell, we asked whether these cases were subject to on-going somatic hypermutation. We randomly selected six cases with mutated VH genes (patients 7, 12, 13, 15, 21, and 22) to study intraclonal variation. Case 7 displayed an apparent low number of sequence differences when compared to the germline VH sequence. Intraclonal heterogeneity was demonstrated in all cases studied, even in case 7. The nucleotide variation observed was in all cases higher than the expected Taq polymerase error rate, indicating on-going mutational activity (chi-square test, P ). The respective genealogical trees of the clonally related sequences are shown in Figure 1. Mutation Status, Clinical Features, Immunophenotype, and Genotype The results are summarized in Table 3. The Kaplan Meier curves did not reveal statistically different survival rates among the mutated and nonmutated cases, but there was a trend toward a shorter survival in the unmutated cases (results not illustrated). These results were basically the same whether cutoff values of 98% or 99% were used to discriminate mutated from nonmutated cases. Those values are somewhat arbitrary, a fact nicely illustrated by the findings of on-going mutation in case 7, a case that is classified with the so-called unmutated group even when a cutoff value of 99% is used. However, case 7 belongs biologically to the group of mutated cases by virtue of its capacity to mutate. There also seems to be a male predominance among the unmutated cases, although this is not statistically significant (P 0.22). We further correlated immunophenotype and genotype with IgV gene mutation status in SMZL. Interestingly, all but 3 of the 16 mutated cases expressed light chains, whereas we see a more usual pattern among the unmutated cases. However, this difference was not statistically significant. In addition, expression of CD5 was observed in three of seven of the unmutated cases, whereas it was present in only 2 of 16 of the mutated cases but also this was not statistically significant (P 0.14). There were few abnormal genotypes observed both in the mutated and nonmutated cases by conventional cytogenetics or fluorescent in situ hybridization analysis for p53 or trisomy 3. p53 deletions were observed in three cases of which one case in the IgH unmutated group and two in the mutated group. None of these genetic findings correlated significantly with mutation status. Discussion SMZL has been recognized as a B-cell lymphoma entity in the new World Health Organization classification. 1 Although the morphology, immunophenotype, and clinical presentation of SMZL have been well documented, the pathogenesis and the cell of origin have not yet been fully elucidated. Mutation analysis of the antigen receptor

6 686 Tierens et al Figure 1. Genealogical trees of the mutations observed with respect to the germline sequence, in the cloned IgH sequences derived from SMZL cases 7, 12, 13, 15, 21, and 22. The numbers above the circles refer to the particular clones that were sequenced, whereas each circle represents a particular nucleic acid sequence. The nucleotide changes with respect to the germline sequence are indicated within the circles. Circles that are connected with a line represent closely homologous sequences that differ only by the nucleic acids indicated in the lowest circle. The figure illustrates the progressive accumulation of mutations or on-going mutation in the sequences.

7 On-Going Somatic Mutations in SMZL 687 Table 3. Disease Features and Ig Mutation Status Feature 99% homology* 99% homology P value Number of cases 7 16 M/F ratio 5/2 7/ Median age 69 years 66 years Lymphadenopathy Lymphocyte count /L /L (median) Kappa Lambda CD Trisomy P53 abnormalities *Refers to the homology with respect to germline genes found in V-BASE, a comprehensive Ig gene database. Result of chi-square test. genes in a limited number of cases have revealed that most cases of SMZL presumably derive from a postgerminal center cell, ie, a cell with somatic hypermutation but without evidence of on-going mutation. However, our previous study on a small number of cases as well as a recent report of 35 cases of SMZL have demonstrated heterogeneity with respect to the somatic mutation pattern including a number of cases without Ig somatic mutation. 13,21 Our findings confirm that SMZL as CLL is heterogeneous with respect to the mutation pattern of the Ig genes. Whereas the majority of the cases studied displayed somatic hypermutations in the Ig genes, one-third had unmutated Ig genes. Thus, SMZL likely derives from different subsets of B cells, ie, naive as well as memory B cells. These data are consistent with the heterogeneity found in the normal splenic marginal zone B-cell population, which comprises functionally heterogeneous B cells. 29,30 Three mutation patterns have been observed in the rearranged IgVH genes of normal splenic marginal zone B cells: cells without somatic hypermutation, cells with somatic hypermutation but without evidence of antigen selection, and cells with somatic hypermutation characteristic of antigen selection. Marginal zone B cells with mutations could be generated through a T-cell-dependent immune response that takes place in the germinal center whereas the unmutated B cells could represent naive B cells that have not been challenged by antigen. Alternatively, some of the marginal zone B cells without mutations could be the result of a T-cell-independent type II immune response. 31,32 The latter hypothesis may be supported by the occurrence of a variant of SMZL developing in patients with hyperreactive malarial splenomegaly. 33 The tropical variant of SMZL has similar morphological, immunophenotypic, and clinical features as the cases occurring in Western countries, but does not show mutations of the Ig genes. 34,35 It has been postulated that repeated malarial infection may induce a chronic T-cell-independent type II immune reaction that then provides a target for malignant transformation. 34,35 Malaria antigens contain short antigenic repeats and predominantly induce IgM synthesis in the absence of T-cell help. 36 Unlike the tropical variant of SMZL, there is no known cause associated with the development of the Western variant of SMZL. Our findings suggest that the Western SMZL arise from marginal zone B cells involved in diverse immunological challenges to T-cell-dependent or T-cell-independent antigens. Interestingly, we found that the IgVH genes in a subset of SMZL undergo on-going somatic mutation. Hitherto, there was no convincing evidence of intraclonal variation of the IgVH genes in these lymphomas. 18,19 Although intraclonal variation of Ig V H genes is the hallmark of follicular lymphoma, it is also described in a limited number of cases of MALT lymphomas and nodal marginal zone B-cell lymphomas Intraclonal heterogeneity in SMZL suggests that the lymphoma cells are prone to continuous mutational activity. Because the germinal center is hitherto the major site of clonal expansion and somatic mutation, it could be deduced that at least a subgroup of SMZL and other marginal zone lymphomas originate from a germinal center cell. However, neither the immunophenotype nor the morphology is compatible with a germinal center cell origin of this lymphoma subtype. Alternatively, the lymphoma cells may acquire somatic hypermutations independent of the germinal center microenvironment. Indeed, it has been suggested that a second B-cell diversification pathway exists separate from the classical cognate T-B cell interaction that takes place in the germinal center. 40 Patients with X-linked hyperigm syndrome who have no functional CD40L and lack germinal centers, generate IgM /IgD /CD27 memory B cells with a low-level of somatic hypermutation We are currently performing a genome-wide gene expression analysis in our cases of SMZL to find out whether SMZL with or without somatic hypermutations share a similar gene expression profile and thereby a common cell of origin or whether substantially different gene expression patterns can be found possibly indicating an origin from different B-cell subsets. The rearranged VH genes of SMZL mostly belong to the VH1 and VH3 gene families, as has also been described for CLL. However, there appears to be an equal distribution of VH1 and VH3 family genes in the mutated versus unmutated SMZL in contrast to what is described in CLL. In CLL, the V1-69 gene