{'fraction_non_alphanumeric': 0.03495377274082911, 'fraction_numerical': 0.023918878616164627, 'mean_word_length': 4.947499113160696, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
CDR3 Sequences of MALT Lymphoma Show Homology with Those of Autoreactive B‐Cell Lines

We have examined the CDR3 sequence and adjacent regions of immunoglobulin genes from B‐cell lymphoma of mucosa‐associated lymphoid tissue (MALT). Twenty‐nine sequences (15 sequences from 13 low‐grade MALT lymphomas, marginal zone B‐cell lymphomas; 7 sequences from 6 highgrade MALT lymphomas; 7 sequences from 7 diffuse large cell lymphomas) were obtained after cloning of the polymerase chain reaction‐amplified segments. In the low‐grade MALT, high‐grade MALT and diffuse large cell lymphomas, the mean length of the CDR3 region was 47.6 ± 10.31 (range 21 to 60), 38.71 ± 10.37 (range 27 to 57) and 40.86 ± 3.34 (range 39 to 48) nucleotides, respectively. The length of the CDR3 region was significantly greater in the low‐grade MALT lymphoma group than in the other two groups. CDR3 sequences in lymphoma cell clones of 14 cases showed 60 to 81% homology with autoantibody‐associated lymphocyte clones including rheumatoid factor. The incidences of these autoantibody‐associated lymphocyte clones were higher in the high‐grade MALT (4/6) and diffuse large lymphomas (5/7) than in the low‐grade MALT lymphoma (5/13). Cases with more than 70% homology at the nucleotide level were found to have 71 to 82% homology with autoantibodies at the protein level in the low‐grade MALT lymphomas (2/13), and 67% homology in the high‐grade MALT lymphomas (2/7). These results indicate that MALT lymphomas may be derived from the malignant transformation of autoreactive B‐cells.

mas occur in extranodal organs such as the stomach, salivary gland, thyroid and lung. 1,2) The organization of MALT is acquired as a result of chronic inflammation caused by factors such as Helicobacter pylori (H. pylori) infection, chronic sialadenitis of Sjögren syndrome, and Hashimoto's thyroiditis. MALT lymphomas are considered to arise from these backgrounds. [3][4][5] In comparison with equivalent nodal B-cell lymphomas, the clinical behavior of MALT lymphoma is one of slow dissemination and the prognosis is more favorable. The participation of autoantigen in MALT lymphomagenesis has been suggested. The immunoglobulin derived from gastric MALT lymphoma cells was shown to be specifically responsive to autotissues. 6,7) Moreover, it was demonstrated that proliferation of low-grade gastric MALT lymphoma cells is indirectly stimulated by H. pylori via specific tumor-infiltrating T cells. 8,9) Low-grade gastric MALT lymphoma regresses after the eradication of H. pylori. 10) Thus antigen stimulation may play a role in the pathogenesis and development of MALT lymphomas. 11) B-Cell lymphomas occurring in certain organs can be classified into low-grade and high-grade MALT lymphomas, and non-MALT high-grade lymphomas. However, it frequently becomes problematic whether a high-grade lymphoma is of MALT type or not, because the characteristic features of low-grade MALT lymphoma, such as the presence of reactive follicles and lymphoepithelial lesions, are readily lost with high-grade transformation. Therefore, analysis of the antigen binding sites of these lymphomas may give clues to the nature of the immune response.
Immunoglobulin heavy chain (IgH) gene has three hypervariable regions, so-called complementarity determining regions (CDR1, 2 and 3). CDR3 directly contacts the antigen and is the most variable region of the Ig molecule. Antigen specificity is highly dependent on the composition of CDR3. CDR3 changes its structure in accordance with the antigen. The nucleotide sequence of CDR3 is developmentally regulated, with its length showing a tendency to increase as the antigenic stimuli increase with the maturation of the individuals. 12,13) The increase of diversity of IgH gene in response to various antigens is generated through the recombination of multiple VH, D and JH segments, increased use of the longer DH and JH gene segments, increased number of D-D fusions, and additions to the N region. Owing to this diversity, the CDR3 region is unique in each rearrangement. Analysis of the CDR3 sequence, therefore, is a useful procedure for studying the clonality of malignant cells and obtaining information about the binding antigen.
