Somatic hypermutation in low-grade mucosa-associated lymphoid tissue- type B-cell lymphoma

The origin of low-grade mucosa-associated lymphoid tissue (MALT)-type B- cell lymphoma is still unclear. Using a novel two-step procedure, we have sequenced the Ig VH genes expressed by cells from four patients with gastric low-grade MALT-type lymphoma. The nucleotide sequences of the complementarity determining region 3 (CDR3) of the genomic DNA were first amplified using consensus oligonucleotide primers, then sequenced. Based on the CDR3 sequence amplified from each MALT lymphoma, individual tumor-specific primers were synthesized and used directly in the polymerase chain reaction (PCR) to analyze the sequences of their Ig heavy-chain variable region. When compared with the germ-line sequence, many nucleotide substitutions, mainly in the CDRs, were found in the variable gene sequences of the four MALT lymphomas. The mutations showed a high replacement-to-silent ratio and were distributed in a way which suggested that the tumor cells had been positively selected through their antigen receptor. Our findings indicate that the MALT-type lymphoma B cells are hypermutated postgerminal center lymphocytes that have undergone antigen selection.     

Salivary gland mucosa-associated lymphoid tissue lymphoma immunoglobulin V(H) genes show frequent use of V1-69 with distinctive CDR3 features

Salivary gland mucosa associated lymphoid tissue (MALT) type lymphomas are B-cell neoplasms that develop out of a reactive infiltrate, often associated with Sj?gren's syndrome. Previous reports from our laboratory involving 10 patients suggested these lymphomas expressed a restricted immunoglobulin (Ig) V(H) gene repertoire with over use of V1-69 gene segments. To better determine the frequency of V1-69 use and whether there may also be selection for CDR3 structures, we sequenced the V(H) genes from 15 additional cases. Over half of the potentially functional V(H) genes (8 of 14) used a V(H)1 family V1-69 gene segment, whereas the other cases used different gene segments from the V(H)1 (V1-46), V(H)3 (V3-7, V3-11, V3-30.3, V3-30.5), and V(H)4 (V4-39) families. The 8 V1-69 V(H) genes used 5 different D segments in various reading frames, but all used a J4 joining segment. The V1-69 CDR3s showed remarkable similarities in lengths (12-14 amino acids) and stretches of 2 to 3 amino acids between the V-D and D-J junctions. They did not resemble CDR3s typical of V1-69 chronic lymphocytic leukemias. This study extends our earlier work in establishing that salivary gland MALT lymphomas represent a highly selected B-cell population. Frequent use of V1-69 appears to differ from MALT lymphomas that develop at other sites. The high degree of CDR3 similarity among the V1-69 cases suggests that different salivary gland lymphomas may bind similar, if not identical epitopes. Although the antigen specificities are presently unknown, similar characteristic CDR3 sequences are often seen with V1-69 encoded antibodies that have anti-IgG or rheumatoid factor activity. (Blood. 2000;95:3878-3884)     

Synthesis of Rh Fv phage-antibodies using VH and VL germline genes

Antibodies to the D antigen of the Rh system use a restricted set of immunoglobulin V and J gene segments, especially VH DP50 and DP63, JH6, Vlambda DPL16 and Jlambda 2/3. These gene segments may confer a natural affinity on the antibodies for the D antigen and this hypothesis has been tested by constructing two single-chain Fv phage-antibody libraries based on the germline gene segments DP50 and DP63; structural variability was obtained by insertion of 11 amino acids in random sequence in the VHCDR3. 10 anti-D antibodies were selected from these libraries, each with a unique VHCDR3. In contrast, selections with the CcEe antigens produced antibodies reacting with the Rh polypeptide molecules but without strict blood group specificity. One of these latter DP50-based antibodies was converted into 12 different antibodies with specificity for E by replacing the original germline light chain with chains from a rearranged L chain library. The CDR1 and CDR2 sequences of the DP50-based antibodies were common to both anti-D and anti-E molecules; differentiation between D and E specificity was dependent on VHCDR3 sequences and their correct pairing with an appropriate L chain.     