belonging to the VH1 family, is overrepresented in the unmutated CLL whereas VH3 family genes such as V3-07 and V3-21 are more frequently used in the mutated CLL. 16,45,46 Unlike the observations in CLL, a biased use of V1-2 in the nonmutated as well as the mutated SMZL cases was seen in the present study. This finding confirms recently published data on SMZL. 13 The preferential usage of certain VH genes in addition to the evidence for antigen selection in approximately half of all SMZL cases with somatic hypermutation in our study, suggests a role for antigen stimulation in the development of this lymphoma. The latter has previously been shown in a subset of the MALT lymphomas of the gut, where chronic Helicobacter pylori infection triggers lymphomagenesis. Which antigens might be involved in the pathogenesis of SMZL is not known but the biased use of the V1-2 gene suggests the involvement of a common antigen. Interestingly, the VH1 and VH3 family genes observed in SMZL have been reported to be as-

8 688 Tierens et al sociated with autoantibodies, such as rheumatoid factors and agglutinins. The latter suggests that SMZL might be derived from autoreactive B cells. 18 We have compared the immunophenotypical differences between the mutated and unmutated SMZL. Although the number of unmutated cases is relatively small, the expression of CD5 in three of seven cases is noteworthy. CD5 expression is characteristic of B-1 B cells, which are polyreactive B cells playing a role in the T-cellindependent immune response. 32,47,48 Interestingly, polyreactive B cells akin to B-1 cells may localize in the splenic marginal zone of rodents. 49 Alternatively, CD5 expression may also reflect differences in activation status of the B cells. It is clear that CD5 engagement modulates the B-cell antigen receptor signaling The significance, if any, of the immunophenotypical differences between mutated and unmutated SMZL is not clear and needs further study. In our series, there was no statistically significant difference in survival between mutated and unmutated SMZL cases as recently described. 13 The reason for this discrepancy is not clear but may be because of the relatively lower number of unmutated cases in our series and/or the relatively short follow-up period in our series compared to the series previously published. In addition, our series of cases may be biased by the fact that all of our cases show circulating villous lymphocytes. In conclusion, we have documented that SMZL shows a heterogeneous pattern of Ig somatic hypermutation, and importantly, that on-going somatic mutation occurs in a substantial number of cases. These features, including the occurrence of on-going mutation have previously been documented in nodal and extra-nodal marginal zone lymphoma. Taken together, the mutation data suggest that marginal zone cell lymphomas, irrespective of their localization, originate from a similar B-cell subset with the capacity to mutate its immunoglobulin genes. Genome-wide gene expression analysis of SMZL as well as normal marginal zone B cells could potentially further elucidate the cell or cells of origin of this lymphoma subtype. References 1. Isaacson PG, Piris MA, Catovsky D, Swerdlow S, Montserrat E, Berger F, Müller-Hermelink HK, Nathwani B, Harris N: Splenic marginal zone lymphoma. Tumours of Hematopoietic and Lymphoid Tissues. Edited by ES Jaffe, NL Harris, H Stein, JW Vardiman. Lyon, IARC Press, 2001, pp Neiman RS, Sullivan AL, Jaffe R: Malignant lymphoma simulating leukemic reticuloendotheliosis. A clinicopathologic study of ten cases. Cancer 1979, 43: Melo JV, Hegde U, Parreira A, Thompson I, Lampert I, Catovsky D: Splenic B-cell lymphoma with circulating villous lymphocytes: differential diagnosis of B-cell leukemias with large spleens. J Clin Pathol 1987, 40: Catovsky D, Matutes E: Splenic lymphoma with circulating villous lymphocytes/splenic marginal-zone lymphoma. Semin Hematol 1999, 36: Isaacson P, Matutes E, Bruke M, Catovsky D: The histopathology of splenic lymphoma with villous lymphocytes. Blood 1994, 84: Matutes E, Owusu-Ankomah K, Morilla R, Garcia-Marco J, Houlihan A, Que TH, Catovsky D: The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia 1994, 8: Troussard X, Mauvieux L, Radford-Weiss I, Rack K, Valensi F, Garand R, Vekemans M, Flandrin G, Macintyre EA: Genetic analysis of splenic lymphoma with villous lymphocytes: a groupe Francais d hematologie cellulaire (GFHC) study. Br J Haematol 1998, 101: Gruszka-Westwood AM, Matutes E, Cognet LJA, Wotherspoon A, Catovsky D: The incidence of trisomy 3 in splenic lymphoma with villous lymphocytes: a study by FISH. Br J Haematol 1999, 104: Mateo M, Mollejo M, Villuendas R, Algara P, Sanchez-Beato M, Martinez P, Piris MA: 7q31-31 allelic loss is a frequent finding in splenic marginal zone lymphoma. Am J Pathol 1999, 154: Corcoran MM, Mould SJ, Orchard JA, Ibbotson RE, Chapman RM, Boright AP, Platt C, Tsui LC, Scherer SW, Oscier DG: Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosomal 7q translocation. Oncogene 1999, 18: Hernandez JM, Garcia JL, Gutierrez NC, Mollejo M, Marinez-Climent JA, Flores T, Gonzalez MB, Piris MA, San-Miguel JF: Novel genomic imbalances in B-cell splenic marginal zone lymphomas revealed by comparative genomic hybridization and cytogenetics. Am J Pathol 2001, 158: Gruszka-Westwood AM, Hamoudi RA, Matutes E, Tuset E, Catovsky D: p53 abnormalities in splenic lymphoma with villous lymphocytes. Blood 2001, 97: Algara P, Mateo MS, Sanchez-Beato M, Mollejo M, Navas IC, Romero L, Sole F, Salido M, Florensa L, Martinez P, Campo E, Piris MA: 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: Stevenson FK, Sahota S, Zhu D, Ottensmeier C, Chapman C, Oscier D, Hamblin T: Insight into the origin and clonal history of B-cell tumours as revealed by analysis of immunoglobulin variable region genes. Immunol Rev 1998, 162: Küppers R, Klein U, Hansmann ML, Rajewsky K: Cellular origin of human B-cell lymphomas. N Engl J Med 1999, 341: Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK: Unmutated immunoglobulin V H genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999, 94: Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buchbinder A, Budman D, Dittmar K, Kolitz J, Lichtmand SM, Schulman P, Vinciguerra VP, Rai KR, Ferrarini M, Chiorazzi N: Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999, 94: 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: Dunn-Walters DK, Boursier L, Spencer J, Isaacson PG: Analysis of immunoglobulin genes in splenic marginal zone lymphoma suggests on-going mutation. Hum Pathol 1998, 29: Miranda RN, Cousar JB, Hammer RD, Collins RD, Vnencak-Jones CL: Somatic mutation analysis of IgH variable regions reveals that tumor cells of most parafollicular (monocytoid) B-cell lymphoma, splenic marginal zone B-cell lymphoma, and some hairy cell leukemia are composed of memory B lymphocytes. Hum Pathol 1999, 30: Tierens A, Delabie J, Pittaluga S, Driessen A, De Wolf-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: Bahler DW, Pindzola JA, Swerdlow SH: Splenic marginal zone lymphomas appear to originate from different B cell types. Am J Pathol 2002, 161: Matutes E, Morillo R, Owusu-Ankomah K, Houlihan A, Catovsky D: The immunophenotype of splenic lymphoma with villous lymphocytes and its relevance to the differential diagnosis with other B-cell disorders. Blood 1994, 83: Küppers R, Zhao M, Hansmann ML, Rajewsky K: Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J 1993, 12:

9 On-Going Somatic Mutations in SMZL Chang WC, Casali P: The CDR1 sequences of a major proportion of germline Ig VH genes are inherently susceptible to amino-acid replacement. Immunol Today 1994, 15: Lossos IS, Tibshirani R, Narasimhan B, Levy R: The inference of antigen selection on Ig genes. J Immunol 2000, 165: Dunn-Walters DK, Spencer J: Strong intrinsic biases towards mutation and conservation of bases in human IgV H genes during somatic hypermutation prevent statistical analysis of antigen selection. Immunology 1998, 95: Corbett SJ, Tomlinson IM, Sonnhammer ELL, Buck D, Winter G: Sequence of the human immunoglobulin diversity (D) segment locus: a systematic analysis provides no evidence for the use of DIR segments, inverted D segments, minor D segments or D-D recombination. J Mol Biol 1997, 270: 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: Tierens A, Delabie J, Michiels L, Vandenberghe P, De