LAUSR

Sir, Mantle cell lymphoma (MCL) is a mature B-cell malignancy characterized by the translocation t(11;14) (q13;q23) resulting in overexpression of the cell cycle protein cyclin D1 (CCND1). It is an extremely heterogeneous disease in terms of clinical course. In 2008, overexpression of the transcription factor SOX11 was also proposed as an identifier for the neoplasm [1]. Chronic lymphocytic leukaemia (CLL) shares many features with MCL. A characteristic indication for CLL is, however, the clonal expansion of both CD5 and CD23 B lymphocytes, leading to accumulation in blood and bone marrow in contrast to CD5 and CD23 in MCL. In CLL, the mutational status of the IGH-V genes envisages the prognosis along with mutations in TP53, NOTCH1, SF3B1 and BIRC3 [2]. Recent exome studies in MCL and CLL have pinpointed a number of genes mutated in B-cell malignancies including the gene ROBO1, which is a member of the Roundabout family consisting of four evolutionary receptors involved in neuronal guidance [3, 4]. ROBO1 was originally found to mediate axon growth, controlling the central nervous system midline crossing [5], but has now also been described as a tumour suppressor gene with down-regulation causing tumour cell migration and metastasizing [6]. In several types of cancer, the SLITROBO1 pathway is described to be important for oncogenesis where low expression is associated with a poor prognosis and promoter hypermethylation of ROBO1 is likewise acknowledged to play a role in tumour development [7–9]. In leukaemia and lymphoma, there has, to our knowledge, only been a single study addressing promoter methylation of ROBO1 [10]. The pathobiological implications of this transmembrane protein are demonstrated by downstream activation of b-catenin and Ecadherin by its ligand SLIT2. This facilitates altered cell adhesion and motility, also induced through the inhibition of CDC42 – an antagonist of MET signalling. Collectively, this clearly suggests that ROBO1 suppression stimulates cell migration and metastasis in human cancers [6, 7]. In order to explore the role of ROBO1 in lymphoid haematologic neoplasm, we analysed mRNA expression levels and methylation status of this gene in MCL and CLL and as control groups also in acute myeloid leukaemia (AML) and healthy individuals. Twenty-five diagnostic (24 blood and one bone marrow) and four relapse (three blood and one bone marrow) samples from patients with MCL were examined by expression analysis of ROBO1, and 24 samples were in addition examined for promoter hypermethylation of ROBO1 and Sanger-sequenced [11]. Additionally, 20 diagnostic samples from patients with CLL were analysed. Nineteen diagnostic samples from patients with AML were included for expression and promoter methylation analysis, and blood samples from 20 healthy individuals were employed for expression and methylation studies where 11 samples also were selected for Sanger sequencing as detailed in Table S1. The project was approved and registered at the ethical committee, Central Denmark Region with the Project Number M-2013-139-13. RNA was purified on a MagNA Pure 96 (Roche Diagnostics GmbH, Basel, Switzerland) followed by concentration of all samples on Amicon Ultra-0.5 centrifugal filters followed by cDNA synthesis using qScriptTM Flex cDNA kit (Quanta Bioscience, Gaithersburg, MD, USA). The qPCR assay Hs00268049_m1 (Applied Biosystems, Foster City, CA, USA) targeting ROBO1 mRNA was applied in triplicate on a MX3005 (Agilent Technologies, Santa Clara, CA, USA), and the quantitative range of the assays was determined. b-Glucuronidase (GUS) and b-2-microglobulin (B2M) were employed as reference genes, and the cell line K562 was used as positive control. DCq was calculated as Ave(Cq, target gene) Ave (Cq, reference genes). DNA was purified on a Maxwell 16 Instrument using the Maxwell 16 Blood DNA Purification Kit (Promega, Fitchburg, WI, USA), and subsequent bisulphite modification of genomic DNA was performed with the EZ DNA methylationTM kit (Zymo Research, Irvine, CA, USA). Methylation-sensitive melting curve analysis (MS-MCA) [11] was performed in a 25-uL reaction mixture containing Brilliant II SYBR Green Master Mix (Agilent Technologies), 500 nM of each primer and approximately