t(14;18) chromosomal translocation in follicular lymphoma: an event occurring with almost equal frequency both at the D to JH and at later stages in the rearrangement process of the immunoglobulin heavy chain gene locus

bcl-2/IgH fusion is considered a genetic error which occurs at the diversity (D) to joining (J_H) stage of the gene rearrangement process in the immunoglobulin heavy chain (IgH) gene locus. Translocations of the bcl-2 protooncogene to the IgH locus at ontogenetically later IgH gene rearrangements are thought to represent exceptions. In the present study we analysed the junctional nucleotide sequence of 18 bcl-2/IgH fusion genes identifiable by polymerase chain reaction performed on DNA extracted from diagnostic lymph node tissue of 14 follicular lymphoma patients. In all clones studied, segments of variable length were found interposed between bcl-2 and J_H gene sequences. Nucleotide sequence data analysis and comparisons performed with the corresponding germline sequences using the GenBank/EMBL database revealed the presence of D segments in most of the bcl-2/IgH fusion genes under study (13/18). By the same kind of computer-aided analysis, previously unrecognized D segments were identified in many published junctional sequences. These results suggest that bcl-2/IgH fusion events are very prevalent in rather more differentiated stages in B-cell ontogeny than previously recognized.     

Concurrent Gastric and Pulmonary Mucosa-Associated Lymphoid Tissue Lymphomas with Pre-Existing Intrinsic Chronic Inflammation: A Case Report and a Review of the Literature

Herein, we report a rare case of concurrent gastric and pulmonary mucosa-associated lymphoid tissue (MALT) lymphomas. A 65-year-old man who had been diagnosed with Helicobacter pylori-positive gastric MALT lymphoma received eradication therapy and achieved complete remission. During follow-up, he developed de novo pulmonary MALT lymphoma as a sequela of pulmonary tuberculosis, accompanied by recurrent gastric MALT lymphoma. Polymerase chain reaction (PCR) products of the CDR3 region of the immunoglobulin heavy chain gene showed an overall polyclonal pattern with bands at 400 base pairs (bp) and 200 bp predominant in the pulmonary tissue, as well as two distinctive bands in the gastric tissue at 400 bp and 200 bp. This case suggests that multiorgan lymphomas are more likely to be independent from each other when they are far apart, involve different organ systems, and have independent precipitating factors.     

Impaired T-cell regulation of B-cell growth in Helicobacter pylori--related gastric low-grade MALT lymphoma.

BACKGROUND & AIMS: Neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma are responsive to T helper cells and sensitive to withdrawal of H. pylori-induced T-cell help. METHODS: The clonal progeny of T cells from the gastric mucosa of 5 patients with MALT lymphoma was compared with that of T-cell clones obtained from 5 H. pylori-infected patients with chronic gastritis. RESULTS: T-cell clones were assessed for specificity to H. pylori, cytokine profile, help for B-cell proliferation, and perforin- or Fas-mediated cytotoxic regulation of B-cell growth. Twenty-eight of 165 CD4(+) gastric clones from MALT lymphoma and 33 of 178 CD4(+) clones from chronic gastritis recognized H. pylori antigens. Cytokine production was similar in the 2 series of clones. All MALT lymphoma-derived clones dose-dependently increased their B-cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1 because of concomitant cytolytic killing of B cells. T-cell clones from MALT lymphoma had both reduced perforin-mediated cytotoxicity and poor ability to induce Fas-mediated apoptosis. These defects were limited to gastric T cells. CONCLUSIONS: H. pylori-induced T cell-dependent B-cell activation and deficient cytotoxic control of B-cell growth may link H. pylori infection, local T-cell response, and genesis of low-grade gastric MALT lymphoma.     

