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(Investigative Ophthalmology and Visual Science. 2001;42:2450-2457.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Immunoglobulin Heavy Chain Gene Analysis of Ocular Adnexal Extranodal Marginal Zone B-Cell Lymphoma

Yoko Hara1,2, Naoya Nakamura1, Tetsuo Kuze1, Yuko Hashimoto1, Yoshikazu Sasaki1, Asumi Shirakawa1, Minoru Furuta2, Keiko Yago2, Keiichiro Kato2 and Masafumi Abe1

1 From the Departments of Pathology and 2 Ophthalmology, Fukushima Medical University School of Medicine, Fukushima-City, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To clarify the cellular origin of extranodal marginal-zone B-cell lymphoma (EZML) of the mucosa-associated lymphoid tissue (MALT) type in ocular adnexa, the somatic mutation was analyzed in the immunoglobulin heavy-chain variable region (VH) gene.

METHODS. Eight cases of EZML in the orbit and four in the conjunctiva were studied. The VH genes were amplified by a seminested PCR and sequenced directly. These were compared with the closest published VH germline segments to determine the somatic mutation frequency. Intraclonal microheterogeneity, which was termed the ongoing mutation frequency in the current study, was estimated by counting the number of single nucleotide substitutions in individual clones and dividing by the total number of nucleotides analyzed. Nine cases of gastrointestinal EMZL were also examined for comparison.

RESULTS. The somatic mutation frequency varied between 2.0% and 12.7%, with a mean value of 7.9%. Ten cases with intraclonal microheterogeneity showed between one and six further substitutions. The average of ongoing mutation frequency was 0.11%, with a range of 0% to 0.25%. In the gastrointestinal EMZLs, the average of somatic mutation frequency was 8.5% (1.5%–14.2%) and of ongoing mutation frequency was 0.51% (0.25%–0.75%).

CONCLUSIONS. The average of ongoing mutation frequency in ocular adnexal EMZL was lower than that in gastrointestinal EMZL. Both ocular adnexal and gastrointestinal EMZLs are derived from postgerminal center memory B cells, but the low ongoing mutation frequencies of ocular adnexal EMZL may result from less antigen stimulation and follicular colonization in the orbit relative to gastrointestinal EMZL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Extranodal marginal-zone B-cell lymphoma (EMZL) of the mucosa-associated lymphoid tissue (MALT) type lymphoma represents a distinct group of non-Hodgkin’s lymphomas, on the basis of histogenesis and biological activity.1 They are most commonly seen in the gastrointestinal tract,2 also appearing in the lungs, thyroid, salivary gland, thymus, and the ocular adnexa (the conjunctiva, eyelid, lacrimal gland, and orbit).1 EMZLs typically arise in sites normally devoid of lymphoid tissue, which have accumulated lymphoid tissue due to chronic inflammation or autoimmune disorders. Thus, EMZLs of the gastrointestinal tract are associated with Helicobacter pylori infection,3 whereas EMZLs of the thyroid gland and salivary gland are associated with Hashimoto’s thyroiditis4 and myoepithelial sialadenitis (characteristic of Sjögren syndrome),5 respectively.

In the ocular adnexa, however, it is not clear whether EMZLs are associated with chronic inflammation or autoimmune disorders such as Wegener granulomatosis, Mikulicz disease, and Sjögren syndrome. Normal conjunctiva and lacrimal glands contain occasional resident lymphocytes, and eyelids also have lymphatic drainage into local lymph nodes.6 The lymphatic tissue that appears in these sites is believed to be entirely MALT, acquired after antigen stimulation.7 It is likely that lymphomas may arise from reactive lymphoid tissue. The dependence of EMZL on specific antigen stimulation explains its tendency to remain localized in the mucosal sites.

