scholarly journals Development of a Liquid Biopsy Based Purely Quantitative Digital Droplet PCR Assay for Detection of MLH1 Promoter Methylation in Colorectal Cancer Patients

2021 ◽  
Author(s):  
Danyi Wang ◽  
Dennis O’Rourke ◽  
Jorge Sanchez Garcia ◽  
Ti Cai ◽  
Juergen Scheuenpflug ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Sensitivity And Specificity ◽  
Promoter Methylation ◽  
Liquid Biopsy ◽  
Methylation Status ◽  
Assay Sensitivity ◽  
Digital Droplet Pcr ◽  
Mlh1 Promoter ◽  
Healthy Donors ◽  
Droplet Pcr

Abstract Background MutL Homolog 1 (MLH1) promotor methylation is associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. As cancer-specific DNA methylation changes in body fluids are limited, it is particularly challenging to develop clinically applicable liquid biopsy methodologies with high sensitivity and specificity. The purpose of this study was to develop a fit-for-purpose methylation sensitive restriction enzyme (MSRE) based digital droplet PCR (ddPCR) assay to examine MLH1 promoter methylation in ctDNA in advanced CRC. Methods Primers and probes were designed to amplify CpG sites of the MLH1 promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to MSRE HpaII digestion. Plasma samples from 20 healthy donors and 28 CRC patients were analyzed with the optimized MSRE procedure using ddPCR. Results Using methylated and unmethylated controls, we optimized the conditions for HpaII enzyme digestion to ensure complete digestion and avoid false positives. Based on the results from the ddPCR assay using 1ng circulating cell-free DNA (cfDNA) input from healthy donors or CRC samples, ROC curves were generated with an area under the curve (AUC) value of 0.965. The optimal assay sensitivity and specificity was achieved when 8 positive droplets were used as acceptance criteria (78% sensitivity and 100% specificity). A tiered-based cutoff (20%, 50%, 80% percentile based) was applied to distinguish CRC samples with different methylation level.Conclusions Our study demonstrated that the liquid biopsy assay for MLH1 promoter methylation detection using purely quantitative ddPCR is a simple and ultrasensitive procedure that provides reliable methylation detection in ctDNA. The MSRE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development.

scholarly journals Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH1 promoter methylation in colorectal cancer patients

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Danyi Wang ◽  
Dennis O’Rourke ◽  
Jorge F. Sanchez-Garcia ◽  
Ti Cai ◽  
Juergen Scheuenpflug ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Sensitivity And Specificity ◽  
Promoter Methylation ◽  
Liquid Biopsy ◽  
Methylation Status ◽  
Area Under The Curve ◽  
Assay Sensitivity ◽  
Digital Droplet Pcr ◽  
Healthy Donors ◽  
Droplet Pcr

Abstract Background MutL Homolog 1 (MLH1) promotor methylation is associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. As cancer-specific DNA methylation changes in body fluids are limited, it is particularly challenging to develop clinically applicable liquid biopsy methodologies with high sensitivity and specificity. The purpose of this study was to develop a fit-for-purpose methylation sensitive restriction enzyme (MSRE) based digital droplet PCR (ddPCR) assay to examine MLH1 promoter methylation in ctDNA in advanced CRC. Methods Primers and probes were designed to amplify CpG sites of the MLH1 promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to MSRE HpaII digestion. Plasma samples from 20 healthy donors and 28 CRC patients were analyzed with the optimized MSRE procedure using ddPCR. Results Using methylated and unmethylated controls, we optimized the conditions for HpaII enzyme digestion to ensure complete digestion and avoid false positives. Based on the results from the ddPCR assay using 1 ng circulating cell-free DNA (cfDNA) input from healthy donors or CRC samples, ROC curves were generated with an area under the curve (AUC) value of 0.965 (95% CI: 0.94, 0.99). The statistically optimal assay sensitivity and specificity was achieved when 8 positive droplets were used as acceptance criteria (78% sensitivity and 100% specificity, 95% CI: 0.45, 0.95). A tiered-based cutoff (20, 50, 80% percentile based) was applied to distinguish CRC samples with different methylation level. Conclusions Our study demonstrated that the liquid biopsy assay for MLH1 promoter methylation detection using purely quantitative ddPCR is a simple and highly sensitive procedure that provides reliable methylation detection in ctDNA. The MSRE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development.

scholarly journals Genome-wide analysis identifies critical DNA methylations within NTRKs genes in colorectal cancer

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Zijian Chen ◽  
Zenghong Huang ◽  
Yanxin Luo ◽  
Qi Zou ◽  
Liangliang Bai ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Promoter Methylation ◽  
Expression Profiles ◽  
Methylation Status ◽  
Gene Promoter ◽  
Normal Mucosa ◽  
Suppressor Gene ◽  
Survival Outcome ◽  
Prognostic Models

Abstract Background Neurotrophic tropomyosin receptor kinases (NTRKs) are a gene family function as oncogene or tumor suppressor gene in distinct cancers. We aimed to investigate the methylation and expression profiles and prognostic value of NTRKs gene in colorectal cancer (CRC). Methods An analysis of DNA methylation and expression profiles in CRC patients was performed to explore the critical methylations within NTRKs genes. The methylation marker was validated in a retrospectively collected cohort of 229 CRC patients and tested in other tumor types from TCGA. DNA methylation status was determined by quantitative methylation-specific PCR (QMSP). Results The profiles in six CRC cohorts showed that NTRKs gene promoter was more frequently methylated in CRC compared to normal mucosa, which was associated with suppressed gene expression. We identified a specific methylated region within NTRK3 promoter targeted by cg27034819 and cg11525479 that best predicted survival outcome in CRC. NTRK3 promoter methylation showed independently predictive value for survival outcome in the validation cohort (P = 0.004, HR 2.688, 95% CI [1.355, 5.333]). Based on this, a nomogram predicting survival outcome was developed with a C-index of 0.705. Furthermore, the addition of NTRK3 promoter methylation improved the performance of currently-used prognostic model (AIC: 516.49 vs 513.91; LR: 39.06 vs 43.64, P = 0.032). Finally, NTRK3 promoter methylation also predicted survival in other tumors, including pancreatic cancer, glioblastoma and stomach adenocarcinoma. Conclusions This study highlights the essential value of NTRK3 methylation in prognostic evaluation and the potential to improve current prognostic models in CRC and other tumors.

scholarly journals P34.06 The Clinical Utility of Liquid Biopsy by Digital Droplet PCR in Patients with Advanced NSCLC

2021 ◽  
Vol 16 (3) ◽  
pp. S417
Author(s):  
V. Lamberts ◽  
M. Aldea ◽  
L. Mezquita ◽  
C. Jovelet ◽  
D. Vasseur ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Clinical Utility ◽  
Advanced Nsclc ◽  
Digital Droplet Pcr ◽  
Droplet Pcr

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

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

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

scholarly journals Methylation in p14ARF is Frequently Observed in Colorectal Cancer with Low-level Microsatellite Instability

2009 ◽  
Vol 37 (4) ◽  
pp. 1038-1045 ◽  
Author(s):  
K Kominami ◽  
T Nagasaka ◽  
HM Cullings ◽  
N Hoshizima ◽  
H Sasamoto ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Microsatellite Instability ◽  
Promoter Methylation ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Gene Promoters ◽  
Braf Mutations ◽  
Low Level ◽  
Methylguanine Dna Methyltransferase ◽  
High Level

Colorectal cancer (CRC) can be classified as high-level microsatellite instability (MSI-H), low-level MSI (MSI-L) and microsatellite stable (MSS) depending on levels of MSI. MSI-H CRC relies on a distinct molecular pathway due to the mismatch repair (MMR) deficiency and shows methylation in multiple gene promoters. The genetic pathway leading to MSI-L is unknown, although higher levels of promoter methylation are observed in this group compared with MSS CRCs. This study explored how promoter methylation affects MSI phenotype, by analysing the methylation status of eight CRC-related promoters, MSI phenotype and KRAS/BRAF mutations in a series of 234 CRCs. Promoter methylation of p14ARF was significantly related to MSI-L CRC with KRAS mutation. The MSI-H phenotype was related to methylation of MLH1 as expected, while the MSS phenotype was related to methylation of p16INK4a and O6-methylguanine-DNA methyltransferase, although this was not statistically significant. Thus, promoter methylation of p14ARF could be a significant alteration leading to CRC with MSI-L.

