1 Departement de Pathologie, 2 Service d'Hépato-Gastroentérologie, 3 Service d'Hématologie Clinique, 4 Service d'Immunologie Biologique, Hôpital Henri Mondor, AP-HP, 51 avenue du Maréchal de Lattre de Tassigny, 94 010 Créteil, France
* Correspondence to: Dr C. Copie-Bergman, Département de Pathologie, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94 010 Créteil, France. Tel: +33-1-49-81-2728; Fax: +33-1-49-81-2733; Email: christiane.copie{at}hmn.ap-hop-paris.fr
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Abstract |
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Patients and methods: Gastric biopsies and gastrectomy resection specimens of four patients who developed GML and early gastric cancer (EGC) were analysed by morphology, immunohistochemistry and molecular biology.
Results: Four patients (three males and one female; mean age 48 years) were diagnosed with GML. Helicobacter pylori infection was observed in three cases. Two patients had localized disease (stages IE and IIE, respectively) and were treated with H. pylori eradication therapy followed by an alkylating agent for one patient. Two patients had disseminated disease (stage IV), and were treated with an alkylating agent. Three cases were t(11;18) positive. All patients achieved initially complete lymphoma remission. Long-term endoscopic surveillance detected an EGC at the same location as the lymphoma in all patients at a mean time of 9.5 years (range 2.517 years) after lymphoma diagnosis. Gastrectomy specimens showed residual GML in all cases.
Conclusion: Prolonged residual GML could constitute an additional risk factor for the development of gastric carcinoma. Long-term endoscopic surveillance is mandatory in patients treated conservatively for gastric MALT lymphoma.
Key words: gastric cancer, gastric MALT lymphoma, Helicobacter pylori, translocation t(11;18)
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Introduction |
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Helicobacter pylori infection is an important risk factor for the development of gastric carcinoma. Gastritis induced by H. pylori causes atrophy and intestinal metaplasia, the precursor lesions of intestinal type of gastric carcinoma. Eradication of H. pylori has been shown to prevent the disease in animal models [6] and high-risk patients [7
]. Helicobacter pylori probably exerts its effect over a long period of time, and whether mucosal injuries induced by this bacterium are reversible after it has been eradicated remains to be determined [8
10
]. In localized H. pylori-associated GML, eradication of H. pylori with antibiotics is now recognized as the first-line therapy of conservative management, and remission of GML is observed in 75% of cases [11
]. However, about 25% of cases are resistant to H. pylori eradication therapy, especially cases harbouring the t(11;18) (q21;q21) translocation that fuses the amino terminal of the API2 gene to the carboxyl terminal of the MALT1 gene [12
]. The clinical management of patients resistant to H. pylori eradication is still a matter of debate.
Gastric MALT lymphoma usually has an indolent natural course, but molecular studies have shown that the tumour is often multifocal and, despite apparent clinical and histological remission, neoplastic clones may persist whose significance remains to be determined [13, 14
]. Therefore long-term follow-up with multiple gastric biopsies is usually recommended.
In this study we report four patients with GML treated with a conservative approach (antibiotics and/or an alkylating agent) who subsequently developed EGC after a mean time of 9.5 years (range 2.517 years) despite H. pylori eradication.
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Materials and methods |
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Histology and immunohistochemistry
Diagnostic material consisted of sequential gastroscopic biopsies and gastrectomy specimens for all patients. Tissue specimens were fixed in buffered formaldehyde or alcoholformalinacetic acid (AFA) fixative, embedded in paraffin and routinely processed for histological studies. When available, gastric biopsy specimens were snap-frozen in liquid nitrogen for genotypic studies. Haematoxylin and eosin stained sections of all cases were reviewed. Diagnosis of GML was made according to the criteria of Isaacson [15]: presence of a diffuse infiltrate of the lamina propria by CD20+ CD5 centrocyte-like cells associated with lymphoepithelial lesions and reactive lymphoid follicles. Presence of H. pylori was assessed on modified Giemsa stained sections. Immunohistochemical studies were performed on paraffin sections using the Ventana automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA), their appropriate diaminobenzidine (DAB) detection kit and antibodies for CD20/L26, CD3, Bcl2, Mib1, (Ki67), CD5, CD10, CD23 (Novocastra Laboratories Ltd, Newcastle upon Tyne, UK) and cytokeratin (KL1, Immunotech-Beckman Coulter, Miami, FL, USA). Where appropriate, the sections were pretreated by heating in a microwave oven to facilitate antigen demonstration.
Helicobacter pylori serologies and Cag status
Serologies from three patients were obtained from stored serum samples that had been collected at the time of diagnosis. Serum IgG antibodies against H. pylori were detected by ELISA (Enzygnost, Berthing). Antibodies against CagA antigen were detected by western blotting using a commercially available kit (Helico Blot 2.0, Genelabs) as previously described [16].
