Tissue Factor Pathway Inhibitor 2 (TFPI2) Is Commonly Methylated in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4617-4617
Author(s):  
Eleftheria Hatzimichael ◽  
Aggeliki Dasoula ◽  
Valentinos Kounnis ◽  
Andreas Katsenos ◽  
Tim Crook ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Genomic Dna ◽  
Tissue Factor Pathway Inhibitor ◽  
Cpg Island ◽  
Methylation Status ◽  
Tissue Factor Pathway ◽  
Beta 2 ◽  
Beta 2 Microglobulin

Abstract Abstract 4617 Background: Tissue Factor Pathway Inhibitor 2 (TFPI2) is a member of the Kunitz-type serine proteinase inhibitor family that regulates extracellular tissue homeostasis by inhibiting matrix metalloproteinases. Transcriptional silencing of TFPI2 due to DNA methylation of the CpG island of its promoter has been reported in several tumors and has been suggested to facilitate tumor cell invasion and angiogenesis. We investigated the DNA methylation status of TFPI2 in multiple myeloma (MM) patients as part of a program of epigenetic profiling of multiple myeloma. Methods: Genomic DNA extracted from two human MM cell lines (U-266 and RPMI-8226) and bone marrow aspirate samples from a well chracterized series of MM patients was modified by sodium bisulphite using the EZ DNA methylation kit, ZymoResearch. Bisulfite modified DNA was used as a template for Methylation Specific PCR with primers specific for unmethylated and methylated alleles. Control methylated (CpG Genome™ Universal Methylated, Chemicon International) and unmethylated genomic DNA was included in each experiment. Chi-square test, logistic regression analysis and unpaired t-test were used to investigate associations between gene methylation and ISS stage, presence of extramedullary disease, bone disease, anemia (hemoglobin ≤10 mg/dL), renal failure, serum albumin and beta (2)-microglobulin level. Kaplan-Meier curves were used to estimate the probabilities of survival and the Log-rank test to assess the statistical significance of differences in event rates. Results: Forty six patients with MM (26 male, 20 female; mean age 64) and two human MM cell lines were studied. According to the International Staging System (ISS) 21 patients were classified as stage I, 10 stage II and 15 as stage III disease. Methylation in the CpG island of the promoter of TFPI2 was detected in 30 patients (65%, 95%CI= 52,6–78,7%) and in both cell lines (100%). Patients with methylated TFPI2 had significantly elevated beta 2 microglobulin levels (5553 vs 3787 mg/L mean values, p=0.0002) but we did not detect a statistically significant difference in overall survival among patients with methylated versus non methylated TFPI2. No other relevant correlations were detected. Conclusion: TFPI2 is commonly methylated in MM patients. Transcriptional silencing of TFPI2 may play a role in the mechanism of myelomatogenesis and its methylation status warrants further evaluation as a diagnostic co-marker for this disease. Disclosures: No relevant conflicts of interest to declare.

BIK (Bcl2-Interacting Killer) CpG Methylation Status in Multiple Myeloma Patients: a Potential Predictor of Relapsed/Refractory Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2397-2397
Author(s):  
Eleftheria Hatzimichael ◽  
Aggeliki Dasoula ◽  
George Dranitsaris ◽  
Amalia Vassou ◽  
Justin Stebbing ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cpg Island ◽  
Performance Status ◽  
Methylation Status ◽  
Cpg Methylation ◽  
Epigenetic Silencing ◽  
Refractory Disease ◽  
Ecog Performance Status ◽  
Beta 2 ◽  
Beta 2 Microglobulin

