Promoter Methylation of Multiple Genes in Germinal Center Hyperplasia and Lymphomas of Germinal Center Origin

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4487-4487
Author(s):  
Jose M Paz-Carreira ◽  
Raquel Losada ◽  
Arantxa Garcia-Rivero ◽  
Augusto Alvarez ◽  
Fernando Bal ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
B Cell ◽  
Promoter Methylation ◽  
Germinal Center ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Promoter Hypermethylation ◽  
Epigenetic Mechanism ◽  
Suppressor Genes

Abstract INTRODUCTION. Germinal centers (GC) are unique sites in peripheral lymphoid tissue where clonal selection of B cells takes place. This occurs as a response to stimulation by various antigens originating, sometimes, follicular hyperplasia (FH). GC have been known as a major source of B-cell lymphomas including follicular (FL) and diffuse large cell (DLCL). DNA methylation of tumor-suppressor genes is a mechanism of gene silencing involved in the pathogenesis of FL and DLCL. Much less is known about the role of methylation in FH. We determined the methylation status of 6 tumor-suppressor genes in 43 patients with FH, 18 patients with FL and 49 patients with DLCL in order to see the differential implication of this epigenetic mechanism in the pathological and the physiological development of GC. MATERIAL AND METHODS. Genomic DNA extracted from paraffin-embedded samples of 43 FH, 18 FL and 49 DLCL after being treated with EZ DNA-Methylation Kit (Zymo Research) with the manufacturer’s instructions, were analyzed by methylation-specific polymerase chain-reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, p14, SHP1, MGMT and PDRM1. All samples were obtained mostly from lymph nodes and tonsils. Diagnosis was based on morphology and immunohistochemistry analysis. All cases were matched for age, sex and ethnic origin. RESULTS: DAP-k promoter methylation occurred with higher frequency in FL(89%) than in DLCL(78%) and FH(40%). SHP1 was methylated in 61% of FL, 58% of FH and 23% of DLCL. RARb was methylated in 67% of FL patients, 30% of DLCL and only 12% of FH. Eight (44%) FL, seventeen (35%) DLCL and four (10%) BFH patients showed MGMT methylation. Promoter hypermethylation of p14 was detected only in 5 (12%) FH, 2 (4%) DLCL and none FL patients. Methylation of PRMD1 was present only in 1 (6%) FL, 2 (6%) DLCL and 1 (4%) FH samples. CONCLUSIONS. Inactivacion of DAP-K and SHP1 is present in B-cell malignancies, DLCL and FL, and BFH. Therefore, it may represent a physiologic event conferring a temporal survival advantage necessary for a GC hyperplastic response. Inactivation of the retinoic acid response through the methylation of Rarâ is significantly more frequent in lymphomas than in FH. As reported in other tumors methylation of MGMT is more frequent in lymphomas than in FH. With our data methylation of Cyclin dependent kinase inhibitors p14 is not a differential pathogenic event in lymphomas of GC origin, in fact it is more frequent in FH. Promoter Methylation of PDRM1 is not the mechanism involved in lymphomagenesis in FL and DLCL, the two FH positive deserve further follow-up to determine its significance.

Prognostic Significance of Promoter Methylation of Multiple Genes in Germinal Center Hyperplasia (GCH) and Lymphomas of Germinal Center Origin.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3950-3950
Author(s):  
Jose M Paz-Carreira ◽  
Arantxa Garcia-Rivero ◽  
Raquel Losada ◽  
Jose C Mendez ◽  
Manuel Albors ◽  
...  
Keyword(s):  
Overall Survival ◽  
Tumor Suppressor ◽  
Promoter Methylation ◽  
Germinal Center ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Prognostic Significance ◽  
Promoter Hypermethylation ◽  
Aberrant Methylation ◽  
Suppressor Genes

