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(American Journal of Pathology. 2003;162:681-689.)
© 2003 American Society for Investigative Pathology

Regular Articles

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

Anne Tierens*, Jan Delabie*, Agnieszka Malecka*, Junbai Wang{dagger}, Alicja Gruszka-Westwood{ddagger}, Daniel Catovsky{ddagger} and Estella Matutes{ddagger}

From the Departments of Pathology* and Tumor Biology,{dagger} The Norwegian Cancer Institute and Radiumhospital, University of Oslo, Oslo, Norway; and Academic Haematology and Cytogenetics,{ddagger} The Royal Marsden Hospital, London, United Kingdom


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
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. Two-thirds 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.

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.7-11 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 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.18-20 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
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
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 {kappa} and {lambda} 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 2x 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 2x 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, 2x SSC, 0.05 mol/L sodium phosphate buffer, pH 7.0). The slides were then quickly rinsed in 2x 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 1x SCC at 45°C, followed by three washes in 0.1x SSC at 60°C and one wash in 4x 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 4x 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 µL of the eluted mRNA were reverse-transcribed using Thermoscript reverse transcriptase and Oligo (dT) (Life Technologies, Inc., Grand Island, NY). Ten µL 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 µl of diethyl pyrocarbonate H2O containing 2 µmol/L of Oligo (dT)25 and 5 µl 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 µl 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 µL 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 µL of a mastermix (200 µmol/L dNTPs, 2.5 mmol/L MgCL2, 100 nmol/L of each primer in PCR buffer, 5 µl of cDNA) was heated at 94°C for 10 minutes before adding 1.5 U Taq polymerase diluted in 10 µl of dH2O. 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 µl of PCR product was size fractionated on a agarose gel 1.5% in 1x TBE buffer. The gel bands containing the monoclonal IgH products were excised and purified using the Concert PCR purification kit (Life Technologies, Inc.).

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 chi-square 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
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
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 19 x 109/L (range, 2.3 to 410) and all had a circulating clonal B-cell population as assessed by immunophenotyping. All patients presented with splenomegaly, but only a minority presented with lymphadenopathy and/or hepatomegaly.


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  		Table 1. Summary of the Clinical Features, Immunophenotype, and Genotype of the SMZL Cases

 
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 CLL6 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 numbers 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 AJ487485 to AJ487510. 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.


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  		Table 2. Sequence and Mutation Analysis of Ig VH Genes in SMZL

 
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 < 0.0001). The respective genealogical trees of the clonally related sequences are shown in Figure 1 .



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  		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.

 
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 {kappa} 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).


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  		Table 3. Disease Features and Ig Mutation Status

 
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
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
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 genes in a limited number of cases have revealed that most cases of SMZL presumably derive from a postgerminal center cell,18-20 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 VH 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.37-39 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.40-44 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 associated 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-cell-independent 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.50-52 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.


   Footnotes
 
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. E-mail: jan.delabie{at}labmed.uio.no

Supported by a grant from the Norwegian Cancer Society.

Accepted for publication November 6, 2002.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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