scholarly journals Droplet Digital PCR for the Quantification of Alu Methylation Status in Hematological Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2612-2612
Author(s):  
Paola Orsini ◽  
Luciana Impera ◽  
Elisa Parciante ◽  
Cosimo Cumbo ◽  
Crescenzio Francesco Minervini ◽  
...  
Keyword(s):  
High Risk ◽  
Methylation Level ◽  
Hematological Malignancies ◽  
Methylation Status ◽  
Risk Groups ◽  
Digital Pcr ◽  
Differential Methylation ◽  
Droplet Digital Pcr ◽  
Alu Repeats ◽  
Alu Sequences

Abstract Introduction. Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. They are located in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu repeats methylation has been associated with tumor aggressiveness and investigated in some solid tumors, but the global Alu methylation level has not yet been investigated in hematological malignancies. Moreover, today, some of the techniques designed to measure global DNA methylation are focused on the methylation level of specific genomic compartments, including repeat elements. In this work we propose a new method for investigating Alu differential methylation, employing droplet digital PCR (ddPCR) technology, applied in patients affected by chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Methods. The study included a total of 46 patients: 30 CLL patients, 7 patients with MDS at intermediate/high risk, and 9 CMML patients. The study also involved acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with azacytidine (AZA) 0.75 µM or decytabine (DEC) 0.75 µM. Four healthy donors (HD) were also included as controls. For each DNA sample two aliquots of 250ng of gDNA were simultaneously digested (with 1 unit of Alu-in/sensitive isoschizomers either MspI or HpaII) and ligated (to a previously prepared synthetic adaptor) in parallel in two separate tubes. Considering that the genomic DNA amount in a human diploid cell is about 6 pg/cell, for each sample we calculated the percentage of methylated consensus Alu sequences as the ratio between the sum of positive droplets obtained from the three wells of both HpaII (MH) and MspI (MM) final dilutions, according to the following formula: [1-(sumMH/sumMM)]x100. The significance level was set at p<0.05 for all analyses. Results. Using our ddPCR assay, we observed a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, and a minor decrease with AZA (p=0.058). Moreover, comparing the global Alu methylation levels at diagnosis and after AZA treatment in MDS patients, we observed a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis. We also extended the assessment of our assay in CLL patients at diagnosis. We observed a significant decrease of the Alu methylation level in CLL patients compared to HD. CLL patients were also classified in the following three cytogenetic risk groups according to the karyotypic alterations identified by Fluorescent In Situ Hybridization (FISH): low (with isolated 13q deletion), intermediate (without 11q, 13q and 17p deletions or with trisomy 12), and high risk (with 11q or, 17p deletions, or more than two chromosomal aberrations). Alu methylation status of the low and high-risk groups was more significantly reduced compared to HD, whereas considering intermediate-risk patients the difference was less evident. Finally, for CMML patients, a significant decrease of Alu sequences methylation was observed in patients harboring the main SRSF2 gene hotspot. However, these preliminary results should be confirmed by extending the analysis to other CMML patients. Conclusions. In our work, we propose a new method to investigate Alu differential methylation based on ddPCR technology. This assay represents an alternative to conventional quantitative-PCR (qPCR), introducing ddPCR as a more sensitive and immediate technique for Alu methylation analysis. Moreover, compared to qPCR, our ddPCR Alu assay may be carried out using very small amounts of digested gDNA (about 6 pg), and does not require a reference gene for the analysis of ddPCR data. To date, this is the first application of ddPCR to study global DNA methylation by inspecting DNA repeats. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose. Disclosures No relevant conflicts of interest to declare.

scholarly journals Droplet digital PCR for the quantification of Alu methylation status in hematological malignancies

2018 ◽  
Vol 13 (1) ◽  
Author(s):  
Paola Orsini ◽  
Luciana Impera ◽  
Elisa Parciante ◽  
Cosimo Cumbo ◽  
Crescenzio F. Minervini ◽  
...  
Keyword(s):  
Hematological Malignancies ◽  
Methylation Status ◽  
Digital Pcr ◽  
Droplet Digital Pcr

scholarly journals Level of Seven Neuroblastoma-Associated mRNAs Detected by Droplet Digital PCR Is Associated with Tumor Relapse/Regrowth of High-Risk Neuroblastoma Patients

2020 ◽  
Vol 22 (2) ◽  
pp. 236-246 ◽  
Author(s):  
Khin K.M. Thwin ◽  
Toshiaki Ishida ◽  
Suguru Uemura ◽  
Nobuyuki Yamamoto ◽  
Kyaw S. Lin ◽  
...  
Keyword(s):  
High Risk ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Tumor Relapse

Identification of Differential Methylation Markers Among Cytogenetic Risk Groups of Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1464-1464
Author(s):  
Min Fang ◽  
Xiaoyu Qu ◽  
Jerry Davison ◽  
Liping Du ◽  
Frederick R. Appelbaum ◽  
...  
Keyword(s):  
High Risk ◽  
Risk Group ◽  
Risk Groups ◽  
High Risk Group ◽  
Differential Methylation ◽  
The Other ◽  
P Value ◽  
Single Region ◽  
The Right ◽  
Methylation Ratio

Abstract Abstract 1464 Aberrant DNA methylation has been shown as an important mechanism in the progression from myelodysplasia (MDS) to acute myeloid leukemia (AML), leading to use of the demethylating agents, 5-azacitidine and decitabine, for treatment of both disorders. While these drugs produce responses, the ability to distinguish potential responders from potential non-responders remains limited. The purpose of this work was to bring new technology to study this problem. Recent studies demonstrated that promoter DNA methylation is not randomly distributed in AML blasts but rather is highly organized and associated with biologically distinct AML subtypes. Because cytogenetics at presentation is the most important prognostic factors in predicting response to therapy, remission duration and overall survival in AML, we aimed to identify differentially methylated genomic regions (DMR) in cytogenetically defined risk groups of AML. Published literature suggested that the new comprehensive high-throughput array-based relative methylation analysis (CHARM) has the highest sensitivity and specificity among all the array-based genome profiling methods and should be the most accurate means to identify methylation markers. It is a customized NimbleGen HD2 array of tiled 50mer-probes typically separated by 30–40 bases covering approximately 4.6 million CpG sites across the genome. This assay is highly quantitative for approximately 100,000 independent CpG sites. We performed methylation profiling with CHARM on 15 age-matched patients divided into 3 groups (n=5 in each): (1) high-risk AML, defined as patients with a complex or monosomal karyotype, inv(3)/t(3;3), t(6;9), or FLT3-ITD; (2) intermediate-risk AML, defined as patients with normal karyotype, trisomy 8, t(9;11), or others; (3) low-risk AML, defined as patients with t(8;21) or inv(16). Five age-matched normal individuals served as control. Randomly fractionated DNA was divided into two equal portions with and without McrBC treatment, which cleaves methylated DNA, then size-fractionated, purified and subject to whole-genome amplification prior to hybridization with the CHARM array. Data analysis was performed with R and Bioconductor. AML patients showed a very strong hypermethylation signature as compared with the normal control blood. A unique set of DMRs was identified which distinguishes between any two risk groups. The number of DMRs and those with p -values < 0.01 are shown in Table 1. There were fewer methylation discriminators between low- and mid-risk groups than between high-risk and the other risk groups. Figure 1 shows the comparison between high-risk group and other risk groups highlighting the 12 top DMRs with lowest p -values. In silico validation of the DMRs identified by CHARM verified previously reported aberrant hypermethylation of tumor suppressor genes like p15CDKN2B, discriminating normal from AML patients, CDH1 promoter hypermethylation in high-risk AML compared with mid-risk AML, and HOXB3 hypomethylation in mid-risk AML compared with both low-risk and high-risk AML, etc. Technical validation using quantitative bisulfite pyrosequencing on 8 genes, including DCC, DUOX2, NEFL, and PITX1, demonstrated an 87.5% concordant rate with CHARM. Testing of additional AML samples with validated markers are underway to confirm the top DMRs identified, which may serve as useful biomarkers to predict response to azacitidine and decitabine.Table 1Number of DMRsNumber with p-value < 0.01Normal blood VS at risk3768311Low-risk VS mid-risk156530Low-risk VS high-risk247573Mid-risk VS high-risk2651107Figure 1.Results from the CHARM analysis on AML patients stratified by cytogenetic risk, highlighting comparison between the high-risk and other risk groups. The top twelve differentially methylated regions ( DMRs) with p -value < 0.01 are shown. Box-and-whisker plots to the left of the dashed vertical line in each panel present the log2 methylation ratio of the high-risk group and the other two risk groups combined, in a single region of differential methylation. To the right of the dashed line the high-risk group is compared with each of the other groups. Each panel's header text identifies the genomic region.Figure 1. Results from the CHARM analysis on AML patients stratified by cytogenetic risk, highlighting comparison between the high-risk and other risk groups. The top twelve differentially methylated regions ( DMRs) with p -value < 0.01 are shown. Box-and-whisker plots to the left of the dashed vertical line in each panel present the log2 methylation ratio of the high-risk group and the other two risk groups combined, in a single region of differential methylation. To the right of the dashed line the high-risk group is compared with each of the other groups. Each panel's header text identifies the genomic region. Disclosures: No relevant conflicts of interest to declare.