Abstract Abstract 3950 Poster Board III-886 INTRODUCTION Germinal centers (GC) are unique sites in peripheral lymphoid tissue where clonal selection of B cells takes place. GC have been known to be a major source of B-cell lymphomas, including follicular (FL) and diffuse large cell (DLCL). DNA methylation of tumor-suppressor genes is a mechanism of gene silencing involved in the pathogenesis of FL and DLCBLs. Much less is known about the role of methylation in GCH. We determined the methylation status of 5 tumor-suppressor genes in 50 patients with lymphoma of GC origin and 50 GCH in order to find any differences between the pathological and the physiological state as well as its prognostic significance. MATERIAL AND METHODS. Genomic DNA extracted from paraffin-embedded samples of 30 DLCL, 20 FL and 50 GCH were analyzed by methylation-specific polymerase chain-reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, p14 and MGMT. Methylation status of each gene was correlated with clinicopathological status. Overall survival (OS) rates were calculated by the Kaplan-Meier method and differences were compared with the log-rank test. RESULTS. Median age was 65 in patients with lymphoma and 19 for GCH. Sex distribution was similar in all entities (60% females). Both lymphoma groups were balanced with respect to the presence of B symptoms, bulky disease, bone marrow infiltration, advanced stage and high IPI/FLIPI. DAP-k promoter methylation was present in more patients with lymphoma (89 and 87%) than with BFH (37%) p<0.0001. RaRB was methylated with higher frequency in FL (60%) than in DLCL (23%) and FH (12%) p<0.0001. SHP1 was more frequently methylated in FL (67%) and GCH (58%) than in DLCL (20%) p=0.01. Promoter hypermethylation of SHP1 was significantly associated with longer OS (p=0.021). Methylation of RaRB, p14 and MGMT were associated with shortened OS but the differences were not statistically significant. Those patients with DAPK methylated live longer but not significantly. In multivariate analysis hypermethylation of none of the genes studied remained an independent prognostic factor. CONCLUSIONS. Inactivation of DAP-K, and Rarβ is present in GC lymphomas with significantly higher frequency than in BFH. Thus, it may have pathogenic significance. SHP1 is methylated more frequently if FL and BFH than in DLCL, therefore that gene may be associated with aggressive disease. Methylation of DAP-k, SHP1, Rarβ, p14 and MGMT has no significant impact on overall survival. Markers for aberrant methylation may represent a promising way to monitor the onset and progression of malignancies but more extensive and prospective trials are needed to precisely define its role. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DNA methylation profiling in MEN1-related pancreatic neuroendocrine tumors reveals a potential epigenetic target for treatment

2018 ◽  
Vol 179 (3) ◽  
pp. 153-160 ◽  
Author(s):  
E B Conemans ◽  
L Lodewijk ◽  
C B Moelans ◽  
G J A Offerhaus ◽  
C R C Pieterman ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Promoter Methylation ◽  
Tumor Suppressor Genes ◽  
Gene Promoter ◽  
Pancreatic Neuroendocrine Tumor ◽  
Promoter Hypermethylation ◽  
Suppressor Gene ◽  
Suppressor Genes ◽  
Frequent Event

ObjectiveEpigenetic changes contribute to pancreatic neuroendocrine tumor (PanNET) development. Hypermethylation of promoter DNA as a cause of tumor suppressor gene silencing is a well-established oncogenic mechanism that is potentially reversible and therefore an interesting therapeutic target. Multiple endocrine neoplasia type 1 (MEN1) is the most frequent cause of inherited PanNETs. The aim of this study was to determine promoter methylation profiles in MEN1-related PanNETs.Design and methodsMethylation-specific multiplex ligation-dependent probe amplification was used to assess promoter methylation of 56 tumor suppressor genes in MEN1-related (n = 61) and sporadic (n = 34) PanNETs. Differences in cumulative methylation index (CMI), individual methylation percentages and frequency of promoter hypermethylation between subgroups were analyzed.ResultsWe found promoter methylation of a large number of potential tumor suppressor genes. CMI (median CMI: 912 vs 876,P = 0.207) was the same in MEN1-related and sporadic PanNETs. We found higher methylation percentages ofCASP8in MEN1-related PanNETs (median: 59% vs 16.5%,P = 0.002). In MEN1-related non-functioning PanNETs, the CMI was higher in larger PanNETs (>2 cm) (median: 969.5 vs 838.5;P = 0.021) and in PanNETs with liver metastases (median: 1036 vs 869;P = 0.013). Hypermethylation ofMGMT2was more frequent in non-functioning PanNETs compared to insulinomas (median: 44.7% vs 8.3%;P = 0.022). Hypermethylation of the Von Hippel–Lindau gene promoter was observed in one MEN1-related PanNET and was associated with loss of protein expression.ConclusionPromoter hypermethylation is a frequent event in MEN1-related and sporadic PanNETs. Targeting DNA methylation could be of therapeutic value in MEN1 patients with advanced PanNETs.