EMZLs of MALT type are believed to arise from marginal-zone B cells, because of the morphologic and immunophenotypic similarity of the cell populations: both EMZL cells and normal marginal zone B-cells are CD5-, CD10-, and CD20+ and are usually IgM+, IgD-, and cyclin D1-.8 Marginal-zone B cells that express IgM appear to be enriched with early-memory B cells, the direct progeny of germinal center (GC) B cells.9 It has been shown that somatic hypermutation of immunoglobulin heavy-chain variable region (VH) genes occurs during B-cell differentiation. When coupled to antigen selection in the GCs, somatic mutation results in the production of antibodies of increased affinity.10 11 Somatic hypermutation appears to be restricted to the B-cells’ proliferating within the microenvironment of the GC. Thus, somatically mutated VH genes are a hallmark of GC B cells and their descendants.12 Mutations occur mainly in the framework regions (FWs) and complementarity-determining regions (CDRs) of the VH genes, which often show a marked accumulation of replacement (R) mutations in their CDRs. The CDR encodes the antigen-binding site13 and clustering of R mutations in the CDR is believed to improve antigen binding, thus contributing to antigen selection.14 Somatic hypermutation has been found to occur in the rearrangement of the VH genes of follicular lymphoma (FL),15 diffuse large B-cell lymphoma,16 multiple myeloma,17 and EMZL,18 but not in mantle cell lymphoma.19 Cases of B-cell chronic lymphocytic leukemia have been reported to include both germline20 and hypermutated configurations.21 Somatic mutations are found in normal IgM+ memory B cells of peripheral blood in the range of 0% to 10.0% (average, 2.0%), and in EMZLs of the MALT type in the range of 1.4% to 10.2% (average, 4.3%). Generally, the average somatic mutation frequency in EMZL is slightly higher than that of normal human B cells.12

Somatic hypermutation of VH genes has been found only in GC B cells22 23 24 ; therefore, gastric EMZLs that show somatic hypermutations without intraclonal variations are most likely derived from post-GC marginal-zone B cells.18 25 However, a recent study has shown intraclonal variations of VH genes in gastric EMZL.26 Ongoing mutation is known to exist in both FLs and EMZLs, which shows genetically that direct antigen stimulation plays an important role in the clonal expansion of these lymphomas.

Several studies have examined somatic mutation in the VH genes of EMZLs, including those in the stomach and ocular adnexa, but to our knowledge, there have not been any studies of intraclonal microheterogeneity in ocular adnexal EMZL.

In this study, we cloned and sequenced the VH genes expressed in 12 cases of ocular adnexal EMZLs, to define intraclonal microheterogeneity, and investigated whether direct antigen stimulation is involved in the development or growth of these tumors. We compared the frequency of somatic mutation and intraclonal microheterogeneity of VH genes of ocular adnexal EMZL to those in eight cases of gastric EMZLs and one of duodenal EMZL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and Samples
This study conformed to the tenets of the Declaration of Helsinki, and informed consent was obtained from the participants. Twenty-one cases of ocular adnexal EMZL that occurred from 1989 to 2000 were retrieved from the archives of the Department of Pathology of Fukushima Medical University. Of these, 12 cases were used in this study. The complete oligonucleotide sequences of the variable region (CDR1, FW2, CDR2, and FW3) and VDJ region (CDR3) were identified using PCR techniques. The principle clinical features of these patients are shown in Table 1 . Our samples consisted of eight women and four men, ranging in age from 32 to 83. Eleven primary lymphomas were unilateral and one (case 12) was bilateral. Eight tumors involved the orbit, three the conjunctiva, and one both conjunctiva. According to the Ann Arbor criteria, all patients considered had stage 1E tumors. Two patients had undergone surgical excision only, nine patients had received localized radiation therapy only, and one patient received chemotherapy. The follow-up periods ranged from 4 to 128 months: seven patients achieved a complete remission, two had a residual tumor, one had metastasis to lung, one died of other causes, and one had no reported follow-up.


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  		Table 1. Clinical Details of Cases of Ocular Adnexal EMZL

 
Records in nine cases of gastrointestinal EMZLs (eight in the stomach, one [case 4] in the duodenum) were taken from our files between 1988 and 1997 for this study. The clinical details of these patients are shown in Table 8 . Gastric EMZLs often involved the antrum, showing a flat infiltrative lesion with several ulcers. Duodenal EMZL (case 4) was present in the bulbus and consisted of numerous small polyps.


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  		Table 8. Clinical Details, Somatic Mutation, and Intraclonal Microheterogeneity Analysis in Gastrointestinal EMZL

 
Fresh, surgically resected materials were fixed with 20% formalin and embedded in paraffin for routine histologic examinations. Portions of the fresh materials were embedded in ornithine carbamyl transferase compound (OCT; Miles, Elkhart, IN), flash-frozen in liquid nitrogen, and stored at -80°C for immunohistochemical and genetic examinations.

All lymphomas were classified according to the Revised European-American Lymphoma (REAL)8 and World Health Organization (WHO) classifications.27 Immunohistochemical staining was performed on the paraffin-embedded sections and frozen sections according to a previously described streptavidin-biotin complex method.28 Monoclonal and polyclonal antibodies used in this study were as follows: Monoclonal IgG, IgA, IgM, and IgD and {kappa}, {lambda}, CD3 (Leu4), CD5 (leu1), CD10 (CALLA; Becton Dickinson, San Diego, CA), CD20 (Dako, Kyoto, Japan), CD21 (C3d fragment) for B cell, anti-bcl-2 (bcl protein; Dakopatts, Grostrup, Denmark), MIB-1 for proliferating cells in the G1, S, G2, and M phases (Immunotech SA, Marseilles, France; cyclin D1; MBL, Nagoyo, Japan).