Abstract 5679: Using droplet digital PCR to analyzeMYCNcopy number in plasma from patients with high-risk neuroblastoma

2017 ◽  
Author(s):  
Marco Lodrini ◽  
Annika Sprüssel ◽  
Kathy Astrahantseff ◽  
Daniela Tiburtius ◽  
Robert Konschak ◽  
...  
Keyword(s):  
High Risk ◽  
Digital Pcr ◽  
Droplet Digital Pcr

scholarly journals Detecting TP53 mutations in diagnostic and archival liquid-based Pap samples from ovarian cancer patients using an ultra-sensitive ddPCR method

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nicolai Skovbjerg Arildsen ◽  
Laura Martin de la Fuente ◽  
Anna Måsbäck ◽  
Susanne Malander ◽  
Ola Forslund ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Diagnosis ◽  
Risk Groups ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Ovarian Cancer Screening ◽  
Assay Sensitivity ◽  
Diagnostic Samples ◽  
Very High ◽  
Generation Sequencing

Abstract High-grade serous ovarian cancer (HGSOC) is the most common subtype of epithelial ovarian cancer and early detection is challenging. TP53 mutations are a hallmark of HGSOC and detection of these mutations in liquid-based Pap samples could provide a method for early diagnosis. Here we evaluate the use of IBSAFE, an ultra-sensitive droplet digital PCR (ddPCR) method, for detecting TP53 mutations in liquid-based Pap samples collected from fifteen women at the time of diagnosis (diagnostic samples) and/or up to seven years prior to diagnosis (archival samples). We analysed tumours for somatic TP53 mutations with next generation sequencing and were able to detect the corresponding mutations in diagnostic samples from six of eight women, while one patient harboured a germline mutation. We further detected a mutation in an archival sample obtained 20 months prior to the ovarian cancer diagnosis. The custom designed IBSAFE assays detected minor allele frequencies (MAFs) with very high assay sensitivity (MAF = 0.0068%) and were successful despite low DNA abundance (0.17–206.14 ng, median: 17.27 ng). These results provide support for further evaluation of archival liquid-based Pap samples for diagnostic purposes and demonstrate that ultra-sensitive ddPCR should be evaluated for ovarian cancer screening in high-risk groups or in the recurrent setting.

The Disease Risk Index Is a Robust Tool for Allogeneic Hematopoietic Stem Cell Transplantation Risk Stratification: An Independent Validation Study on a Large Cohort of the European Society for Blood and Marrow Transplantation (EBMT)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 988-988 ◽  
Author(s):  
Roni Shouval ◽  
Joshua Fein ◽  
Myriam Labopin ◽  
Nicolaus Kroger ◽  
Rafael F. Duarte ◽  
...  
Keyword(s):  
Overall Survival ◽  
High Risk ◽  
Cumulative Incidence ◽  
Research Funding ◽  
Hematological Malignancies ◽  
Disease Risk ◽  
Risk Index ◽  
Risk Groups ◽  
Advisory Committees ◽  
Very High

Abstract Background: Allogeneic stem cell transplantation is a potentially curative procedure to a long list of hematological malignancies, but involves substantial risk of morbidity and mortality. Means for accurately predicting outcome and assessing risk are thus greatly needed. The Disease Risk Index (DRI) is a prognostic tool developed and validated by Armand et al. across a wide range of hematological malignancies (Blood 2012, Blood 2014) on cohorts of American patients. The Index stratifies patients into 4 distinct risk groups (low, intermediate, high, very high) and has yet to be validated in an international cohort. We sought to evaluate the validity of the DRI in a large cohort of European patients. Methods: This was a retrospective validation study on an independent cohort of patients undergoing allogeneic HSCT and reported the European Society for Blood and Marrow Transplantation (EBMT). Patients included had a hematological malignancy and underwent allogeneic transplantation between the years of 2000 and 2015. Risk groups were coded in accordance with the refined DRI (Blood, 2014). Outcomes were evaluated 4 years after the allogeneic HSCT. Overall survival (OS) was calculated with the Kaplan-Meier method. The log-rank test was used for comparisons of Kaplan-Meier curves. Cumulative incidence curves for nonrelapse mortality (NRM) and relapse with or without death were constructed reflecting time to relapse and time to NRM, respectively, as competing risks. The difference between cumulative incidence curves in the presence of a competing risk was tested with the Gray method. The prognostic effect of the DRI strata was estimated using a Cox proportional hazard model for OS and a Fine and Gray model for NRM and relapse. Results: A total of 89,061 patients from 423 transplantation centers were included in the analysis. Median age was 48.3 (IQR 36.2-57.5). The most frequent indication for transplantation was AML (39,530 patients) followed by ALL (16,206) and MDS (9,750); other indications spanned the spectrum of hematological malignancies. The majority of patients were in 1st or 2nd complete remission (54%). The median follow-up period was 3.6 years. Approximately 63% of patients were classified as intermediate risk by DRI, suggesting that this group could be further partitioned. The 4 year overall survival (95% CI) of the low, intermediate, high, and very high risk groups was 60.8% (59.9-61.8), 51.3% (50.8‐51.8), 27.0% (26.1‐27.8), 18.4% (17.1-19.8) (Figure 1). The same groups corresponded with increasing cumulative incidence of relapse; 8.9% (8.3-9.4), 19.3% (18.9-19.7), 39.0% (37.8-39.6), 45.1% (43.4-46.7), respectively. The DRI groups also showed increasing hazard between strata in the overall survival setting; intermediate risk was associated with a hazard ratio of 1.32, high risk 2.67 and very high risk 3.71 relative to low risk. Relapse showed a similar pattern. NRM was less strongly stratified by DRI (Table 1). The DRI groups maintained a similar risk, regardless of whether the transplantation was performed prior or after 2008. DRI was the strongest determinant of overall survival and relapse when introduced to a multivariable model with additional covariates. AUC for the index at 4 years was 62.5 for OS, 58.5 for NRM and 68.2 for relapse. Conclusions: We have validated the Disease Risk Index in a massive European data set. The groupings suggested by the DRI corresponded with distinct risk groups for overall mortality and relapse. Overall, our results indicate the international applicability of this robust prognostic tool. Figure 1. Kaplan-Meyer survival curves for overall survival, stratified by DRI Figure 1. Kaplan-Meyer survival curves for overall survival, stratified by DRI Table 1 Table 1. Disclosures Bader: Medac: Consultancy, Research Funding; Riemser: Research Funding; Neovii Biotech: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Bonini:Molmed SpA: Consultancy; TxCell: Membership on an entity's Board of Directors or advisory committees. Dreger:Gilead: Consultancy; Janssen: Consultancy; Novartis: Speakers Bureau; Gilead: Speakers Bureau; Novartis: Consultancy; Roche: Consultancy. Kuball:Gadeta B.V,: Membership on an entity's Board of Directors or advisory committees. Montoto:Roche: Honoraria; Gilead: Research Funding.

scholarly journals Droplet digital PCR is an accurate method to assess methylation status on FFPE samples

Epigenetics ◽  
2018 ◽  
Vol 13 (3) ◽  
pp. 207-213 ◽  
Author(s):  
Liesbeth Van Wesenbeeck ◽  
Leen Janssens ◽  
Hanne Meeuws ◽  
Ole Lagatie ◽  
Lieven Stuyver
Keyword(s):  
Methylation Status ◽  
Accurate Method ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Ffpe Samples

Suicide in Nógrád County, Hungary, 1970-1994

Crisis ◽  
1999 ◽  
Vol 20 (2) ◽  
pp. 64-70 ◽  
Author(s):  
Tamás Zonda
Keyword(s):  
High Risk ◽  
Psychiatric Disorders ◽  
Suicide Rate ◽  
Risk Groups ◽  
The Elderly ◽  
Female Ratio ◽  
Suicide Notes ◽  
Suicidal Intention ◽  
Male Female Ratio

The author examined completed suicides occurring over a period of 25 years in a county of Hungary with a traditionally low (relatively speaking) suicide rate of 25.8. The rates are clearly higher in villages than in the towns. The male/female ratio was close to 4:1, among elderly though only 1.5:1. The high risk groups are the elderly, divorced, and widowed. Violent methods are chosen in 66.4% of the cases. The rates are particularly high in the period April-July. Prior communication of suicidal intention was revealed in 16.3% of all cases. Previous attempts had been undertaken by 17%, which in turn means that 83% of suicides were first attempts. In our material 10% the victims left suicide notes. Psychiatric disorders were present in 60.1% of the cases, and severe, multiple somatic illnesses (including malignomas) were present in 8.8%. The majority of the data resemble those found in the literature.

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