Abstract Abstract 2397 Poster Board II-374 Background-Aim: BIK (bcl2-interacting killer) is the founding member of the BH3-only family proapoptotic proteins and is implicated in the selection of B cells in humans. The expression of BIK in cancer is prevented by chromosomal deletions or by epigenetic silencing. On the other hand, BIK upregulation is induced by proteasome inhibitors such as bortezomib and MG132. Although it has been shown that BIK is a target of epigenetic silencing in the IL-6 dependent multiple myeloma cell line KAS-6/1, no studies exist regarding the CpG methylation status of BIK in primary tumors. We wished to examine the CpG methylation status of BIK in patients with multiple myeloma (MM). Patients and methods: Bone marrow aspirate samples from individuals with MM were obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study methylation in the BIK CpG island. Genomic DNA was isolated and bisulphite modification performed using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, ZymoResearch respectively). Control methylated (CpG GenomeTM Universal Methylated, Chemicon International) and unmethylated genomic DNAs were included in each experiment. Ten bone marrow samples from individuals with borderline thrombocytopenia, that proved to have no haematological malignancy, served as negative controls. Logistic regression analyses were used to measure the association between gene methylation and age, ISS stage, ECOG performance status, extramedullary disease, bone disease, anemia (Hb 10 mg/dl), serum albumin and beta 2 microglobulin levels and relapsed/refractory disease. Results: Methylation in the BIK CpG island was studied in 40 MM patients (21 male, 19 female, mean age 66). ISS staging were as follows: stage I 14 patients (36%), II 12 patients (31%), III 12 patients (31%). Methylation of the BIK CpG island was founded in 16 patients (40%) and men were more likely to be methylated (OR=3.08, p=0.098). No correlation was found between BIK CpG methylation and age, ECOG performance status, ISS staging, anemia, beta 2 microglobulin levels, albumin levels, and overall survival. Patients methylated for BIK had a 2-fold tendency to have bone disease (p=0.35) and a 3-fold tendency to have extramedullary disease (p=0.14). Notable, patients with methylated BIK had a higher risk of relapsed/refractory disease (OR=5.4, p=0.033). Conclusions: To the best of our knowledge, this is the first demonstration of aberrant methylation in the BIK CpG island in MM patients. Interesting associations between BIK CpG methylation and some relevant clinical parameters in patients with MM were suggested by the data. Most importantly, BIK CpG methylation in this series of patients was found to be strongly associated with relapsed/refractory disease. These findings warrant further evaluation in a larger sample of patients in order to better define the prognostic and clinical utility of BIK methylation in MM. Patients with refractory/relapsed disease could possibly benefit from agents enhancing BIK expression. Disclosures: No relevant conflicts of interest to declare.

DNA methylation status of Smurf2 promoter in patients with multiple myeloma

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e19537-e19537
Author(s):  
E. Hatzimichael ◽  
A. Dasoula ◽  
J. Stebbing ◽  
G. Dranitsaris ◽  
T. Crook ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Serum Albumin ◽  
Cpg Island ◽  
Methylation Status ◽  
Risk Of Death ◽  
Extramedullary Disease ◽  
Increased Risk ◽  
Beta 2 ◽  
Beta 2 Microglobulin

e19537 Background: Multiple myeloma (MM) is an incurable interleukin (IL)-6 dependent plasma-cell malignancy. Transforming growth factor-β (TGF-β) is the major inducer of IL-6 secretion by bone marrow stromal cells. The signaling responses to TGF-b are mediated by the Smad proteins. The Smurf2 gene (Smad ubiqitiniation regulatory factor 2) encodes a Smad-specific E3 ubiquitin ligase and targets Smad2 and Smad3 for proteasome-dependent degradation. Methods: Bone marrow samples from individuals with MM were obtained at diagnosis and in 5 cases at disease progression as well. Genomic DNA was isolated and bisulphite modification was performed using commercially available kits. The methylation-specific polymerase chain reaction was employed to study the methylation status of the CpG island. Logistic regression analysis was used to measure the association between gene methylation and the development of advanced disease (DS≥ II), extramedullary disease, bone disease, anemia (Hb 10 mg/dl), serum albumin and beta 2 microglobulin levels. Results: We analysed the methylation of Smurf2 in 45 cases of MM (24 male, 21 female, mean age 66.4 years). No sample from the control population was found methylated. The Smurf2 gene promoter was found to be methylated in 11/45 MM patients (24%). Interesting trends were noted where patients with methylated Smurf2 promoter had an increased risk of death (HR = 1.3; p = 0.68), anemia (OR=2.1, p=0.2) and advanced stage (OR=1.3, p=0.6) and a reduced risk of extramedullary disease (OR= 0.2, p=0.2). No association was found between Smurf2 methylation status and bone lytic lesions, serum albumin levels or beta-2 microglobulin levels. Conclusions: Interesting associations between Smurf2 methylation and some relevant clinical parameters in patients with MM were suggested by the data. These findings warrant further evaluation in a larger sample of patients in order to enhance our statistical power and better define the prognostic and clinical value of Smurf2 methylation in MM. No significant financial relationships to disclose.

Snk/Plk2 Methylation Is a Frequent Event in Patients with Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4479-4479
Author(s):  
Eleftheria Hatzimichael ◽  
George Dranitsaris ◽  
Aggeliki Dasoula ◽  
Nelofer Syed ◽  
Justin Stebbing ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Serum Albumin ◽  
Median Survival ◽  
Cpg Island ◽  
Methylation Status ◽  
Extramedullary Disease ◽  
Frequent Event ◽  
Beta 2 ◽  
Beta 2 Microglobulin

Abstract Background-Aim: The polo like kinases (Plk) are highly conserved in evolution and have critical functions in the regulation of proliferation and cell cycle checkpoints. We previously showed that polo like kinase 2 (Snk/Plk2) is subject to methylation-dependent transcriptional silencing in B lymphomas, with a very high frequency in Burkitt lymphomas, implying that Snk/Plk2 may have a tumour suppressor function in B lymphocytes. However, no study has examined epigenetic changes in plasma cell dyscrasias. Here, we have examined CpG methylation in Snk/Plk2 in a well-characterised series of multiple myeloma (MM) patients. Patients and Methods: Bone marrow samples from individuals with MM were obtained at diagnosis and disease progression. Genomic DNA was isolated and bisulphite modification was performed using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). The methylation-specific polymerase chain reaction (MSP) with primers for methylated and unmethylated alleles of the Snk/Plk2 gene promoter was employed to study its methylation status. Control methylated (CpG Genome™ Universal Methylated, Chemicon International) and unmethylated genomic DNAs were included in each experiment. Ten bone marrow samples from individuals with borderline thrombocytopenia, that proved to have no haematological malignancy, served as negative controls. Survival curves were generated using the method of Kaplan-Meier and compared with the log-rank test. Logistic regression analyses were also used to measure the association between gene methylation and the development of advanced disease (DS³II), extramedullary disease, bone disease, anemia (Hb≤10 mg/dl), serum albumin and beta 2 microglobulin levels. Results: We analysed the methylation of Snk/Plk2 in 45 cases of multiple myeloma (MM) (24 male, 21 female, mean age 66.4 years ±12.4). Using the Durie and Salmon staging system MM patients’ disease stages were as follows: smoldering MM 7/45, IA 9/45, IIA 13/45 patients, IIIA 7/45 patients and IIIB 9/45 patients. Classical cytogenetic analysis was available in 30/45 patients and none of them was found to have chromosome 13 abnormalities. No sample from the control population was found methylated. The Snk/Plk2 promoter was found to be methylated in 27/45 (60%) MM patients. Median survival of patients in whom the Snk/Plk2 CpG island was methylated was 7.1 years compared to a median survival of 8.8 years for unmethylated. However Snk/Plk2 methylation was not a predictor of excess mortality (HR=0.6, p=0.5), bone lytic lesions (OR=0.56, p=0.3), anemia (OR=0.5, p=0.3) or advanced stage as defined above (OR=0.8, p=0.7). A trend was noted where patients with methylated Snk/Plk2 had a reduced risk of developing extramedullary disease (OR=0.3, p=0.1). No association was found between the methylation and serum albumin (p=0.3) or beta 2 microglobulin levels (p=0.8). Conlusions: We examined for the first time the methylation status of Snk/Plk2 in MM patients and showed that it is a common event in these patients implying that loss of function in this gene is frequently involved in the pathogenesis of MM. However there was lack of association between the methylation status of the gene and relevant clinical parameters. Further evaluation in a larger sample of patients is needed in order to better define the prognostic and clinical value if any of Snk/Plk2 methylation in MM.

scholarly journals Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but not Decreased Promoter Methylation in Colorectal Cancer

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 539 ◽  
Author(s):  
Alexei J. Stuckel ◽  
Wei Zhang ◽  
Xu Zhang ◽  
Shuai Zeng ◽  
Urszula Dougherty ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Cell Lines ◽  
Promoter Methylation ◽  
Cpg Island ◽  
Methylation Status ◽  
Cxcr4 Expression ◽  
Normal Colonic Mucosa ◽  
Gene Body ◽  
Expression Levels