The aim of the present study was to determine the CDR3 nucleotide sequence of MALT lymphoma cell clones and to evaluate the properties of their antigen bind-ing sites, and, moreover, to understand the origin and the role of antigen in the clonal selection of the lymphoma cells on the basis of its primary structure.

MATERIALS AND METHODS
Pathologic samples Specimens were obtained from patients who were clinically diagnosed as having primary B-cell lymphoma and who underwent tumor resection. Transbronchial lung biopsy (TBLB) specimens from two patients with chronic bronchitis served as reactive controls, because bronchial mucosa is exposed to various antigens. A portion of the specimen was snap-frozen in liquid nitrogen for immunohistochemical analysis. Histopathologic studies and immunohistochemical analysis Routinely processed formalin-fixed, paraffinembedded samples were sectioned at 3 µm and stained with hematoxylin-eosin. Classification of non-Hodgkin's lymphomas was based on the MALToma 14,15) and REAL classifications. 16) High-grade lymphomas were classified according to the criteria of Hsi et al. 17) The immunological phenotype of each lymphoma was determined by immunohistochemical methods using the standard avidin-biotin complex technique, and the results were considered in conjunction with those of the histopathologic studies. The following antibodies were used: CD19 (B4, Coulter Immunology, Hialeah, FL), CD20 (B1, Coulter Immunology; L26, Dakopatts, Glostrup, Denmark), CD21 (B2, Coulter Immunology), CDw75 (LN-1, Nichirei, Tokyo), CD3 (Leu4, Becton Dickinson, Mountain View, CA), CD4 (Leu3a, Becton Dickinson), CD5 (Leu1, Becton Dickinson), CD8 (Leu2a, Becton Dickinson), CD43 (MT-1, Bio-Science, Emmenbrücke, Switzerland) and CD45RO (UCHL-1, Dakopatts). DNA preparation DNA extraction and microdissection from surgically resected tissues were performed according to the previously described methods. 18) A part of the frozen specimen was also homogenized mechanically and centrifuged at 35,000 rpm for 18 h at 20°C by the guanidinium/cesium chloride method. Isolated genomic DNA was incubated for 2 h at 50°C with 200 µg/ml proteinase K and 0.5% sodium dodecyl sulfate, extracted with phenol/chloroform, and subsequently dialyzed. Polymerase chain reaction (PCR) for IgH genes IgH gene from paraffin-embedded or frozen samples was amplified according to the two-step PCR method of Wan et al. 19) The primers used were: 5′ ACACGGC[C/T][G/ C]TGTATTACTGT 3′ (Fr3A), 5′ TGAGGAGACGGTGA-CC 3′ (LJH), and 5′ GTGACCAGGGT[A/G/C/T]CCTT-GGCCCCAG 3′ (VLJH). Fr3A is the oligomer for the third framework portion of the V region. LJH and VLJH are based on the consensus sequence from the J region. For the first step of amplification, 1 µl of extracted DNA was subjected to PCR amplification with 2.5 units of Taq polymerase and with the primers for Fr3A and LJH at a final concentration of 0.25 µM in 100 µl of standard buffer. Each PCR experiment contained a sample without the DNA template as a negative control, and a sample with DNA extracted from Burkitt lymphoma (lymph node) as a positive control in which IgH gene rearrangement had been detected by Southern blotting. Denaturing was carried out for 2 min at 94°C, annealing for 2 min at 60°C, and extension for 2 min at 72°C for 30 cycles. The second step of 20 cycles with Fr3A and VLJH was performed with 10 µl of a 1/1000 dilution of the first step PCR product as template. The PCR product (40 µl) was extracted with phenol/chloroform and precipitated with ethanol. The precipitate was then dissolved in 5 µl of Tris-EDTA (TE) buffer and electrophoresed on 2% agarose gel, and the gel was stained with ethidium bromide to visualize the DNA under a short-wavelength UV light. The expected size of the amplified products was about 100 bp. Sequence analysis The PCR product was ligated to the PCR TM vector and the ligation mixture was transformed into One Shot TM competent cells by using a TA Cloning Kit (Invitrogen Corp., San Diego, CA). The subcloned DNAs were picked up at random and DNA was purified. Sequencing was performed using a Taq Dye Primer Cycle Sequencing Core Kit (Applied Biosystems, Foster City, CA).