Gastric relapse of stage I ocular adnexal mucosa-associated lymphoid tissue lymphoma

A 57-year-old male with right ocular adnexal mucosa-associated lymphoid tissue lymphoma (MALT lymphoma) was successfully treated with local radiation therapy. The gastroendoscopic examination revealed a slight inflammatory change of the gastric mucosa, and the urease test was positive. Eradication therapy against Helicobacter pylori was successfully done, however, the patient developed gastric MALT lymphoma two years after the initial treatment. Southern blot analysis of the immunoglobulin heavy chain gene rearrangement revealed that the lymphoma cells from the ocular adnexal and gastric MALT lymphomas were identical, indicating that the gastric MALT lymphoma was not the primary but the metastatic region from the ocular adnexal MALT lymphoma. Further, immunohistochemical staining demonstrated the expression of integrin alpha4beta 7 on ocular adnexal MALT lymphoma cells, which is essential for the adhesion of lymphocytes to gastrointestinal mucosa. These results indicate that ocular adnexal MALT lymphoma cells can metastasize to the stomach, depending on the adhesional function of integrin alpha4 beta7.     

Utilization of Ig heavy chain variable, diversity, and joining gene segments in children with B-lineage acute lymphoblastic leukemia: implications for the mechanisms of VDJ recombination and for pathogenesis

Sequence analysis of the immunoglobulin heavy chain genes (IgH) has demonstrated preferential usage of specific variable (V), diversity (D), and joining (J) genes at different stages of B-cell development and in B-cell malignancies, and this has provided insight into B-cell maturation and selection. Knowledge of the association between rearrangement patterns based on updated databases and clinical characteristics of pediatric acute lymphoblastic leukemia (ALL) is limited. We analyzed 381 IgH sequences identified at presentation in 317 children with B-lineage ALL and assessed the V(H)D(H)J(H) gene utilization profiles. The D(H)J(H)-proximal V(H) segments and the D(H)2 gene family were significantly overrepresented. Only 21% of V(H)-J(H) joinings were potentially productive, a finding associated with a trend toward an increased risk of relapse. These results suggest that physical location at the V(H) locus is involved in preferential usage of D(H)J(H)-proximal V(H) segments whereas D(H) and J(H) segment usage is governed by position-independent molecular mechanisms. Molecular pathophysiology appears relevant to clinical outcome in patients who have only productive rearrangements, and specific rearrangement patterns are associated with differences in the tumor biology of childhood ALL.     

Intestinal dissemination of gastric mucosa-associated lymphoid tissue lymphoma

Despite increasing identification of concurrent gastric and intestinal lymphomas of mucosa-associated lymphoid tissue (MALT), the clonal relationship between the two tumors and their sequential development are poorly understood. It is also unknown whether the development of these concurrent tumors is closely associated with direct antigen stimulation, which is thought to play an important role in the clonal expansion of low-grade MALT lymphomas. To investigate these, we have studied six cases of concurrent gastric and intestinal MALT lymphomas by polymerase chain reaction (PCR) amplification, cloning, and sequencing of the rearranged Ig gene, a strategy that has been widely used for analysis of clonality and antigen-driven properties of B-cell malignancies. In each case, an identical or nearly identical complementarity determining region (CDR) 3 sequence was observed between the dominant clones of concurrent gastric and intestinal MALT lymphomas. In four of six cases examined, sufficient Ig variable region sequence information was obtained to permit analysis of somatic mutations. The mutation patterns in one case suggest that the intestinal lesion is secondary to the gastric tumor, and the mutation patterns in two cases indicate that the gastric and intestinal lesions are derived from different tumour subclones, which emerge after expansion of a common early tumor clone. Furthermore, three of four cases showed ongoing Ig mutations among different PCR clones at each site. These results show that concurrent gastric and intestinal MALT lymphomas are derived from the same clone and suggest that the intestinal lesions result from dissemination of gastric tumours. Antigen stimulation may play a role in tumor evolution, particularly at an early stage.     

