Please cite this article in press as: Hatzimichael E, et al. Absence of methylation-dependent transcriptional silencing in TP73 irrespective of the methylation status of the CDKN2A CpG island in plasma cell neoplasia. Leuk Res (2009), doi:10.1016/j.leukres.2009.04.009 ARTICLE IN PRESS G Model LR-3446; No. of Pages 4 Leukemia Research xxx (2009) xxx–xxx Contents lists available at ScienceDirect Leukemia Research journal homepage: www.elsevier.com/locate/leukres Brief communication Absence of methylation-dependent transcriptional silencing in TP73 irrespective of the methylation status of the CDKN2A CpG island in plasma cell neoplasia Eleftheria Hatzimichael a,∗ , Leonidas Benetatos a , Aggeliki Dasoula a , George Dranitsaris b , Stavroula Tsiara a , Ioannis Georgiou a , Maria Syrrou c , Justin Stebbing d , Helen M. Coley e , Tim Crook d , Konstantinos L. Bourantas a a Department of Haematology, University Hospital of Ioannina, St. Niarchou Av., 45 500 Ioannina, Greece b Department of Medical Oncology, Princess Margaret Hospital, Toronto, Canada c Laboratory of General Biology, University of Ioannina, Ioannina, Greece d Imperial College, Department of Medical Oncology, Charing Cross Hospital, London, UK e Postgraduate Medical School, University of Surrey, Guildford, Surrey GU2 7XH, UK article info Article history: Received 30 July 2008 Received in revised form 12 March 2009 Accepted 3 April 2009 Available online xxx Keywords: Multiple myeloma Methylation TP73 CDKN2A Waldenström’s macroglobulinemia abstract Few studies exist regarding the methylation status of the TP73 CpG island in plasma cell dyscrasias. We have tested whether CpG methylation of both CDKN2A and TP73 occurs in 45 individuals with multiple myeloma (24 male and 21 female, mean age 66.4 years) and in 4 patients (2 male and 2 female, mean age 61.7 years) with Waldenström’s macroglobulinemia. No patient was found to be methylated for the promoter of TP73 while CDKN2A promoter was found to be methylated in 12/45 MM patients (26.6%) at diagnosis and in 1/4 WM patients. To verify the absence of detectable methylation observed using MSP, we performed bisulphite sequence analysis on a subset of the cases and confirmed the absence of methylation. Interesting trends were identified where patients with methylated CDKN2A had an increased risk of death (HR = 1.9, p = 0.32), advanced stage disease (DS ≥ II) (OR = 1.9, p = 0.3) and anemia (OR = 1.4, p = 0.6). TP73 CpG methylation is an infrequent event in patients with MM and WM. Further evaluation in a larger sample of patients is needed in order to enhance our statistical power and to test our hypothesis that CDKN2A methylation status can become a useful prognostic biomarker. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Transcriptional silencing is increasingly implicated in the patho- genesis of human cancer, including haematological malignancies [1]. Plasma cell (PC) neoplasms range from indolent disorders that can be managed expectantly, to more aggressive variants that require immediate treatment. Although the cytogenetic abnormal- ities for the development and progression of PC neoplasms are becoming better understood the role of epigenetic changes remains incompletely characterised. The TP73 gene is a homologue of the p53 tumor suppressor gene which functions in the regulation of cell cycle, apoptosis and development. Despite structural and functional similarities with p53, TP73-null mice do not show the increased susceptibil- ity to spontaneous tumorigenesis of p53 null animals, although there are defects in neuronal development. A variety of pro- and anti-apoptotic isoforms of TP73 is often expressed in neoplasia, with changes in expression between normal and tumor tissues ∗ Corresponding author. Tel.: +30 2651099729; fax: +30 2651046617. E-mail address: me00350@cc.uoi.gr (E. Hatzimichael). [2,3]. These changes occur through differential splicing at both C- and N-termini, alternative promoter use, and (in some cancers) aberrant, cancer-associated, CpG methylation with subsequent transcriptional silencing. The latter mechanism of inactivation of pro-apoptosis TAP73 isoforms occurs commonly in B lymphopro- liferative disorders, including acute lymphoblastic leukaemia (ALL) [4], non-Hodgkin’s lymphomas [5] and, strikingly, natural killer cell lymphomas [6]. Aside from TP73, a number of other genes have been shown to be transcriptionally silenced by aberrant CpG methylation in B lymphoproliferative disorders, of which CDKN2A is amongst the most common and simultaneous methylation of both CDKN2A and TP73 has been reported in some B cell neoplasms [6]. In PC neopla- sia, hypermethylation of CDKN2A is a frequent, reproducible finding [7], although the prognostic significance has not been definitively established and the role of methylation in regulating expression of CDKN2A has been questioned [8]. In contrast, few studies have addressed methylation of TP73. Here, we have tested whether CpG methylation of both CDKN2A and TP73 occurs in PC neoplasia. 2. Patients and methods Bone marrow samples from individuals with multiple myeloma (MM) and Waldenström’s macroglobulinemia (WM) were obtained at diagnosis. MM diag- 0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.04.009