(2004) 18, 156–160 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $25.00 www.nature.com/leu Primary pulmonary MALT show frequent and heterogeneous cytogenetic abnormalities, including and translocations involving API2 and MALT1 and IGH and MALT1 ED Remstein1, PJ Kurtin1, RR Einerson2, SF Paternoster2 and GW Dewald2

1Divisions of Anatomic Pathology and Hematopathology, Mayo Clinic, Rochester, MN, USA; and 2Division of Laboratory Genetics, Mayo Clinic, Rochester, MN, USA

API2-MALT1 fusion and aneuploidy are common chromosomal As most MALT lymphomas arise in the stomach, the majority of abnormalities in MALT . In studying their incidence cytogenetic research has been performed on gastric MALT and relationship in primary pulmonary MALT lymphomas, a translocation involving MALT1 and IGH was also identified. In lymphomas. We chose to study primary pulmonary MALT all, 28 primary pulmonary MALT lymphomas were studied by lymphomas because although they have been relatively infre- fluorescence in situ hybridization using an API2-MALT1 probe quently studied, the incidence of t(11;18) is thought to be highest and multiple centromeric probes, as well as IGH-BCL2, IGH- in this site10,12 and aneuploidy has also been shown to be MALT1, and MALT1 breakapart probes in selected cases. Seven common in this site.10 The initial aim of the study was to (25%) had API2-MALT1 fusion; all seven lacked aneuploidy determine the incidence of and relationship between t(11;18) and except for two with 3 in a small clone. Three (11%) had IGH-MALT1 fusion; two also showed trisomy 3 and 12. A total of aneuploidy in a series of primary pulmonary MALT lymphomas 11 (39%) had aneuploidy only, with trisomy 3 and 18 being the using fluorescence in situ hybridization (FISH) performed on most common. Ectopic nuclear bcl-10 expression, which has nuclei isolated from paraffin-embedded tissue. In the course of been previously associated with API2-MALT1, was seen this investigation, a translocation involving MALT1 and the by in 86% of API2-MALT1 fusion- immunoglobulin heavy chain gene (IGH) was also identified in positive cases, one IGH-MALT1 fusion-positive case, two three cases. While this manuscript was being prepared, two other aneuploidy-only cases, and two normal cases. In primary reports identifying the same translocation in MALT lymphoma pulmonary MALT lymphomas, cytogenetic abnormalities are 13,14 common (75%) and heterogeneous, encompassing API2- were published. However, to our knowledge this is the first MALT1 and IGH-MALT1, which are mutually exclusive, as well report of IGH-MALT1 fusion in pulmonary MALT lymphomas. as aneuploidy, which may be present in the latter but is rare in the former. Ectopic nuclear bcl-10 expression is associated with API2-MALT1 but may also be seen in IGH-MALT1 fusion- Materials and methods positive, aneuploidy-only, and normal cases. Leukemia (2004) 18, 156–160. doi:10.1038/sj.leu.2403185 Published online 23 October 2003 Patient samples Keywords: MALT lymphoma; IGH-MALT1; t(14;18); t(11;18); FISH; cytogenetics In all, 28 patients who were diagnosed with pure low-grade primary pulmonary MALT lymphoma at Mayo Clinic between 1981 and 1994 and who had suitable paraffin blocks available Introduction were identified. In all cases, rigorous clinical staging confirmed that the site of involvement was limited to one or both lungs. t(11;18)(q21;q21), which results in the production of a fusion Each case was previously studied clinically, morphologically, protein composed of the apoptosis inhibitor API2 on chromo- and phenotypically by one of the authors (PJK),15 and five cases some 11q21 and the paracaspase MALT1 on chromosome were also previously studied by FISH in our laboratory.10 All 18q21, is thought to be the most common structural chromo- patients consented to research use of their tissue, and the study somal abnormality in MALT lymphoma, occurring in 20–50% 1–9 was approved by the Mayo Clinic Institutional Review Board. of cases. This translocation is found exclusively in MALT Each lymphoma met the morphologic and phenotypic criteria lymphomas, and has not been identified in primary splenic or 1–3 (by paraffin or frozen section immunohistochemistry or flow primary nodal lymphomas. When present, cytometry) of the World Health Organization Classification of it is usually the sole cytogenetic abnormality and is primarily Haematopoietic and Lymphoid Tissues,16 and in each case associated with low-grade disease, having been identified sufficient paraffin-embedded material was available for isolation to date in only two cases of diffuse large B-cell lymphoma of nuclei for FISH. transformation of low-grade MALT lymphoma.10,11 Aneuploidy, particularly trisomy 3, 7, 12, and/or 18, is also common in MALT lymphoma but is rarely associated with Immunohistochemistry t(11;18), suggesting that MALT lymphomas with t(11;18) and those with aneuploidy develop along different pathogenetic Immunoperoxidase stains were applied to paraffin sections of all pathways.10 cases but one (due to insufficient tissue) according to previously published methods,17 using antibodies directed against Bcl-10 Correspondence: Dr ED Remstein, Divisions of Anatomic Pathology (Zymed, South San Francisco, CA, USA). and Hematopathology, Mayo Clinic, Hilton Building, Room 1166, 200 First Street SW, Rochester, MN 55905, USA; Fax: þ 1 507 284 1599 This work was presented in part at the 44th Annual Meeting of the American Society of , Philadelphia, PA, USA, December FISH analysis 8, 2002 Received 12 May 2003; accepted 11 August 2003; Published online FISH was performed using a slight modification of a previously 23 October 2003 described method,18 in that either 50-mm sections were cut or FISH in pulmonary MALT lymphomas ED Remstein et al 157 two tissue sample cores were collected using a 20-gauge 1.5- Statistical analysis inch blunt needle from the paraffin block. The method used was otherwise identical to the previously described method. Briefly, FISH specimens were studied in random order in an indepen- to isolate nuclei, the samples were deparaffinized with xylene, dent and blinded manner by two microscopists (RRE and SFP). rehydrated, manually disaggregated, enzymatically digested Each microscopist scored 100 consecutive qualifying interphase with a proteinase K solution, washed, resuspended, and placed nuclei from different areas of the same slide. The normal ranges on a charged glass slide. The slides were dried and sequentially were calculated in previous published10 and unpublished treated with hot citric acid, 2 Â standard saline citrate, and studies using lymph nodes and from patients without a pepsin. The slides were then dehydrated, probe was added to malignant hematological disorder. The upper bound of the each slide, the DNA and probe were co-denatured and normal range was determined using a one-sided 95% con- hybridized, and the slides were washed and counterstained. fidence interval for observing the maximum number of nuclei Cells were viewed with a fluorescent microscope equipped with for each false-positive signal pattern seen in 200 scoreable appropriate filter sets. nuclei using the binomial distribution.

FISH probes Results

Centromere-specific a-satellite (CEN) probes for chromosomes 3 FISH analysis and 12 were directly labeled with SpectrumOranget, and those for chromosomes 7 and 18 were directly labeled with The FISH results are summarized in Table 1. API2-MALT1 fusion SpectrumGreent (Vysis, Inc., Downers Grove, IL, USA). DNA was identified in seven of 28 primary pulmonary MALT probes for BCL2 (telomeric to the MALT1 gene at 18q21) and lymphomas (25%). The mean percent and standard deviation IGH (at 14q32) were directly labeled with SpectrumOranget of nuclei per case with an abnormal FISH pattern was and SpectrumGreent, respectively (Vysis, Inc., Downers Grove, 62.6714.2 with a range of 40–85. FISH patterns included IL, USA). DNA probes for API2 and MALT110 were directly 1R1G2F (n ¼ 4), 2R2G1F þ 1R1G2F (n ¼ 1), 2R1G1F (n ¼ 1), labeled with SpectrumOranget and SpectrumGreent, respec- and 1R2G1F (n ¼ 1). The 1R1G2F (Figure 1a) and 2R2G1F tively. DNA probes for MALT110 and IGH (consisting of the IGH patterns are indicative of API2-MALT1 fusion, with the latter component of the Vysis, Inc. BCL2-IGH probe set) were directly pattern likely resulting from technical splitting of one of the two labeled with SpectrumOranget and SpectrumGreent, respec- fusion signals. The 2R1G1F (Figure 1b) and 1R2G1F (Figure 1c) tively. The MALT1 breakapart probe is composed of Spectru- patterns also represent API2-MALT1 fusion, with the 2R1G1F mOranget- and SpectrumGreent-labeled DNA probes that pattern resulting from a breakpoint within the API2 hybridiza- hybridize to either side of the MALT1 breakpoint (prototype tion site and a breakpoint outside the MALT1 hybridization site, version of the Vysis, Inc. MALT1 probe). As the signals were and the 1R2G1F pattern resulting from a breakpoint outside the observed with filters through which the SpectrumOranget API2 hybridization site and a breakpoint within the MALT1 fluorophore appears red and the SpectrumGreent fluorophore hybridization site. Alternatively, it is possible that loss of DNA appears green, the colors will be referred to as red (R) and green around the break and fusion point has occurred in either API2 or (G) in this article. Yellow fusion signals will be referred to as F. MALT1. The API2, IGH, MALT1, and BCL2 probes span API2, IGH, All seven API2-MALT1 fusion-positive MALT lymphomas had MALT1, and BCL2, respectively. Thus, a D-FISH (dual fusion) a normal FISH pattern using centromeric probes to chromo- pattern (1R1G2F) is produced using the appropriate probe set if somes 7, 12, and 18. Five of seven API2-MALT1 fusion-positive IGH-BCL2, API2-MALT1,orIGH-MALT1 fusion is present. The MALT lymphomas also had a normal FISH pattern using a API2-MALT1, CEN3/CEN7, and CEN12/CEN18 probe sets were centromeric probe to chromosome 3, and the remaining two applied in all cases. The IGH-BCL2 and IGH-MALT1 probe sets cases showed trisomy 3 in a subset of cells (mean 16% of cells). and the MALT1 breakapart probe were applied in selected In three cases (11%), an extra MALT1 signal was seen using cases. the API2-MALT1 probe (Figure 1g), but a normal signal pattern

Table 1 Summary of FISH results and ectopic nuclear Bcl-10 expression

FISH results Number of % abnormal nuclei Number of cases (mean, range) cases with ectopic nuclear bcl-10 expression

API2-MALT1 (total) 7 62.6, 40–85 6 API2-MALT1 with aneuploidy (trisomy 3) 2 Trisomy 3 – 16, 6–26 2 API2-MALT1 without aneuploidy 5 Aneuploidy – 0 4 IGH-MALT1 (total) 3 80.7, 66–89 1 IGH-MALT1 with aneuploidy (trisomy 3 and 12) 2 Trisomy 3 and 12 – 71.8, 56–81 0 IGH-MALT1 without aneuploidy 1 Aneuploidy – 0 1 Aneuploidy only (total) 11 27.2, 8–65 2 Trisomy 3 and 18 4 26.8, 9–53 1 Trisomy 3 3 8.7, 8–9 0 Trisomy 3, 12, and 18 2 42.2, 13–65 1 Trisomy 3 and 12 1 10 0 Trisomy 18 1 31 0 No cytogenetic abnormality 7 0 2

Leukemia FISH in pulmonary MALT lymphomas ED Remstein et al 158 consistent with IGH-MALT1 fusion. In addition, both trisomy 3 and trisomy 12 were seen in two of the three cases in a majority of the cells. The remaining case had a normal FISH pattern using centromeric probes to chromosomes 3, 7, 12, and 18. In all, 11 cases (39%) that lacked both API2-MALT1 and IGH- MALT1 fusion had aneuploidy involving one or more of the chromosomes tested (Figures 1d–f). Trisomy 3 (n ¼ 10) and trisomy 18 (n ¼ 7) were most frequently seen, and trisomy 12 (n ¼ 3) was also identified. None of the cases showed trisomy 7. The most common combination was trisomy 3 and trisomy 18. As expected, all cases with trisomy 18 had an extra CEN18 signal as well as an extra MALT1 signal using the API2-MALT1 probe. Finally, seven cases (25%) lacked cytogenetic abnorm- alities using this panel of FISH probes.

Immunohistochemistry

Bcl-10 expression was tested because ectopic nuclear localiza- tion of bcl-10 has been associated with t(11;18)(q21;q21).6,19,20 In all, 11 of 27 cases (41%) showed ectopic nuclear localization of bcl-10 (Table 1). By cytogenetic category, ectopic nuclear localization of bcl-10 was seen in six of seven of cases (86%) with API2-MALT1 fusion, one of three cases (33%) with IGH- MALT1 fusion, two of 10 cases (20%) with aneuploidy alone, Figure 1 Representative FISH patterns in abnormal interphase and two of seven cases (29%) without a detectable cytogenetic cells. (a–c) Abnormal FISH patterns in t(11;18)(q21;q21)-positive MALT lymphomas using the API2-MALT1 probe. (a) 1R1G2F (dual abnormality. fusion (D-FISH)) pattern. (b) 2R1G1F (extra signal-red (ES-R)) pattern. (c) 1R2G1F (extra signal-green (ES-G)) pattern. (d–f) Abnormal FISH patterns in an aneuploidy-only MALT lymphoma. (d) Trisomy 3, no Discussion trisomy 7. 3R2G pattern using CEN3 (R) and CEN7 (G) probes. (e) Trisomy 12 and trisomy 18. 3R3G pattern using CEN12 (R) and CEN18 To date, three recurrent structural chromosomal abnormalities (G) probes. (f) Extra MALT1 signal consistent with trisomy 18. 2R3G pattern using the API2-MALT1 probe. (g–l) Abnormal FISH patterns in have been described in MALT lymphoma. The first, an IGH-MALT1 fusion-positive case. (g) 2R3G (extra MALT1 signal) t(11;18)(q21;q21), is thought to be the most common structural pattern using the API2-MALT1 probe. (h) Trisomy 12, no trisomy 18. abnormality in MALT lymphoma, being present in 20–50% of 3R2G pattern using CEN12 (R) and CEN18 (G) probes. (i) Trisomy 3, cases.1–9 It has previously been demonstrated in 38–55% of no trisomy 7. 3R2G pattern using CEN3 (R) and CEN7 (G) probes. (j) pulmonary MALT lymphomas10,12,19 and was identified in 25% 2R3G (extra IGH signal) pattern using the IGH-BCL2 probe. (k) of primary pulmonary MALT lymphomas in the present study. 1R1G2F (D-FISH) pattern using the IGH-MALT1 probe. (l) 1R1G1F (breakapart) pattern using the MALT1 breakapart probe. This translocation results in the production of a fusion protein composed of the apoptosis inhibitor API2 on chromosome 11q21 and the paracaspase MALT1 on chromosome 18q21. t(11;18) has been shown to be prognostically and therapeuti- was seen using a centromeric probe for cally significant in gastric MALT lymphomas, being associated (Figure 1h). This FISH pattern is incompatible with trisomy 18 with tumors that extend deeply into the gastric wall and/or but could be consistent with either partial trisomy 18 disseminate beyond the stomach,6 and also being associated (isochromosome 18 or a involving 18q) with gastric MALT lymphomas that are - or a translocation involving MALT1 and a partner gene other negative21 or fail to respond to H. pylori eradication ther- than API2. To distinguish between these two possibilities, a FISH apy.22,23 probe for IGH-BCL2 was applied. BCL2 is just telomeric to The second translocation, t(1;14)(p22;q32), is thought to be MALT1 (at 18q21), but the FISH probes for MALT1 and BCL2 do uncommon in MALT lymphomas, although its incidence is not overlap. Therefore, the presence of cells with three BCL2 presently unknown. It results in juxtaposition of BCL10 at 1p22 signals would indicate partial trisomy 18, while the presence of with the IGH promoter region at 14q32. A signal transduction cells with two BCL2 signals would suggest a translocation pathway in which bcl-10 and MALT1 bind together to activate involving MALT1. In each case, two BCL2 signals were seen per the IkB kinase complex, resulting in increased translocation into cell, and three IGH signals were also identified in a mean of the nucleus and hence activation of NF-kappa B, has recently 70.3% of cells per case (Figure 1j). The presence of a normal been described.24 It has been postulated that both the t(1;14) BCL2 signal pattern suggested a translocation involving MALT1 and the t(11;18) can upregulate this pathway, the former due to and a partner gene other than API2, and the presence of an extra increased bcl-10 expression binding to normal MALT1 and the IGH signal indicated that the partner gene might in fact be IGH. latter due to expression of the API2-MALT1 fusion protein, Using an IGH-MALT1 FISH probe, a 1R1G2F pattern was which can self-oligomerize and activate NF-kappa B indepen- identified in each case involving a mean of 80.7% of cells dent of bcl-10.24,25 Therefore, this pathway may represent the (s.d. ¼ 12.7) with a range of 66–89% of cells (Figure 1k), unifying feature between these two seemingly unrelated indicating that this was indeed IGH-MALT1 fusion. A MALT1 cytogenetic abnormalities in MALT lymphoma. breakapart probe also showed splitting of the probe in each case A third structural abnormality, t(14;18)(q32;q21), which (mean 74%, range 61–85% of cells per case) (Figure 1l), juxtaposes the IGH promoter region at 14q32 with MALT1 at

Leukemia FISH in pulmonary MALT lymphomas ED Remstein et al 159 18q21, was recently identified.13,14 Molecular cloning of the necessary to investigate the pathogenetic and clinical signi- translocation breakpoints performed by Sanchez-Izquierdo ficance of IGH-MALT1 fusion and aneuploidy in MALT et al14 using a long-distance inverse polymerase chain reaction lymphoma. technique on two cases demonstrated that the MALT1 break- In contrast to cytoplasmic expression in normal B-cells, bcl- points lie immediately 50 to the first exons or are within the 50 10 is expressed in the nucleus in MALT lymphomas harboring noncoding regions. This translocation is thus analogous to other t(1;14) as well as in a subset of MALT lymphomas that lack this translocations involving IGH in that the coding regions of the translocation.32 Several studies have demonstrated an associa- partner gene remain intact, and it may represent a third tion between t(11;18) and ectopic nuclear bcl-10 expression, independent chromosomal structural abnormality that potenti- with nuclear bcl-10 expression being seen in 81–100% of ates MALT lymphoma progression by upregulating the NF-kappa t(11;18)-positive cases.6,19,20 However, each study also identi- B signal transduction pathway. fied a smaller group of t(11;18)-negative cases that also showed Streubel et al13 identified t(14;18)(q32;q21) by two-color FISH nuclear bcl-10 expression (16–30%). Our study showed similar analysis in 12 of 66 MALT lymphomas (18%) arising in sites findings, with nuclear bcl-10 expression being seen in 86% of such as liver (four of four), skin (three of 11), ocular adnexa API2-MALT1 fusion-positive cases and 25% of API2-MALT1 (three of eight), and salivary gland (two of 11), but not in MALT fusion-negative cases (one IGH-MALT1 fusion-positive case, lymphomas arising in other sites such as stomach, intestine, lung two cases showing aneuploidy only, and two cases without (n ¼ 7), thyroid, or breast. In our laboratory, IGH-MALT1 fusion apparent cytogenetic abnormality). This supports the concept was detected by two-color FISH analysis in three of 28 primary that there is an association between API2-MALT1 fusion and pulmonary MALT lymphomas (11%). As this translocation is nuclear bcl-10 expression, but suggests that nuclear bcl-10 relatively uncommon, we postulate that the discrepancy expression may also be associated with a variety of cytogenetic regarding its presence in pulmonary MALT lymphomas simply abnormalities, albeit at a lower frequency. reflects the difference in sample size (n ¼ 7 versus n ¼ 28), The discovery of t(14;18)(q32;q21) involving IGH and MALT1 although the possibility of geographic variability cannot be is also of diagnostic importance for routine clinical cases. excluded. Also, it is not clear whether the pulmonary MALT Follicular lymphoma, which is usually associated with lymphomas in the Streubel et al study represented primary or IGH-BCL2, and MALT lymphoma, which may be associated recurrent disease. In a previous study of cytogenetic abnorm- with IGH-MALT1, may be difficult to distinguish from alities of MALT lymphomas by FISH performed in our one another by standard morphologic and immunophenotypic laboratory,10 no abnormal FISH signal patterns that would have techniques, and cytogenetic analysis is sometimes employed. led us to suspect the presence of IGH-MALT1 fusion were However, due to MALT1’s close proximity to BCL2 on identified in 37 specimens from diverse sites such as lung, chromosome 18 at band q21, IGH-MALT1 is indistinguishable parotid, orbit, stomach, thyroid, colon, small bowel, lacrimal from IGH-BCL2 by standard cytogenetics. For this reason, both gland, kidney, paravertebral region, and skin of scalp, suggest- MALT lymphoma and follicular lymphoma must be considered ing that this translocation is indeed relatively uncommon. in the differential diagnosis when t(14;18)(q32;q21) is identified Similar to the findings of Streubel et al, in the present study by routine karyotyping. In at least one case in the literature, a API2-MALT1 and IGH-MALT1 fusions were mutually exclusive tumor that appeared to have the morphologic and phenotypic events. features of a MALT lymphoma was diagnosed as follicular The present study also provides additional support for the lymphoma, at least in part based on the presence of observation that API2-MALT1 fusion and aneuploidy are usually t(14;18)(q32;q21).33 In order to differentiate IGH-MALT1 from mutually exclusive events in MALT lymphoma. Similar to our IGH-BCL2, additional techniques such as FISH or PCR must be previous study,10 all API2-MALT1 fusion-positive MALT lym- utilized. phomas lacked trisomy 7, 12, or 18, and all but two lacked trisomy 3, with the two exceptions showing trisomy 3 in only a subset of cells. Four MALT lymphomas have been previously Acknowledgements described in which the major clone contained t(11;18)/API2- MALT1 fusion while a second, minor clone or subclone The authors thank Ms Stephanie Brockman for her technical contained trisomy 3.10,26,27 In contrast, two of the three IGH- assistance in the fluorescence in situ hybridization laboratory, and MALT1 fusion-positive MALT lymphomas in our study showed Ms Kristin Rieger for her expert secretarial assistance. aneuploidy (both trisomy 3 and trisomy 12) in the majority of cells. An association between IGH-MALT1 fusion and aneu- References ploidy was also demonstrated by Streubel et al, who used centromeric probes for chromosomes 3 and 18 and found 1 Rosenwald A, Ott G, Stilgenbauer S, Kalla J, Bredt M, Katzenberger trisomy 37 trisomy 18 by FISH in four of 12 (33%) IGH-MALT1- T et al. Exclusive detection of the t(11;18)(q21;q21) in extranodal positive MALT lymphomas.13 Furthermore, 39% of our API2- marginal zone lymphomas (MZBL) of MALT type in contrast MALT1 fusion-negative and IGH-MALT1 fusion-negative cases to other MZBL and extranodal large B cell lymphomas. Am J Pathol showed aneuploidy, usually involving chromosomes 3 and 18. 1999; 155: 1817–1821. This is similar to our previous study that showed aneuploidy 2 Remstein ED, James C, Kurtin PJ. Incidence and subtype specificity of API2-MALT1 fusion translocations in extranodal, nodal, and (most commonly trisomy 3 and/or 18) in 41% of API2-MALT1 splenic marginal zone lymphomas. Am J Pathol 2000; 156: 1183– fusion-negative (and apparently IGH-MALT1 fusion-negative) 1188. MALT lymphomas.10 Although aneuploidy has been suggested 3 Maes B, Baens M, Marynen P, De Wolf-Peeters C. The product of to be associated with recurrent disease in MALT lymphoma,10 its the t(11;18), an API2-MLT fusion, is an almost exclusive finding in pathogenetic significance is presently unknown. One could marginal zone cell lymphoma of extranodal MALT-type. Ann speculate that aneuploidy may be associated with other as yet Oncol 2000; 5: 521–526. 4 Baens M, Maes B, Steyls A, Geboes K, Marynen P, De Wolf-Peeters unidentified structural chromosomal abnormalities such as C. The product of the t(11;18), an API2-MLT fusion, marks nearly t(1;14), which by karyotypic studies has almost always been half of gastric MALT type lymphomas without large cell prolifera- associated with aneuploidy.28–31 Additional studies will be tion. Am J Pathol 2000; 156: 1433–1439.

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