Analysis of immunoglobulin V genes suggests cutaneous marginal zone B-cell lymphomas recognise similar antigens

Extranodal marginal zone B-cell lymphomas (EMZL) are thought to develop from reactive infiltrates that represent immune responses to external or auto-antigens. Except for gastric EMZL, the antigenic triggers of EMZL development are mostly unknown, although a subset of cutaneous EMZL have been associated with Borrelia burgdorferi infections. To further evaluate whether a common antigen may be promoting the development of cutaneous EMZL, the immunoglobulin heavy chain variable (V(H)) genes from eight USA cases were sequenced and analysed. All used V(H)3 family gene segments, with 2/8 using the same V3-30 segment, 2/8 using the closely related V3-30.3 or V3-33 segments, 6/8 containing mutations and 2/7 showing evidence of ongoing mutation. Many of the complimentarity-determining region 3s (CDR3s) also showed similarities in length and displayed conserved amino acid motifs in the non-templated areas between the diversity and joining segments. The use of similar V(H) gene segments and conserved CDR3 amino acid motifs suggests that some of these cutaneous EMZL may bind the same or similar antigen via their surface immunoglobulin receptor. Analysis of the somatic mutations present in many of the V(H) genes was also consistent with antigen directly stimulating the growth of cutaneous EMZL.     

Features of the overexpressed V1-69 genes in the unmutated subset of chronic lymphocytic leukemia are distinct from those in the healthy elderly repertoire

Chronic lymphocytic leukemia (CLL) comprises 2 subsets, distinguished by expression of unmutated or mutated V(H) genes, with the former having a worse prognosis. Biased usage of the V1-69 gene is found in unmutated cases and is combined with selected D gene segments and J(H)6. It is controversial whether this is a CLL-associated feature or mirrors the normal B-cell pattern. Since CLL is a disease of the elderly, where changes in the B-cell repertoire may occur, we have analyzed V1-69 usage in the elderly (older than 75 years) population. Using monoclonal antibody (MoAb) G6, specific for 51p1-related V1-69 alleles, we found no increased expression with age. In 51p1-encoded immunoglobulin M (IgM), complementarity-determining region 3 (CDR3) length and frequency of D and J(H) genes were similar to those in the healthy young and distinct from those in CLL. These findings support the concept that CLL arises from B cells driven by antigen/superantigen and is not a stochastic event in the elderly B-cell population.     

Immunological and molecular analysis of B lymphocytes in low-grade MALT lymphoma of the stomach. Are there any useful markers for predicting outcome after Helicobacter pylori eradication?

Background: Background: Although Helicobacter pylori eradication is effective in treating low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma, the condition in some patients deteriorates even after the eradication. Therefore, it is important to predict the disease outcome before starting H. pylori eradication. We investigated the usefulness of flow cytometry, quantifying CD19- and CD20-positive B lymphocytes in MALT lymphoma tissue, for predicting the disease outcome after H. pylori eradication. Methods: Tissue specimens from 14 patients with H. pylori-positive low-grade gastric MALT lymphoma were examined by histology, Southern blotting, and flow cytometry before therapy. Serum levels of soluble interleukin (IL)-2 receptor were also measured. The relationship between the data and the prognosis after H. pylori eradication was analyzed. Results: Remission occurred in 10 of the 14 patients. The condition in the 4 remaining patients deteriorated even after H. pylori eradication. The percentages of CD19- and CD20-positive cells in MALT lymphoma tissue from the patients in remission were both significantly lower than those in the tissue from patients not in remission. Indeed, 4 of the 5 patients in whom both CD19- and CD20-positive cells accounted for more than 50% of the total number of lymphocytes had gastrectomy, whereas all patients in whom both CD19- and CD20-positive cells accounted for less than 50% of the total number of lymphocytes achieved remission. Although immunoglobulin gene rearrangement was present in all patients operated on, there were also 6 patients whose MALT lymphoma was ameliorated in spite of the presence of gene rearrangement. The serum level of soluble IL-2 receptor was in the normal range in all patients tested. Conclusions: Analysis of mature B-cell markers in MALT lymphoma tissue is more useful than the examination of immunoglobulin gene rearrangement or serum levels of soluble IL-2 receptor in predicting the outcome of low-grade gastric MALT lymphoma after H. pylori eradication. Received: January 5, 2001 / Accepted: November 2, 2001     

Restricted V(H) gene usage in lamina propria B cells producing anticolon antibody from patients with ulcerative colitis.

