DNA methylation status of Smurf2 promoter in patients with multiple myeloma

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.

Download Full-text

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

Blood ◽

10.1182/blood.v112.11.4479.4479 ◽

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.

Download Full-text

Methylation Status of SMURF2 and Correlation with Clinical Parameters in Patients with Multiple Myeloma

Blood ◽

10.1182/blood.v112.11.4472.4472 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4472-4472

Author(s):

Eleftheria Hatzimichael ◽

Aggeliki Dasoula ◽

Justin Stebbing ◽

George Dranitsaris ◽

Konstantinos Bourantas ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Serum Albumin ◽

Methylation Status ◽

Gene Promoter ◽

Clinical Parameters ◽

Risk Of Death ◽

Extramedullary Disease ◽

Beta 2 ◽

Beta 2 Microglobulin

Abstract Background–Aim: Multiple myeloma (MM) is an incurable interleukin (IL)-6 dependent plasma-cell malignancy. It has been previously suggested that 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 a heteromeric complex of two types of transmembrane serine threonine kinase receptors at the cell surface and their intracellular substrates, the Smad proteins. The Smurf2 gene (Smad ubiqitiniation regulatory factor 2) encodes a Smad-specific E3 ubiquitin ligase. Smurf2 targets Smad2 and Smad3 for proteasome-dependent degradation and enhances the inhibitory activity of Smad7. In addition to its role in regulating TGF-beta signalling, Smurf2 is up-regulated during replicative senescence in response to telomere shortening and induces senescence when ectopically expressed. Together these properties imply that Smurf2 may have tumour suppressor potential and may be involved in MM pathogenesis, but no studies exist regarding the smurf2 methylation status in human neoplasia. Here we have investigated for the first time the methylation status of Smurf2 in patients with MM. Patients and methods: Bone marrow samples from individuals with multiple myeloma (MM) were obtained at diagnosis and in some cases at 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 Smurf2 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 analysis was 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 SMURF2 in 45 cases of multiple myeloma (MM) (24 male, 21 female, mean age 66.4 years). 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. 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: To the best of our knowledge, this is the first demonstration of Smurf2 methylation in human neoplasia. Also, 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.

Download Full-text

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

Blood ◽

10.1182/blood.v114.22.2397.2397 ◽

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.

Download Full-text

Snk/Plk2 CpG methylation in patients with multiple myeloma

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e19532 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood.v120.21.4617.4617 ◽

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.

Download Full-text

Impact of Serum Albumin and B2-Microglobulin level on 2-Year Overall Survival among Newly Diagnosed Multiple Myeloma Patients: A retrospective Study

RAS Medical Science ◽

10.51520/2766-5240-5 ◽

2020 ◽

Vol 1 (1) ◽

Author(s):

Sherian Salama ◽

Rodaina Yousef ◽

Asma Al Olama ◽

Mahmoud Marashi ◽

Hana Salama ◽

...

Keyword(s):

Multiple Myeloma ◽

Serum Albumin ◽

United Arab Emirates ◽

Staging System ◽

Albumin Level ◽

Age At Diagnosis ◽

Newly Diagnosed ◽

International Staging System ◽

Beta 2 ◽

Beta 2 Microglobulin

Background: Multiple myeloma accounts for 1% of all cancers and approximately 10% of all hematologic malignancies. Evaluation and initial staging of the disease is made once the diagnosis is confirmed. The recommended staging system is the International Staging System (ISS). Which determines the Myeloma prognosis by 2 factors: beta-2 Microglobulin and Serum albumin. Goal and Objective: The main goal of this study is to assess the effect of Beta-2 microglobulin and Serum albumin on patient’s survival rate with Multiple Myeloma. The secondary objective is to compare the age at diagnosis with other literature. Methodology: The current study was carried out in Hematology Unit, Dubai Hospital, Dubai, Dubai Health Authority (DHA), United Arab Emirates. Chart review was done retrospectively for 49 newly diagnosed patients with Multiple Myeloma diagnosed between the period 2012-2016. Purposive sample was used to those patients who met the inclusion criteria of this study, to be diagnosed and treated in DH. diagnosed and received regular treatment in Dubai Hospital. Results: Medina follow-up of the patients in this study was (12.8) months. The 2-year overall survival rate for patients with Multiple Myeloma (n = 49) was approximately 80%. While, the 2-year OS rate based on Albumin level. Patients with albumin level > 3.5 mg\dl was significantly higher compared to those who had an albumin level <3.5 mg\dl. 100%, 65% respectively, P = 0.033. Moreover, the 2-year OS rate in terms B2MG level. Patients who had a B2MG < 3.5 mg\dl OS was slightly higher compared to those who had (3.5-5.5 and 5.5 mg\dl). OS rate approximately 85 %, 80 % and 75 respectively, P = .737 Conclusion: Multiple myeloma (MM) is a very heterogeneous disease. For this reason, various prognostic factors and staging systems have been developed to predict the disease outcome. International Staging System (ISS) is very useful in determine the survival based on serum β2- microglobulin and serum albumin levels. The age at diagnosis in Dubai hospital, United Arab Emirates is much younger compared to other studies conducted worldwide. The sample used in the study was also highly diverse in terms of culture and nationality. Such diversity is largely typical in Gulf especially in United Arab Emirates. Therefore, this can play important role in age at diagnosis.

