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Long-Term Persistence of Monoclonal B Cells After Cure of Helicobacter pylori Infection and Complete Histologic Remission in Gastric Mucosa–Associated Lymphoid Tissue B-Cell Lymphoma

From the Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus der Technischen Universität, Dresden; Abteilung für Hämatologie, Onkologie und Immunologie, Philipps Universität, Marburg; Institut für Immunologie der Technischen Universität, Dresden; and Institut für Pathologie, Klinikum Bayreuth, Bayreuth, Germany.

Address reprint requests to Christian Thiede, MD, Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus der Technischen Universität, Dresden, Germany; email: thiede{at}oncocenter.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Cure of Helicobacter pylori infection is associated with remission induction in the majority of patients with low-grade gastric mucosa associated lymphoid tissue (MALT) lymphoma in localized stages; however, limited data exist as to whether these patients may be cured of their lymphoma. The present study was performed to investigate whether the polymerase chain reaction (PCR) for the rearranged immunoglobulin heavy chain region may be used to define "molecular" remission.

PATIENTS AND METHODS: Ninety-seven patients who suffered from low-grade gastric MALT lymphoma stage I_E were observed with central pathology and molecular biology after cure of H pylori infection. PCR was performed with the use of consensus primers for the framework regions 1, 2, and 3 and monoclonality was corroborated by sequence analysis. In selected cases, microdissection was performed to study the origin of the monoclonal B cells.

RESULTS: Of the 97 patients, 77 obtained complete endoscopic and histologic remission (CR). Twenty of 44 patients with PCR monoclonality at diagnosis and with sufficient molecular follow-up displayed monoclonal bands for a median time of 20.5 months after CR (range, 0 to 50.4 months). These B cells were related to the original lymphoma clone by sequence analysis. Microdissection analysis identified basal lymphoid aggregates as the source of these monoclonal B cells. Local relapse occurred in and was observed by PCR in four patients. All four patients displayed monoclonal PCR before relapse, and three of these four showed ongoing PCR monoclonality throughout their course, indicating the persistence of malignant cells.

CONCLUSION: Half of all patients with gastric MALT lymphoma show long-term PCR monoclonality up to several years after cure of H pylori infection and CR. Patients with monoclonal PCR should be observed closely, whereas long-term PCR negativity may indicate cure of the disease.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
INFECTION WITH Helicobacter pylori has been identified as one of the most frequent infections worldwide. H pylori has been implicated in different human diseases such as gastritis, gastric and duodenal ulcer, gastric cancer, and low-grade gastric lymphomas of the mucosa-associated lymphoid tissue type.^1-6 Infection with H pylori results in the damage of gastric epithelium; and, within the gastric mucosa, which normally is devoid of lymphoid follicles, the development of gastric mucosa associated lymphoid tissue (ie, MALT) is induced.⁷ Gastric lymphomas of MALT type are thus regarded as the latest stage of a chronic active H pylori-induced gastritis.^8,9 Data from epidemiological, biologic, and molecular-immunologic studies^3,5,10 have implicated H pylori in gastric MALT lymphomas.

The ultimate proof that chronic H pylori-induced gastritis is a prerequisite for development of low-grade gastric MALT lymphomas has come from therapeutic studies in which patients with low-grade gastric MALT lymphomas experienced complete histologic and endoscopic remission (CR) after cure of the infection.^11-15 However, these studies have shown that this therapy is efficient in limited stages only.¹⁶ Although several hundred patients have been treated using eradication of H pylori, these studies have not provided sufficient evidence as to whether the patients showed long-lasting remission. In one investigation, it was shown that most patients remained in CR over a median follow-up time of 24 months.¹

The polymerase chain reaction (PCR) for the rearranged immunoglobulin heavy chain region has been shown helpful in the differential diagnosis of gastric MALT lymphoma;^17-19 however, there is still controversy with regard to the meaning of PCR monoclonality in the absence of clear histologic signs of lymphoma. Few data exist on the role of the PCR in the follow-up of patients after a complete remission (CR). Two studies have reported that ongoing B-cell monoclonality can be observed; however, this was demonstrated with only with a short follow-up and a limited number of patients investigated.^14,17

The prevalence and clinical implications of this finding as well as the origin of these monoclonal B cells have not yet been elucidated. The aim of the present study was to investigate whether detection of monoclonal B cells is a frequent and lasting phenomenon in patients with gastric MALT-lymphomas who obtain a CR after cure of the infection. In addition, we asked whether patients with detectable monoclonal B cells, after entering a CR, have a different clinical outcome as compared with patients whose biopsies revealed the presence of polyclonal B cells during follow-up. Finally, we studied the topographic origin of monoclonal B cells within the gastric mucosa by microdissection.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
The 97 patients prospectively analyzed in this study were part of an ongoing multicenter trial to investigate the role of curing H pylori infection in low-grade gastric MALT lymphomas in clinical stage I_E.^12,14 The clinical study has been previously described in detail.¹⁴ Staging consisted of clinical examination, upper endoscopy with central histologic investigation (M.S.), CT scan of thorax and abdomen, and, wherever possible, endosonography. Cure of H pylori infection was achieved with the use of a dual regimen that consisted of amoxicillin (750 mg tid) and omeprazole (40 mg tid) for 2 weeks (n = 93 patients), or with metronidazole (400 mg bid), clarithromycin (250 mg bid), and omeprazole (20 mg bid) for 7 days (n = four patients). All patients were endoscoped 4 weeks after the end of treatment. Thereafter, patients obtaining complete histologic and endoscopic remission were endoscoped every 6 months in the first 2 years, whereas patients responding partially were investigated every 4 weeks. Patients who did not show any sign of clinical or histologic response after 3 months of follow-up, as well as patients in partial remission at 6 months, underwent surgery and/or chemotherapy. The study was approved by the local ethical committees of Humboldt University (Berlin, Germany) and University Erlangen (Nuremberg, Germany).

Histopathologic Analysis
Histopathologic analysis of the biopsy samples was performed as previously described.¹⁴ A median number of eight biopsies were analyzed at each time point. The criteria for the diagnosis of low-grade gastric MALT lymphoma were unequivocal evidence of lymphoepithelial destruction and replacement of gastric glands by uniform centrocyte-like cells. Definition of complete histologic regression was rendered whenever the posttreatment biopsies showed no sign of remaining lymphoma but instead an empty tunica propria with small basal clusters of lymphocytes and scattered plasma cells. Partial histologic regression was defined as posttreatment biopsy samples revealing either focal atypical lymphoid cells or lymphoepithelial destruction and an empty tunica propria as signs of lymphoma regression. Relapse of the underlying gastric MALT lymphoma was diagnosed whenever lymphoepithelial destructions were again present after the patient had entered CR. For the present study, CR was defined as one time point without lymphoma signs as defined above, and relapse was consequently stated to have occurred whenever lymphoma was again present.

DNA Extraction
High-molecular-weight DNA was extracted from bioptic specimens collected at diagnosis and during follow-up by standard protocols using phenol/chloroform extraction after overnight sodium dodecyl sulfate/proteinase K digestion. To avoid a possible sample error, several (n = 3 to 5) gastric biopsies were pooled. In some cases, DNA was obtained from routinely processed paraffin embedded tissues. Therefore, 100 µm tissue sections were deparaffinized with xylene, washed with 100% ethanol, and dried. DNA was subsequently extracted using a commercially available kit as recommended by the manufacturer (QiaAmp DNA Tissue Kit, Qiagen, Hilden, Germany).

PCR Amplification, Cloning, and Sequencing
The protocols used for PCR amplification, cloning, and sequencing have been previously described.²⁰ To avoid cross-contamination, strict separation of DNA extraction, PCR setup, and post-PCR manipulations was maintained and aerosol-resistant pipet tips were used for all steps. In the majority of the cases, a semi-nested PCR-strategy was used for amplification of rearranged variable, diversity, and joining (VDJ) sequences as recently reported.^14,20 First-round PCR was performed in 50 µL, containing 200 µmoles/l deoxynucleoside-triphosphates, 0.001% gelatin, 50 mmoles/l KCL, 1.5 mmoles/l MgCl₂, 10 mmoles/l TrisHCl, 500 nmoles/l of FR2a and LJH-primers,²⁰ and 100 ng of DNA. The reaction mixture was covered with 2 drops of paraffin oil. Hot start was performed by addition of Taq polymerase (1 U, Applied Biosystems, Foster City, CA) after 5 minutes incubation at 94°C. First-round PCR consisted of 29 cycles with 94°C for 60 seconds, 55°C for 60 seconds, and 72°C for 60 seconds, with 180 seconds of denaturation in the first cycle followed by a final extension cycle of 94°C for 60 seconds and 60°C for 10 minutes. One µl of the first reaction diluted 1:100 was used for second-round PCR, under identical conditions, but the VLJH primer substituting the LJH primer and a total of 22 cycles were performed. Framework region (FR) 1 PCR was used according to the protocol published by Aubin et al²¹ using a FR1 consensus primer and an equimolar mixture of three joining region primers (JH1245, JH3, and JH6). PCR products were separated on 3% agarose gels and stained with ethidium bromide (0.5 µg/mL).

To corroborate monoclonality, PCR bands were subcloned. In several cases it was necessary to separate the predominant monoclonal bands from a polyclonal background. For this purpose, monoclonal bands were cut from the gel and PCR amplificates were recovered using a silica matrix (QIAEX II, Qiagen). Purified PCR products were cloned into PCR II vector (TA-Cloning Kit, Invitrogen, Leek, Netherlands). After transformation of competent Escherichia coli (One Shot INV F’, Invitrogen) and plating on selective agar (50 µg/mL ampicillin; 40 µg/mL X-gal), between eight to 20 white colonies were selected per sample and minipreps prepared from 5 mL overnight liquid cultures using the Mini Plasmid Spin Kit (Qiagen). DNA sequencing of plasmids was performed on 373A and 377 automatic DNA sequencers (Applied Biosystems, Foster City, CA) using Dye Terminator Cycle sequencing kits (Applied Biosystems) as recommended by the manufacturer. T7 and M13 reverse primers were used for forward and backward sequencing.

Microdissection of Gastric Mucosa Specimens
Five samples from three patients in CR after cure of the H pylori infection showing ongoing PCR monoclonality were investigated by microdissection. These individuals were randomly chosen from the group of patients with PCR monoclonality on availability of appropriate sample material. The samples were removed 0 to 3 years after obtaining a histologic CR.

Four to eight serial 3 to 4 µm sections of archival, formalin-fixed, paraffin-embedded gastric biopsies were cut on glass slides, deparaffinized, stained briefly with Mayer’s hematoxylin, and kept in 1x Tris buffered saline until microdissection. Using an automated micromanipulator unit (Eppendorf, Hamburg, Germany), lymphoid cells of defined topographic origin (basal lymphoid aggregates, upper mucosal layer) were selectively resected under buffer solution (Qiagen Buffer ATL, Qiagen Tissue DNA kit) and recovered with a drawn-out glass capillary using a cell traim unit (Eppendorf). The detached cell groups of two to three serial sections, yielding a total of about 50 to 100 lymphoid cells, were transferred to an Eppendorf cap.

For the subsequent DNA extraction, the QIAamp DNA Tissue Kit (Qiagen) was used; however, the deparaffinization step was omitted and the DNA elution volume was reduced to 30 µL to increase the final DNA concentration. To confirm the quality of the extracted DNA, 2.5 µL were amplified using primers for the p53 or ß-IFN-gene as recently described.^22,23 Five µl of the eluted DNA was used for PCR amplification with the FR2 and FR3 PCR setup as previously described. To achieve a higher sensitivity, the number of cycles was increased to 34 in the first and 28 in the second PCR and the product of the first PCR was diluted only 1:10 for the semi-nested PCR. Cloning and sequence analysis was performed as described above. Appropriate positive and negative controls were run with each experiment.

