INTRODUCTION

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Lymphomas of mucosa-associated lymphoid tissue (MALT) represent a distinct group of commonly occurring extranodal non-Hodgkin's lymphomas that often remain localized at their sites of origin for many years (1). Because malignant B cells share features with normal splenic marginal zone B cells, they are classified as marginal zone B-cell lymphoma in the revised European-American classification of lymphoid neoplasms (REAL) classification (2). Recent studies provid evidence that antigen may play a role in the pathogenesis of low-grade MALT lymphomas (3).

The immunoglobulin heavy chain (IgH) locus on chromosome 14 contains an estimated 100 to 150 variable (V_H) genes, 30 diversity (D) genes, and six junctional (J_H) gene segments. During the maturation of a normal B cell, the unique combination of single germline V_H, D, and J_H gene fragments gives rise to a functional V_H-D-J_H unit (7). Added diversity occurs through the junctional insertion of nucleotides at the boundaries of these fragments, and hypermutation, where the nucleotide sequences of the germline fragments are altered. Somatic hypermutation of Ig V_H genes is generally believed to occur in the germinal centers, and replacement mutations in CDRs have been used as indicators of antigen selection (8). The positive or negative selective pressure of somatic mutation in B cell malignancies can pinpoint the developmental stage of neoplastic cells. Therefore, somatic mutations with the positive selective pressure have been found in postgerminal center cell lymphomas such as follicular lymphoma (9), but not in pregerminal center cell lymphomas, such as mantle cell lymphoma (10).

In this study, we used a novel 2-step polymerase chain reaction (PCR) and sequence procedure to analyze the Ig V_H region genes of biopsy samples from 11 patients with low-grade pulmonary MALT lymphomas. Analysis of the tumor-derived Ig V_H genes of low-grade pulmonary MALT lymphomas indicates that low-grade pulmonary MALT lymphoma B cells are postgerminal center lymphocytes with or without intraclonal variation.

	METHODS

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Patients

The study was performed on tissue samples obtained by surgical resection or open-chest lung biopsy (OC) from patients with a histopathologic diagnosis of low-grade pulmonary MALT lymphoma. For the purpose of this study, 13 cases were collected from our own files and from those of other hospitals for the years 1982 to 1997. Among the 13 cases, we found 11 low-grade pulmonary MALT lymphomas, one small lymphocytic (consistent with CLL) lymphoma, and one diffuse large cell lymphoma without evidence of pulmonary MALT lymphoma. The latter two were excluded from our study. Materials obtained from these 11 patients by surgical resection or open-chest biopsy were used for the study (Table 1). Nine patients underwent complete resection of the lymphoma tissue, but the remaining two patients (patients 4 and 10) underwent only open-chest biopsy. In these two patients, mass shadows expanded very slowly, and subsequent percutaneous needle biopsy (PCNB) was done 10 or 8 yr after the open-chest biopsy procedure.

Histopathologic Studies

Lobectomy or open-chest biopsy specimens were fixed in 10% formalin and embedded in paraffin wax. The sections were subjected to hematoxylin-eosin (H-E) and immunohistochemical stainings. Immunohistochemical studies were performed on the following markers: leukocyte-common antigen (LCA) (Dakopatts, Globstrup, Denmark), CD20 (L26; Dakopatts), CD45RA (MB1 Euro-Diagnostica, Malmo, Sweden), CD43 (MT1; Euro-Diagnostica), CD45RO (UCHL1; Dakopatts), and anti-kappa and lambda light chains (Dakopatts). The criteria used to identify primary pulmonary lymphoma of the lung were a lack of evidence of disseminated lymphoma at the time of diagnosis, as well as no such evidence for 3 mo after diagnosis (11). When a diagnosis of pulmonary MALT lymphoma was reached, the lesion was subclassified into the REAL classification (2).

Microdissection and DNA Extraction

To increase the concentration of neoplastic DNA over the total tissue DNA, surgically resected materials were microdissected in the following way. An H-E section was visualized under a ×5 objective and the nonlymphoid tissue was carefully scraped off leaving a zone as small as 25 mm² rich in the infiltrate of interest. Four serial sections 5 µm thick were laid on top of the first H-E section, and the corresponding zones were selected; the rest was scraped off. Microdissected areas from four sections were then scraped into one tube. DNA was obtained from the microdissected materials as described by Wan and colleagues (12).

PCR

The two-step seminested PCR method was performed in a GeneAmp PCR 9600 system (Perkin Elmer-Cetus, Norwalk, CT). The primer sequences were: 5' TGG(A/G)TCCG(A/C)CAG(G/C)C(T/C)(T/C)C- (A/G/T/C)GG 3' (termed Fr2) for the second framework portion of the V_H region; 5' TGAGGAGACGGTGACC 3' (termed LJH), or 5' GTGACCAGGGT[A/G/C/T]CCTTGGCCCCAG 3' (termed VLJH) for the J_H region (13, 14). Each PCR experiment included a sample without DNA template as a negative control and a sample with DNA extracted from a Burkitt's lymphoma lymph node (as a positive control) whose Ig V_H gene rearrangement was detected by Southern blotting. One PCR cycle consisted of denaturation for 1 min at 94° C, annealing for 1 min at 60° C, and extension for 1 min at 72° C. The first-step consisted of 30 cycles with primers FR2 and LJH and 5 µl of template DNA; the second step consisted of 20 cycles with FR2 and VLJH, with 10 µl of a 1 to 1,000 dilution of the first step PCR product as a template. The amplified DNA was precipitated from the PCR product (40 µl) and dissolved in TE buffer (5 µl). This mixture was electrophoresed on 2% agarose gel and stained with ethidium bromide to visualize the DNA under short-wavelength UV light.

