¹Department of Oncology and Hematology, University Hospital Eppendorf, Hamburg, Germany

²Department of Internal Medicine, Kochi Medical School, Kochi, Japan

³Center for Human Genetics and Flanders Interuniversity Institute for Biotechnology, University of Leuven, Belgium

Correspondence to: J Dierlamm, Department of Oncology and Hematology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; Fax: 0049-40-42803-2186

Leukemia (2002) 16, 1863-1864. doi:10.1038/sj.leu.2402683

TO THE EDITOR

The chromosomal translocation t(11;18)(q21;q21) characterizes about one-third of extranodal marginal zone B cell lymphomas of the MALT-type.^1,2,3 We and others have recently shown that this translocation leads to a fusion of the apoptosis inhibitor gene API2 on chromosome 11 and the novel MLT/MALT1 gene, which encodes a human paracaspase, on chromosome 18.⁴ The pathogenetic relevant event involves the derivative chromosome 11 and leads to linkage of the three baculovirus IAP repeat (BIR) domains present in the N terminus of API2 and a variable part of MLT/MALT1, which always contains the caspase p20-like domain.^1,3,4,5The chimeric protein effectively activates NF-B, a potential pro-survival signal in B cells.⁶

We here describe the first variant translocation of the t(11;18), a novel three-way translocation t(11;12;18)(q21;q13;q21), occurring in a MALT lymphoma of the lung. All analyses were performed on material from a pulmonary MALT lymphoma, stage I_EA. Fifty analyzed metaphases showed the t(11;12;18)(q21;q13;q21) as the sole chromosomal abnormality. The detailed clinical, histological and cytogenetic data have previously been published.⁷ However, the cytogenetic breakpoints, which have been described in the previous report as t(11;12;18)(q13;q13;q12), had to be revised to t(11;12;18) (q21;q13;q21) according to the molecular genetic results of this study (Figure 1). This indicates that the chromosomal breakpoints of the t(11;12;18) may be difficult to assess by conventional cytogenetic analysis and that the relationship to the t(11;18) associated with MALT lymphomas could therefore be missed (Figure 1). The recognition of the t(11;18) and related aberrations is of particular importance since they characterize the clinically distinct low-grade MALT lymphomas and especially those which might respond poorly to Helicobacter pylori eradication therapy.⁸

Interphase FISH was performed according to standard methods using cytospin preparations of cells isolated from the snap-frozen lung tumor. Five hundred interphase cells were analyzed for each probe. For the detection of the 5'API2-3'MLT/MALT1 fusion resulting from the t(11;18)(q21;q21), P1 artificial chromosome (PAC) clones 59N7 and 116G16 were used as previously described.² PAC 59N7 contains genomic sequences derived immediately downstream of the MLT/MALT1 gene. PAC clone 166G16 spans approximately 100 kb and contains the complete API2 gene. The cut-off level for diagnosing the t(11;18) was 4.4%.² The FISH analysis showed fusion signals in 15% of analyzed cells, which indicates the presence of a 5'API2-3'MLT/MALT1 fusion.

To demonstrate the exchange of genetic material between chromosomes 18 and 12, PAC clone 117B5 containing genomic sequences derived immediately upstream of MLT/MALT1² and yeast artificial chromosome (YAC) clones hybridizing to the chromosomal regions 12q12-13 (YAC 919G8), 12q21 (YAC 753A8) and 12q24 (YAC 839F6), respectively, were applied. The cut-off values for diagnosing a fusion or colocalization of chromosomes 18 and 12 were 3-4%. By applying this FISH assay, an exchange of genetic material between chromosomes 18 and 12 was clearly demonstrated. Twelve to 16% of interphase cells showed a colocalization of genomic sequences derived immediately upstream of MLT/MALT1 and sequences of the chromosomal regions 12q12-13, 12q21 and 12q24, respectively.

To further characterize the breakpoint regions of the t(11;12;18), RT-PCR and DNA sequencing was performed as previously described.^1,4,5 For detection of the 5'API2-3'MLT/MALT1 fusion transcript a forward primer specific for exon 7 of API2 (API2-7f1; 5'-ATTAATGCTGCCGTGGAAAT-3') and a reverse primer specific for exon 11 of MLT/MALT1 (MLT-11r; 5'-ATGGATTTGGAGCATCAACG-3') were used.¹ The reciprocal MLT/MALT1-API2 fusion was analyzed with primers specific for exon 3 of MLT/MALT1 (MLT-3f; 5'-CAGTCTTGGCTGGACAGTTTGTGA-3') and exon 8 of API2 (API2-8r; 5'-GAGAGTTTCTGAATACAGTGGCTGCAA-3').

RT-PCR and DNA sequencing revealed an in-frame fusion of exon 7 of API2 with exon 5 of MLT/MALT1. In addition, a minor transcript, likely resulting from alternative splicing, fusing exon 7 of API2 and exon 6 of MLT/MALT1 was found (Figure 2). Similar findings are observed in the standard t(11;18), where a 5'API2-3'MLT/MALT1 fusion encoded on the derivative chromosome 11 represents the pathogenetic relevant event and leads to NF-B activation.⁶ The breakpoints in API2 occurred in the vast majority of reported cases with t(11;18) in intron 7 of API2, whereas the breakpoints within the MLT/MALT1 gene have been located upstream of exons 3, 5, 8 and 9, respectively. In all cases, the caspase p20-like domain of MLT/MALT1 was preserved and linked to the three baculovirus IAP repeat (BIR) domains present in the N terminus of API2.^1,3,4,5 In a considerable proportion of t(11;18)-positive cases the reciprocal MLT/MALT1-API2 transcript encoded on the derivative chromosome 18 was not expressed due to cryptic deletions in API2 or MLT .^1,4,5 Similarly, MLT/MALT1-API2 is not expressed in the t(11;12;18) as shown by RT-PCR. This is likely due to a translocation between chromosomes 18 and 12. This exchange of genetic material is clearly demonstrated by our FISH analyses. In order to identify the unknown region of chromosome 12 attached to 3'API2 and 5'MLT/MALT1, respectively, we performed 5' and 3'RACE-PCR.

For 3'-RACE-PCR, oligonucleotide 465 was used to synthesize first-strand cDNA from 1 g of total RNA with MuMLV-reverse transcriptase.⁹ Nested PCR was performed with primers MLT-4f1 (5'-GCAGGCTTTTA TGTCTGTCG-3') and 467 and MLT-4f2 (5'-TTGAATTCAGCCA GTGGTCA-3') and 468.^5,9 Amplification products were cloned and colonies were screened by PCR with universal primers for the vector. Six hundred and sixty-two positive colonies were sequenced.

For 5'-RACE-PCR, reverse transcription was performed with oligonucleotide API2-9r (5'-CTGAAACATCTTCTGTGGGA-3'). A polyA tail was added with dATP and terminal transferase. Double-stranded cDNA was generated using Klenow DNA polymerase I with the primer 466.⁹ First-step PCR was performed with primers API2-8r1 (5'-AACACAGCTTCAG CTTCTTGC-3') and 467 and second-step with API2-8r2 (5'-TTAATAATTCCGGCAGTTAG TAGAC-3') and 468.^1,9 Colonies were cloned and screened as described above. Clones containing the PCR products were further analyzed with API2 primers specific for exon 7 and exon 8, namely API2-7f3 (5'-TGAAATAAGGGAAGAGGAGAGA-3') and API2-8r3 (5'-GTCAAATGTTGAAAAAGTGCCATT-3'). Eight hundred and twenty-two negative clones were sequenced.

Despite sequencing a large number of clones, exclusively wild-type API2 and MLT/MALT1 sequences could be detected. Therefore, the fusion products of 3'API2 and 5'MLT/MALT1, respectively, with chromosome 12 might not be expressed.

Our results further underline the pathogenetic importance of the 5'API2-3'MLT/MALT1 fusion and demonstrate a close relationship between the novel t(11;12;18) and the standard t(11;18).

Acknowledgements

This work was supported by grant 70-2644Di2 from the Deutsche Krebshilfe/Dr Mildred Scheel Stiftung für Krebsforschung. M Baens and P Marynen were supported by a grant of the 'Belgische Federatie tegen Kanker'.

1 Baens M, Maes B, Steyls A, Geboes K, Marynen P, De Wolf-Peeters C. The product of the t(11;18), an API2-MLT fusion, marks nearly half of the gastro-intestinal MALT type lymphomas without large cell proliferation. Am J Pathol 2000; 156: 1433-1439. MEDLINE

2 Dierlamm J, Baens M, Stefanova-Ouzounova M, Hinz K, Wlodarska I, Steyls A, Maes B, Driessen A, Verhoef G, Gaulard P, Hagemeijer A, Hossfeld DK, De Wolf-Peeters C, Marynen P. The t(11;18)(q21;q21) characterizes a subgroup of extranodal MALT lymphomas: a two-color fluorescence in situ hybridization study using API2 and MLT specific probes. Blood 2000; 96: 2215-2218. MEDLINE

3 Remstein ED, James CD, Kurtin PJ. Incidence and subtype specifity of API2-MALT1 fusion translocations in extranodal, nodal, and splenic marginal zone lymphomas. Am J Pathol 2000; 156: 1183-1188. MEDLINE

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5 Baens M, Steyls A, Dierlamm J, De Wolf-Peeters C, Marynen P. Structure of the MLT gene and molecular characterization of the genomic breakpoint junctions in the t(11;18)(q21;q21) of marginal zone B-cell lymphomas of MALT type. Genes Chromosom Cancer 2000; 29: 281-291. MEDLINE

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7 Kubonishi I, Sugito S, Kobayashi M, Asahi Y, Tsuchiya T, Yamashiro T, Miyoshi I. A unique choromosome translocation t(11;12;18)(q13;q13;q12) in primary lung lymphoma. Cancer Genet Cytogenet 1995; 82: 54-56. MEDLINE

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9 Cools J, Bilhou-Nabera C, Wlodarska I, Cabrol C, Talmant P, Bernard P, Hagemeijer A, Marynen P. Fusion of a novel gene, BTL, to ETV6 in acute myeloid leukemias with a t(4;12)(q11-q12;p13). Blood 1999; 94: 1820-1824. MEDLINE

Figure 1 A Giemsa-banded partial karyotype of the t(11;12;18)(q21;q13;q21) and the corresponding chromosomal ideograms are shown.

Figure 2 The photograph of the gel shows the API2-MLT/MALT1 product obtained by RT-PCR from the MALT lymphoma with t(11;12;18)(q21;q13;q21) (middle lane). A major transcript resulting from a fusion of exon 7 of API2 and exon 5 of MLT/MALT1 is seen at 832 bp. In addition, a smaller band, likely resulting from alternative splicing of MLT/MALT1, is evident at 653 bp and indicates a fusion of exon 7 of API2 and exon 6 of MLT/MALT1. A size marker (M) is shown on the left and and a negative control (H₂O) is shown on the right. On the right side of the figure, a schematic representation of the localization of the breakpoints in API2 and MLT/MALT1 and the corresponding fusion product is demonstrated.