Molecular biology
For immunoglobulin gene rearrangement studies, DNA was extracted from frozen gastric biopsies when available, or from material fixed in formalin and embedded in paraffin wax, by standard proteinase K digestion and a phenolchloroform extraction procedure. Immunoglobulin gene rearrangement studies were carried out using PCR as previously described [17].
Detection of the API2MALT1 fusion transcript was performed using RTPCR. Total RNA was extracted from frozen tissue sections and reverse transcribed with superscript II (Gibco-BRL). cDNA was amplified with ß-actin primers to verify the RNA quality and reverse transcription efficiency, and with different API2 MALT1 primer sets in parallel with known positive and negative controls [18].
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Results |
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One year later, follow-up gastric biopsies showed recurrence of the disease and revealed low-grade dysplasia of the antrum. In October 2000, 30 months after the initial diagnosis of GML, endoscopy revealed an ulcer of the antrum. EUS showed a moderate thickening of the gastric wall (5 mm) without a lymph node. Histological examination of gastric biopsies revealed high-grade dysplasia. A total gastrectomy was performed. Histological analysis of the surgical specimen disclosed an EGC of intestinal type, limited to the mucosa, developed in an area of severe chronic atrophic gastritis and intestinal metaplasia. Residual MALT lymphoma was observed beneath the adenocarcinoma throughout the antrum but sparing the corpus. Genotypic studies of frozen gastric biopsies showed clonal rearrangement of the immunoglobulin gene. The t(11;18) MALT lymphoma fusion transcript was not detected by RTPCR in frozen gastric biopsies. Three years after the gastrectomy, the patient is well without recurrence of disease.
Patient 2
A 65-year-old woman was diagnosed with H. pylori-associated GML in July 1998. She had a past history of gastric ulcer since 1960. Gastric biopsies of adjacent mucosa did not show any intestinal metaplasia nor glandular atrophy. Molecular studies showed clonal rearrangement of the immunoglobulin gene, and the t(11;18) fusion transcript was detected retrospectively in a frozen tumour sample taken in 1999. She did not respond to H. pylori eradication therapy and was treated with an alkylating agent for a period of 1 year. Follow-up gastric biopsies disclosed minimal residual disease for 2 years but she relapsed in January 2002, when intestinal metaplasia and low-grade dysplasia of the corpus were observed.
Because of massive gastric bleeding she underwent a total gastrectomy and splenectomy in December 2002. GML was observed in the deep part of the corpus and extended throughout the gastric wall. An EGC of intestinal type limited to the mucosa was diagnosed, overlying the lymphoma. Eighteen months after the gastrectomy, she remains well without recurrence of the disease.
Patient 3
A 40-year-old man presented in June 1983 with dyspepsia. Endoscopy showed multiple ulcers of the corpus and H. pylori-associated GML was diagnosed. Gastric biopsies of adjacent mucosa did not show any intestinal metaplasia or glandular atrophy. The staging procedure demonstrated lung involvement. The bone marrow was not involved. The patient received single-agent chemotherapy with an alkylating agent for 2 years and achieved complete clinical and histological remission.
Follow-up endoscopy showed focal gastric relapse in 1987. The disease remained stable until June 1992 where X-ray examination disclosed pulmonary opacities. The patient received an additional course of an alkylating agent for 1 year and was in complete remission at the end of the treatment. Gastric relapse with H. pylori infection was demonstrated in May 1995. The patient received anti-H. pylori therapy with successful eradication but only partial remission of the lymphoma.
In July 1996, 13 years after the initial diagnosis of GML, endoscopic control showed a polypoid mass of the corpus and diffuse thickened mucosal folds. EUS showed a moderate thickening of the gastric wall with no detectable lymph node. Histological examination of gastric biopsies revealed high-grade dysplasia. The patient underwent a total gastrectomy. Analysis of the surgical specimen disclosed an EGC of intestinal type limited to the mucosa in a area of chronic atrophic gastritis with proliferative intestinal metaplasia overlying MALT lymphoma (Figure 1). Disseminated GML was found in the antrum, the corpus (Figure 2) and the duodenum. Genotypic studies performed on initial frozen gastric biopsies showed clonal immunoglobulin gene rearrangement. RTPCR analysis of frozen gastric biopsies taken in 1987, 1992 and 1996 demonstrated the presence of the MALT-lymphoma specific t(11;18) fusion transcript. Eight years after the gastrectomy, the patient remains well without recurrence of disease.
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Eleven years later, in November 1990, endoscopy and gastric biopsies showed similar features. Staging procedure demonstrated bone marrow and lung involvement. The patient received monochemotherapy with an alkylating agent for 18 months and achieved complete remission.
In February 1996, 17 years after the initial diagnosis of GML, endoscopy showed a superficial ulceration of the antrum and biopsies revealed high-grade dysplasia. A total gastrectomy was performed. An EGC of intestinal type limited to the mucosa was diagnosed. Although the patient was considered in complete remission of the lymphoma, residual GML was found next to the carcinoma, extending throughout the antrum and the corpus. Genotypic studies revealed clonal rearrangement of the immunoglobulin gene. RTPCR cytogenetic analysis of frozen gastric biopsies taken in 1991 and 1995 demonstrated the presence of the MALT-lymphoma-specific t(11;18) fusion transcript. H. pylori-like microorganisms were not detected and serology was negative. Eight years after the gastrectomy, the patient is well and has no recurrence of the disease.
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Discussion |
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Helicobacter pylori is recognized as a carcinogen and is involved in the development of both EGC and GML. In our series, three patients were H. pylori positive and one patient was H. pylori negative. Despite eradication of H. pylori, all patients developed EGC after a mean of 9.5 years (range 2.517 years). Only one patient (patient 1) in our series showed premalignant histological changes of the adjacent mucosa at the time of lymphoma diagnosis. These were characterized by mild intestinal metaplasia without glandular atrophy. Recent studies have shown that H. pylori eradication significantly retards progression of intestinal metaplasia, but 2.3% of H. pylori-infected patients may still develop gastric cancer, whether or not the infection has been eradicated [10]. In addition, Wong et al. [19
] showed that prevention of gastric cancer by H. pylori eradication in high-risk population is only significantly efficient in patients who did not have any precancerous lesions at inclusion. Therefore it is possible that ECG could have occurred in patient 1 irrespective of the presence of GML. Interestingly, patients 2, 3 and 4 developed gradually extensive intestinal metaplasia and glandular atrophy, although patient 4 was H. pylori negative. Additional factors independent of H. pylori probably contributed to the development of premalignant histological changes and subsequent EGC in these cases.
Variable virulence factors of H. pylori strains such as CagA may reinforce the risk of cancer in infected patients [20]. Interestingly, although we previously demonstrated a low CagA prevalence in our series of GML [16
], H. pylori strains of patients 1 and 2 were found to harbour the CagA pathogenicity island that is known to be significantly associated with an increased risk of gastric cancer [21
]. However, the number of reported cases in the present study is too small to allow definite conclusions to be drawn.
It is also possible that alkylating agents exert a carcinogenic effect; however, one patient (patient 1) in our series and the three cases reported by Morgner et al. [5] did not receive chemotherapy. Long-term persistent GML may favour the development of chronic atrophic gastritis, intestinal metaplasia which then progresses to intraepithelial neoplasia, and could represent an additional risk factor for the development of EGC. It is noteworthy that in our series, EGC developed at the same location as the lymphoma several years before, and that residual MALT lymphoma was observed in the deep mucosal layers of all gastrectomy specimens, although one patient was considered to be in complete remission for 6 years (patient 4). Most cases in our series harboured the GML-specific t(11;18) translocation. Although this is a small series, these data suggest that t(11;18)-positive cases may be more likely to have residual GML in the deeper layers of the stomach, which may be underestimated by upper conventional endoscopic biopsies, and may be more at risk of subsequent development of an EGC. In addition, these observations show that although two patients (patients 3 and 4) had stage IV t(11;18)-positive GML, they lived for a prolonged period of time with residual disease (up to 17 years) and had an indolent clinical course.
Treatment of GML has been controversial. It is now widely accepted that a conservative approach may provide an alternative to gastrectomy which is associated with significant morbidity. Antibiotic therapy of localized H. pylori-associated GML is advisable, and in cases of antibiotic failure single-agent chemotherapy is an efficient alternative therapy in some patients [22]. However, the present data show that, despite apparent clinical and histological response to H. pylori eradication and/or chemotherapy with an alkylating agent, residual MALT lymphoma may persist for several years in the deep part of the mucosa and may favour the occurrence of gastric carcinoma.
In conclusion, these observations show that GML is an indolent disease that may persist at the site of origin for a prolonged period of time. Persistent residual disease may represent an additional risk factor for the development of gastric carcinoma. In our opinion, development of conservative treatments that aim to induce complete remission without any residual disease should be considered in order to prevent cancer development, and long-term endoscopic follow-up with multiple gastric biopsies is mandatory.
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Acknowledgements |
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Received for publication July 22, 2004. Revision received March 3, 2005. Accepted for publication March 9, 2005.
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