BACKGROUND AND AIMS: Autoimmune responses against colonic epithelium may play a role in the development of colonic inflammation associated with ulcerative colitis (UC). In this study, we established and characterized B-cell lines and clones that produced anticolon antibody from inflamed colonic mucosa of UC subjects. METHODS: B-cell lines were generated through Epstein-Barr virus transformation of lamina propria lymphocytes (LPLs) from colonic mucosa and peripheral blood lymphocytes, and these lines were screened for the production of anticolon antibodies. B-cell lines were then cloned by limiting dilution culture, and messenger RNA expression of immunoglobulin heavy-chain variable region (V(H)) was assessed. RESULTS: V(H) gene families used in B-cell lines established from LPLs of normal controls were diverse, and B-cell lines from UC LPLs expressed a restricted V(H)3 family usage. All 15 clones from UC used a restricted V(H)3 gene family, whereas diverse V(H) gene families were used by 24 clones from normal controls. The analysis of nucleotide sequences indicated that these clones were derived from various germline gene segments. CONCLUSIONS: The restricted V(H) gene usage in anticolon autoantibodies producing B-cell clones suggests that a particular antigenic stimulus contributes to the pathogenesis of UC.     

T-cell receptor delta/alpha rearrangements in lymphoid neoplasms

Rearrangements within the T-cell receptor (TCR)delta/alpha locus were analyzed in a wide variety of lymphoid neoplasms by eight DNA probes specific for TCR J delta, J alpha and C alpha segments. In all 11 T- cell malignancies, rearrangement and/or deletion of TCR delta was detected irrespective of the stage of maturation of the tumor. The organization of TCR delta correlated with the phenotype of the tumor: In "prethymic" T-cell acute lymphocytic leukemia (ALL), TCR delta was the only TCR gene to be rearranged. More mature T cell malignancies expressing CD4 together with CD3 showed deletion of both alleles of TCR delta, suggestive of TCR V alpha-J alpha rearrangement. All 43 B-cell tumors expressing surface immunoglobulin (sIg), including two cases of adult B-cell ALL, had germline configuration of TCR delta/alpha. In contrast, all 17 B-cell precursor ALLs (null, common, and pre-B-cell ALLs) had rearrangement and/or deletion of TCR delta/alpha. A single case of "histiocytic" lymphoma also showed biallelic deletion of TCR delta. Oligoclonal rearrangements of Ig and TCR genes were observed in two cases of B-cell precursor ALL and in one case of T-cell lymphoblastic lymphoma. Patterns of such "aberrant" TCR rearrangement were similar to those observed in T-lineage malignancies. In particular, seven of eight cases of B-cell precursor ALL and the histiocytic lymphoma which demonstrated biallelic TCR delta deletion, (suggestive of a V alpha-J alpha rearrangement) had clonal TCR beta rearrangement. These data support the hypothesis that supposedly aberrant rearrangements of the TCR genes may follow the same developmental controls as found in T-cell differentiation, despite the lack of evidence for further commitment to the T-cell lineage. TCR delta rearrangement is a useful marker of clonality of immature T-cell tumors which may have only this gene rearranged but is not specific to the T-cell lineage.     

Stochastic rearrangement of immunoglobulin variable-region genes in chicken B-cell development.

The molecular mechanism by which immunoglobulin (Ig) gene rearrangement occurs is highly conserved between mammalian and avian species. However, in avian species, an equivalent to the mammalian pre-B cell, which has undergone Ig heavy-chain gene rearrangement and expresses mu heavy chains in the absence of Ig light-chain rearrangement, has not been convincingly demonstrated. It is consequently unclear whether an ordered progression of gene rearrangement events leading to functional Ig expression occurs in avian species. To examine the sequence of Ig gene rearrangement events in chicken B-cell development, we transformed day 12 embryo bursal cells with the REV-T(CSV) retrovirus. More than 100 clones were analyzed by Southern blotting and polymerase chain reaction for the presence of Ig gene rearrangements. The majority of these clones contained only germline Ig sequences. Several clones contained complete heavy- and light-chain rearrangements and 13 clones contained only heavy-chain rearrangements analogous to stages of mammalian B-cell development. However, 5 clones contained rearrangements of light-chain genes in the absence of complete heavy-chain rearrangement. Consequently, we conclude that rearrangement of chicken Ig light-chain genes does not require heavy-chain variable-region rearrangement. This observation suggests that chicken Ig gene rearrangement events required for Ig expression occur stochastically rather than sequentially.     