In colorectal cancer (CRC), upregulation of the C-X-C motif chemokine receptor 4 (CXCR4) is correlated with metastasis and poor prognosis, highlighting the need to further elucidate CXCR4’s regulation in CRC. For the first time, DNA methylation and 5-hydroxymethylcytosine aberrations were investigated to better understand the epigenetic regulation of CXCR4 in CRC. CXCR4 expression levels were measured using qPCR and immunoblotting in normal colon tissues, primary colon cancer tissues and CRC cell lines. Publicly available RNA-seq and methylation data from The Cancer Genome Atlas (TCGA) were extracted from tumors from CRC patients. The DNA methylation status spanning CXCR4 gene was evaluated using combined bisulfite restriction analysis (COBRA). The methylation status in the CXCR4 gene body was analyzed using previously performed nano-hmC-seal data from colon cancers and adjacent normal colonic mucosa. CXCR4 expression levels were significantly increased in tumor stromal cells and in tumor colonocytes, compared to matched cell types from adjacent normal-appearing mucosa. CXCR4 promoter methylation was detected in a minority of colorectal tumors in the TCGA. The CpG island of the CXCR4 promoter showed increased methylation in three of four CRC cell lines. CXCR4 protein expression differences were also notable between microsatellite stable (MSS) and microsatellite instable (MSI) tumor cell lines. While differential methylation was not detected in CXCR4, enrichment of 5-hydroxymethylcytosine (5hmC) in CXCR4 gene bodies in CRC was observed compared to adjacent mucosa.

Methylation Profile of B-Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2951-2951
Author(s):  
Jun Fan ◽  
Asou Norio ◽  
Masao Matsuoka
Keyword(s):  
Dna Methylation ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Mononuclear Cells ◽  
Cpg Island ◽  
Methylation Status ◽  
Lymphocytic Leukemia ◽  
Dna Hypomethylation ◽  
Peripheral Blood Mononuclear

Abstract DNA methylation plays an important role in the development and aging of mammalian cells, and its dysregulation has been frequently observed in cancer cells. The purpose of this study is to investigate the involvement of aberrant DNA methylation in B chronic lymphocytic leukemia (B-CLL) cells. We compared methylation status of B-CLL cells isolated from patients with that of normal CD19+ cells isolated from health donors by methylated CpG island amplification/representative difference analysis method. 5 hypermethylated and 27 hypomethylated DNA regions were identified in B-CLL sample. Among the 27 hypomethylated regions, 5 located on chromosome 9q34, 3 on 10q25-26 and 4 on 19q13. Methylation status was confirmed by sequencing using sodium bisulfite-treated DNA samples. By comparing DNA samples from same patients at different clinical stages, we found that lower methylation density in these regions is linked with disease progression. Expression of 15 genes surrounding hypomethylated regions was studied by RT-PCR. Expression of laminin beta3 gene and melanotransferrin gene was found to be upregulated in all B-CLL cell lines as well as lymphoma cell lines comparing with normal CD19+ peripheral blood mononuclear cells. B-cell CLL/lymphoma 11b gene showed increased expression in only 2 B-CLL cell lines. For other genes, no transcriptional change was found regardless of changed DNA methylation. This study showed the predominance of DNA hypomethylation in B-CLL cells compared with hypermethylation. Hypomethylated regions clustered in a limited number of chromosomes and methylation density appeared to be inversely correlated with disease progress. Figure Figure

Genome-Wide Identification of Aberrant Promoter Associated CpG Island Methylation in Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2127-2127
Author(s):  
Shao-qing Kuang ◽  
Weigang Tong ◽  
Hui Yang ◽  
Mathew K. Lee ◽  
Zhi-Hong Fang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
B Cell ◽  
Cell Lines ◽  
Cpg Island ◽  
Trichostatin A ◽  
Methylation Status ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Leukemia Cell Lines ◽  
Aberrant Dna Methylation

