Characterization of TET and IDH gene expression in chronic lymphocytic leukemia: comparison with normal B cells and prognostic significance

Van Damme et al. Clinical Epigenetics (2016) 8:132 DOI 10.1186/s13148-016-0298-y RESEARCH Open Access Characterization of TET and IDH gene expression in chronic lymphocytic leukemia: comparison with normal B cells and prognostic significance Michaël Van Damme1*, Emerence Crompot1, Nathalie Meuleman2, Marie Maerevoet2, Philippe Mineur3, Dominique Bron2, Laurence Lagneaux1 and Basile Stamatopoulos1 Abstract Background: Chronic lymphocytic leukemia (CLL) is the most common hematological malignancy in western countries, characterized by a heterogeneous clinical course. Although genetic studies have identified chromosomal aberrations or specific mutations, epigenetic changes have been poorly characterized in CLL. Methods: We assessed ten-eleven translocations (TET) 1, 2, and 3, isocitrate dehydrogenase (IDH) 1, and 2 messenger RNA (mRNA) expression using real-time PCR on purified leukemic B cells from 214 CLL patients (median follow-up = 75 months, range 1–380), normal peripheral blood B cells (n = 20), and umbilical cord blood B cells (n = 21). The microenvironment influence was assessed after 24 h co-culture of CLL cells with bone marrow mesenchymal stromal cells (BMSC). Finally, 5-hydroxymethylcytosine level (%5-hmC) was assessed by ELISA in CLL cells alone or with microenvironment stimuli. Results: TET 1 and 3 and IDH2 were decreased in CLL cells compared with healthy B cells (P = 0.0221, 0.0013, <0.0001, respectively), while IDH1 was overexpressed (P = 0.0037). TET2 and IDH1 were significantly correlated with treatment-free survival (TFS); patients with high TET2/IDH1 expression had a higher median TFS (111 months) than patients with low expression (78 months, P = 0.0071/0.0123). Moreover, TET1 expression decreased (P = 0.0371), while TET3 and IDH2 expression increased (P = 0.0273/0.0039) in co-cultures. However, %5-hmC was not correlated with clinical data and was unchanged following microenvironment stimuli. Conclusions: Despite a slight deregulation in CLL cells compared with normal B cells, we identified a significant association between TET/IDH gene expression and prognosis, suggesting that epigenetic changes could potentially be associated with disease progression. Moreover, despite an identical %5-hmC, TET gene expression was influenced by contact with BMSC confirming the crucial role of the microenvironment in CLL pathogenesis. Keywords: Chronic lymphocytic leukemia, TET, IDH, 5-Hydroxymethylcytosine, Prognosis * Correspondence: 1 Laboratory of Clinical Cell Therapy, ULB Cancer Research Center (U-CRC), Institut Jules Bordet, Université Libre de Bruxelles (ULB), Route de Lennik, 808, 1070 Brussels, Belgium Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Van Damme et al. Clinical Epigenetics (2016) 8:132 Background Chronic lymphocytic leukemia (CLL) is the most common hematological malignancy in the west and is characterized by a heterogeneous clinical course [1]; some patients will live several decades without any symptoms, while others will rapidly require a treatment and will have a decreased overall survival (OS). Clinical and molecular factors, such as Binet stage, lymphocyte doubling time (LDT), mutational status of the immunoglobulin heavychain variable-region (IgHV), zeta-chain-associated protein kinase 70 (ZAP70), lipoprotein lipase (LPL) or CD38 expression, and serum levels of soluble CD23 (sCD23) and beta-2-microglobulin (B2M), can be used to classify patients into different prognostic subgroups [2]. Moreover, increasing evidence suggests a role for the microenvironment in CLL pathogenesis. Our group previously demonstrated that bone marrow mesenchymal stromal cells (BMSC) protect CLL but not normal B cells from apoptosis through direct contact [3]. While genetic lesions, such as chromosomal aberrations [4] or specific mutations, [5–8] are involved in CLL physiopathology, in recent years, growing evidence has suggested that epigenetic characteristics are key factors in leukemic processes. Recent studies have investigated epigenetic features and demonstrated the importance of DNA methylation [9] or histone post-translational modifications in prognosis, oncogene regulation, or therapeutic targeting [10–12]. We demonstrated in previous papers that histone deacetylase (HDAC) mRNA expression was associated with poor (HDAC7, HDAC10, and SIRT5) or good prognosis (HDAC6, SIRT3, and SIRT6) [13] in CLL patients. Moreover, global HDAC enzymatic activity is a strong predicator of poor prognosis in CLL which can refine well-known prognostic factors [14]. In 2009, Tahiliani and colleagues discovered 5hydroxymethylcytosine (5-hmC) as the sixth base of the DNA in mammalian cells [15]. Ten-eleven translocation proteins (TET) are the dioxygenases responsible for the oxidation of 5-methylcytosine (5-mC) to form 5-hmC. There are three known TET isoenzymes (TET1, 2, and 3), and they require oxygen, Fe(II), and 2-oxoglutarate for their activity. This last cofactor is produced in the Krebs cycle by the isocitrate dehydrogenases (IDH) 1 and 2. Other subsequent studies suggested that the 5-hmC marker could be a step in the demethylation process [16–21] and/or a pattern allowing specific enzymes to bind hydroxymethylated regions of the genome [22–26]. Hydroxymethylation enzyme defects have previously been associated with hematological malignancies; mutations in TET2 were found in acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CMML) and induced loss of hydroxymethylation and were linked with poor prognosis [27–30]. However, reports on TET2 mutations in B cell neoplasms are Page 2 of 11 rare [31], and little is known about DNA hydroxymethylation in CLL. In the present study, we measured the mRNA expression of TET1, 2, and 3 and IDH1 and 2 by quantitative real-time PCR (qPCR) in highly purified CD19+ B cells from a large cohort of CLL patients (n = 214) and correlated these data with clinical outcome. We also investigated the potential association between the global DNA 5-hmC rate and microenvironment stimuli, especially BMSC. Methods Patients and samples This study was approved by the Bordet Institute Ethics Committee and conducted according to the principles expressed in the Declaration of Helsinki. All samples were collected at the time of diagnosis before any treatment, and after, written informed consent was obtained from 21 (...truncated)