Stage IV intramucosal gastric marginal zone B cell lymphoma of mucosa-associated lymphoid tissue type

A 45-year-old woman with no symptoms underwent upper gastrointestinal endoscopy. A discolored area was noted at the greater curvature of the gastric upper body. Endoscopic ultrasonography demonstrated thickening of the second sonographic layer indicating that the depth of invasion was confined to the mucosa. A urea breath test and anti-Helicobacter pylori antibody test were negative. A computed tomography scan showed a consolidation at the right lung. Gastric biopsy and transbronchial lung biopsy (TBLB) demonstrated a monotonous proliferation of atypical small lymphocytes. A diagnosis of gastric marginal zone B cell lymphoma of mucosa-associated lymphoid tissue type (MALT lymphoma) was made. The clinical stage was stage IV. A genetic analysis showed rearrangement of the joining region of the immunoglobulin heavy chain gene and identical clones in both lesions. An API2–MALT1 fusion gene was detected in the gastric lesion. After H. pylori eradication treatment, combination treatment with rituximab plus CHOP (R-CHOP) was performed; 6 months later an endoscopy revealed complete disappearance of the lesion. Multiple gastric biopsies showed no infiltrating atypical lymphocytes. Similarly, the lesion in the lung showed complete remission (CR) on CT and TBLB. This report shows that a gastric MALT lymphoma located in the mucosa and disseminated to the lung maintained CR by R-CHOP.     

V(H) gene family utilization in different B-cell lymphoma subgroups

V(H) gene family specific polymerase chain reaction (PCR) amplification was performed in 87 B-cell lymphoma samples from 4 different subgroups. No apparent restriction in the VH gene usage was found in follicular lymphomas, lymphoplasmacytoid lymphomas or large B-cell lymphomas, whereas a biased VH1 utilization was shown in patients with chronic lymphocytic leukemia. Eleven of 18 chronic lymphocytic leukemia cases utilized the VH1 gene family, and nucleotide sequencing of the VH1 gene rearrangements revealed that a majority utilized the DP10 (51p1) germline gene, which has been reported to be strongly associated with autoimmune disease. No VH5 or VH6 rearrangements were amplified in the chronic lymphocytic leukemia subgroup, 2 gene families which previously have been found to be over-represented in these patients. In a high proportion (40%) of large B-cell lymphomas, VH gene family-specific PCR failed to amplify any rearrangement. Using primers hybridizing to the framework regions 2 and 3 and Southern blot analysis of the immunoglobulin heavy chain locus, clonal rearrangements were displayed in two-thirds of these PCR negative cases. However, the rearrangement status could not be elucidated in 5 of 35 patients with large B-cell lymphoma.     

Primary Hepatic Low-Grade B-Cell Lymphoma of the Mucosa-Associated Lymphoid Tissue Type: A Case Report and Review of the Literature

Primary hepatic lymphoma, mostly diffuse large B-cell lymphoma, is a rare disease. We describe an extremely rare case of low-grade B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type occurring in the liver. A 61-year-old man with a history of hepatitis A presented with early gastric cancer and a liver mass. Needle biopsy of the liver tumor suggested low-grade B-cell lymphoma by histology and polymerase chain reaction of the immunoglobulin heavy chain gene. The tumor (3.4 x 2.8 x 2.4 cm) was completely resected from the anterior segment of the right lobe of the liver. Atypical lymphoid cells of small to intermediate size proliferated in the tumor, and lymphoepithelial lesions were recognized. Immunohistochemically, lymphoma cells were positive for CD20 and negative for CD5, CD10, and cyclin D1. Staging procedures showed no lymphoma lesion other than the liver tumor. Thus, the patient was diagnosed with low-grade hepatic marginal zone B-cell lymphoma of the MALT type. The patient has been followed up for 1.5 years since surgical resection with no recurrence. The clinicopathologic characteristics and management of this rare disease are discussed.