scholarly journals Consistent DNA Hypermethylation Patterns in Laryngeal Papillomas

2010 ◽  
Vol 1 (2) ◽  
pp. 69-77 ◽  
Author(s):  
Josena K Stephen ◽  
Kang Mei Chen ◽  
Veena Shah ◽  
Vanessa G Schweitzer ◽  
Glendon Gardner ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Promoter Hypermethylation ◽  
Aberrant Methylation ◽  
Dna Hypermethylation ◽  
Suppressor Genes ◽  
Multiple Tumor ◽  
Specific Pcr

Abstract Introduction This study examined the contribution of promoter hypermethylation to the pathogenesis of respiratory papillomatosis (RP), including recurrences (RRP) and progression to squamous cell carcinoma (SSC). Materials and methods A retrospective cohort of 25 laryngeal papilloma cases included 21 RRP, two of which progressed to SCC. Aberrant methylation status was determined using the multigene (22 tumor suppressor genes) methylation-specific multiplex ligationdependent probe amplification assay and confirmed using methylation specific PCR. Results Twenty genes had altered DNA methylation in 22 of 25 cases. Aberrant methylation of CDKN2B and TIMP3 was most frequent. Promoter hypermethylation of BRCA2, APC, CDKN2A and CDKN2B was detected in 2 RRP cases with subsequent progression to SCC. Of the 25 cases, 22 were positive for HPV-6, 2 for HPV-11 and 1 for HPV-16 and 33. Conclusion Consistent aberrant methylation of multiple tumor suppressor genes contributes to the pathogenesis of laryngeal papillomas. Persistent aberrant DNA methylation events in 2 RRP cases that progressed to cancer indicate an epigenetic monoclonal progression continuum to SCC.

Association Between Cyclin D1 Polymorphism with CpG Island Promoter Methylation Status of Tumor Suppressor Genes in Gastric Cancer

2010 ◽  
Vol 55 (12) ◽  
pp. 3449-3457 ◽  
Author(s):  
Tomomitsu Tahara ◽  
Tomoyuki Shibata ◽  
Masakatsu Nakamura ◽  
Hiromi Yamashita ◽  
Daisuke Yoshioka ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Tumor Suppressor ◽  
Cyclin D1 ◽  
Promoter Methylation ◽  
Tumor Suppressor Genes ◽  
Cpg Island ◽  
Methylation Status ◽  
Suppressor Genes ◽  
Promoter Methylation Status

Promoter Methylation of Multiple Genes in Diffuse Large Cell Lymphoma (DLCL), Follicular Lymphoma (FL) and Bening Follicular Hyperplasia (BFH).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2608-2608
Author(s):  
Jose M. Paz-Carreira ◽  
Raquel Losada ◽  
Arantxa Garcia-Rivero ◽  
Augusto Alvarez ◽  
Fernando Bal ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Promoter Methylation ◽  
Tumor Suppressor Genes ◽  
Kinase Inhibitors ◽  
Promoter Hypermethylation ◽  
Cyclin Dependent Kinase ◽  
B Cell Malignancies ◽  
Follicular Hyperplasia

Abstract Transcriptional silencing of tumor suppressor genes, associated with DNA methylation, is a common epigenetic event in leukemias and myelodisplastic syndromes. Much less is known about the specific methylation changes that occur in DCLC, FL and their normal counterpart BFH. Methylation of cyclin dependent kinase inhibitors is more common in high grade lymphomas and may be an important step in the progression and transformation of follicular lymphoma. We determined the methylation status of 7 tumor suppressor genes in 31 patients with B-cell malignancies. Seven patients with benign follicular hyperplasia were used as normal controls. The target genes chosen are potentially involved in B-cells malignancies and encoding proteins implicated in apoptosis regulation (death associated protein kinase, DAP-K), Jak/STAT3 signalling pathway (SHP1), hormonal response (RARb), DNA repair (O6-methilguanine-DNA methyltranferase, MGMT), cell cycle control (p14ARF and p15INK4b) and detoxification of environmental xenobiotics such as doxorubicin (glutathione S-transferase P1, GSTP1). Genomic DNA extracted from paraffin-embedded samples of thirty one B-cell malignancies (twenty six DLCL and five FL) were analyzed by methylation-specific polymerase chain reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, MGMT, p14, p15 and GSTP1. Samples were obtained mostly from lymph nodes; spleen, stomach, skin, and parathyroid gland biopsies were also included. Specimens were collected at diagnosis before specific therapy. Diagnosis was based on morphology and immunohistochemistry analysis. All cases were matched for age, sex and ethnic origin. DAP-k promoter methylation occurred with similar frequency in DLCL (100%), FL (100%) and BFH (86%). SHP1 was methylated in 75% of DLCL, all FL and the 7 patients with BFH. RARb was methylated in all DLCL and FL patients and 85% of the BFH. Sixteen (50%) DLCL, three (60%) FL and none BFH patients showed MGMT methylation. Promoter hypermethylation of p14 and p15 was detected in 11 (42%) and 13 (50%) DLCL, 2 (40%) and 3(60%) FL, 3(42%) and 5(71%) BFH patients respectively. Methylation of GSTP1 was absent in all samples. Inactivacion of DAP-K, SHP1 and Rarβ is present in B-cell malignancies, DLCL and FL, and BFH. Therefore, it may represent a physiologic event conferring a temporal survival advantage necessary for a BFH response. With our data methylation of Cyclin dependent kinase inhibitors such as p14 and p15 is not a differential pathogenic event in DLCL with respect to FL or BFH. Finally, the absence of GSTP1 methylation in DLCL and FL questions its relevance in B-cell neoplasia development.

Aberrant Gene Promoter Methylation of Tumor Suppressor Genes in Plasma Cell Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2487-2487
Author(s):  
Carmen Stanganelli ◽  
Jorge Arbelbide ◽  
Juliana Zimerman ◽  
Dorotea Beatriz Fantl ◽  
Claudia Corrado ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Peripheral Blood ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Genetic Alterations ◽  
Negative Regulator ◽  
Epigenetic Mechanism ◽  
Gene Methylation ◽  
Suppressor Genes ◽  
Number Of Genes

Abstract There is increasing evidence that, in addition to genetic aberrations, epigenetic processes play a major role in carcinogenesis. Particularly, hypermethylation of CpG islands of the promoter regions of tumor suppressor genes (TSG) is now considered as an important epigenetic mechanism for gene inactivation. Multiple myeloma (MM) is characterized by neoplastic proliferation of monoclonal plasma cells. The natural course of disease may progress through monoclonal gammopathy of undetermined significance (MGUS) to MM. During this process, multiple genetic alterations are sequentially acquired and aberrant promoter hypermethylation might be one of the steps involved in this progression. In this study, we have evaluated methylation status of the following TSG: p15INK4b, p16INK4a, p14ARF, SOCS-1, p27KIP1, RASSF1A and p73 genes, in order to determine if they were involved in the evolution of MGUS to MM. Forty four MM (21 males; mean age 67.5 years; Durie-Salmon clinical stages: I: 20%, II:14% and III: 66%) and 21 MGUS patients (6 males; mean age 68 years) were study. All patients gave informed consent and the study was approved by the Ethics Committee of our Institution. Peripheral blood samples from 10 normal individuals and CpGenome Universal Methylated DNA (Chemicon International) were used as negative and positive controls, respectively. DNA was extracted from bone marrow cells of patients and peripheral blood lymphocytes of controls using phenol/chloroform method. Methylation status was performed using Methylation Specific PCR (MSP) technique. For statistical analysis, Student t and Fisher exact tests were used. The methylation index (MI; ratio between the number of genes methylated and the number of genes analyzed) was also calculated. SOCS-1 gene methylation was significantly more frequent in MM (52%) than in MGUS patients (14%) (p=0,006). Frequencies of methylation of p14ARF, p15INK4b, p16INK4a and RASSF1A were comparable in both entities: 29%, 32%, 7% and 2%, respectively, for MM; and 29%, 29%, 5% and 0%, respectively, for MGUS. TP73 gene showed a tendency of higher methylation in MM (45%) than in MGUS (33%). All patients lacked methylation at p27KIP1 gene. Whereas the percentage of MM with at least one gene methylated (84%) did not showed differences to that of MGUS (66%), the mean MI of MGUS was lower (0.16; range 0.14-0.43) than that of MM (0.24; range 0.14-0.71) (p&lt;0.05). None of the target genes were methylated in normal samples. No statistical significant correlation with clinical characteristics: gender, age, isotype, level of M-component, type of light chain, stage of the disease, haemoglobin, serum albumin level, calcium, β2 microglobulin and LDH, were observed. To our knowledge, this is the first report of methylation in MM and MGUS from Argentina. The similar frequency of p14ARF, p15INK4b, p16INK4a and RASSF1A gene methylation observed in MM and MGUS would suggest that they are probably not involved in the progression of MGUS. However, SOCS1 gene methylation was significantly more frequent in MM than in MGUS suggesting that methylation of this gene might be involved in clonal evolution of MGUS to MM. SOCS1 is a negative regulator of cytokine signaling, being important in normal lymphocyte development and differentiation. Silencing of SOCS1 may result in greater responsiveness to cytokines, which may favour the neoplastic development.

scholarly journals Chrysin Modulates Aberrant Epigenetic Variations and Hampers Migratory Behavior of Human Cervical (HeLa) Cells

2022 ◽  
Vol 12 ◽  
Author(s):  
Ritu Raina ◽  
Abdulmajeed G. Almutary ◽  
Sali Abubaker Bagabir ◽  
Nazia Afroze ◽  
Sharmila Fagoonee ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Histone Modification ◽  
Hela Cells ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Global Dna Methylation ◽  
Dependent Manner ◽  
Suppressor Genes ◽  
Protein Levels

Purpose: Plant-derived phytochemicals have shown epigenetic modulatory effect in different types of cancer by reversing the pattern of DNA methylation and chromatin modulation, thereby restoring the function of silenced tumor-suppressor genes. In the present study, attempts have been made to explore chrysin-mediated epigenetic alterations in HeLa cells.Methods: Colony formation and migration assays followed by methylation-specific PCR for examining the methylation status of CpG promoters of various tumor-suppressor genes (TSGs) and the expression of these TSGs at the transcript and protein levels were performed. Furthermore, global DNA methylation; biochemical activities of DNA methyltransferases (DNMTs), histone methyl transferases (HMTs), histone deacetylases (HDACs), and histone acetyl transferases (HATs) along with the expression analysis of chromatin-modifying enzymes; and H3 and H4 histone modification marks analyses were performed after chrysin treatment.Results: The experimental analyses revealed that chrysin treatment encourages cytostatic behavior as well as inhibits the migration capacity of HeLa cells in a time- and dose-dependent manner. Chrysin reduces the methylation of various tumor-suppressor genes, leading to their reactivation at mRNA and protein levels. The expression levels of various chromatin-modifying enzymes viz DNMTs, HMTs, HDACs, and HATS were found to be decreased, and H3 and H4 histone modification marks were modulated too. Also, reduced global DNA methylation was observed following the treatment of chrysin.Conclusion: This study concludes that chrysin can be used as a potential epigenetic modifier for cancer treatment and warrants for further experimental validation.

Detection and Analysis of RNAs Expression Profile for Methylated Candidate Tumor Suppressor Genes in Nasopharyngeal Carcinoma

2019 ◽  
Vol 19 (6) ◽  
pp. 772-782
Author(s):  
Shuang Zhao ◽  
Ye Zhang ◽  
Xujun Liang ◽  
Maoyu Li ◽  
Fang Peng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Nasopharyngeal Carcinoma ◽  
Tumor Suppressor ◽  
Promoter Methylation ◽  
Tumor Suppressor Genes ◽  
Enrichment Analysis ◽  
Pathway Enrichment Analysis ◽  
Suppressor Genes ◽  
Multiple Tumor

Background:DNA methylation, which acts as an expression regulator for multiple Tumor Suppressor Genes (TSGs), is believed to play an important role in Nasopharyngeal Carcinoma (NPC) development.Methods:We compared the effects of 5-aza-2-deoxycytidine (decitabine, DAC) on gene expression using RNA sequencing in NPC cells.Results:We analyzed Differentially Expressed Genes (DEGs) in NPC cells using DAC demethylation treatment and found that 2182 genes were significantly upregulated (≥ 2-fold change), suggesting that they may play a key role in cell growth, proliferation, development, and death. For data analysis, we used the Gene Ontology database and pathway enrichment analysis of the DEGs to discover differential patterns of DNA methylation associated with changes in gene expression. Furthermore, we evaluated 74 methylated candidate TSGs from the DEGs in NPC cells and summarized these genes in several important signaling pathways frequently disrupted by promoter methylation in NPC tumorigenesis.Conclusion:Our study analyzes the DEGs and identifies a set of genes whose promoter methylation in NPC cells is reversed by DAC. These genes are potential substrates of DNMT inhibitors and may serve as tumor suppressors in NPC cells.

Prognostic Impact of the Number of Methylated Genes in Myelodysplastic Syndromes and Acute Myeloid Leukemias Treated with 5-Azacytidine

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1717-1717
Author(s):  
María Abáigar ◽  
Fernando Ramos ◽  
Rocío Benito ◽  
María Díez-Campelo ◽  
Javier Sánchez-del-Real ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Overall Survival ◽  
Tumor Suppressor ◽  
Myelodysplastic Syndromes ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Prognostic Impact ◽  
Suppressor Genes ◽  
Myeloid Leukemias ◽  
Acute Myeloid

Abstract Abstract 1717 Background: Aberrant DNA methylation of tumor suppressor genes is a common event in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). Therefore hypomethylating agents like azacytidine (AZA) and decitabine seem to be a good therapeutic approach for the treatment of these diseases. Clinical experience and recent published data have demonstrated that AZA is effective for the treatment of MDS and AML patients. However, the prognostic impact of the aberrant hypermethylation on response and outcome to AZA treatment remains to be determined. For this reason the influence of the methylation status of a selected set of tumor suppressor genes on the overall survival and clinical response in MDS and AML patients, prior to treatment with AZA was studied. Patients and Methods: A total of 78 patients with MDS or AML who had been treated with AZA were evaluated. Among these, 25 were excluded because they had received less than 4 cycles, AZA was used after allogeneic stem cell transplantation, response was not assessable, or there was not enough quality DNA available. So finally, the study was focused in 53 patients: 36 MDS and 17 AML. Most of the AML included in the study had low blast count (20–30%). Responses were assessed according to the IWG MDS criteria in accordance to Fenaux et.al, and IWG AML criteria following European LeukemiaNet recommendations. DNA methylation status was analyzed using the Methylation Specific Multiplex Ligation Probe Amplification (MS-MLPA), with a panel of 24 different tumor suppressor genes related to cell cycle control, apoptosis regulation, DNA repair, cell adhesion and cell growth. Results: In the study cohort 47% of patients had cytogenetic alterations prior to AZA treatment, 4 with isolated -5/del(5q), 7 with isolated −7/del(7q), 4 with trisomy 8, 4 with not isolated -5/del(5q), 1 with trisomy 14, and 5 with complex cytogenetics. Methylation analysis showed that most patients (74%) had at least one methylated gene, but only 10% of patients displayed more than 3 methylated genes. The most frequently methylated genes were IGSF4 (28.3%), CDKN2B (24.5%), ESR1 (22.6%), CDH13 (17%) and CDKN1B (11.3%). The presence of a high number (≥2) of methylated genes (p=0.02), an adverse cytogenetics (p=0.03) or anemia (p=0.04) were independent prognostic factors associated with shorter overall survival. Moreover, the analysis of those patients displaying “no methylation”, patients with 1 methylated gene, patients with 2 methylated genes and those with more than 3 methylated genes, showed that as the number of methylated genes increases, the survival was shorter. The patients displaying the highest level of methylation (more than 3 genes), had a very short survival (median OS of 9.3 months, p<0.001). Forty-nine per cent of all patients responded to azacytidine. The statistical analysis revealed that the number of methylated genes did not correlate in general with the clinical response to AZA, although four of the five patients with more than 3 genes methylated did not respond. By contrast, cytogenetics was the only factor that independently influenced the response to AZA. Thus 64.7% of patients with favorable cytogenetics responded to AZA, while only 28.6% of those with adverse cytogenetics responded (p=0.03). Interestingly, our study included 3 MDS patients with isolated chromosome 7 alterations, and all of them responded. Conclusion: In summary, these results suggest that the analysis of the methylation status before AZA treatment by a feasible methodology such as MS-MLPA could be used in a clinical setting in order to identify a group of patients with poor survival, in which other alternative therapeutic approaches might be considered. Disclosures: Ramos: Celgene, Novartis, Amgen, Pfizer, and Merck Sharp & Dohme: Honoraria. Hernández:Celgene: Research Funding.

Detecting Aberrantly Methylated Genes in HNSCC Saliva

2008 ◽  
Vol 139 (2_suppl) ◽  
pp. P89-P90
Author(s):  
Seema Sethi ◽  
Kang-Mei Chen ◽  
Michael S Benninger ◽  
Maria J Worsham
Keyword(s):  
Tumor Suppressor ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Simultaneous Detection ◽  
Promoter Hypermethylation ◽  
Early Event ◽  
Gene Probe ◽  
Aberrant Methylation ◽  
Suppressor Genes ◽  
Normal Controls

Problem Aberrant promoter hypermethylation of multiple tumor suppressor genes is an early event in carcinogenesis of HNSCC. Saliva is a non-invasive sample which has immense potential for use in molecular screening and early diagnosis of head and neck cancers due to ease of collection, patient compliance allowing repeatabililty. We evaluated saliva DNA for simultaneous detection of promoter hypermethylation in multiple genes in HNSCC cases and compared them with normal controls. Methods Saliva was collected in a prospective cohort of 37 study subjects: 27 HNSCC patients and 10 normal controls. Saliva DNA was interrogated for aberrant methylation status using a multicandidate gene probe panel. 35 unique genes related to HNSCC were simultaneously examined of which 22 were tumor suppressor genes. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay was used. Results Of the 22 tumor suppressor genes examined simultaneously in the saliva DNA of the study subjects, four HNSCC cases showed aberrant promoter hypermethylation in one or more genes. Aberrantly methylated genes included TIMP3, APC, MLH1, RARB and CDKN1B, CDKN2B, GSTP1, CD44, and ESR. aberrant methylation of CDKN2B was observed in 3/4 cases and GSTP1 in 2/4 cases. Only one of the 10 controls had aberrant methylation of CDKN2B. Conclusion In this exploratory study, MS-MLPA identified multiple aberrantly methylated genes simultaneously in the same assay run. The most important advantage is its utility as a high throughput multi-gene screening approach requiring very small amounts of DNA, unlike more laborious single gene methods such as MSP. Significance Epigenetic signatures from MS-MLPA profiling, upon subsequent validation as diagnostic or prognostic biomarkers, can become reduced to a more definitive candidate gene panel of only a few key genes for early detection and screening of HNSCC. Support HFHS A10236; NIH R01 DE15990.

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