Microsatellite instability in gastric MALT lymphomas and other associated neoplasms

Annals of Oncology, Jul 1999

Background: Microsatellite instability (MSI), caused by a reduced efficacy of the DNA mismatch repair (MMR) machinery, represents a type of genomic instability frequently detected in HNPCC spectrum cancers and in a subset of sporadic carcinomas. The involvement of MSI in the pathogenesis of gastric lymphoma of mucosa-associated lymphoid tissue (MALT) has never been conclusively investigated. In this study, we tested the presence of MSI in tumor samples of patients harboring both MALT lymphomas and other types of malignancies. Materials and methods: We examined 10 microsatellite loci (D3S11, D3S1261, D3S1265, D6S262, D6S193, BAT-26, BAT-25, D17S250, APC, D2S123) out of a total of 34 primary tumors from 14 patients with MALT lymphomas and one or more additional neoplasms. The patients' MSI results were also tested for an association with a positive family history of cancer. Results: MSI, defined by the presence of microsatellite alterations in more than 40% of the examined loci, was scored negative in all tumors studied, and pedigree analysis failed to identify any condition of familial cancer among the patients examined. Conclusions: The present study suggests that defects in DNA mismatch repair do not contribute significantly to the molecular pathogenesis of MALT lymphomas and associated neoplasms.

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Microsatellite instability in gastric MALT lymphomas and other associated neoplasms

D. Furlan 0 F. Bertoni R. Cerutti 0 M. Taborelli 0 G. Pinotti E. Roggero F. Cavalli M. Bonato 0 E. Zucca C. Capella 0 0 Dipartimento di Scienze Cliniche e Biologiche e Servizio di Anatomia Patologica, Universila dell'Insubria e Ospedale di Circolo, Varese; 'Istituto Oncologico delta Svizzera Italiana, IOS1, Divisione di Oncologia Medica, Ospedale San Giovanni , Bellinzona , Switzerland 1 Centro inlerdisciplinare di Oncologia, Ospedale di Circolo , Varese , Italy Summary Background: Microsatellite instability (MSI), caused by a reduced efficacy of the DNA mismatch repair (MMR) machinery, represents a type of genomic instability frequently detected in HNPCC spectrum cancers and in a subset of sporadic carcinomas. The involvement of MSI in the pathogenesis of gastric lymphoma of mucosa-associated lymphoid tissue (MALT) has never been conclusively investigated. In this study, we tested the presence of MSI in tumor samples of patients harboring both MALT lymphomas and other types of malignancies. Materials and methods: We examined 10 microsatellite loci (D3S11, D3S1261, D3S1265, D6S262, D6S193, BAT-26, BAT25, D17S250, APC, D2S123) out of a total of 34 primary tumors from 14 patients with MALT lymphomas and one or more additional neoplasms. The patients' MSI results were also tested for an association with a positive family history of cancer. Results: MSI, defined by the presence of microsatellite alterations in more than 40% of the examined loci, was scored negative in all tumors studied, and pedigree analysis failed to identify any condition of familial cancer among the patients examined. Conclusions: The present study suggests that defects in DNA mismatch repair do not contribute significantly to the molecular pathogenesis of MALT lymphomas and associated neoplasms. Introduction B-cell lymphomas of mucosa-associated lymphoid tissue (MALT) comprise a distinct clinico-pathological entity among the extra-nodal non-Hodgkin's lymphomas [1]. Gastric lymphoma is the most common type of MALT lymphoma and there is strong evidence that Helicobacter pylori infection is critical in the pathogenesis of this tumor [2]. However, it is not clear whether H. pylori alone causes the evolution from chronic gastritis to MALT lymphoma, or if other factors interfering with pathogenetic mechanisms of these tumors are required. The genetic background underlying the pathogenesis of MALT lymphoma is still poorly understood. Some authors have identified trisomy 3 as the most frequent numerical chromosome aberration in lowgrade MALT lymphomas [3-6]. In a recent report Ott et al. [7], studying a large series of low- and high-grade MALT lymphomas with a sensitive bicolor fluorescence in situ hybridization (FISH), identified aneusomies of chromosomes 3, 7, 12 and 18 only in high-grade cases. In addition, reciprocal translocation such as t(ll;18)(q21;q21) [8] and genomic rearrangement of bcl-6 [9] have been reported to be the most common aberrations in low-grade MALT lymphomas. In 1993, the discovery of microsatellite instability (MSI) in colorectal cancer and its link to hereditary nonpolyposis colorectal cancer (HNPCC) highlighted a new oncogenic mechanism [10]. MSI, defined as the presence of tumor-associated alterations in the germline size of microsatellite repeats, is the result of a reduced efficacy of the DNA mismatch repair (MMR) machinery. MSI has also been found in a wide variety of human sporadic cancers [11-13] and may be the most likely cause of multiple primary tumors in non-HNPCC patients [14]. Although MSI is rarely involved in lymphomas [15,16], Peng et al. in 1996 [17] reported a surprisingly high frequency of MSI in both low- (50%) and high- (56%) grade MALT lymphomas and suggested that MMR defects may cause the accumulation of mutations in cancer-related genes such as TP53 [17]. Du et al. [18] proposed that MSI may also explain the increased incidence of additional tumors (such as carcinomas or other types of lymphomas) observed in patients with MALT lymphomas [19] and represent additional evidence that MALT tumors are a distinct clinico-pathological and genetic entity [20]. In sharp contrast to these findings, other studies [21, 22] failed to identify MSI as a frequent aberration in gastric MALT lymphomas. The aim of this work was to define the frequency of Rinopharynx (68) Stomach (68) Lymph node (75) Stomach (75) Stomach (69) Stomach (52) Stomach (48) Stomach (62) Stomach (55) Rinopharynx (48) Stomach (70) Adrenal gland (62) LungDLBCL(71) Oropharynx DLBCL Breast DCa (43) Pancreas DCa (48) Breast DCa (64) Bladder PTCa (62) Larynx Ca (50) Rectum Ca (59) Stomach Ca (70) Stomach Ca (62) Bilateral kidney PCa (62) Patient MALT lymphoma site MALT lymphoma grade Additional lymphomas Additional carcinomas Abbreviations: LG - low-grade MALT lymphoma; HG - high-grade MALT lymphoma; DLBCL - diffuse large B-cell lymphoma; DCa - ductal carcinoma; PTCa - papillary transitional carcinoma; Ca - carcinoma; PCa - papillary carcinoma; CCa - clear cell carcinoma. The ages of tumor onset are indicated in brackets. MSI in tumor samples of patients harboring both MALT lymphomas and other types of malignancies. According to the literature, these patients with different multiple malignancies might have an acquired and/or inherited risk for defects in their DNA mismatch repair systems [14]. The patients' MSI results were also tested for an association with a positive family history. Materials and methods Case selection and histological investigations A total of 34 primary tumors from 14 patients with MALT lymphomas and one or more additional malignancies from which material for MSI analysis was available were selected from a consecutive series of MALT lymphomas observed in our institutions betweeen 1986 and 1998 (Table 1). The clinical data of some of the patients were analysed in a previous paper [19]. All patients were from a geographic area that is characterized by a high incidence of MALT lymphomas (southern Switzerland and northern Italy). Microdissection and DNA extraction For each patient. 10 um-thick sections were obtained from paraffin blocks of formalin-fixed tumor tissues and from at least two blocks of histologically normal tissue including gastric mucosa, bone-marrow, lymph nodes and spleen. We performed a careful dissection of all of the pathology specimens under microscopic guidance to ensure the presence of at least 70% of malignant cells in the selected area. Genomic DNA was obtained by xylene deparaffinization followed by proteinase 1C digestion and purification by MicroSpin S-200 Columns (Pharmacia Biotech, Italy). Microsatellite markers and MSI analysis We analyzed MSI status by examining two mononucleotide and eight dinucleotide microsatellite markers: five of these (D3SI1, D3S1261, D3S1265, D6S262) were selected for the high rates of instability in MALT lymphomas reported by Peng et al. [17] and for chromosome location (D6S193; [23]). In addition, in accord with the recent guidelines for MSI analysis [24], we included in our study the panel of markers (BAT-26, BAT-25, D17S250, APC, D2SI23) recommended by the National Cancer Institute Workshop of Bethesda 1997 [25] for their high sensitivity and specificity in the detection of MSI tumors. The PCR mixture (50 ul) included 100-200 ng of genomic DNA, 10 mM Tris. 50 mM KC1, 1-2 mM MgCl2, 200 uM dNTPs and 50 pmol of each primer, 2U Taq Polymerase (Perkin-EImer, Italy). Samples were denatured at 94 "C for 5 minutes followed by 35 cycles of denaturation (94 =C, 30'), annealing (55 C, 30'), extension (72 C, 30') and a final extension of 5 minutes at 72 C in a PerkinEImer Gene Amp Thermal 2400. PCR products were electrophoresed on 6% denaturing polyacrilamide sequencing gels for two hours at 1600 V, 40 mA, 50 "C. Polyacrilamide gels, fixed on glass plates with methacryloxypropyltrimethoxysane, were silver-stained using the DNA Silver Staining System (Promega, Italy). Silver-stained gels were reproduced on EDF films (Kodak, USA). Larynx Ca Rectum Ca Stomach Ca Stomach Ca Stomach Ca D3S1265 D6S262 Pedigree analysis A three-generation family history of cancer was analyzed during the genetic counselling of all 14 patients. Results Fourteen patients with MALT lymphomas and one or more additional malignancies (Table 1) were tested for repeat instability by microsatellite analysis. A total of 34 tumors (21 MALT lymphomas, 11 carcinomas, 2 diffuse large B-cell lymphomas) were studied at 10 microsatellite loci including two mononucleotide repeats and eight dinucleotide repeats. The results of microsatellite analysis are summarized in Table 2 and representative cases are illustrated in Figures 1 and 2. Microsatellite alterations at three loci on chromosome 3 were observed in both gastric MALT lymphoma and lung DLBCL of patient 5 and a novellength allele for a single locus was detected in the gastric MALT lymphomas of patients 4, 9 and 10. In one carcinoma (a papillary transitional carcinoma of the urinary bladder) size alterations at loci D2S123 and D3S1265 were observed (case 10, Table 2). All microsatellite alterations showed only a single new band resulting from slight expansion or contraction of the original allele and were classified as type II according to Thibodeau et al. [26]. None of the MALT or associated tumors exhibited alterations at more than 40% of the ten loci tested and, as a consequence, MSI was scored negative in all tumors studied according to the Bethesda guidelines [25]. In contrast, according to the criteria for MSI adopted by reported in other families but no typical association with well known familial cancer syndromes was identified. Discussion Italy E-mail:


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D. Furlan, F. Bertoni, R. Cerutti, M. Taborelli, G. Pinotti, E. Roggero, F. Cavalli, M. Bonato, E. Zucca, C. Capella. Microsatellite instability in gastric MALT lymphomas and other associated neoplasms, Annals of Oncology, 1999, 783-788,