Sequence Analysis

The other portion of the PCR product was ligated to the PCR vector, and the ligation mixture was transformed into One Shot competent cells using a TA Cloning Kit (Invitrogen Corp., San Diego, CA). Ligated clones were chosen at random and phage DNA was purified. The inserts in the PCR vector were sequenced by the Dye Primer method using a Taq Dye Primer Cycle Sequencing Core Kit (Applied Biosystems, Norwalk, CT). All clones were sequenced from both directions using the M13 forward and reverse primers. The Ig V_H gene sequences were aligned to EMBL/GenBank databases with DNASIS system (Hitachi Software Enginerring, Yokohama, Japan).

Analysis of Mutations

The probability (q) that a replacement (R) mutation will localize to CDR was calculated using the formula CDR Rf × CDR_rel, where CDR Rf is the replacement frequency inherent to CDR sequence, and CDR_rel is the relative size of the CDR. We calculated the probabilities (p) that an excess or scarcity of R mutations arose by chance using the binomial distribution model P = [n!/k! (n  k)!] q^k (1  q)^n-k, where n is the total number of observed mutations and k is the number of observed R mutations in the CDR (9, 15).

	RESULTS

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Histopathology of Low-Grade Pulmonary MALT Lymphomas

Eleven cases of low-grade pulmonary MALT lymphoma were classified as extranodal marginal zone B-cell lymphoma (low-grade B-cell lymphoma of MALT type) (Table 1). In all cases, reactive follicles were sparsely present and surrounded by tumor cell infiltration consisting of small- to medium-sized lymphoid cells. These 11 cases showed lymphoepithelial lesions, which were diagnosed as low-grade pulmonary MALT lymphomas. All 11 cases were classified as B-cell lymphoma on the basis of the expression of at least two B-cell markers.

In Patients 4 and 10, subsequent PCNB specimens (obtained 10 and 8 yr later) demonstrated monomorphous lymphocytic infiltration suggestive of low-grade pulmonary MALT lymphomas, whose lymphoma cells were similar to previous open-chest biopsy specimens without high-grade transformation.

Two-Step PCR and Sequencing Analysis of the Ig V_H Regions of Low-Grade Pulmonary MALT Lymphomas

All cases of low-grade pulmonary MALT lymphoma showed a sharp monoclonal band (about 250 base pairs) on electrophoresis (Table 1 and Figure 1). The results of sequencing are presented as the incidence rates of the predominant sequences out of the total number of vector clones analyzed. All sequences from 11 cases of low-grade pulmonary MALT lymphomas showed closely related sequences, indicating amplification from dominant lymphoma cell clones with a small proportion of normal reactive lymphocyte clones. Other sequences obtained had different individual CDR3 sequences characteristic of contaminating normal reactive B cells (data not shown). The closest germline V_H gene and its degree of similarity to each case are shown in Table 2. For all 11 cases of primary pulmonary lymphoma, a single major Ig V_H sequence was identified with rates from 5/10 to 8/10.

View larger version (64K): [in this window] [in a new window]   Figure 1.   PCR analysis of Ig VH gene rearrangement. The numbers above the lanes correspond to the case numbers in Table 1. Arrow ( ) = 200 base pairs; double arrow ( ) = 300 base pairs; S = surgical biopsy specimen; P = percutaneous needle biopsy specimen; Po = positive control; N = negative control.

The distribution of the somatic mutations in the tumor- derived V_H genes are shown in Table 3. Eight lymphoma clones expressed V_H genes derived from the V_H3 family, whereas the other three used members of the V_H4 family. For CDR, there were more R mutations than expected in all cases, with significant (p < 0.01) clustering indicative of antigen detection in all cases. Eight lymphoma Ig V_H genes showed evidence of intraclonal heterogeneity. In one (Patient 4) 11 nucleotide substitutions of major Ig V_H genes derived from lymphoma clones were found between the open-chest biopsy and the PNCB performed 10 yr later. In contrast, the other three lymphoma Ig V_H genes (Patients 5, 10, and 11) demonstrated lack of intraclonal heterogeneity. In Patient 10, Ig V_H genes of the lymphoma cell clones derived from the open-chest and PCNB (8 yr later) were identical. The most frequently found J_H segments were J_H4 (45.5%), J_H6 (18.2%), and J_H5 (18.2%), with no major contribution from the D region.

	DISCUSSION

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As there are some reports demonstrating that MALT lymphomas share some similarities with marginal zone B cells, they have been classified as marginal zone B cell lymphomas in the REAL classification (2). Gastric MALT lymphomas use positive selectively mutated V_H genes without intraclonal variation, consistent with a derivation from postgerminal center marginal zone memory B cells (16, 17). However, recent studies have demonstrated that the process of hypermutation is still ongoing in MALT lymphomas because of the presence of intraclonal variation in the Ig V_H genes (18). There have been some studies its on Ig V_H genes that include a few pulmonary MALT lymphomas, but hypermutation of Ig V_H genes has not been analyzed at some points over a long time period. In this study, we investigated the Ig V_H genes derived from 11 low-grade pulmonary MALT lymphoma to gain an insight into the nature of lymphoma cell clones and to determine whether these lymphoma cell clones remain in a state of hypermutation over a long time period.

Some nucleotide substitutions may have resulted from replication errors of Taq polymerase during PCR. However, this explanation is inadequate because the ongoing mutation rate of approximately 5.8% in our study is greater than the Taq error rate, which has been reported to be about 0.03%, (19). In all cases, specific nucleotide substitutions occurring in more than one clone have been observed by sequencing in both orientations. Ig V_H gene hypermutation seems to be a general feature of low-grade pulmonary MALT lymphomas.

We observed frequent somatic mutations of the rearranged Ig V_H genes in all 11 cases of low-grade pulmonary MALT lymphoma. A higher CDR R:S mutation ratio (> 2.9, calculated for somatic mutations occurring randomly in a gene encoding a protein whose structure need not be preserved) reflects the positive selective pressure of an antigen (affinity-maturation) on those gene products that come into close contact with an antigen, whereas a lower FR R:S mutation ratio (< 2.9) reflects the negative pressure of structural components that need to be conserved (20). The accumulation of R mutations in CDR indicates a role for an antigen in the selection of lymphoma cell clones and that the cell of origin has been exposed to the hypermutation mechanism in the germinal center. For all lymphoma cases, CDR showed more R mutations than expected, with significant (p < 0.01) clustering indicative of antigen selection. In addition, four patients (Patients 1, 6, 8, and 9) showed Ig D-D recombination, which is believed to be an important mechanism for antigen-driven selection and is very rare (< 1/33,000) in pre-B-cells.

Two-step PCR and sequencing analysis of Ig V_H genes in low-grade pulmonary MALT lymphomas have given mixed results, showing that the genes can be divided into two groups, "hypermutated with intraclonal variation" and "hypermutated without intraclonal variation" types.

Eight cases (Patients 1 through 4 and 6 through 9) demonstrated intraclonal variations and belonged to "hypermutated with intraclonal variation" type. In addition, we were able to show continuous somatic mutations occurring in the first and second (10 yr later) biopsy materials obtained from Patient 4. These findings indicate that most low-grade pulmonary MALT lymphoma cells are still under the influence of the hypermutation mechanism. Considering the fact that Ig V_H gene hypermutation requires the microenvironment of the germinal center and will not occur in marginal zones (21), these lymphomas might have originated in postgerminal center marginal zone B cells and the ongoing Ig mutations might reflect reentry into a germinal center pathway to further mutation. The reentry of normal B cells into germinal centers has been demonstrated for rat splenic marginal zone cells after antigenic stimulation (22, 23). Accumulation of affinity maturations over a long time period in Patient 4 might suggest that germinal center reentry of lymphoma cell clones might be followed continuously by further Ig V_H gene hypermutation.

The other three patients (Patients 5, 10, and 11) demonstrated only one major clone with hypermutation (hypermutated without intraclonal variation type). These lymphoma cells might have originated in pure postgerminal center marginal zone B cells and might not undergo reentry into a germinal center environment. These clones might no longer be susceptible to the hypermutation mechanism and their Ig V_H genes seemed to be stable. In this type, proliferative expansion outside the germinal center seems to take place without further hypermutation. Sequencing of the PCR products from open-chest biopsy materials and the subsequent PCNB specimens obtained from Patient 10 confirmed this possibility. It may be important to point out that splenic lymphoma with villous lymphocytes, belonging to marginal zone B-cell lymphoma in the REAL classification, has also been reported to show somatic hypermutation without intraclonal heterogeneity (24).

The chronic inflammation associated with MALT lymphoma such as Hashimoto's thyroiditis, Sjogren's syndrome, and Helicobacter pylori gastritis is autoimmune in nature (25- 27). Autoimmunity may play a role in the pathogenesis of low-grade MALT lymphomas (Patients 3 and 4). It is interesting that four (Patients 1, 7, 10, and 11, DP50/hv3019b9, V_H26, V_H4.21) of the 11 Ig V_H genes of low-grade pulmonary MALT lymphomas studied here are frequently found in a variety of autoantibodies such as cold agglutinins, rheumatoid factors, and anti-DNA antibodies (28, 29). These findings are consistent with the fact that autoimmunity may be involved in the pathogenesis of low-grade pulmonary MALT lymphoma.

In summary, we have shown that low-grade pulmonary MALT lymphoma clones have undergone somatic hypermutations in their Ig V_H genes and can be divided into two groups, "hypermutated with intraclonal variation" and "hypermutated without intraclonal variation" types.