ORIGINAL ARTICLE Differential DNA methylation patterns of small B-cell lymphoma subclasses with different clinical behavior FB Rahmatpanah 1 , S Carstens 1 , J Guo 1 , O Sjahputera 1 , KH Taylor 1 , D Duff 1 , H Shi 1 , JW Davis 2,3 , SI Hooshmand 1 , R Chitma-Matsiga 1 and CW Caldwell 1 1 Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri School of Medicine, Columbia, MO, USA; 2 Department of Health Management and Informatics, University of Missouri, Columbia, MO, USA and 3 Department of Statistics, University of Missouri, Columbia, MO, USA Non-Hodgkin’s lymphoma (NHL) is a group of malignancies of the immune system with variable clinical behaviors and diverse molecular features. Despite the progress made in classification of NHLs based on classical methods, molecular classifications are a work in progress. Toward this goal, we used an array- based technique called differential methylation hybridization (DMH) to study small B-cell lymphoma (SBCL) subtypes. A total of 43 genomic DMH experiments were performed. From these results, several statistical methods were used to generate a set of differentially methylated genes for further validation. Methy- lation of LHX2, POU3F3, HOXC10, NRP2, PRKCE, RAMP, MLLT2, NKX6.1, LRP1B and ARF4 was validated in cell lines and patient samples and demonstrated subtype-related prefer- ential methylation patterns. For LHX2 and LRP1B, bisulfite sequencing, real-time reverse transcriptase-polymerase chain reaction and induction of gene expression following treatment with the demethylating agent, 5 0 -aza-2 0 -deoxycytidine, were confirmed. This new epigenetic information is helping to define molecular portraits of distinct subtypes of SBCL that are not recognized by current classification systems and provides valuable potential insights into the biology of these tumors. Leukemia (2006) 20, 1855–1862. doi:10.1038/sj.leu.2404345; published online 10 August 2006 Keywords: CpG island; microarray; DNA methylation; non- Hodgkin’s lymphoma; small B-cell lymphoma Introduction Every year, approximately 54 000 new cases of non-Hodgkin’s lymphoma (NHL) are diagnosed in the US. B-cell chronic lymphocytic leukemia (B-CLL)/small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL) and follicular lymphoma (FL), referred to as small B-cell lymphoma (SBCL), compromise nearly one-third of all NHL cases. 1 Current classification systems are based on clinical staging, chromosomal abnormal- ities 2 and cell surface antigens, and offer important diagnostic information. However, there is still considerable overlap in biology, clinical behavior and genetic and epigenetic alterations among the SBCL subtypes. DNA methylation within the CpG island (CGI) in promoter (and other) regions plays an important role in cancers by potentially silencing a broad spectrum of genes. 3 Aberrant patterns of CGI methylation are not random, but tumor type-specific, and can influence the gene expression profile. 4 DNA hypermethylation of some genes such as p57(KIP2), p15(INK4B), 5,6 DAPK, 7 SHP-1 8 and p73 9 are frequent occurrences in lymphoid malignancies. The SBCL subtypes are B-cell malignancies that correspond to different stages of normal B-cell differentiation and differ in clinical behavior. Whereas B-CLL/SLL and FL are generally indolent, MCL is more rapidly progressive. B-CLL/SLL is not one disease but is comprised of at least two biological subtypes representing pre-germinal center and post-germinal center B cells. 10 MCL is a pre-germinal center-derived malignancy, and FLs are of germinal center derivation. Expression microarray studies have provided information to assess clinical aggressive- ness and guide the choice of treatment in FL. 11 Alizadeh et al. 12 used a lymphochip to monitor gene expression signatures of diffuse large B-cell lymphoma subgroups derived from distinct stages of B-cell differentiation. In addition, tumor classification can also be achieved by microarray-based DNA methylation profiling. 13 Few published reports have focused on the identification of genes whose methylation profiles differ between currently recognized SBCLs. Rush et al., 14 using Restriction Landmark Genomic Scanning, studied global CGI methylation in 10 CLL patient samples. Secreted Frizzled Related Protein gene family, a negative regulator of the Wnt signaling pathway, was found to be frequently methylated in CLL patients. 15 In this study, we used a high throughput approach to classify SBCL subtypes (43 patient samples). Hierarchical clustering 16 of the DNA methylation data was used to group each subtype on the basis of similarities in their DNA methylation patterns. Our data revealed that there is diversity in DNA methylation among the different SBCL subtypes, and some genes were preferentially methylated in a subtype-related manner. The main purpose of this study was to move closer to determination of epigenetic signatures of SBCLs based on DNA gene promoter methylation profiling. Materials and methods Patient samples Tissue and blood samples were obtained from patients following diagnostic evaluation, in compliance with the Local Institutional Review Board. DNA was isolated from a total of 43 patient samples and control DNA was isolated from peripheral blood collected from seven healthy male and seven healthy female volunteers with mean age o30 years. These samples were pooled separately as NL1 (female) and NL2 (male). The QIAamp DNA Blood Minikit (Qiagen, Valencia, CA, USA) was used to purify genomic DNA. Samples from 15 patients with FL, 12 with MCL and 16 with B-CLL/SLL were used in this study. All cases of Received 28 November 2005; revised 16 May 2006; accepted 12 June 2006; published online 10 August 2006 Correspondence: Dr CW Caldwell, Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri, 115 Business Loop I-70 West, Columbia, MO 65203, USA. E-mail: caldwellc@health.missouri.edu Leukemia (2006) 20, 1855–1862 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu