Leukemia (2006) 20, 1080–1088 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu ORIGINAL ARTICLE Deregulated expression of fat and muscle genes in B-cell chronic lymphocytic leukemia with high lipoprotein lipase expression M Bilban1,2,8, D Heintel3,8, T Scharl4, T Woelfel4, MM Auer2,3, E Porpaczy3, B Kainz3, A Kro¨ber5, VJ Carey6, M Shehata2,3, C Zielinski2, W Pickl7, S Stilgenbauer5, A Gaiger2,3,6, O Wagner1,2,UJa¨ger2,3 and the German CLL Study Group 1Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria; 2Ludwig Boltzmann Institute for Clinical and Experimental Oncology, Vienna, Austria; 3Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna; Vienna, Austria; 4Department of Statistics and Probability Theory, Vienna University of Technology, Vienna, Austria; 5Department of Internal Medicine III, University of Ulm, Ulm, Germany; 6Department of Medicine, Harvard Medical School, Boston, MA, USA and 7Institute of Immunology, Medical University of Vienna, Vienna, Austria Lipoprotein lipase (LPL) is a prognostic marker in B-cell studies was the identification of a novel prognostic marker, the chronic lymphocytic leukemia (B-CLL) related to immunoglo- ZAP-70 protein, which has already entered routine diagnos- bulin VH gene (IgVH)mutational status. We determined gene tics.3,4,9,26–31 However, microarray analysis has identified a expression profiles using Affymetrix U133A GeneChips in two groups of B-CLLs selected for either high (‘LPL þ ’, n ¼ 10) or number of other potential prognostic or therapeutic targets that low (‘LPLÀ’, n ¼ 10) LPL mRNA expression. Selected genes have not yet been validated for their clinical importance. were verified by real-time PCR in an extended patient cohort One of the genes most closely related to IgVH mutation status (n ¼ 42). A total of 111 genes discriminated LPL þ from LPLÀ B- is lipoprotein lipase (LPL). High expression of this central CLLs. Of these, the top three genes associated with time to first enzyme of lipid metabolism was associated with unmutated B- treatment were Septin10, DMD and Gravin (Pp0.01). The CLL in almost all profiling studies, regardless of methodology or relationship of LPL þ and LPLÀ B-CLL gene expression 1,3,4,32,33 signatures to 52 tissues was statistically analyzed. The LPL þ B-cell selection process. We and others have recently B-CLL expression signature, represented by 64 genes was shown that mRNA expression of LPL predicts treatment-free as significantly related to fat, muscle and PB dendritic cells well as overall survival in B-CLL patients.32,33 High intracellular (Po0.001). Exploration of microarray data to define functional levels of LPL protein can be detected in unmutated CLL cells.32 alterations related to the biology of LPL þ CLL identified two Lipoprotein lipase is normally produced and secreted by functional modules, fatty acid degradation and MTA3 signaling, adipocytes, macrophages, cardiac and skeletal muscle cells as being altered with higher statistical significance. Our data and catalyzes the hydrolysis of the triacylglycerol component of show that LPL þ B-CLL cells have not only acquired gene 34–36 expression changes in fat and muscle-associated genes but chylomicrons and very low-density lipoproteins (VLDL). also in functional pathways related to fatty acid degradation The protein is bound to the surface of endothelial cells as well as and signaling which may ultimately influence CLL biology and B-CLL cells.37 Additional non-catalytic binding functions lead to clinical outcome. increased accumulation and cellular uptake of lipoproteins.35 Leukemia (2006) 20, 1080–1088. doi:10.1038/sj.leu.2404220; Interestingly, LPL is regulated by cytokines including tumor published online 13 April 2006 necrosis factor-alpha (TNF-a) which also plays an important role Keywords: B-CLL; lipoprotein lipase; gene expression profiling; 38–40 prognostic markers; septin10; dystrophin in the pathophysiology of B-CLL. Although the function of LPL in B-CLL is still not understood, its effects on metabolism, energy homeostasis and microenvironment suggest a consider- able biological relevance for the disease. We hypothesized that microarray analysis of two subsets of B- Introduction CLLs differing greatly in their LPL expression should reveal gene patterns not only associated with prognosis but also with Gene-expression profiling has introduced a new dimension into biology, particularly in regard to fatty acid metabolism and our understanding of biology as well as clinical behavior of adipose tissue. B-cell chronic lymphocytic leukemia (B-CLL).1–10 Initial studies Using this single gene denominator approach we were able to showed that all B-CLL cases have common features resembling identify known as well as novel genes related to IgVH mutational those of a memory B cell.1,3,11,12 Subsequently, considerable status, lipid metabolism, or both. We show that the expression differences in gene expression were linked to various discri- signature of unmutated/LPL-high B-CLL is closely related to that minators between good and poor risk B-CLL: these in- of adipose tissue and muscle cells. Moreover, we determined clude cytogenetic aberrations,13,14 expression of the CD38 the prognostic power of the most prominent factors by real-time protein,15,16 the expression of microRNAs,17,18 and most PCR using RNA from a well-defined patient cohort. These importantly, immunoglobulin VH gene (IgVH) mutational sta- include novel genes as well as several genes, previously tus.15,16,19–25 The first practical result emerging from these identified by expression profiling in the context of mutational status, that have never been validated. Besides the intriguing Correspondence: Professor U Ja¨ger, Department of Internal Medicine I, biological questions regarding the origin, regulation, or plasti- Division of Hematology and Hemostaseology, Medical University of city of B-CLL cells raised in this study, we now provide evidence Vienna, Wa¨hringer Gu¨rtel 18-20, A-1090 Vienna, Austria. for the clinical importance of some of the discriminatory genes E-mail: [email protected] 8These authors contributed equally to this work. commonly found in expression profiling analysis. Received 8 September 2005; revised 21 February 2006; accepted 27 Using this approach it was possible to stratify CLL subgroups February 2006; published online 13 April 2006 by LPL-expression and to recover prognostic factors known from LPL expression profiling in B-CLL M Bilban et al 1081 the studies using IgVH mutational status as well as novel targets from the Weizmann Institute of Science (http://bioinfo.weiz- in high-risk B-CLL. mann.ac.il/bioinfo.html), and a previously published classifica- tion scheme for biological functions.44 Hierarchical clustering was performed using Pearson’s correlation coefficient as Patients, materials and methods distance measure and Ward’s optimization criterion to cluster genes and samples, respectively. To visualize relationships and cRNA synthesis and gene expression profiling to judge the clustering quality, we presented samples and genes Total RNA from peripheral mononuclear cells (PBMC) of 20 in two-dimensional principal component space of the corre- well-characterized B-CLL patients (diagnosed at the Division of sponding variance-covariance matrix. Hematology at the Medical University of Vienna) was isolated Publicly available Affymetrix U133A expression data for 32 as described. Patient characteristics are given in Table 1. 52 tissues were used from SymAtlas (http://symatlas.gnf.org/ Informed consent was obtained from all patients according to SymAtlas/) for a phenotypic analysis. GeneChip files were study protocols approved by the local ethics committee (EK nos. preprocessed as described above for CLL samples. 38/1998, 505/2002, 495/2003). Total RNA was repurified with Gene set enrichment analysis (GSEA)45 is a computational RNeasy MinElute kit as per the manufacturer’s instructions method that determines if a given set of genes (e.g. known (Qiagen Valencia, CA, USA) and checked for integrity pathways, specific areas of the genome or clusters from a cluster by agarose gel electrophoresis. A measure of 5 mg total RNA analysis) shows statistically significant differences between two was then used for GeneChip analysis. Preparation of phenotypic states (i.e. LPL þ or LPLÀ CLLs). Briefly, the GSEA cRNA, hybridization to human U133A GeneChips (Affymetrix, calculation involves three steps: calculation of an enrichment Santa Clara, CA, USA) and scanning of the arrays were carried score (ES) followed by estimation of the significance level of ES out according to the manufacturer’s protocols (https://www. and adjustment for Multiple Hypothesis Testing. All GSEA 41 affymetrix.com; Bilban et al. ). calculations were done in ‘R’. Bioinformatic analysis RNA signal extraction, normalization and filtering (to eliminate Real-time PCR genes with extremely low expression) was performed as The significance of selected genes was validated by real-time described42 (http://www.bioconductor.org/). We identified sig- PCR in 42 B-CLL PBMNC patient samples (20 mutated, 22 nificant differences between sample groups using a previously unmutated, 25 Binet A, 7 Binet B, 10 Binet C; 17 female, 25 described method:43 we calculated a t-statistic of the two groups male). Total RNA was extracted from CLL and PBM cells and of CLLs for each gene. We addressed the multiple comparisons analyzed by RT-PCR for LPL mRNA expression as described problem by estimating the false discovery rate (FDR) in a simple previously32 using RNA-Beet (Tel-Test Inc., Friendswood, TX, manner as the ratio of the expected number of false positives at a USA) and first strand cDNA synthesis kit (Amersham Pharmacia given P-value threshold to the number of positives actually Biotech, Inc., Piscataway, NJ, USA). Real-time PCR was found. Using this statistical approach comparisons of gene performed with the ABI Prism 7000 Sequence Detector (Applied expression with absolute fold changes of at least 1.5-fold Biosystems, Foster City, CA, USA) according to the manufac- (increase or decrease) were selected at qo0.01.