Statistical Analysis
The Mann-Whitney test for unpaired samples was used for calculation of the significance of the distribution between different groups. Correlations were calculated using a nonparametric model (Spearman’s rank order correlation) using a two-sided design. The statistical analysis was performed using the GraphPad PRISM software package version 2.01 (San Diego, CA).

	RESULTS

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Clinical Data
Of the 97 patients, 77 went into complete endoscopic and histologic remission, whereas nine patients responded only partially and another 11 patients showed no response. Of the nine partial responders, five were referred for surgery, whereas three received chemotherapy (five cycles of 750 mg/m² cyclophosphamide, 50 mg/m² doxorubicin, 2 mg vincristine, and 100 mg oral prednisone for 5 days every 21 days [CHOP]). One 84-year-old patient who was in partial remission died of a stroke. In 11 patients, no change was seen and 10 of these patients were therefore referred for surgery (N = 8) or chemotherapy (N = 2; five cycles of CHOP). One patient in this group requested that no further therapy be performed. After 52 months, this patient showed lymphoma progression with enlargement of thoracic lymph nodes, in addition to the gastric lymphoma. In the group of patients with CR, the remissions were observed after a median time of 5.1 months (range, 1 to 25.2 months) from start of therapy to the first diagnosis of CR.

The 77 patients with CR have been observed in continuous CR for a median of 33 months (range, 0 to 65 months). In this group of 77 patients experiencing CR, eight relapses were observed, seven of which were in the gastric mucosa (ie, "local" relapses). There was one distant relapse in the nasal cavity. This was a high-grade lymphoma in a patient presenting primarily with low-grade gastric MALT lymphoma. By molecular analysis, the high-grade lymphoma was not related to the original clone.¹⁴ The relapses occurred between 4.2 and 23.3 months (median, 11.3 months) after patients entered a CR. Four of the seven patients with local relapse achieved a second CR without any further therapy. One patient had a reappearance of the lymphoma after reinfection with H pylori and achieved a second CR after cure of the infection. One patient was treated with radiotherapy and in another patient a gastrectomy was performed.

Analysis of Monoclonal B Cells in Gastric Mucosa at Diagnosis and During Follow-Up
A total of 442 samples from the 97 patients in this study were analyzed. Sufficient material (ie, good amplification of reference genes) was available in 402 samples from 90 patients, of which 64 (71%) revealed monoclonal bands at first presentation. All eight patients with monoclonal PCR at diagnosis, who showed only partial (n = 6) or no response (n = 2) after cure of the H pylori infection, remained monoclonal during the follow-up.

Of the 77 patients obtaining complete histologic remission, samples of 70 patients were available at diagnosis, of which 49 (70%) displayed B-cell monoclonality with at least one of the primer combinations used. In 44 of these 49 patients, sufficient material was present for follow-up investigations. This material was collected during a period of 0 to 50.4 months with a median follow-up time of 25.8 months after CR.

Polyclonal bands were observed in the last analysis in 24 (55%) of these 44 patients, whereas in 20 patients (45%) PCR still indicated the presence of monoclonal B cells during follow-up. Changing PCR patterns, with monoclonal PCR switching to polyclonal PCR and later again indicating monoclonal B cells, were found in several patients. Some typical PCR patterns are presented in Figs 1 and 2.

View larger version (37K): [in this window] [in a new window]   Fig 1. Examples of molecular follow-up. (A) Patient no. 12 with monoclonal (mc) PCR at diagnosis (Ly) and in first. control in partial remission (pR) PCR analysis performed in cR revealed no amplification or polyclonal (pc) bands. (B) Patient no. 81 obtained a cR but showed mc PCR during the follow-up. (C) Patient no. 87 obtained cR, but relapsed after reinfection with H pylori. He obtained 2nd cR after successful eradication. Abbreviations: mc co, monoclonal control (B-cell line Raji); pc co, polyclonal control (peripheral blood leukocytes); NTC, no template control.

View larger version (42K): [in this window] [in a new window]   Fig 2. Follow-up of 44 patients with lymphoma samples displaying mc PCR at diagnosis. Patients in whom the PCR (A) became polyclonal and (B) remained monoclonal. Ticks indicate 1 year. (denote the histologic follow-up (filled, lymphoma/relapse; half filled, pR; open, CR). () indicate PCR results (filled, mc PCR; open, pc PCR).

View larger version (12K): [in this window] [in a new window]   Fig 3. Comparison of histologic response time and molecular clearance of monoclonal B cells. In the 17 patients analyzed, the interval between the histologic examinations was 10 months and for the molecular analyses 12 months.

Monoclonal B Cells Derive From Lymphoid Aggregates Typical for Ex-H pylori Gastritis
To study the origin of monoclonal B cells in the patients reaching CR after cure of the H pylori infection, we performed microdissection analysis in three randomly selected patients with follow-up samples that displayed B-cell monoclonality in the PCR after CR. In all three patients analyzed, the fraction dissected from basal lymphoid aggregates contained monoclonal cells with PCR bands of identical size as compared with the original MALT-B-cell-lymphoma clone ( Table 1). In contrast, six of seven microdissected specimens, obtained from the upper mucosal layers containing plasma cells and lymphocytes, revealed no PCR bands or monoclonal products of different sizes (Table 1 and Fig 4). The PCR band obtained from a single sample from the upper mucosal layer had a similar size, however sequence analysis performed on this and all other samples from the upper mucosa with PCR amplification of a V_H-rearrangement revealed different CDR3-sequences (Table 1, Fig 4c). In contrast, identical or closely-related V_H sequences were found in seven of 10 sequenced DNA samples extracted from the basal lymphoid aggregates (Table 1 and Fig 4c). The other three microdissection samples derived from the basal aggregates of one sample contained sequences not related to the initial lymphoma B-cell clone, which is in accordance with the observation that these basal aggregates are not specific per se for residual disease. Detailed analysis revealed that, compared with the original lymphoma clone, two patients displayed clonal variance. In patient no. 127, the sequences found had the same VDJ rearrangement, but the D-segment used was in germline configuration, whereas in patient no. 81 sequencing revealed evidence of ongoing mutations. A more extensive molecular follow-up for this patient has been recently published.²⁰ Compared with these data, although sharing the majority of aberrations, the clone found in the microdissection analysis represented a novel variant with two unique silent mutations. Little variation was observed within this clone (data not shown).

View larger version (99K): [in this window] [in a new window]   Fig 4. (A) Microdissection, (B) PCR, and (C) sequence analysis in patient no. 56 with ongoing PCR monoclonality. Abbreviations: NTC, no template control; PCR, polymerase chain reaction. Pos. co. DNA control, 100 ng of human placenta DNA; controls VH-PCR (Fig 1). In 4c, (––––) indicate sequence identity.

	DISCUSSION

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In recent years, molecular markers have been used successfully to identify patients with minimal residual disease (MRD) who are at a higher risk for relapse. For instance, in acute lymphoblastic leukemia, detection of clonal B cells as MRD after reaching CR is a highly significant marker for worse outcome.²⁵ In chronic lymphocytic leukemia and non-Hodgkin’s lymphoma, molecular detection of MRD as assessed by the presence of B cells belonging to the malignant clone has been shown to be associated with an increased relapse rate after autologous and allogeneic bone marrow transplantation.^26,27

In the present study, we used a sensitive PCR-technique to detect monoclonal B cells in the gastric mucosa during follow-up of patients after obtaining a complete histologic remission. We took advantage of the rearranged V_H-genes coding for the variable region of the heavy chain of the immunoglobulin molecule, which is a specific marker for a given B-cell population. PCR amplification of this rearrangement allows detection with higher sensitivity as compared with conventional methods such as histology or immunohistochemistry.

The results indicate that patients in complete lymphoma remission after H pylori eradication can be subdivided into two major groups according to the results of the PCR follow-up. In approximately 50% of the patients, a switch to a polyclonal pattern will be observed and no monoclonal B cells found at the end of follow-up within the gastric biopsies at a level of sensitivity of 1% to 5%; whereas, in the other half of the patients, monoclonal PCR products remain detectable for up to 4 years, although these patients have also achieved a complete endoscopic and histologic remission.