Wolf-Peeters C: Marginal zone B cells in the lymph node and the spleen show somatic hypermutation and display clonal expansion. Blood 1999, 93: Amlot PL, Hayes AE: Impaired human antibody response to the thymus-independent antigen, DNP-Ficoll, after splenectomy. Implications for postsplenectomy infections. Lancet 1985, I: Martin F, Oliver AM, Kearney JF: Marginal zone and B1 B cells unite in the in the early response against T-independent blood-borne particulate antigens. Immunity 2001, 14: Zhu D, Thompsett AR, Bedu-Addo G, Stevenson FK, Bates I: VH gene sequences from a novel tropic splenic lymphoma reveal a naive B cell as the cell of origin. Br J Haematol 1999, 107: Bates I, Bedu-Addo G, Bevan DH, Rutherford TR: Use of immunoglobulin gene rearrangements to show clonal lymphoproliferation in hyper-reactive malarial splenomegaly. Lancet 1991, 337: Bates I, Bedu-Addo G, Rutherford TR, Bevan DH: Splenic lymphoma with villous lymphocytes in tropical West-Africa. Lancet 1992, 340: Kumar N, Zheng H: Evidence for epitope-specific thymus independent response against a repeat sequence in a protein antigen. Immunology 1998, 94: Bahler DW, Miklos JA, Swerdlow SH: Ongoing Ig gene hypermutation in salivary gland mucosa-associated lymphoid tissue-type lymphomas. Blood 1997, 89: Du M, Diss TC, Xu CC, Peng H, Isaacson PG, Pan L: Ongoing mutation in MALT lymphoma immunoglobulin gene suggests that antigen stimulation plays a role in the clonal expansion. Leukemia 1996, 10: Conconi F, Bertoni F, Pedrinis E, Motta T, Roggero E, Luminari S, Capella C, Bonato M, Cavalli F, Zucca E: Nodal marginal zone B-cell lymphomas may arise from different subsets of marginal zone B lymphocytes. Blood 2001, 98: Weller S, Faili A, Garcia C, Braun MC, LeDeist FF, de Saint Basile GG, Hermine O, Fischer A, Reynaud CA, Weill JF: CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans. Proc Natl Acad Sci USA 2001, 98: Facchetti F, Appiani C, Salvi L, Levy J, Notarangolo LD: Immunohistologic analysis of ineffective CD40-CD40 ligand interaction in lymphoid tissues from patients with X-linked immunodeficiency with hyper-igm. Abortive germinal center cell reaction and severe depletion of follicular dendritic cells. J Immunol 1995, 154: Korthauer U, Graf D, Mages HW, Briere F, Padayachee M, Malcolm S, Ugazio AG, Notarangelo LD, Levinsky RJ, Kroczek RA: Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-igm. Nature 1993, 361: Aruffo A, Farrington M, Hollenbaugh D, Li X, Milatovich A, Nonoyama S, Bajorath J, Grosmaire LS, Stenkamp R, Neubauer M: The CD40 ligand, gp 39, is defective in activated T cells from patients with X-linked hyper-igm syndrome. Cell 1993, 72: Allen RC, Armitage RJ, Conley ME, Rosenblatt H, Jenkins NA, Copeland NG, Bedell MA, Edelhoff S, Disteche CM, Simoneaux DK: CD40 ligand gene defects responsible for X-linked hyper-igm syndrome. Science 1993, 259: Schroeder HW, Dighiero D: The pathogenesis of chronic lymphocytic leukemia: analysis of the antibody repertoire. Immunol Today 1994, 15: Fais F, Ghiotto F, Hashimoto S, Sellers B, Valetto A, Schulman P, Vinciguerra VP, Rai K, Rassenti LZ, Kipps TJ, Dighiero G, Schroeder Jr HW, Ferrarini M, Chiorazzi N: Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J Clin Invest 1998, 102: Hardy RR, Hayakawa K: Developmental origins specificities and immunoglobulin gene biases of murine Ly-1 B cells. Intern Rev Immunol 1992, 8: Kearney JF: CD5 B-cell networks. Curr Opin Immunol 1993, 5: Chen X, Martin KA, Forbush KA, Perlmutter RM, Kearney JF: Evidence for selection of a population of multireactive B cells into the splenic marginal zone. Int Immunol 1997, 9: Pers JO, Jamin C, Predine-Hug P, Lydyard P, Yuoinou P: The role of CD5-expressing B cells in health and disease. Int J Mol Med 1999, 3: Gary-Gouy H, Schmitt P, Dalloul A, Daeron M, Bismuth G: The pseudo-immunoreceptor tyrosine-based activation motif of CD5 mediates its inhibitory action on B-cell receptor signaling. J Biol Chem 2000, 275: Simarro M, Calvo J, Vila JM, Places L, Padilla O, Alberola-Ila J, Vives J, Lozano F: Signaling through CD5 involves acidic sphingomyelinase, protein kinase C-zeta, mitogen-activated protein kinase, and c-jun NH2-terminal kinase. J Immunol 1999, 162: