Leukemia (2009) 23, 574–580 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE Recurrent translocations involving the IRF4 oncogene locus in peripheral T-cell lymphomas AL Feldman, M Law, ED Remstein, WR Macon, LA Erickson, KL Grogg, PJ Kurtin and A Dogan Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Oncogenes involved in recurrent chromosomal translocations In this paper, we show for the first time the presence of serve as diagnostic markers and therapeutic targets in translocations involving the multiple myeloma oncogene-1/ hematopoietic tumors. In contrast to myeloid and B-cell neoplasms, translocations in peripheral T-cell lymphomas interferon regulatory factor-4 (IRF4) locus on 6p25 in PTCLs. On (PTCLs) are poorly understood. Here, we identified recurrent the basis of the observation of a PTCL, unspecified (PTCL-U), translocations involving the multiple myeloma oncogene-1/ with a karyotype including t(6;14)(p25;q11.2), we studied 169 interferon regulatory factor-4 (IRF4) locus in PTCLs. IRF4 PTCLs and found 12 with IRF4 translocations (including the translocations exist in myeloma and some B-cell lymphomas, index case). Two had t(6;14)(p25;q11.2) translocations that but have not been reported earlier in PTCLs. We studied 169 fused the IRF4 and T-cell receptor-alpha (TCRA) gene loci. Both PTCLs using fluorescence in situ hybridization and identified 12 cases with IRF4 translocations. Two cases with t(6;14) were cytotoxic PTCL-Us that presented in the bone marrow and (p25;q11.2) had translocations between IRF4 and the T-cell rapidly developed skin lesions. Such tumors might represent a receptor-alpha (TCRA) locus. Both were cytotoxic PTCLs, distinct clinicopathologic entity. unspecified (PTCL-Us) involving bone marrow and skin. In Eight cutaneous ALCLs (C-ALCLs) had translocations invol- total, 8 of the remaining 10 cases were cutaneous anaplastic ving IRF4 but not TCRA (57% of C-ALCLs tested). One systemic large-cell lymphomas (ALCLs) without TCRA rearrangements ALK-negative ALCL and an additional PTCL-U showed translo- (57% of cutaneous ALCLs tested). These findings identified IRF4 translocations as a novel recurrent genetic abnormality in cations involving IRF4 but not TCRA. IRF4 translocations were PTCLs. Cytotoxic PTCL-Us involving bone marrow and skin and not detected in ALK-positive ALCLs. C-ALCL is an indolent containing IRF4/TCRA translocations might represent a distinct disease that currently lacks specific pathologic features to clinicopathologic entity. Translocations involving IRF4 but not distinguish it from skin involvement by systemic ALK-negative TCRA appear to occur predominantly in cutaneous ALCLs. ALCL,7,8 which is aggressive and often fatal.1,2 Testing for IRF4 Detecting these translocations may be useful in lymphoma translocations may have clinical diagnostic utility in C-ALCL. diagnosis. Further, due to its involvement in translocations, Although molecular pathways associated with ALK transloca- MUM1/IRF4 protein may play an important biologic role in some 9 PTCLs, and might represent a possible therapeutic target. tions in ALK-positive ALCLs have been studied extensively, Leukemia (2009) 23, 574–580; doi:10.1038/leu.2008.320; corresponding pathways in ALCLs lacking ALK translocations published online 6 November 2008 are poorly understood. MUM1/IRF4 protein could play a Keywords: MUM1; IRF4; PTCL; ALCL; translocation; cutaneous biologic role in PTCLs with IRF4 translocations, as IRF4 is lymphoma oncogenic in vitro.10 Utilization of MUM1/IRF4 as a therapeutic target in patients with PTCL merits exploration. Materials and methods Introduction Cases Peripheral T-cell lymphomas (PTCLs) are malignant neoplasms We studied specimens from 169 patients with PTCL diagnosed of mature (peripheral) T lymphocytes, and represent approxi- by WHO (World Health Organization) criteria.1 There were 104 mately 10% of non-Hodgkin lymphomas.1 Despite treatment men and 65 women (M:F ratio, 1.6:1), with a mean age of 58 with conventional chemotherapy, the majority of patients with years (range, 5–92 years). Cases included 23 angioimmuno- PTCLs die of their disease.2 New, targeted therapies might be blastic T-cell lymphomas (13%), 72 PTCL-Us (43%), 18 ALK- aimed at genes involved in recurrent chromosomal transloca- positive ALCLs (11%), 24 ALK-negative ALCLs (14%), 14 tions,3–5 but, in contrast to myeloid neoplasms and B-cell C-ALCLs (8%) and 18 other PTCLs (Table 1). The study was lymphomas,1 translocations in most PTCLs remain poorly approved by the Institutional Review Board and the Biospeci- understood. Identifying novel recurrent translocations in PTCLs mens Committee of Mayo Clinic. may also help define specific disease entities and serve as the basis for clinical diagnostic testing, as they have in the case of Fluorescence in situ hybridization chromosomal translocations involving ALK in ALK-positive 6 IRF4 and TCRA fluorescence in situ hybridization (FISH) probes anaplastic large-cell lymphomas (ALCLs). However, ALK- 11–13 were developed as described earlier. BAC clones (Supple- positive ALCLs represent only about 6% of PTCLs.2 mentary Table S1) were identified using the University of California Santa Cruz Genome Browser (http://www.genome. Correspondence: Dr AL Feldman, Department of Laboratory Medicine UCSC.edu) and ordered from ResGen Invitrogen (Carlsbad, CA, and Pathology, Mayo Clinic, 200 1st St SW, Hilton 1152D, Rochester, USA). As telomeric IRF4 clones showed minimal cross- MN 55905, USA. E-mail: [email protected] hybridization to 16p11, positive cases were confirmed using a Received 7 May 2008; revised 28 August 2008; accepted 18 second breakapart IRF4 probe (probe no. 2; Supplementary September 2008; published online 6 November 2008 Table S1). BAC DNA was isolated using the Qiagen (Valencia, IRF4 translocations in PTCLs AL Feldman et al 575 Table 1 IRF4 translocations and MUM1/IRF4 protein expression in peripheral T-cell lymphomas Diagnosis FISH Immunohistochemistry No. of positivesa (%) No. of positivesa (%) Angioimmunoblastic T-cell lymphoma 0/19 (0) 0/23 (0) PTCL, unspecified 3/64 (5) 20/72 (28) CD30 positive 1/17 (6) 13/18 (72) CD30 negative 2/47 (4) 7/54 (13) Anaplastic large cell lymphoma, ALK positive 0/18 (0) 16/17 (94) Anaplastic large cell lymphoma, ALK negative 1/23 (4) 20/22 (91) Cutaneous anaplastic large cell lymphoma 8/14 (57) 13/14 (93) T-cell large granular lymphocyte leukemia 0/4 (0) 0/4 (0) Hepatosplenic T-cell lymphoma 0/3 (0) 0/3 (0) Subcutaneous panniculitis-like T-cell lymphoma F 0/1 (0) Enteropathy-associated T-cell lymphoma 0/2 (0) 0/2 (0) Extranodal NK/T-cell lymphoma, nasal type 0/8 (0) 0/8 (0) Total 12/155 (8) 69/166 (42) Abbreviations: FISH, fluorescence in situ hybridization; PTCL, peripheral T-cell lymphoma. aIncludes informative cases only, of 169 total cases tested. CA, USA) Plasmid Maxi Kit and fluorescently labeled using tissue or tissue microarray sections were pretreated in 1 mM SpectrumOrange-dUTP or SpectrumGreen-dUTP and the Abbott EDTA buffer at pH 8.0 for 30 min at 98 1C (PT Module; Lab Molecular (Des Plaines, IL, USA) Nick Translation Kit. Cen- Vision, Fremont, CA, USA), then stained for MUM1/IRF4 using a tromeric and telomeric BAC DNA were labeled with different monoclonal mouse anti-human antibody (MUM1p, 1:50; Dako). fluorophores for breakapart probes, and with the same Detection was with Dual Link Envision þ /DAB þ (Dako). fluorophore for dual-fusion (D-FISH) probes. Probe validation Scoring was performed in correlation with hemotoxylin and was conducted based on earlier described ‘familiarization’ eosin and appropriate immunostains (for example, CD20 and procedures.14 Specificity of hybridization was confirmed on CD3). MUM1/IRF4 was considered positive when 430% of metaphases from a splenic marginal zone lymphoma with IRF4/ tumor cells demonstrated nuclear staining. Technical factors IGH fusion,13 a PTCL with a TCRA translocation,11 and normal precluded scoring in rare cases. Diagnosis of C-ALCL required samples. TCRB and TCRG probes were purchased from Dako CD30 positivity in 475% of tumor cells, as per WHO criteria.1 (Carpinteria, CA, USA). The upper limit of the normal range for This cutoff was also used to define CD30 positivity in cases of each probe was determined using a 95% confidence interval as PTCL-U. described earlier.11,14 Upper limits of normal for IRF4, TCRA, TCRB and TCRG were 6, 9, 5 and 6%, respectively. Paraffin tissue microarrays were constructed as described.3,11,12 Conventional cytogenetics In cases with insufficient tissue, whole-tissue sections were Results of karyotype analysis prepared at the time of biopsy analyzed. B5- and formalin-fixed cases were included, as we using earlier described methods19 were reviewed retrospectively have shown similar FISH results with both fixatives.15,16 FISH was when available. performed as described earlier.11 Sections were sequentially immersed in Citrisolve, Lugol solution and sodium thiocyanate. Slides were microwaved for 5 min in citrate buffer, then digested Results and discussion in 0.4% pepsin solution at 37 1C. FISH reagent (10 ml; 7 mlLSI buffer and 3 ml probe) was placed on each slide and slides were We identified 12 PTCLs with IRF4 translocations among 155 cover-slipped, denatured and incubated in a humidified chamber PTCLs with informative FISH results (8%; Tables 1 and 2). These at 37 1C for 12 h. Slides were washed, counterstained with 40, included 3/64 PTCL-Us (5%), 1/23 ALK-negative ALCLs (4%) 6-diamidino-2-phenylindole dihydrochloride, and analyzed by and 8/14 C-ALCLs (57%). IRF4 translocations were not seen in a microscopist (ML) using a fluorescent microscope with ALK-positive ALCLs, angioimmunoblastic T-cell lymphomas or appropriate filter sets. A minimum of 50 cells and a maximum other PTCL subtypes. MUM1/IRF4 protein was detected in the of 200 cells were scored per case.