Two issues need to be stressed in this context: (a) whether or not a polyclonal PCR is a real indicator of molecular remission and (b) what is the meaning of ongoing PCR monoclonality in the stomach?

With respect to the first question, we cannot rule out that in some patients with polyclonal PCR a so-called sample error may be the reason for a negative PCR result. As indicated by the fact that the molecular remission took about 1 year longer than the histologic and endoscopic remission of the lymphoma (Fig 3), the clearance of monoclonal B cells may be delayed. This is in accordance with other studies.¹⁵ During this period, it may be possible to detect changing patterns in the PCR. Indeed, we have found several patients with monoclonal PCR at diagnosis that turns into a polyclonal state after some time and then again becomes monoclonal (Fig 2a and b). However, in nine patients, three or more consecutive biopsies were found to contain polyclonal B cells only, and a reappearance of PCR monoclonality was observed in none of these patients. This suggests that although there is a time period where sampling error might occur, it is not an important factor once the lymphoma B cells are completely eliminated. We have further corroborated this finding using clone-specific PCR with oligonucleotides specifically binding the CDR3 region in three of these patients, where we could not find the initial B-cell clone with an even higher sensitivity (1:1,000 to 1:10,000, data not shown). Thus, it might be that at least some of these patients have been cured of their disease solely by eradication therapy.

In contrast to this group, the PCR detected monoclonal B cells at the last analysis in 45% of the patients, although these patients had also achieved a histologic and endoscopic remission. In several of these patients, B-cell monoclonality was detectable for up to 4 years after a CR. Of special interest was the question of whether or not a monoclonal PCR was able to predict a local relapse. In our study, a relapse was defined as a reappearance of lymphoma after at least one endoscopy showing a CR. In seven patients, the follow-up examinations revealed microscopic evidence of lymphoma after a CR had been diagnosed endoscopically and histologically. None of these patients had macroscopic signs of disease at the time of relapse. However, as discussed above, in this specific situation, sampling error might have been a problem. Therefore, at least some of these samples referred to as a relapse may represent partial remissions where residual lymphoma was missed. This would explain why four of the seven patients achieved an ongoing second CR (with follow-up occurring at 6.8, 20.4, 30.6, and 46.1 months, respectively) without any further therapy. A similar case was recently reported in a small series of six patients by Isaacson et al.²⁸ Three of the seven patients in whom a gastric relapse was diagnosed in our study received a specific treatment. One of the local relapses was associated with an H pylori reinfection. This patient was given a second course of eradication therapy (7 days of triple therapy, see Patients, Materials, and Methods). He became H pylori negative again and subsequently achieved a second CR (molecular follow-up shown in Fig 1c). The second patient was treated with surgery, which revealed low-grade disease stage I_E. A third patient received radiotherapy. Two of these three patients were in the group of patients with persistent B-cell monoclonality, whereas in the third patient monoclonality could not be demonstrated even at diagnosis using the PCR technique described. Thus, ongoing monoclonal PCR may indicate a higher risk for local relapse. Future studies with larger patient numbers are clearly needed to finally answer this question. To avoid potential misinterpretations due to sample error, we currently recommend at least two consecutive examinations without macroscopic or microscopic evidence of lymphoma to state that a patient is in CR of the lymphoma.

We also looked for the origin of monoclonal B cells within the gastric mucosa. In microdissection studies, basal clusters of small lymphoid cells were identified as the source for monoclonal B cells in three of our patients with ongoing PCR monoclonality. These aggregates are composed of morphologically unsuspicious lymphocytes and are located in the basal gastric mucosa (Fig 4). By immunohistochemistry these cells strongly expressed BCL-2 , showed weak expression of the proliferation marker Ki67, and rarely contained apoptotic cells as detected by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling staining (Wündisch T, Thiede C et al unpublished results). Similar B-cell aggregates are frequently found in patients with gastritis after the cure of H pylori infection, thus, from a morphologic point of view, have no significance for remaining lymphoma cells. However, sequence analysis of microdissected lymphocytes from the aggregates of our patients revealed clonal identity with the initial lymphoma cells in most instances, suggesting a link between the original lymphoma clone and these basal lymphoid aggregates. In light of this finding, it seems important to point out the difference between PCR monoclonality found in cases of gastritis without previous or present signs of lymphoma and persisting PCR monoclonality in patients treated for frank lymphoma. Others and we have been able to demonstrate PCR monoclonality in patients with gastritis without any signs of lymphoma.^18,29,30 In a recent study of 110 patients with H pylori-associated gastritis (Wotherspoon grades I to IV), PCR monoclonality was found in 18% (Wündisch T et al, unpublished results.). The basis of this phenomenon is not fully understood. Technical artifacts due to preferential amplification of B cells enriched in lymphoid follicles may account for the majority of these results.³¹ Thus, although B-cell monoclonality clearly precedes the development of MALT lymphoma,^9,19 the clinical relevance of a monoclonal PCR without histologic evidence of lymphoma seems questionable. In contrast, our data in patients with persistent B-cell PCR monoclonality after obtaining CR indicate that a B-cell population closely related to the lymphoma remains in a resting state in basal lymphoid cell clusters. These cells can be found several years after the disappearance of the lymphoma and can give rise to a relapse of the tumor on reinfection with H pylori, as shown in one patient. The genetic origin of this phenomenon is not yet clear. The fact that the immunoglobulin molecules produced by the lymphoma B cells recognize autoantigens,^32,33 as well as the finding that in some patients the process of somatic mutation and clonal evolution was ongoing even after eradication of H pylori, points to the relevance of autoreactivity.²⁰ An interesting question is the impact of novel genetic markers like mutations in BCL10 gene³⁴ or the API2 -MLT fusion³⁵ associated with the t(11;18)in this context. Du et al recently reported BCL10 mutations in 3 of 11 lymphomas unresponsive to H pylori eradication;³⁶ however, some of these cases were already disseminated at the time of treatment (Du et al, personal communication). We have looked for BCL10 alterations in biopsy samples of five MALT lymphoma patients showing only partial remission or no change after H pylori eradication. None of these patients showed somatic mutations in the coding region of BCL10 (Thiede et al, unpublished results). Thus, based on these limited data, BCL10 mutations might be more relevant in later stages of disease and may not account for the persistence of monoclonal B cells in early lymphomas. More relevant to these early cases might be the API2 -MLT fusion, which seems to be able to distinguish H pylori-responsive from unresponsive cases.³⁷ However, the relevance of both alterations in cases with persistent PCR monoclonality remains to be determined.

In conclusion, the data presented here suggest that the use of the PCR in the follow-up of patients with primary low-grade gastric MALT lymphoma is a useful method for the identification of two groups of patients. In about one half of the patients, the lymphoma B cells can no longer be detected, which might indicate that at least some of these patients are cured of their disease. In contrast, in the other half of the patients, long-term B-cell monoclonality was found. Because only a subgroup of these patients suffered from evident relapse, longer follow-up investigations are necessary to answer the question as to whether patients with ongoing B-cell monoclonality are at a significantly higher risk for relapse. In contrast, our data suggest that the PCR might identify patients with continuous polyclonal states, who may be cured from their disease. Based on these data, we suggest that patients who are consistently PCR negative (in three or more consecutive analyses) for the detection of monoclonal B cells may enter a less intense follow-up protocol with fewer endoscopies, whereas patients with ongoing monoclonal PCR should be monitored more closely. This stratification could potentially increase the acceptance and safety of this treatment, which is still to be considered experimental.

APPENDIX
The following physicians participated in the German Mucosa–Associated Lymphoid Tissue Lymphoma Study: D. Allmendinger, Esslingen; W. Bachmann, Kronach; F. Baghdadi, Neustadt; J.H. van de Berg, Burglengenfeld; W. Berges, Aachen; P. Bethke, Eutin; G. Blüm, Goch/Niederrhein; T. Bock and T. Bock, Wittenberge; J. Bode, Stuttgart; D.Böhm, Brandenburg; T. Botzkurt, Koblenz; R. Breuer, R. Keuser, and G. Hermesdorf, Koblenz; R. Bucher, Zorneding; A. Buteanu and D.-N. Bujorica, Heilbronn; H. Diehl, Diepholz; R. Dyck, W. Dillman, and K. Metz, Aschaffenburg; C. Endres, Lehnin; W. Fortelny, Waldsassen; J. Freise, Mülheim; G. Freudenhammer, Köln-Merheim; M. Frevel, Bitburg; K.-P. Gail, Heppenheim; B. Geiter, Karlsruhe; U. Gieseler, Speyer; H.-J.Giesen, L. Schmitz, and M. Besta, Korschenbroich; H. Gigglberger, Regensburg; M. Greiner, Dorsten; R. Gugler, Karlsruhe; S. Hierlmeier, Kelheim; J. Hotz, Celle; W.-H. Itzen, Norden; L. Jostarndt, Dortmund; G. Judmaier, Innsbruck, Austria; A. Jung, W. Lupberger, and W. Deiß, Augsburg; M. Kaps, München; H. Klann, Passau; D. Klein and J. Habbig, Köln; R. Kobes, Werdau; H. Koppenhoefer and M. Hörner, Walldorf; W. Krämer, G. Walker, and A. Lessenich, Pirmasens; B. Krüger, Selb; W. Kühner and L. Schneider, Ebersberg; D. Leykam, Hildesheim; W. Longdong, Berlin; A. Lütke and J. Weismüller, Koblenz; Ch. Lunsken and U. Stets de Fries, Mülheim; P. Malfertheiner, Magdeburg; C. Manegold, Holzminden; J. Merkt and H. Heidt, Heilbronn; B. Mücke, Halle; G. Mülhoff, Kevelaer; B. Müller, Merseburg; W. Neuefeind, Aachen; M. Neumeyer, Oldenburg; W. Ordnung, Bad Kissingen; C. Ott, Zapfendorf;P. Otto, Burgwedel; K. Paulini and M. Karhoff, Limburg; S. Pape, Paderborn; C. Pechlivanis, Herten; G. Pommer, Oldenburg; P. Rodewyk, Dortmund; P. Rohde, Hamm; M. Sackmann, München; W. Sommer and E. Bästlein, Köln; B. Schaller and H. Kolthoff, Nagold; B. Schmack and K. Wagner, Bayreuth; K. Schmeding, Wilhelmshaven; R. Schmitz, Bergisch-Gladbach; L. Schlömann, Essen; A. Schramm, Bayreuth; H.-J. Schreiber, Bamberg; F. Schuhmacher, Schillingsfürst; H. Schulz, Bremen; H. Schweikert, Münchberg; W. Spitzer, Neustadt; W. Simon and W. Barina, Würzburg; O. Stadelmann, Fürth; M. Sulliga, Essen; J. Stumpf, Scheßlitz; H.-W. Tessen, G. Wilhelms, and C. Engelhardt, Goslar; R. Teupke, Osterode; F. Trump, Pegnitz; A. Utzig, Limburg; S. Yücel, Holzminden; J. Zeus and J. Schenk, Erlangen.

	ACKNOWLEDGMENTS

 
Supported in part by grant no. 70-225 I from the Deutsche Krebshilfe, Bonn, Germany.

We thank Manuela Neumann and Cornelia Grosse for expert technical assistance. Julia Roebekke provided excellent help in data management. We appreciate the help of the physicians (Appendix) and their patients who participated in the clinical study.

	NOTES

 
Presented in part at the 1999 Annual Meeting of the American Gastroenterological Association, Orlando, FL, May 15-19 and at the Forty-First Annual Meeting of the American Society for Hematology, New Orleans, LA, December 3-7, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted January 10, 2000; accepted December 7, 2000.

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