The character of the CDR3 region was identified by comparison of the sequences with those in the GenBank and EMBL databases using the FASTA program. For D genes, minimal homology consisted of six matches in a row or seven matches interrupted by one mismatch. 20) Homology search to published B-cell clones was done both at nucleotide and protein levels. Statistical analysis Statistical analysis of differences between low-grade MALT and high-grade MALT or diffuse large lymphoma groups was performed using the onetailed Student's t test.

DISCUSSION
MALT lymphomas often occur in association with autoimmune disorders, and are believed to produce autoreactive immunoglobulin, as shown in gastric MALT lymphoma. This specific nature of MALT lymphoma cells must be reflected in the properties of CDR3 in IgH gene. So far, VH analyses suggest that low-grade MALT lymphomas are derived from post-germinal center B cells, 34) as well as a large proportion of diffuse large cell lymphomas. Therefore, precise analysis of the CDR3 sequence derived from each lymphoma clone may shed some light on the relation of MALT lymphomas and "de novo" large B-cell lymphomas occurring in mucosal organs.
As the same nucleotide sequence of the IgH CDR3 region is shared by only one in 20,000 circulating B cells, 35) it is a useful marker for determining the clonality of the malignant clone in lymphomas. The sequencing of the CDR3 regions revealed a single dominant clone in 23 cases, and two major sequences in the other 3 cases (6, 19 and 24) (Table I). In cases 6, 19 and 24, either these two major sequences were derived from different cells, or the IgH gene of both alleles from the same cell was expressed. Concerning the latter possibility, there is recent evidence that IgH allelic exclusion is not absolute and that a subset of B-CLL expresses more than one functional Ig heavy chain. 36) The D region of the sequences in all cases had significant homology with one of the presently known germline D genes. Among them, eight cases used the DXP′1 gene. The DXP group gene is reported to be the most frequently used D segment both in fetal liver lymphocytes 13) and in adult peripheral B cells. 35) DHQ52, which is preferentially used at the fetal stage, 37) was observed in one case each of the high-grade MALT and diffuse large cell lymphoma groups, but in none of the low-grade MALT group. Ten of the 26 cases used two germline D genes, possibly by D-D fusion. This D-D fusion was frequently observed in lowgrade MALT, especially in pulmonary MALT lymphoma (5 of the 8 cases). Immunoglobulin D-D fusion seemed to be central to the generation of antibody diversity. Although lymphocytes in both organs are likewise exposed to various antigens, the present study indicates that the diversity of IgH gene is higher in the lung than in the stomach.
The nucleotide sequence of CDR3 is developmentally regulated. The N sequence is found in 68% of fetal B cells, 86% of neonatal B cells, and 91 to 100% of mature adult B cells. 12) The average length of the N-D-N region in the fetal liver lymphocytes, neonatal and adult peripheral B cells is 22 (range 12 to 45), 24 (range 9 to 56) and 31 (range 13 to 54) nucleotides, respectively, showing a tendency to increase with age. 13) Therefore, the presence of the N sequence and the length of CDR3 can act as indicators of immunological selections on B-cell maturation. As the present study shows, the length of the CDR3 region was significantly greater in the low-grade MALT lymphoma group than in the other two groups. In the lowgrade MALT, high-grade MALT and diffuse large cell lymphomas, the mean length of N-D-N was 30.2±8.84 (range 7 to 40), 21.71±7.99 (range 7 to 30) and 24.86± 4.10 (range 19 to 32) nucleotides, respectively. Together with the usage of DHQ52, these findings indicate that the properties of CDR3 in high-grade MALT lymphoma resemble those in diffuse large cell lymphoma rather than in low-grade MALT lymphoma. The maturation stage of low-grade MALT lymphoma is similar to ontogenetic mature B cells. Diffuse large cell lymphoma does not show a distinct difference from high-grade MALT lymphoma and therefore is at an ontogenetic early maturation stage.
The differentiating B cell undergoes positive selection directed by the presence of surface IgH with low affinity for self-antigen. Thus, B cells that express self-reactive receptors mature into functional B cells before entering the germinal center. In the germinal center of the lymphoid follicle, IgH gene causes somatic mutation for an adaptive response to foreign antigen. The B cells whose antigen receptors are bound to this antigen selectively proliferate under the influence of helper T cells. 12,38) Positive ligand selection by low-affinity interaction with self antigen is also considered to shape the primary repertoire, which includes natural autoantibodies. Thus, the formation of some repertoires is performed by antigen-driven selection. Accumulating evidence has indicated that usage of V gene segment is preferential in some repertoires. [39][40][41][42][43] Although VH segment usage may be influenced by the ability of VH segments to facilitate certain ligand interactions, CDR3 is likely to play a major role in specific ligand interaction and repertoire development. 44) The specificity of antibody to antigen is highly dependent on the composition of CDR3. Therefore, similarities in the nucleotide sequence of the CDR3 region may be useful for detecting the origin of lymphoma cells and for determining whether a specific antigen participates in the clonal proliferation of lymphoma cells. Accordingly, we investigated the particular properties of the CDR3 region derived from tumor cell clones. The lymphoma cell clones of 14 cases showed 60 to 81% homology with autoantibodyassociated lymphocyte clones, which include natural autoantibodies, anti-DNA antibodies, Hashimoto thyroiditis-related antibody and rheumatoid factor. 21,22,24,25,27,28,[31][32][33] The incidences of these autoantibody-associated lymphocyte clones were higher in the high-grade MALT (4/ 6, 67%) and diffuse large cell lymphomas (5/7, 71%) than in low-grade MALT lymphoma (5/13, 39%). We also investigated the sequences of VH segment in 9 gastric lymphomas. Seven cases (2 from 3 low-grade MALT lym-phomas; 2 from 3 high-grade MALT lymphomas; 3 from 3 diffuse large lymphomas) showed 85 to 98.3% homology with those of autoantibodies (data not shown). There are 4 cases in which the deduced amino acid sequences of CDR3 in low-and high-grade MALT lymphomas show a high degree (67 to 82%) of homology with natural autoantibody and rheumatoid factor. Some authors have suggested that the specificity of autoantibodies can be influenced not only by the structure of heavy chain CDR3, but also by mutation of that of CDR2 and light chain CDR1, and furthermore, reactivity of autoantibodies can be changed by several amino acid substitutions in IgH CDR3. 45,46) It seems highly significant that our lymphoma cells showed homology with CDR3 of a known autoantibody clone. Therefore, our findings suggest that the cells of MALT lymphoma are autoantigen-related, and that some diffuse large cell lymphomas may also be derived from selected autoreactive B-cell clones, even in the absence of histological evidence of MALT lymphoma.
Concerning the histogenesis, low-grade MALT lymphomas are considered to arise from acquired lymphoid tissues formed in response to insults such as H. pylori infection. Moreover, H. pylori-associated MALT lymphomas have been shown to result from both polyclonal and monoclonal B-cell proliferations. 47) High-grade MALT lymphoma has been considered to arise from transformed low-grade MALT lymphoma cells. Indeed, the findings that the range of length of CDR3 showed a narrowing tendency with high-grade malignant transformation and that the incidence of autoantibody-associated lymphocyte clones was higher in high-grade lymphomas than in lowgrade MALT lymphoma support the hypothesis that highgrade lymphoma B cells are derived from cells highly selected by autoantigen, leading to a monoclonal proliferation of B cells, within the milieu of polyclonal MALT lymphoma cells. Thus, high-grade lymphoma cells may have acquired immaturity of differentiation and the ability to destroy auto-tissues.