Chromosome instability and translocation t(11;18) in primary gastric marginal zone B-cell lymphoma of MALT-type

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1 Hematological Oncology Hematol. Oncol. 2007; 25: Published online 2 July 2007 in Wiley InterScience ( Research Article Chromosome instability and translocation t(11;18) in primary gastric marginal zone B-cell lymphoma of MALT-type Maria Grazia Tibiletti 1 *, Katia Milani 1, Vittoria Martin 1,3, Emanuele Zucca 4, Teresio Motta 6, Sergio Cortelazzo 7, Graziella Pinotti 2, Luca Mazzucchelli 3, Giancarlo Pruneri 8, Giovanni Martinelli 9, Renzo Barbazza 10, Carlo Capella 1 and Francesco Bertoni 5 On behalf of the International Extranodal Lymphoma Study Group (IELSG) 1 Anatomic Pathology Unit, University of Insubria, Ospedale di Circolo, Varese, Italy 2 Oncology Unit, University of Insubria, Ospedale di Circolo, Varese, Italy 3 Istituto Cantonale di Patologia, Locarno, Switzerland 4 Lymphoma Unit, Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland 5 Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland 6 Anatomia Patologica e Citologia, Ospedali Riuniti, Bergamo, Italy 7 Ematologia, Ospedali Riuniti, Bergamo, Italy 8 Division of Pathology, Laboratory Medicine, European Institute of Oncology, Milan, Italy 9 Department of Haematoncology, European Institute of Oncology, Milan, Italy 10 Servizio di Anatomia Patologica, Ospedale Civile di Feltre, Feltre, Italy *Correspondence to: Dr Maria Grazia Tibiletti, U.O. Anatomia Patologica, Ospedale di Circolo-Università dell Insubria, Viale Borri 57, Varese, Italy. mgtibiletti@hotmail.com Received: 16 April 2007 Revised: 30 May 2007 Accepted: 1 June 2007 Abstract The prognosis for patients with mucosa-associated lymphoid tissue (MALT) lymphomas is good; these tumours have usually an indolent course with overall survival rates that are greater than 80% at 5-year, but some rare cases with histological transformation in aggressive diffuse large cell lymphoma have also been diagnosed. Here, we present cytogenetic results on endoscopic bioptic material of 42 cases of primary gastric extranodal marginal zone B-cell lymphoma (EMZL) using fluorescence in situ hybridization (FISH) approach with API2, MALT1 and centromeric probes for chromosome 3 and 18, and their impact on the clinical outcome. Copyright # 2007 John Wiley & Sons, Ltd. Keywords: API2; MALT1; antibiotics; FISH; stomach; MALT; marginal zone Introduction The stomach is the most common primary site of extranodal marginal zone B-cell lymphoma (EMZL) of mucosaassociated lymphoid tissue (MALT), also known as MALT lymphoma [1,2]. Over 10 years ago Helicobacter pylori has been identified as an etiologic factor in gastric MALT lymphomas following the demonstration of the tumour regressions in the majority of early-stage cases treated with anti-helicobacter antibiotic therapy. The prognosis for patients with MALT lymphomas is good; these tumours have usually an indolent course with overall survival rates that are greater than 80% at 5-year, but rare cases with histological transformation in aggressive diffuse large cell lymphoma have also been diagnosed. Eradication of H. pylori with antibiotics is the standard first-line therapy as the sole initial treatment of localized gastric MALT lymphoma [2,3]. Four main recurrent chromosomal translocations have been associated with the pathogenesis of EMZLs: t(11;18) (q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21) and t(3;14) (p14.1;q32) (1,4,5,6). The latter is the most recently described and establishes the juxtaposition of the transcription factor FOXP1 next to the enhancer region of the immunoglobulin heavy chain (IgH) genes [7]; its pathogenetic role is still unclear. The other three translocations appear to affect the same signalling pathway, determining the activation of nuclear factor kappa B (NF-kB), a transcription factor with a central role in immunity, inflammation and apoptosis [1]. The t(1;14) (p22;q32) translocation is the most rare and determines an over-expression of the BCL10 gene due to the juxtaposition to the IgH promoter region. The t(14;18)(q32;q21) translocation juxtaposes the MALT1 gene (also known as MLT, MLT1) to the IgH promoter region with subsequent MALT1 over-expression [6]. The t(11;18)(q21;q21) translocation is the most common, occurring in 15 40% of cases [1,4,9 11]. It results in the reciprocal fusion of cellular inhibitor of apoptosis protein 2 (ciap2) (also known as AIP1, API2, BIRC3, HAIP1, HIAP1, MALT2, MIHC) on 11q21 with MALT1 on 18q21. The creation of the fusion protein encoded by API2-MALT1 on the derivative chromosome 11 is the pathogenetic event. In addition to these chromosomal translocations, EMZL can present additional chromosomal aberrations, often targeting chromosomes 3, 7, 12 and 18 [12 14]. Copyright ß 2007 John Wiley & Sons, Ltd.

2 Chromosome instability and translocation t(11;18) 185 Here, we present cytogenetic results on endoscopic bioptic material of 42 cases of primary gastric EMZL using fluorescence in situ hybridization (FISH) approach with API2, MALT1 and centromeric probes for chromosomes 3 and 18. Material and methods Cases of primary gastric EMZL with tissue blocks available have been selected from the participating institutions. Twenty-four cases had been enrolled in the LY03 trial [15,16]. Histologic diagnosis was made according to the Revised European-American Lymphoma (REAL)/World Health Organization (WHO) classification [17]. H. pylori infection was assessed at histologic examination (Giemsa stain or other specific methods) or by serology (presence of IgG anti-h. pylori antibodies). Histologic examination of follow-up biopsies was performed applying the score proposed by Wotherspoon et al. [18]. Score 0 2 was defined as complete response (CR). Interphasic FISH was performed on 3 mm thick paraffin blocks. The presence of tumour cells was evaluated in each tissue block on hematoxylin and eosin-stained slides cut before and after the section used for FISH analysis. FISH experiments were carried out following the method previously described by Tibiletti [19]. The probes used were the following: (1) MALT1 break-a-part strategy for screening of rearrangements; (2) API2/MALT1 dual colour dual fusion for the definition of chromosome rearrangement; (3) centromeric probes for detection of copy number changes of chromosomes 3 and 18. In an additional group of 12 primary gastric EMZL, interphasic FISH analysis was performed on isolated nuclei from 15 mm sections using centromeric probes for chromosomes 3, 6, 7, 12, 17, 18 and X. All types of probes (purchased by Oncor, Cedex, France) were directly labelled with red and green fluorochromes. In each case more than 100 nuclei were examined from at least five to eight different areas selected for wellpreserved cellular and nuclear morphology to ensure representative samples and to avoid a nuclear truncation in paraffin-embedded sections. Only experiments with 90% hybridization efficiency were considered. Each sample showing more than two cells with the same chromosome rearrangement was considered positive for the presence of translocation as recommended by the International System for Human Cytogenetic Nomenclature (ISCN) [20]. The threshold values for monosomy, trisomy and polysomy of chromosomes 3 and 18 were evaluated for each probe on a panel of paraffin embedded sections of normal gastric mucosa. The cut-off levels were calculated as the mean value plus three standard deviations of nuclei showing one and three spots, respectively; the standard deviation was calculated assuming a binomial distribution of the spots. With the same strategy the cut-off levels for the interphasic FISH on isolated nuclei were calculated on a panel of control isolated nuclei. Chromosomal instability (CIN) as referred by Lengauer et al. [21], was defined as the presence of aneuploidy involving both chromosomes 3 and 18, in contrast to normal diploid status showing disomy for both chromosomes. Binomial exact 95% confidence intervals (95%CI) were calculated for percentages using the STATA software package (Computing Resource Center, Santa Monica, CA, USA). Results Forty-two cases have been analyzed by FISH. Table 1 shows the clinical features. A t(11;18)(q21;q21) translocation was observed in seven cases (17%; 95% CI, 7 31%). Using centromeric probes for both chromosomes 3 and 18, we identified mixed cell populations showing clones with monosomies, disomies, trisomies and tetrasomies for both chromosomes in the vast majority of the cases (Table 2). In particular, thirty cases (71%; 95% CI, 55 84%) were CINþ, twelve (29%; 95% CI, 16 45%) were classified as diploid showing a disomy of both chromosomes 3 and 18. Also, only one of the seven cases bearing MALT1 translocations showed a stable karyotype. The data were validated on a subset of 12 EMZL with a panel of 5 probes (Table 3). Aneuploidies of more than three different chromosomes were observed in 9 of the 12 cases; the chromosomes more involved were 12,18 and 6. Among the CINþ cases, 23 had an antibiotic eradication therapy and 17 experienced a complete remission (74%; 95% CI, 52 90%) (Figure 1). Five (56%; 95% CI, 21 86%) complete remissions were observed among nine patients treated with antibiotics and showing disomic chromosome assessment. Extra-copies of chromosomes 3 Table 1. Clinical characteristics of the 42 analyzed cases with median age (range): 64 (34 97) years Clinical feature No. of cases % Sex Male Female Unknown 2 Stage I II 3 8 IV 2 5 Unknown 3 H. pylori infection Positive Negative 3 9 Unknown 10 First-line therapy Antibiotics Chemotherapy 1 3 Observation alone 1 3 Unknown 7 Response to antibiotics CR PR 3 9 NC 3 9 PD 2 6 Unknown 0

3 186 M. G. Tibiletti et al Table 2. Chromosomes 3 and 18 assessment and MALT1 translocation in 42 EMZL Case MALT1 translocation Chr 3 Chr 18 1 D D,T 2 D,T D,T 3 D,T,Te M,D 4 D,T M,D 5 D D,T 6 D,T D 7 D D 8 D D 9 D,T D,T 10 D D,M 11 D D 12 D D 13 D D 14 D D,T 15 D D 16 D D,T,Te 17 D D,T,Te 18 D D 19 D,T,Te D,T,Te 20 D D 21 D D 22 D,T D,T 23 M,D M,D 24 M,D M,D 25 D,T M,D 26 D D 27 D,T D 28 D,T,Te D,T 29 D D,T 30 D,T,Te D,T 31 D D,T 32 D D 33 D,T D,T 34 D,T,Te D,T 35 M,D M,D 36 t(11;18) D,T D,T,Te 37 t(11;18) D,T D 38 t(11;18) D,T D,T 39 t(11;18) D nv 40 t(11;18) D,T D,T 41 t(11;18) M,D D 42 t(11;18) D D,T D: disomy. T: trisomy. Te: tetrasomy. or 18 did not affect the response rate to antibiotics. While only one (17%, 95% CI, 0 64%) of the seven cases with MALT translocation and treated with antibiotics obtained a CR, 21 of the 28 (75%; 95% CI, 55 89%) with no translocations achieved the CR. Discussion We analyzed 42 cases of primary gastric EMZL by FISH. A t(11;18)(q21;q21) translocation was observed in seven cases (17%), a percentage within the range reported in the literature [8,9]. Using centromeric probes for both chromosomes 3 and 18 with a very strict cut-off level (mean number of observations on a panel of paraffin embedded sections of normal gastric mucosa plus three standard deviations), we showed that CIN, even if shown as trisomy of single chromosomes, is a common cytogenetic feature of EMZL: 30 cases could be defined CINþ, 12 as diploid. In contrast to what previously shown from most reports [8,9,22], our data underlined that even t(11;18)-positive cases can have additional chromosomal abnormalities. These data were validated by the FISH analyses done on isolated nuclei from a subset of cases, and they were in accordance with a recent publication of EMZL tissue microarrays studied using FISH analysis for chromosomes 1, 3, 12, 18 and X [23]. The latter work indicates that this type of neoplasia is genetically unstable showing aneuploidies of different chromosomes [23]. Since API2 MALT1 fusion and aneuploidy are not mutually exclusive events, the t(11;18) translocation per se cannot be assumed as an indicator of a stable karyotype as generally considered [8,9]. The API2 MALT1 chimeric protein has been demonstrated to amplify the activation of NF-kB transcription factors and consequently to antagonize p53 function and to sensitize the cells for genomic instability [24,25]. The identification of CIN in a large subset of EMZL suggests that disruption of cell-cycle checkpoints may be involved in the evolution of these lymphomas. Table 3. Chromosome assessment in 12 EMZL Case Chr 3 Chr 6 Chr 7 Chr 12 Chr17 Chr18 Chr X 1 m D D,T D D,T D D M,D,T,Te 2 f D Te D,T,Te D Te D,T D M,D,T M 3 m D M,D,T,Te D Te D T M,D D,T M 4 m D D,T,Te D,Te M,D,T,Te D,T D M,D 5 f D,T D,T D D,T,Te D,T D,T,Te D,Te 6 f D D,T D,Te D,T D D,T D,Te 7 m D,Te M,D D D,T D D,T M,T 8 f D D D D,T D D,T D 9 m D D,T,Te D D,T D,T D,T M 10 m D D,Te D D,T D,T,Te D,T M 11 f D,T,Te D D,Te D,T D,T D,T D,T 12 m D D D D,T D D,T M D: disomy. T: trisomy. Te: tetrasomy. m: male. f: female.

4 Chromosome instability and translocation t(11;18) 187 Figure 1. Complete remission rate in the presence or absence of chromosome abnormalities We evaluated the clinical impact of the presence of chromosomal aberrations. Extra copies of chromosomes 3 or 18 did not affect the response rate to antibiotics. In agreement with the notion that cases bearing the chromosomal translocation t(11;18) have a lower response rate to H. pylori eradication therapy [26], only one of the seven cases with MALT translocation and treated with antibiotics obtained a CR, whilst 21 of the 28 with no translocations achieved the CR. A longer follow-up will be necessary to assess the clinical significance of karyotype instability in gastric EMZL. Since our data underlined that also t(11;18)- positive cases can bear additional chromosomal abnormalities, it is worth noting that the routine use of FISH probes specifically targeting the API2/MALT1 uncoupled with the use of centromeric probes for detection of copy number change could underestimate the presence of these CIN, possibly hiding the presence of prognostically separate groups among EMZL patients. Acknowledgements This work was partially supported by Oncosuisse grant OCS References 1. Isaacson PG, Du MQ. MALT lymphoma: from morphology to molecules. Nat Rev Cancer 2004; 4: Zucca E, Bertoni F, Cavalli F. Marginal zone B-cell lymphomas. In The Lymphomas (2nd edn), Canellos GP, Lister TA, Young B (eds). Saunders Elsevier: Philadelphia, PA, USA, 2006; Bertoni F, Zucca E. State-of-the-art therapeutics: marginal-zone lymphoma. J Clin Oncol 2005; 23: Dierlamm J, Baens M, Wlodarska I, et al. The apoptosis inhibitor gene API2 and a novel 18q gene, MLT, are recurrently rearranged in the t(11;18)(q21;q21) associated with mucosaassociated lymphoid tissue lymphomas. Blood 1999; 93: Willis TG, Jadayel DM, Du MQ, et al. Bcl10 is involved in t(1;14)(p22;q32) of MALT B cell lymphoma and mutated in multiple tumor types. Cell 1999; 96: Streubel B, Lamprecht A, Dierlamm J, et al. T(14;18)(q32;q21) involving IGH and MALT1 is a frequent chromosomal aberration in MALT lymphoma. Blood 2002; 101: Streubel B, Vinatzer U, Lamprecht A, Raderer M, Chott A. T(3;14)(p14.1;q32) involving IGH and FOXP1 is a novel recurrent chromosomal aberration in MALT lymphoma. Leukemia 2005; 19(4): Bertoni F, Zucca E. Delving deeper into MALT lymphoma biology. J Clin Invest 2006; 116: Farinha P, Gascoyne RD. Molecular pathogenesis of mucosaassociated lymphoid tissue lymphoma. J Clin Oncol 2005; 23: Murga Penas EM, Hinz K, Roser K, et al. Translocations t(11;18)(q21;q21) and t(14;18)(q32;q21) are the main chromosomal abnormalities involving MLT/MALT1 in MALT lymphomas. Leukemia 2003; 17: Ye H, Liu H, Attygalle A, et al. Variable frequencies of t(11;18)(q21;q21) in MALT lymphomas of different sites: significant association with CagA strains of H. pylori in gastric MALT lymphoma. Blood 2003; 102(3): Dierlamm J, Pittaluga S, Wlodarska I, et al. Marginal zone B-cell lymphomas of different sites share similar cytogenetic and morphologic features. Blood 1996; 87: Dierlamm J, Rosenberg C, Stul M, et al. Characteristic pattern of chromosomal gains and losses in marginal zone B cell lymphoma detected by comparative genomic hybridization. Leukemia 1997; 11: Auer IA, Gascoyne RD, Connors JM, et al. t(11;18)(q21;q21) is the most common translocation in MALT lymphomas. Ann Oncol 1997; 8: Bertoni F, Conconi A, Capella C, et al. Molecular follow-up in gastric mucosa-associated lymphoid tissue lymphomas: early analysis of the LY03 cooperative trial. Blood 2002; 99: Hancock B, Linch D, Delchier J, et al. Chlorambucil versus observation after anti-helicobacter therapy in low-grade gastric lymphoma: results of the international LY03 trial. Ann Oncol 2005; 16(Suppl 5): V Isaacson PG, Muller-Hermelink HK, Piris MA, et al. Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). In World Health Organization Classification of Tumours Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, Jaffe ES, Harris NL, Stein H, Vardiman JW (eds). IARC Press: Lyon, 2001; Wotherspoon AC, Doglioni C, Diss TC, et al. Regression of primary low-grade B-cell gastric lymphoma of mucosa- associated lymphoid tissue type after eradication of Helicobacter pylori. Lancet 1993; 342:

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