PCR Amplification
PCR amplification of VH genes and direct sequencing of the PCR products were performed according to previously described methods.29 DNA samples obtained from flash-frozen samples were digested with proteinase K (PCR-grade; Roche Molecular Biochemicals, Indianapolis, IN), extracted with phenol-chloroform, and precipitated with ethanol.

Seminested PCR was performed with either of two systems (GeneAmp 9700 System; PE Applied Biosystems, Chiba, Japan; or a DNA Thermal Cycler; Perkin Elmer-Cetus, Norwalk, CT), as described previously.16 Briefly, the first amplifications were performed using an upstream consensus V region primer (FR1C and FR2A) and a low-stream primer joining (J) consensus V region primer (LJH). For reamplification, the LJH was replaced by a nested consensus J region primer (VLJH), and the first round amplification product was transferred and used as a template. We used both FR1C and FR2A for all 12 cases, and then sequenced products of both. This enabled us to confirm that clones were the same in 11 cases, but for case 7, we were unable to isolate a band by using FR1C. Primers (FR1C, FR2A, LJH, VLJH) used for seminested PCR amplification were as follows: 5'-AGGTGCAGCTG[G/C] [A/T]G[G/C]AGT C[G/A/T]G G-3' (FR1C) and 5'-TGG[A/G]TCCG[C/A]CAG[G/C]C[T/C] [T/C]C[A/C/G/T]GC-3' (FR2A), as upstream consensus V region primers; 5'-TGAGGAGACGGTGACC-3', as a consensus J region primer (LJH); 5'-GTGACCAGGGT[A/C/G/T]CCTTGGCCCCAG-3', as a consensus J region primer (VLJH). Ten microliters of each amplification product was separated by electrophoresis on a 2% agarose gel and visualized with ethidium bromide.

DNA Sequence Analysis of the VH Gene
DNA sequence analysis was performed with an automated sequencer (SQ5500; Hitachi, Tokyo, Japan), using the dideoxy-chain termination method with fluorescent dyes and a core sequencing kit (Thermo Sequenase; Amersham International, Plc., Little Chalfont, UK). The most homologous sequences were chosen for comparison with the published VH region germline sequence, using the BLASTIN and GenBank databases (provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD, and available at http://www.ncbi.nlm.nih.gov/blast/blast.cgi). Intraclonal diversity was examined as described previously.30 PCR products were ligated into the pCR 2.1 vector and transformed into TOP10F' cells as directed (original TA cloning kit, Invitrogen, Carlsbad, CA). The colony direct PCR assay was used to determine whether colonies included the correct PCR product. At least 10 colonies were chosen and put into 50 µL premix colony direct PCR kit (Insert Check Ready; Toyobo Co. Ltd., Osaka, Japan). The PCR protocol consisted of 30 cycles of 95°C for 20 seconds, 60°C for 5 seconds, and 72°C for 30 seconds. The presence of correct PCR products in 10 samples was confirmed by check electrophoresis in each case and then sequenced by using the same method.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pathologic Findings
All cases if ocular adnexal EMZL showed a diffuse infiltration of lymphoma cells with features of centrocyte-like cell or monocytoid B cells, and in three cases lymphoma cells infiltrated or surrounded atrophic reactive GCs. Several transformed blasts and plasma cells intermingled with lymphoma cells as well. In cases 1 and 6, plasmacytoid cells with Dutcher bodies were found. Follicular colonization and lymphoepithelial lesions (LELs) were not observed.

Gastrointestinal EMZLs were intensely infiltrated by lymphoid cells, predominantly in mucosa propria and submucosa. The proliferating lymphocytes were mainly small to intermediate in size. LEL were seen in all cases of gastrointestinal EMZLs.

Immunohistochemical Studies
The results of immunohistochemical studies are summarized in Table 2 . Lymphoma cells in all cases were positive for CD20and bcl-2, but were negative for CD3, CD5, CD10, CD21, IgD, and cyclin D1. Nine (75%) of the 12 cases expressed surface IgM. Distribution of follicular dendritic cells (FDCs) detected by an immunostaining of CD21 varied from case to case. A diffuse, loose network of FDCs in diffuse areas containing lymphoma cells were observed in three cases (2, 5, and 10), small networks in diffuse areas of lymphomas were observed in five cases (1, 3, 8, 11, and 12). FDCs in atrophic GC only were observed only in case 7.


