Progress Report

Global Gene Methylation Profile of CLL Patients and Its Implication in Therapy

Weigang Tong, Guillermo Garcia-Manero

Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX 77030

1. During the past 6 months, we have been working on the characterization of 280 genes we have identified that are aberrantly methylated in CLL patients compared with normal B cells. Our recent updated data has been presented at the American Society of Hematology (ASH) annual meeting as an oral presentation in Atlanta in December of 2007. The following is the abstract we submitted to ASH. We have analyzed 22 most important genes out of the list and analyzed their methylation profile in 78 CLL patients. We also correlated the methylation status of these genes with patient prognosis and survival. At the moment, we are in the process of preparing the manuscript for submission to the journal “Cancer Research”. Hopefully, the manuscript will be submitted by the end of this month.
2. At the mean time, we are also trying to screen these 22 genes in a large cohort of CLL patients treated with standard FCR therapy. Dr. Wierda from the Leukemia Department and CLL Research Consortium will provide us with these patient samples. We are already in the process of acquiring these samples and analyzing their methylation profile.
3. Using the same MCA/microarray technique, we have also identified around 500 genes that are methylated in ALL patients. We are comparing this list of genes in ALL with the CLL genes, trying to identify common genes that are methylated in both ALL and CLL. We have identified 48 of these common genes and are trying to characterize their methylation and expression status in CLL. Since ALL and CLL have very different biology and maybe have different cells of origin. By doing this, we hope to identify genes that are important in the CLL pathogenesis.

Title: Genome-wide DNA methylation profile of CLL with 17p del allowed identification of multiple epigenetically inactivated molecular pathways with prognostic value in human leukemia

Authors: Wei-Gang Tong, William G Wierda, Neby Bekele, Shao-Qing Kuang, Michael J. Keating and Guillermo Garcia-Manero. Deaprtment of Leukemia and Biostatistics, UT/MD Anderson Cancer Center, Houston, TX, United States, 77030

Body: Aberrant DNA methylation of multiple promoter associated CpG islands is a very prevalent phenomenon in human leukemias. Data from our laboratory indicates that methylation profiling allows the identification of leukemia patients with different risk and prognosis. Despite the advances in the understanding of the molecular biology of CLL, few studies of DNA methylation have been performed in CLL. In the current study, we have developed a new assay combining MCA (Methylated CpG island Amplification) with the Agilent promoter CpG array to identify simultaneously hundreds of abnormally methylated CpG islands in CLL. To perform this, we compared DNA from two CLL patients with 17p del (tester) with that of CD19+ B cells from two age-matched controls (driver). We identified 280 promoter CpG islands differentially methylated in CLL compared to normal controls. Most of these genes are located on chromosomes 19 (16%), 16 (11%), 17 (10%) and 11 (9%). We also performed interaction pathway and functional analysis of these 280 genes using the online Ingenuity Pathway Analysis tools. The initial analysis divided these genes into 25 functional networks, with the majority of genes fall into top 10 networks. The major functions of genes in these interaction networks involve cancer, organ development, cell death, drug metabolism, DNA replication and repair. We validated 22 of these genes (ADCY5, R-spondin1, LHX1, GALGT2, TFAP2C, ING1, SOX11, SOX14, SALL1, LTBP2, APP, DXL1, DLX4, KLK10, BCL11B, NR2F2, FAM62T, HAND2, BNC1, SPOCK, Prima1 and MLL1) in samples from 78 CLL patients and 10 age-matched normal controls. The characteristics of the 78 patients are: median age 59 (range 39-79), male 70%, Rai stage 0-II/III-IV (83%/17%), IgV_H unmutated 49%, ZAP-70 positive 33%. Our results indicate that most of the genes identified by the array are frequently hypermethylated in CLL patients compared with healthy controls. Methylation frequency ranged from 20%-100% in CLL patients. Expression analysis of four selected genes (LHX1, GALGT2, TFAP2C and Prima1) in human leukemia cell lines and CLL patient samples by real-time PCR further confirmed methylation associated gene silencing, and treatment of these cell lines with hypomethylating agent 5-aza-2-deoxycitidine with or without the HDAC inhibitor Trichostatin A resulted in gene re-expression and induction of DNA hypomethylation. We also analyzed the association of methylation status of these genes with IgV_H mutation status, ZAP70 expression and patient survival. Unmutated IgV_H was associated with increased methylation levels of LINE (p<0.0001), which is a marker for global gene methylation and SALL1 (p=0.00008). Expression of ZAP-70 (>20%) was associated with increased methylation levels of LINE (p<0.00001), MLL (p=0.02) and SALL1(p=0.048). Further analysis showed that methylation status of LINE (p=0.007), SALL1 (p=0.019), ADCY5 (p=0.021), R-spondin1 (p=0.002) and APP (p=0.002) correlated with survival. In conclusion, our studies indicate that MCA/promoter array technique allows the identification of large number of promoter CpG islands aberrantly methylated in CLL and also the identification of novel tumor suppressors and signaling pathways that could be important in the tumorigenesis of CLL and other hematological malignancies.