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

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.

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Sensitivity for detecting PIK3CA mutations in early-stage breast cancer with droplet digital PCR.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.11019 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 11019-11019 ◽

Cited By ~ 1

Author(s):

Julia A. Beaver ◽

Sasidharan Balukrishna ◽

Danijela Jelovac ◽

Michaela Jane Higgins ◽

Stacie Jeter ◽

...

Keyword(s):

Breast Cancer ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Wild Type ◽

Pik3ca Mutations ◽

Tumor Specimen ◽

Ffpe Samples ◽

Exon 9 ◽

Hotspot Mutations ◽

Exon 20

11019 Background: PIK3CA is mutated in up to 30% of breast cancers. Classically somatic mutations are identified by Sanger sequencing of the primary tumor specimen. However third generation droplet digital PCR technologies offer a novel platform for quantitative mutation detection with improved sensitivity. Methods: Thirty stage I-III breast cancer patients were consented on an IRB-approved prospective repository study at Johns Hopkins for collection of their primary breast tumor specimen. Formalin-fixed paraffin embedded (FFPE) samples were analyzed by standard sequencing for three PIK3CA hotspot mutations. The DNA from these samples was then analyzed using the RainDrop digital PCR platform with TaqMan probes in a triplex format to simultaneously detect and quantitate hotspot mutations and genome equivalents. Results are expressed as a percentage of mutant to wild-type PIK3CA molecules for each sample. Results: Standard sequencing of all tumors (n=30) identified seven PIK3CA Exon 20 mutations (H1047R) and three Exon 9 mutations (E545K). Samples were scored as PIK3CA mutation positive by digital PCR if the tumor DNA contained at least 5% mutant molecules. All ten mutations identified by sequencing were verified by digital PCR with quantities of mutant molecules ranging from 20.3-55.6% in a given sample. Digital PCR identified additional PIK3CA mutations that were wild type by standard sequencing including three mutant Exon 20 samples, two mutant Exon 9 samples and one sample with an Exon 20 and Exon 9 mutation. Quantities of mutant molecules in these additional samples ranged from 5-28.9%. Conclusions: RainDrop digital PCR offers improved sensitivity and quantification for detecting PIK3CA mutations in FFPE samples using nanograms of DNA. Additional mutations identified by digital PCR may reflect genetic heterogeneity or possibly tissue contamination. The clinical utility of identifying a small proportion of mutations is unknown but may impact eligibility for targeted therapies and clinical trials. Ongoing studies will also address whether the identification of solid tumor mutations in circulating cell-free plasma DNA by digital PCR can improve diagnostics and aid in therapeutic decisions.

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Breast Cancer Subtype Classification Using 4-Plex Droplet Digital PCR

Clinical Chemistry ◽

10.1373/clinchem.2019.302315 ◽

2019 ◽

Vol 65 (8) ◽

pp. 1051-1059 ◽

Cited By ~ 3

Author(s):

Wenwen Chen ◽

Jiaying Zheng ◽

Chang Wu ◽

Shaoxiong Liu ◽

Yongxin Chen ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Progesterone Receptor ◽

Rna Binding ◽

Dynamic Range ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Tyrosine Kinase 2 ◽

Ffpe Samples ◽

Estrogen Receptor 1

Abstract BACKGROUND Infiltrating ductal carcinoma (IDCA) is the most common form of invasive breast cancer. Immunohistochemistry (IHC) is widely used to analyze estrogen receptor 1 (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) that can help classify the tumor to guide the medical treatment. IHC examinations require experienced pathologists to provide interpretations that are subjective, thereby lowering the reproducibility of IHC-based diagnosis. In this study, we developed a 4-plex droplet digital PCR (ddPCR) for the simultaneous and quantitative analyses of estrogen receptor 1 (ESR1), progesterone receptor (PGR), erb-b2 receptor tyrosine kinase 2 (ERBB2), and pumilio RNA binding family member 1 (PUM1) expression levels in formalin-fixed paraffin-embedded (FFPE) samples. METHODS We evaluated the sensitivity, reproducibility, and linear dynamic range of 4-plex ddPCR. We applied this method to analyze 95 FFPE samples from patients with breast IDCA and assessed the agreement rates between ddPCR and IHC to evaluate its potential in classifying breast cancer subtypes. RESULTS The limits of quantification (LOQ) were 25, 50, 50, and 50 copies per reaction for ERBB2, ESR1, PGR, and PUM1, respectively. The dynamic ranges of ESR1, PGR, and PUM1 extended over 50–1600 copies per reaction and those of ERBB2 from 25 to 1600 copies per reaction. The concordance correlation coefficients between 4-plex ddPCR and IHC were 96.8%, 91.5%, and 85.1% for ERBB2, ESR1, and PGR, respectively. Receiver operating characteristic curve area under the curve values of 0.991, 0.977, and 0.920 were generated for ERBB2, ESR1, and PGR, respectively. CONCLUSIONS Evaluation of breast cancer biomarker status by 4-plex ddPCR was highly concordant with IHC in this study.

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

Diagnostic Pathology ◽

10.1186/s13000-018-0777-x ◽

2018 ◽

Vol 13 (1) ◽

Cited By ~ 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

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Droplet digital PCR as an alternative to FISH for MYCN amplification detection in human neuroblastoma FFPE samples

BMC Cancer ◽

10.1186/s12885-019-5306-0 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Dinesh Babu Somasundaram ◽

Sheeja Aravindan ◽

Zhongxin Yu ◽

Muralidharan Jayaraman ◽

Ngoc T. B. Tran ◽

...

Keyword(s):

Digital Pcr ◽

Droplet Digital Pcr ◽

Mycn Amplification ◽

Human Neuroblastoma ◽

Ffpe Samples

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Enumeration of Viable Non-Culturable Vibrio cholerae Using Droplet Digital PCR Combined With Propidium Monoazide Treatment

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.753078 ◽

2021 ◽

Vol 11 ◽

Author(s):

Shuo Zhao ◽

Jingyun Zhang ◽

Zhe Li ◽

Yu Han ◽

Biao Kan

Keyword(s):

Vibrio Cholerae ◽

Quantitative Pcr ◽

Bacterial Species ◽

Accurate Method ◽

Single Copy ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Bacterial Cells ◽

Propidium Monoazide ◽

Vbnc State

Many bacterial species, including Vibrio cholerae (the pathogen that causes cholera), enter a physiologically viable but non-culturable (VBNC) state at low temperature or in conditions of low nutrition; this is a survival strategy to resist environmental stress. Identification, detection, and differentiation of VBNC cells and nonviable cells are essential for both microbiological study and disease surveillance/control. Enumeration of VBNC cells requires an accurate method. Traditional counting methods do not allow quantification of VBNC cells because they are not culturable. Morphology-based counting cannot distinguish between live and dead cells. A bacterial cell possesses one copy of the chromosome. Hence, counting single-copy genes on the chromosome is a suitable approach to count bacterial cells. In this study, we developed quantitative PCR-based methods, including real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), to enumerate VBNC V. cholerae cells by counting the numbers of single-copy genes in samples during VBNC-state development. Propidium monoazide (PMA) treatment was incorporated to distinguish dead cells from viable cells. Both PCR methods could be used to quantify the number of DNA copies/mL and determine the proportion of dead cells (when PMA was used). The methods produced comparable counts using three single-copy genes (VC1376, thyA, and recA). However, ddPCR showed greater accuracy and sensitivity than qPCR. ddPCR also allows direct counting without the need to establish a standard curve. Our study develops a PMA-ddPCR method as a new tool to quantify VBNC cells of V. cholerae. The method can be extended to other bacterial species.

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Comparison of a quantitative Real-Time PCR Assay and droplet digital PCR Assay for detection of Streptococcus agalactiae

10.21203/rs.2.16922/v1 ◽

2019 ◽

Author(s):

Xu-Guang Guo ◽

Yi-Fan Zeng ◽

Chu-Mao Chen ◽

Xiao-Yan Li ◽

Jun-Jiang Chen ◽

...

Keyword(s):

Real Time ◽

Streptococcus Agalactiae ◽

Culture Method ◽

Accurate Method ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Detection Sensitivity ◽

Detection Methods ◽

Pcr Assay ◽

Accuracy And Repeatability

Abstract Background :Streptococcus agalactiae(GBS) is pathogenic bacterium that causes puerperal sepsis in pregnant women and meningitis in newborns. Reliable detection methods for GBS included culture-based approaches, antigen-antibody method and real-time quantitative PCR. However, these methods have low sensitivity and are time consuming. In this study, we established a new detection approach, i.e. droplet digital PCR (ddPCR), for more accurate detection of GBS. Materials and Methods : Clinical specimens infected with GBS were analyzed using culture method. QPCR and ddPCR were used to quantify GBS related genes (CpsE and Sip gene). All experiments were run in triplicate using the same positive strain to verify the repeatability of the methodology. Results: The ddPCR showed outstanding accuracy, with 100% sensitivity, 100% specificity and coefficient of variation =4.5%. Among the detected strains, only positive results were obtained for the CpsE gene by ddPCR, and the detection sensitivity was 5pg/μL. Compared to the level of individual nanogramme-sized(0.5ng/μL) of qPCR assay, ddPCR resulted as a more significantly accurate method for detection of GBS in precision medical area. Conclusions : Droplet digital PCR offers high sensitivity, accuracy and repeatability, and is a suitable approach for detection of positive GBS samples.

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