Detection of partial trisomy 18q21 in cell nuclei isolated from one case with low grade marginal zone lymphoma of the mucosa associated lymphoid tissue (MALT) type by dual colour fluorescence in situ hybridisation (FISH) using MALT1 flanking probes. In red, phage 1 artificial chromosomes (PACs) 83A16 and 119K19 proximal to MALT1; in green, PACs 628B12 and 124N11 distal to MALT1. Several DAPI stained nuclei (blue) with three copies of MALT1 (mixed red/green or yellow signal) are shown, whereas FISH with the centromere 18 probe on nuclei from this same case showed normal disomy (not shown). 

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... patients presented with stage I disease, 15 with stage II, and two with stage IV disease. Apart from surgical resection, one patient was treated with radiotherapy, 16 with chemotherapy (plus Helicobacter pylori eradication in two cases), and two with both chemotherapy and radiotherapy (table 1). Of the six patients with low grade marginal zone lymphoma, three received chemotherapy according to COP and two patients according to COP/BLAM protocols. One patient was treated with cyclophosphamide, vincristin, and prednisolone. The CHOP regimen was administered to all 12 patients with GI DLBCL. Two of these patients were also treated with mitoxantron/prednimustin during the course of their disease. Immunohistochemical characterisation was performed as reported previously. 14 For fluorescence in situ hybridisation (FISH) analysis, we prepared single nuclear suspensions from 20 m m sections of the tumour blocks. After dewaxing with xylene and rehydration in a graded series of ethanol, four sections were digested in 0.005% proteinase K, pH 7.5, for 30–60 minutes at 37 ̊ C and mechanically disaggregated by pressing through a nylon mesh. The isolated nuclei were sedimented by centrifugation, washed, and suspended in phosphate buf- fered saline, and finally spotted on to slides and air dried. Sections were washed for 30 minutes in 2 6 saline sodium citrate at 37 ̊ C and dehydrated in ethanol before hybridisation. For interphase cytogenetic analysis, a centromere specific probe for chromosome 18 (QBIOgene, Illkirch, France) and an 18q21 specific probe set with four phage 1 artificial chromosome (PAC) clones flanking MALT1 were used in each case. The dual colour probe set consisted of the red labelled PACs 83A16 and 119K19, which map proximally to MALT1, and the green labelled PACs 628B12 and 124N11, which map distally. This probe set allowed the highly sensitive detection of trisomies (three yellow or mixed signals), in addition to rearrangements (separated red and green signals) involving MALT1, indicating the occurrence of t(11;18), t(14,18), or variants. 17 18 Hybridisation was performed according to standard methods. Experiments were evaluated using an epifluorescence microscope connected to a CCD camera. In each case, at least 100 cell nuclei were counted. As controls, five samples from normal gastric mucosa and 10 cases of H pylori associated gastritis were studied. Statistical analysis was performed using SPSS software for Windows, version 9.0. Correlations between trisomy 18 and stage were analysed by means of the Pearson x 2 test. Overall survival (OS), disease specific survival (DSS), and failure free survival (FFS), defined as the time from the primary diagnosis to the last follow up or death, to death because of lymphoma, and to disease relapse or death because of lymphoma, respectively, were analysed by means of the Kaplan–Meier method and compared by the log rank test. 19 Cox regression analysis was performed including the variables age, stage, grade, treatment, and trisomy 18q21 (including all cases with trisomic status at the MALT1 locus independent of whether derived from complete or partial trisomy 18). p Values , 0.05 were considered significant. 20 The presence of complete and partial trisomy 18 was studied by interphase FISH applying a centromere 18 probe and a dual colour probe mix for the MALT1 gene at chromosome band 18q21. In five samples of normal gastric mucosa and 10 samples of H pylori associated gastritis, more than two fluorescence signals for the MALT1 probe mix or the centromere 18 probe were detected in a mean of 3.4% (SD, 3.7%) and 0.9% (SD, 1.6%) of the interphase nuclei, respectively. The cut off values for both probes, defined as mean of false positive + 3 SD, was 14.5% and 5.7% for the centromere 18 and MALT1 probes, respectively. Based on these results, and assuming a tumour cell content of at least 50% in all samples based on morphological assessment for both probes, a cut off value of 15% of nuclei with more than two signals was set for the detection of complete or partial trisomy 18 to prevent false positive results. Only one case of low grade marginal zone lymphoma of MALT showed a break apart pattern with the MALT1 probe, probably because of a t(11;18) translocation. Of the remaining 29 GI B cell lymphomas studied, seven showed at least 23% of cells with a signal pattern indicating trisomy 18(q21) and, thus, clearly above the detection limit. A mean of 40% (range, 17–55%) and 46% (range, 23–73%) of the nuclei displayed more than two signals for the centromere 18 and the MALT1 probes, respectively. The seven cases with trisomy 18(q21) comprised two