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  		Table 2. Immunohistochemical Findings in Cases of Ocular Adnexal EMZL

 
PCR and Sequence Analysis
The 12 cases of ocular adnexal EMZL examined in the present study showed VH gene rearrangements and frequent somatic mutations of the rearranged VH genes. The number of mutations, total nucleotides, mutation frequencies (somatic mutation number/total nucleotides, expressed as a percentage) and VH gene family usage are shown in Tables 3 and 4 . All cases but one were found with an in-frame sequence, with one exception (case 5) that had an out-of-frame sequence with 2-bp deletions in CDR2 region (see Table 5 ). This case had two stop codons and surface Ig was not immunohistochemically detected. The average of somatic mutation frequency was 7.9% (ranging from 2.0% to 12.7%). In our study, the average of replacement mutation/silent mutation (R/S) ratio of the CDR1, FW2, CDR2, and FW3 regions were 2.6, 1.4, 1.6, and 1.9, respectively. The VH3 germline family was most frequently used in our series, in 75% of the cases. The other cases consisted of two cases with the VH1 germline family, and one case with the VH4 germline family.


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  		Table 3. Somatic Mutation Analysis in Ocular Adnexal EMZL

 

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  		Table 4. Summary of Somatic Mutation Analysis

 

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  		Table 5. Comparison of Nucleotide Sequence of VH Gene between Case 5 and the Closest Germline DP-31

 
Intraclonal microheterogeneity was analyzed in 11 cases. The nucleotide sequences of 12 cloned PCR products of the representative case 3 are shown in Table 6 . Further single-nucleotide substitutions in unmutated nucleotides were identified, and intraclonal microheterogeneity, also referred to as the ongoing mutation frequency, was estimated by dividing the number of these substitutions by the total nucleotides analyzed. For example, in case 3, four single-nucleotide substitutions and two oligonucleotides that changed from consensus sequence to another oligonucleotide were found; thus, the ongoing mutation rate was 0.25% (6 further mutations/204 nucleotides x 12 clones). The results for each patient are summarized in Table 7 . The average of ongoing mutation frequency was 0.11% (range, 0%–0.25%).


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  		Table 6. Intraclonal Microheterogeneity Analysis of Case 3

 

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  		Table 7. Intraclonal Microheterogeneity Analysis in Ocular Adnexal EMZL

 
Somatic mutation frequency and ongoing mutation frequency in gastrointestinal EMZLs are summarized in Table 8 . Somatic mutation and intraclonal microheterogeneity were analyzed in nine cases and five cases, respectively. The average of somatic mutation frequency was 8.5% (range, 1.5%–14.2%) and of ongoing mutation frequency was 0.51% (range, 0.25%–0.75%). The average of ongoing mutation frequency in gastrointestinal EMZLs was significantly greater than that in ocular adnexal EMZLs (P < 0.0001, Student’s t-test, Tables 7 8 and 9 ).


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  		Table 9. Comparison of Ocular Adnexal EMZL and Gastric EMZL: Somatic Mutation, Ongoing Mutation, and Intraclonal Microheterogeneity

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The cellular origin of human lymphomas has been studied by various approaches including histology, immunophenotyping, and molecular genetics. Recently, molecular studies of somatic mutations in VH genes suggested the cellular origin in some types of B-cell lymphomas.

In the present study, the first to report ongoing mutations (intraclonal microheterogeneity), we analyzed somatic mutations of VH genes in 12 cases of EMZL in the ocular adnexa. The average rate of somatic mutation was 7.9%, with a range from 2.0% to 12.7% (CDR1, FW2, CDR2, and FW3 in 11 cases, CDR2 and FW3 in 1 case). The number of mutations was comparable to that observed in VH genes in EMZLs in the other sites described previously: 8.5% in the gastrointestinal tract (in our series), 5.7% in the lung,31 and 4.7% in the salivary gland.32 The data were nearly consistent with that of ocular adnexal EMZL described in Couplaud et al.33 (8.2% in the ocular adnexa including primary and secondary lesion). These mutations showed much higher R/S ratios in the CDRs than in the FRs, the hallmark characteristic of B cells that have undergone positive antigen selection in the GC.

In addition to high mutation in the VH genes, ongoing mutation (intraclonal microheterogeneity) was observed in 10 of 11 cases, ranging from 0% to 0.25% (Table 7) . These findings suggest that most low-grade ocular adnexal EMZLs are still under the influence of the hypermutation mechanism. Somatic mutations and ongoing mutations are known to occur in GCs but not in post-GC B cells (i.e., memory B cells)34 ; however, recent studies have revealed that gastric EMZLs derived from post-GC B cells show ongoing mutations. The data suggest that these EMZLs respond to direct antigen stimulation through re-entry to GCs, as re-entry of normal B cells into GCs has been demonstrated with rat splenic marginal-zone cells (after antigenic stimulation).35

To evaluate a difference in intraclonal microheterogeneity, we compared the ongoing mutation frequency in ocular adnexal EMZL to that of gastric EMZL (Table 9) . The average frequency of ongoing mutation was lower in ocular adnexal EMZLs than in gastric EMZLs (0. 11% vs. 0. 51%). In ocular adnexal EMZL, the cases with and without loose networks of follicular dendritic cells (FDCs) or atrophic GCs showed average ongoing mutation frequencies of 0.12% and 0.08%, respectively (P = 0.5205, Student’s t-test; Tables 2 7 ) Ongoing mutation is linked to the GCs in the normal immune reaction. The GC of secondary lymphoid follicles provides a microenvironment for B cells to undergo clonal expansion and selection before differentiation into memory B cells. The GC reaction is initiated by rapid proliferation of few antigen-stimulated B cells in association with FDCs.36 FL has an intraclonal microheterogeneity resulting from the ongoing mutation, because FL tends to display a similar architecture (e.g., rich FDCs, T-cells) to the normal reactive follicles.37

In EMZLs, the lymphoma cells occasionally recirculate to reactive GCs, a process termed follicular colonization, and may show intraclonal microheterogeneity. Follicular colonization is frequently observed in gastrointestinal EMZL, but rarely in ocular adnexal EMZL. This may be one of the main reasons for the different ongoing mutation rate between gastrointestinal and ocular adnexal EMZLs. In ocular adnexal EMZL, ongoing mutation frequency in cases with FDC networks was relatively higher than that in cases without FDC networks; therefore, the difference in ongoing mutation frequency between them may reflect the differences in microenvironment. Our data suggest that ongoing mutations are associated with antigen-presenting cells such as FDCs. In other words, the quality and quantity of antigen stimulation through FDCs may affect the ongoing mutation frequency between ocular adnexal and gastrointestinal EMZLs.

It has been reported that, in some instances of ocular adnexal EMZL, some expressed VH genes were associated with autoantibodies: DP-8, DP-10 (also known as 51p1), DP-53, DP-63, and DP-49.33 Autoantibodies using the germline DP-53 and DP-49 have been described in cases of Alzheimer disease and rheumatoid arthritis, respectively. DP-63 has been associated with various diseases, including idiopathic cold agglutinin disease, systemic lupus erythematosus, and rheumatoid arthritis. Autoantibody association has also been observed in EMZLs arising in other sites.31 32 38 Among our series, the closest germline sequence of case 7 was DP-53, which suggests that the epitope possibly responsible for direct stimulation could be a self-antigen.

Recently, t(11;18)(q21;q21), that is API2-MALT1 fusion has been identified as a recurring cytogenetic abnormality in EMZL, particularly in those that arise in extranodal sites.39 After discovery of the API2-MALT1 fusion in EMZLs, nearly 100 cases of lymphoma were examined for this fusion, and one third of the cases were found to be positive by various methods.40 41 42 43 44 45 46 The API 2-MALT1 fusion was frequently found in EMZLs of the lungs (44.4% to ~100%), and stomach (12.5% to ~47.8%), in which EMZLs arise from chronic infectious inflammation, and were rarely detected in EMZLs of the thyroid and parotid glands associated with autoimmune disease. Ocular adnexal EMZLs with this novel gene have not been reported. It is likely that EMZLs involving the lacrimal gland are related to autoimmune disease. Although it has not been clear what kind of chronic antigenic stimulation, such as H. Pylori infection in gastrointestinal EMZLs, are associated with ocular adnexal EMZLs, we suggest the possibility that conjunctival EMZL arises from chronic inflammation. Epstein-Barr virus (EBV) was not detected in any cases of ocular adnexal EMZL by EBER1 RNA in situ hybridization (details not shown).

In summary, we analyzed VH genes in 12 cases of ocular adnexal EMZL. The somatic mutation frequency of ocular adnexal EMZL was comparable to that of EMZL in other sites, which suggests that ocular adnexal EMZL is derived from post-GC memory B cells. The differences in ongoing mutation between ocular adnexal and gastrointestinal EMZLs may result from the quality and quantity of antigen stimulation through FDCs in the primary sites.


   Footnotes
 
Submitted for publication December 19, 2000; revised June 11, 2001; accepted June 27, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Yoko Hara, Department of Pathology, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima-City, 960-1295, Japan. yhara{at}fmu.ac.jp


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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