Download Full-text

The Prolyl-3-Hydroxylase Leprecan Like 1 (Leprel1) Is a Selective Target for Epigenetic Silencing In Multiple Myeloma.

Blood ◽

10.1182/blood.v116.21.3645.3645 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3645-3645

Author(s):

Eleftheria Hatzimichael ◽

Evangelia Xirofotou ◽

Aggeliki Dasoula ◽

Cristiana Lo Nigro ◽

Laura Lattanzio ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Solid Tumour ◽

Cpg Island ◽

Cpg Islands ◽

Structure And Function ◽

Gene Methylation ◽

Extramedullary Disease ◽

Multiple Treatments ◽

And Function

Abstract Abstract 3645 Background–Aim: Gene silencing is a major mechanism of tumour suppressor gene inactivation in neoplasia, including multiple myeloma (MM). Prolyl hydroxylation is an important post-translational modification that affects the structure and function of collagen. The human genome contains 3 prolyl 3 hydroxylases (P3H) which are closely related in structure and function: Leprecan (P3H1), Leprecan Like 1 (Leprel1, P3H2) and Leprecan Like 2 (Leprel2, P3H3). Leprel2 is methylated in multiple solid tumour types, whereas methylation in Leprel1 appears specific for breast cancer. Leprecan is not detectably methylated in any solid tumour we have analysed. Here, we examine the CpG methylation status of the P3H genes in patients with MM. Patients and Methods: Bone marrow aspirate samples from 38 MM patients (22 males, 16 females) were obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study methylation in the Leprel 1, Leprel2 and Leprecan CpG islands. Genomic DNA was isolated and bisulphite modification performed using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. We used 2 sets of independent primers, which map to different areas of the leprel 1(P3H2) CpG island. Pyrosequencing is ongoing to confirm the sensitivity and specificity of MSP for analysis of P3H gene methylation in our study population. Ten bone marrow samples from patients with border line thrombocytopenia that were proved to have no neoplasia served as negative controls. Logistic regression analyses were used to measure the association between gene methylation and age, ISS stage, extramedullary disease, bone disease, anemia, renal failure, multiple treatments and relapsed/refractory disease. Results: None of the three genes under study was methylated in the control bone marrow samples, but methylation in the CpG island of Leprel 1 was detected in 20 cases (52%). No methylation was detected in the CpG islands of Leprel2 or Leprecan. Trends noted were that patients with methylated leprel 1 were more likely to have ISS stage 3 (OR 1.5, p=0.6), to have received multiple treatments (OR=1.7, p=0.4) and to have received radiotherapy for either bone lytic lesions or extramedullary disease (OR=5.6, p=0.1). Overall no significant association was found with any clinical parameter although the study might have been underpowered due to the small population size. Conclusions: This is the first demonstration of methylation of genes encoding collagen, the prolyl hydroxylases in MM, suggesting that this may be a novel class of myeloma suppressors. Interestingly, Leprel1 was methylated at high frequency, whereas Leprecan and Leprel2 were unmethylated, implying a striking specificity in MM for methylation in Leprel1. These findings warrant further evaluation in a larger sample of patients in order to better define the prognostic and clinical utility of leprel1 methylation in MM. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Methylation Status of the Collagen Prolyl-3 Hydroxylases (C-P3H) and C-P4H Genes in Multiple Myeloma: P3H2 Is Selectively Methylated

Blood ◽

10.1182/blood.v118.21.4639.4639 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4639-4639

Author(s):

Eleftheria Hatzimichael ◽

Aggeliki Dasoula ◽

Konstantinos Lagos ◽

Georgianna Kartalou ◽

George Dranitsaris ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Cpg Island ◽

Methylation Status ◽

Cpg Islands ◽

Clinical Parameter ◽

Small Population ◽

Cpg Methylation ◽

Post Translational Modification

Abstract Abstract 4639 Background–Aim: Prolyl hydroxylation is an important and the most common post-translational modification that affects the structure and function of collagen. Enzymes that participate in this process are the collagen prolyl-3 hydroxylases (C-P3H) and the C-P4H. We have previously showed that P3H3 is methylated in multiple solid tumour types, whereas methylation in P3H2 appears to be specific for breast cancer and multiple myeloma (MM). In this study we assessed the CpG methylation status of the P3H genes (P3H1, P3H2, P3H3) in a larger cohort of MM patients and for the first time the CpG methylation status of the P4H genes (P4HA1, P4HA2, P4HA3) and investigated for clinical relevance. Patients and Methods: Bone marrow aspirate samples from 47 MM patients (28 males, median age 66 years) were obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study the CpG methylation in P3H1, P3H2, P3H3, P4HA1, P4HA2 and P4HA3 CpG islands. Genomic DNA was isolated and bisulphite modified using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. For the study of the P3H2 CpG methylation status we used 2 sets of independent primers, which map to different areas of P3H2 CpG island. Ten bone marrow samples from patients with border line thrombocytopenia that were proved to have no neoplasia served as negative controls. Logistic regression analyses were used to measure the association between gene methylation and age, b2 microglobulin, serum albumin, hypercalcemia, ISS stage, extramedullary disease, bone disease, anemia and renal failure (eGFR< 50 ml/min). Results: None of the six genes under study was methylated in the control bone marrow samples. Methylation in the CpG island of P3H2 was detected in 44 % of patients (95% CI 27.8–62.8%) No methylation was detected in the CpG islands of P3H1, P3H3, P4HA1, P4HA2 and P4HA3. Trends noted were that patients with methylated P3H2 were more likely to have ISS stage 3 (OR 2.2, p=0.1). Overall no significant association was found with any clinical parameter although the study might have been underpowered due to the small population size. Conclusions: P3H2 is methylated at high frequency in MM, whereas P3H1, P3H3, P4HA1, P4HA2, P4HA3 were unmethylated, implying a striking specificity for methylation in P3H2 in MM. These findings warrant further evaluation in a larger sample of patients in order to better define the prognostic and clinical utility of P3H2 methylation in MM. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Epigenetic Inactivation Targets the Arginine Biosynthetic Pathway At Two Levels in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.4640.4640 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4640-4640

Author(s):

Eleftheria Hatzimichael ◽

Aggeliki Dasoula ◽

Nelofer Syed ◽

Peter Szlosarek ◽

George Dranitsaris ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Disease ◽

Methylation Status ◽

Cpg Islands ◽

Bioactive Molecules ◽

Arginine Biosynthesis ◽

Down Regulation ◽

Extramedullary Disease ◽

Arginine Deprivation

Abstract Abstract 4640 Background. Arginine is critical for the growth of certain human cancers and is an intermediary in the synthesis of various bioactive molecules. Argininosuccinate synthetase-1 (ASS1) catalyses the rate-limiting step in arginine biosynthesis, the conversion of citruline to arginine. Argininosuccinate lyase (ASL) is an enzyme that catalyses the reversible breakdown of arginosuccinate producing arginine and fumerate. Down-regulation of ASS1 has been demonstrated in several solid cancers and has been shown to confer sensitivity to arginine deprivation (arginine auxotrophic tumors). Methylation-dependent silencing of the ASS1 promoter may account for ASS1 down-regulation and arginine auxotrophy. Arginine deprivation using arginine deiminase (ADI-PEG20) is currently considered as a novel therapeutic intervention for cancer, and early clinical trials have shown promising results in solid tumors. In this perspective, we investigated for the first time the methylation status of the ASS1 and ASL CpG islands in multiple myeloma (MM) and analyzed for clinical relevance. Methods. Genomic DNA was extracted from bone marrow aspirate samples from 46 MM patients (28 male, 18 female, median age 64 years) obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study the methylation in the ASS1 and ASL CpG islands. DNA was isolated and bisulphite modified using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. Ten bone marrow samples, with no neoplastic cells, from patients with borderline thrombocytopenia served as negative controls. Logistic regression analysis was used to measure the association between gene methylation and sex, age, ISS stage, presence of extramedullary disease, renal failure (eGFR<50 ml/min) and bone disease. Results. Methylation in the CpG island of ASS1 was detected in 71.7% patients (95% CI 57–82%) and of ASL in 37% patients (95% CI 24–51%), while simultaneous methylation in both genes was present in 10 patients. None of the two genes were detectably methylated in the control group. Patients with ASS1 methylation were less likely to have bone disease (p=0.04, OR=0.22) and extramedullary disease (p=0.05, OR=0.22). There was no statistically significant difference in overall survival by methylation status of the studied genes. Conclusions. Arginine biosynthesis genes ASS1 and ASL are methylated in MM. Methylation of ASS1 was found to be more frequent and negatively associated with bone or extramedullary disease. Further evaluation of ASS1 and ASL methylation is warranted in MM in the perspective of expanding arginine deprivation trials in these patients Disclosures: No relevant conflicts of interest to declare.

Download Full-text