of the 10 low grade marginal zone lymphomas and five of the 19 GI MALT lymphomas with a diffuse large B cell component, indicating a similar frequency of 18q21 gains in both morphological subtypes. Four and three cases displayed signal patterns indicating complete trisomy 18 or partial trisomy 18q21 including the MALT1 locus (fig 1), respectively. In one case with super- numerary signals for the centromere 18 probe, greatly increased numbers of nuclei with three signals for the MALT1 probe suggested coexistence of cells with complete and partial trisomy 18. FISH with centromere and locus specific probes for other chromosomes (CEP3, CEP7, MYC (8q24), CEP12, IGH (14q32), and TP53 (17p13)) ruled out hyperploidy as the cause of the trisomy 18(q21) in the seven cases with increased copy numbers of the MALT1 locus (data not shown). Trisomy of the MALT1 locus, derived from either complete or partial trisomy 18, was associated with disease stage, although the results did not reach significance (p = 0.051); six of 17 patients who presented with at least stage II disease were trisomic, but only one of 12 patients with stage I disease was trisomic. The median follow up time of the complete study population was 56 months (mean, 69; range, 1–290). During the period evaluated, 16 patients died, eight from the underlying GI lymphoma and eight from other causes without clinical evidence of residual lymphoma. One patient with active disease was alive after 78 months and the remaining patients were alive without evidence of disease (table 1). Increased copy number of the MALT1 locus did not influence FFS (p = 0.153) or OS (p = 0.469) when compared by means of the log rank test, but was significantly associated with a decreased DSS (DSS for patients with a normal copy number of the MALT1 locus, 82%; mean survival time, 234 months; 95% confidential interval (CI), 184 to 284 months; DSS for patients with an increased copy number of the MALT1 locus, 43%; mean survival, 83 months; 95% CI, 23 to 144 months; p = 0.0458; fig 2A). There was no association between increased MALT1 gene dosage and decreased DSS time in the subgroup of low grade marginal zone lymphoma of MALT type (p = 0.722), although such an association was seen in the subgroup of GI lymphomas with large cell component—that is, de novo extranodal DLBCL and low grade marginal zone lymphoma with transformation into DLBCL (DSS for patients with a normal copy number of the MALT1 locus, 86%; mean survival time, 245 months; 95% CI, 189 to 302; DSS for patients with an increased copy number of the MALT1 locus, 40%; mean survival time, 77 months; 95% CI, 5–149 months; p = 0.0447; fig 2B). Applying Cox regression analysis to detect the independent prognostic value of age, stage (I/II v III/IV), grade (low v high), treatment (surgical resection v surgical resection + chemotherapy), and trisomy at the MALT1 locus with regard to OS, FFS, or DSS, only stage with regard to OS was significant (relative risk, 4.793; p = 0.029). Nevertheless, because of the small population size, the power of these Cox regression analyses was restricted and results have to be interpreted with caution. Our study detected an unfavourable prognosis for patients with trisomy 18(q21) including the MALT1 locus in surgically resected t(11;18) negative GI B cell lymphomas using FISH analysis. In particular, GI B cell lymphomas with a large cell component showed a decreased DSS compared with patients with classic low grade marginal zone lymphoma of MALT type. Most frequently, trisomy 18(q21) was found in stage II of the disease. The t(11;18) translocation occurs in approximately 30% of cases of low grade MALT lymphoma according to recent series, 1–9 but in our entire group of 11 patients with low grade marginal zone lymphoma only one carried a break point in the MALT1 locus, which is in agreement with the findings of Barth et al (12%). 11 Interestingly, studies with a higher frequency of pulmonary marginal zone lymphoma showed an increased rate of up to 48%. 13 An approximately equal sensitivity between FISH analysis and reverse transcriptase polymerase chain reaction was found when both techniques were compared. 2 9 This was confirmed in our study by external control examinations (data not shown). Wotherspoon and co-workers were the first to report increased occurrence of trisomy 18, as detected by interphase FISH, in nine patients in a large series of 70 classic low grade marginal zone lymphoma of MALT type. 21 In the GI tract, the authors detected trisomy 18 in 27% of patients. These findings were extended by other authors to extranodal DLBCL. 12 21–23 In addition, comparative genomic hybridisation analysis also revealed chromosome 18 aberrations in extranodal marginal zone lymphomas and extranodal DLBCL, but to a lesser extent. 11 In a careful analysis with discrimination of t(11;18) positive lymphomas, an increased trisomy 18 rate of 48% was reported for pulmonary lymphomas, similar to the t(11;18) translocation and to the initial data of Wotherspoon. 13 21 We examined lymphomas of the GI tract exclusively and found chromosome 18 aberrations in almost one quarter of the samples, with approximately equal percentages in low grade marginal zone lymphomas (two of 10) and in GI B cell lymphomas with a large cell component ...