Abstract Aberrant DNA methylation is a common molecular feature of both pediatric and adult ALL. Specific methylation patterns predict for poor prognosis (Shen et al Blood 2004), and reactivation of epigenetically inactivated molecular pathways results in induction of leukemia cell death (Kuang et al. Oncogene 2007). Until now most studies of methylation in ALL have been based on arbitrary gene selection methods. To overcome this limitation and to study hundreds of promoter CpG islands simultaneously, we have developed a method that combines MCA (Methylated CpG Island Amplification) with either RDA (Representational Difference Analysis) or the Agilent Promoter Microarray platform. With these methods differentially methylated DNA treated with bisulfite is generated after mixing tester DNA (in our case DNA from de novo refractory Ph negative and MLL negative ALL patients) with driver DNA (normal B cell controls) and using specific restriction enzymes and several rounds of PCR. DNA fragments thus generated are either cloned (RDA) or labeled and spotted on the Agilent Array. Using this technology, that can potentially interrogate up to 17K promoters, we have identified 932 promoters targets of aberrant DNA methylation in poor risk ALL from patients that cannot be currently identified by standard molecular methods (Ph and MLL negative). The genes associated with these promoters are distributed through the human genome but an overrepresentation of methylated promoters located in chromosomes 3, 9, 11 and 19 was detected. Using molecular pathway clustering analysis, 404 of these genes are grouped together in 29 specific functional pathways. We have validated the methylation of 31 of these 923 genes by bisulfite pyrosequencing. Of these, 27 (87%) were confirmed to be hypermethylated in 23 human leukemia cell lines but not in normal controls (N=15). Methylation status analysis of these 27 genes allowed for the segregation of T cell versus B cell leukemia cell lines. Fifteen of these genes (GIPC2, RSPO1, MAGI1, CAST1, ADCY5, HSPA4L, OCLN, EFNA5, MSX2, GFPT2, GNA14, SALL1, MYO5B, ZNF382 and MN1) were also frequently hypermethylated in primary ALL samples. Expression analysis of 6 of these genes (GIPC2, MAGI1, ADCY5, HSPA4L, OCLN and GNA14) in leukemia cell lines further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with 5′-aza-2′-deoxycytidine and trichostatin A resulted in gene re-expression, further confirming the role of DNA methylation in their silencing. In summary, we have identified in excess of 900 targets of aberrant DNA methylation in ALL. The study of the epigenetically suppressed pathways represented by these genes should allow us to further understand the molecular pathogenesis of ALL and develop new prognostic biomarkers for patients with Ph and MLL negative disease.

UTX, a Histone Demethylase, Is Inactivated through Homozygous Deletion, Mutation, and DNA Methylation in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1798-1798
Author(s):  
Brian A Walker ◽  
Paola E. Leone ◽  
Nicholas J Dickens ◽  
Kevin D Boyd ◽  
David Gonzalez ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Cpg Island ◽  
Chromatin Condensation ◽  
Methylation Status ◽  
Significant Proportion ◽  
Histone Demethylase ◽  
Homozygous Deletions

Abstract Abstract 1798 Poster Board I-824 Histone modifications are known to mediate transcriptional regulation through changes in chromatin condensation and as such can lead to aberrant transcriptional patterns resulting in malignant transformation. Modulation of chromatin structure via histone modification is becoming recognised as an important pathogenic mechanism in myeloma and has been suggested by the over-expression of MMSET, a histone methyltransferase, by the t(4;14) chromosomal rearrangement. More recently inactivation of UTX, a histone demethylase, has also been suggested to have a role in myeloma pathogenesis and both UTX and MMSET are mediators of transcriptional repression. UTX is inactivated in a number of different cancer cell lines but importantly, mutations and deletions have been detected in myeloma cell lines and we wished to follow up on this observation in uniformly treated clinical cases. UTX is a large gene found on the X chromosome covering 240 kb of genomic DNA and consists of 29 exons encoding a protein with both JmjC-domains and tricopeptide repeats responsible for histone demethylation and polycomb protein interactions. Inactivation of UTX occurs through deletions of individual exons through to large whole gene deletions as well as by mutations scattered throughout the 29 exons. A further mechanism of UTX inactivation which has not been looked for to date is via DNA methylation of the CpG island upstream of the transcriptional start site. We set out to determine the status of UTX in our dataset which includes expression, mapping, and methylation array data from presenting myeloma samples entered into the MRC Myeloma IX clinical trial. The gene expression of UTX was measured on 272 samples using Affymetrix U133 Plus 2.0 arrays and showed that 80% of samples do not express UTX transcripts but using expression quartile analysis we could not detect an effect on overall survival. The mechanism underlying the abrogation of expression was investigated further using the Affymetrix 500K SNP mapping array on a subset of 114 samples to detect copy number alterations. UTX was hemizygously deleted in 21 (42%) female samples and was completely deleted in 1 male sample, at the resolution of the arrays. In order to determine if individual exons were deleted, at a resolution below that detectable by mapping arrays, we performed quantitative PCR coupled with high resolution melting (HRM) analysis using the Rotor-gene Q real-time cycler (Qiagen). Exons were amplified, over 40 cycles, to obtain products of ∼200 bp using LC Green Plus mastermix (Idaho Technologies) in a 10 μl reaction on the Rotor-gene Q with a final HRM step from 72-95 °C with increments of 0.1 °C. Amplification plots combined with the HRM step allows us to identify both homozygous deletions and mutations within the exons. We screened all 114 samples for micro-deletions and mutations and found homozygous deletions in ∼7% of samples and identified a significant proportion of mutations using the HRM method which accounted for a total of ∼10% of gene inactivation. In order to determine if methylation could be responsible for inactivation of the remaining allele we used the Illumina Infinium humanmethylation27 array to study the methylation status at the UTX locus. This array interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. The results of this analysis are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Samples were analyzed using Illumina GenomeStudio and the custom differential methylation algorithm. In samples with a diploid copy number of UTX the methylation signals covered 2 ranges: hemi-methylated (0.35-0.55, n=7) and hyper-methylated (0.73-0.89, n=14). In samples with 1 copy of UTX, which includes all males, there were 3 ranges: hypomethylated (0.08-0.21, n=5), hemi-methylated (0.35-0.51, n=3), and hypermethylated (0.66-0.88, n=48). All of the hypomethylated samples with a single copy of UTX were male, and at least 1 of these samples contained an inactivating exonic deletion resulting in complete loss of function. These data indicate that methylation of the residual allele contributes significantly to the inactivation of UTX along with interstitial deletions and mutations. We will go on to present data on the interaction of UTX with variation at the UTY locus and how this modulates behaviour of the myeloma clone. Disclosures No relevant conflicts of interest to declare.

Snk/Plk2 CpG methylation in patients with multiple myeloma

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e19532-e19532
Author(s):  
A. Dasoula ◽  
E. Hatzimichael ◽  
G. Dranitsaris ◽  
L. Benetatos ◽  
N. Syed ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Median Survival ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Methylation ◽  
Clinical Value ◽  
Very High Frequency ◽  
Frequent Event ◽  
Beta 2 Microglobulin

e19532 Background: We previously showed that polo like kinase 2 (Snk/Plk2) is subject to methylation-dependent transcriptional silencing in a very high frequency in Burkitt lymphomas. Here, we have examined CpG methylation in Snk/Plk2 in a well-characterized series of multiple myeloma (MM) patients. Methods: Bone marrow samples from individuals with MM were obtained at diagnosis and in 5 cases at disease progression as well. Genomic DNA was isolated and bisulphite modification was performed using commercially available kits. The methylation-specific polymerase chain reaction (MSP) was employed to study its methylation status. Control methylated and unmethylated genomic DNAs were included in each experiment. Ten bone marrow samples from individuals proven to have no haematological malignancy, served as negative controls. Logistic regression analyses were used to measure the association between gene methylation and the development of advanced disease (DS≥II), extramedullary disease, bone disease, anemia (Hb 10 mg/dl), serum albumin and beta 2 microglobulin levels. Results: We analyzed the methylation of Snk/Plk2 in 45 cases of MM (24 male, 21 female, mean age 66.4 years ±12.4). Classical cytogenetic analysis was available in 30/45 patients and none of them was found to have chromosome 13 abnormalities. No sample from the control population was found methylated. The Snk/Plk2 promoter was found to be methylated in 27/45 patients (60%). Median survival of patients in whom the Snk/Plk2 CpG island was methylated was 7.1 years compared to a median survival of 8.8 years for unmethylated. However Snk/Plk2 methylation was not a predictor of excess mortality (HR=0.6, p=0.5), bone lytic lesions (OR=0.56, p=0.3), anemia (OR=0.5, p=0.3) or advanced stage as defined above (OR=0.8, p=0.7). Conclusions: Snk/Plk2 DNA Methylation is a frequent event in patients with multiple myeloma. There was no association between the methylation status of the gene and relevant clinical parameters. Further evaluation in a larger sample of patients is needed in order to better define the prognostic and clinical value if any of Snk/Plk2 methylation in MM. No significant financial relationships to disclose.

Genome-Wide DNA Methylation Profile of CLL with 17p del Allowed Identification of Multiple Epigenetically Inactivated Molecular Pathways with Prognostic Value in Human Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 492-492
Author(s):  
Wei-Gang Tong ◽  
William G. Wierda ◽  
Neby Bekele ◽  
Shao-Qing Kuang ◽  
Michael J. Keating ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Cpg Island ◽  
Trichostatin A ◽  
Methylation Status ◽  
Cpg Islands ◽  
Human Leukemia ◽  
Dna Hypomethylation ◽  
Line P ◽  
Normal Controls

Abstract 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%), IgVH 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 IgVH mutation status, ZAP70 expression and patient survival. Unmutated IgVH 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.

A Sensitive Molecular Method for Detection of Disseminating Hematopoietic Tumor Cells by Using Specific Epigenetic DNA Methylation Biomarkers

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4850-4850
Author(s):  
Michael X Wang ◽  
Xiaohui Zhao ◽  
Srilatha Nalluri ◽  
Huan-You Wang ◽  
Kristen H. Taylor ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Genomic Dna ◽  
Cpg Island ◽  
Lymphoblastic Leukemia ◽  
Single Step ◽  
Tumor Cell Lines

Abstract Background: Most cancer deaths are caused by hematogenous spread and subsequent metastasis. Emerging data indicates that the presence of circulating tumor cells in peripheral blood (CTC) and disseminating tumor cells in bone marrow (DTC) is an early event in tumorigenesis. These circulating or disseminating rare tumor cells may represent a distinct clone with cancer stem cell properties (metastatic stem cells). Clinically, the presence of CTC and DTC is relevant to overt metastases. Early detection and characterization of these rare biologically distinct tumor cells with sensitive and specific methods provide vital information for cancer biology and early clinical intervention and thus improve patients’ survival. Method Design: Genomic DNA was extracted from patient blood, bone marrow aspirate specimens and cancer cell lines prior to digestion with 4 methylation sensitive restriction enzymes. Hypermethylated CpG island regions in tumors, in contrast to their counterpart in normal cells which are completely digested, remain resistant to digestion and therefore can be differentially amplified by PCR. Thus, the specific PCR products on the gel represent the specific methylation loci in the tumor cells in the patient specimens. Results: First, we tested a total of 26 cancer cell lines including 17 hematopoietic tumors and 9 solid tumors. The hematopoietic tumor cell lines represent a spectrum of B-cell malignancies, T-cell lymphoblastic leukemia and acute myeloid leukemia. The solid tumor cell lines represent the major anatomic sites of human carcinoma including lung, colon, breast, prostate, ovarian and skin (melanoma). We found multiple DNA methylation markers specific for lymphoid, myeloid and solid tumors. By using DLC-1 (deleted in liver cancer-1), a tumor suppressor gene, as a single marker, CpG island methylation was detected in 13 out of 17 hematopoietic tumor cell lines (76%) and 6 out of 9 solid tumor cell lines (67%). We then used B-cell acute lymphoblastic leukemia (B-ALL) as a testing case to verify the method in a total of 135 clinical specimens. DLC-1 methylation was detected in 64% and 54% of diagnostic bone marrow aspirates and peripheral blood specimens, respectively, but none was detected in normal or non-leukemic bone marrow or blood control samples. By adding two additional methylation markers PCDHGA12 and CDH1, greater than 90% of B-ALL patients can be detected. We also traced 4 B-ALL cases and 4 follicular lymphoma cases up to 10 years retrospectively and found that the DLC-1 methylation is exactly correlated with patient clinical status. Lastly, by mixing normal and leukemic cell genomic DNA, analytic sensitivity was determined as 0.1% or 10−3 in a single-step PCR and 0.00001% or 10−6 in a nested PCR suggesting that this method is capable of detecting as few as 5 leukemic cells in a single-step PCR. Conclusion: By utilizing tumor specific DNA methylation marker(s), we have developed a simple, highly sensitive and specific gel-based PCR assay, namely Multiple Methylation Sensitive Enzyme Restriction PCR (MSR-PCR) for detection of CTC and DTC from blood and bone marrow of majority of hematopoietic malignancies. The method can potentially be used for early diagnosis and molecular monitoring in vast majority of clinical cancer patients.

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