Primary Pulmonary MALT Lymphomas Show Frequent And
Total Page：16
File Type：pdf, Size：1020Kb
Leukemia (2004) 18, 156–160 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $25.00 www.nature.com/leu Primary pulmonary MALT lymphomas show frequent and heterogeneous cytogenetic abnormalities, including aneuploidy 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 lymphoma. 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 trisomy 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 immunohistochemistry 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 marginal zone 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 Hematology, 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 tonsils 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.