scholarly journals ADAMTS9 is Silenced by Epigenetic Disruption in Colorectal Cancer and Inhibits Cell Growth and Metastasis by Regulating Akt/p53 Signaling

2017 ◽  
Vol 44 (4) ◽  
pp. 1370-1380 ◽  
Author(s):  
Ling Chen ◽  
Jun Tang ◽  
Yixiao Feng ◽  
Shuman Li ◽  
Qin Xiang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Promoter Methylation ◽  
Methylation Status ◽  
Cancer Cell Lines ◽  
Colorectal Cancer Cell ◽  
Matrix Assembly ◽  
Colorectal Cancer Cell Lines ◽  
Cancer Tissues

Background/Aims: ADAMTS (disintegrin-like and metalloproteinase with thrombospondin motifs) proteins are extracellular zinc metalloproteinases that play an important role in extracellular matrix assembly and degradation, connective tissue structuring, angiogenesis, and cell migration. Multiple studies suggest that ADAMTS proteins (e.g. ADAMTS9) can act as tumor suppressors. In gastric, esophageal, and nasopharyngeal carcinomas ADAMTS9 is frequently down-regulated by promoter methylation. Whether ADAMTS9 can function as a tumor suppressor gene (TSG) in colorectal cancer is still unclear. Methods: We performed immunohistochemistry, RT-PCR, and qRT-PCR, to examine the expression of ADAMTS9 in colorectal cancer cell lines and primary colorectal cancer tissues. Methylation-specific PCR was also carried out to investigate the promoter methylation status of ADAMTS9. We also explored the functions of ADAMTS9 in colorectal cancer cell lines through in vitro experiments. Results: ADAMTS9 expression was down-requlated or silenced in 83.3% (5/6) of colorectal cancer cell lines, and frequently repressed in 65.6% (21/32) of colorectal cancer tissues. Down-regulation of ADAMTS9 was partially due to promoter methylation. Exogenous expression of ADAMTS9 in colorectal cancer cell lines inhibited cell proliferation and migration through the regulation of cell cycle and apoptosis. In addition, ADAMTS9 prevented the activation of Akt, and its downstream targets in colorectal cancer cell lines. Conclusion: Our findings suggest ADAMTS9 is a TSG in colorectal cancer.

scholarly journals Non-invasive prenatal testing of fetal aneuploidies using a new method based on digital droplet PCR and cell free fetal DNA

2020 ◽  
Author(s):  
Wang Haidong ◽  
Yang Zhijie ◽  
Elena Picchiassi ◽  
Federica Tarquini ◽  
Giuliana Coata ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Trisomy 21 ◽  
Trisomy 13 ◽  
Clinical Validation ◽  
Analytical Sensitivity ◽  
Non Invasive ◽  
Digital Droplet Pcr ◽  
Fetal Dna ◽  
Cell Free Fetal Dna ◽  
Droplet Pcr

ABSTRACTBackgroundCurrent next generation sequencing (NGS) and microarray based Non-Invasive Prenatal Tests (NIPT), used for the detection of common fetal trisomies, are still expensive, time consuming and need to be performed in centralized laboratories. To improve NIPT in clinical routine practice as universal prenatal screening, we have developed a digital droplet PCR (ddPCR) based assay called iSAFE NIPT using cell free fetal DNA (cffDNA) for detection of fetal trisomies 13, 18 and 21 in a single reaction with advantage of high diagnostic accuracy and reduced cost.Materials and MethodsWe first used artificial DNA samples to evaluate analytical sensitivity and specificity of the iSAFE NIPT. Next, we analysed 269 plasma samples for the clinical validation of iSAFE NIPT. Fifty-eight of these, including five trisomies 21, two trisomies 18 and one trisomy 13 were utilised to establish the assay cut-off values based on ratios between chromosome counts. The remaining 211 plasma samples, including 10 trisomies 21, were analysed to evaluate iSAFE NIPT clinical performance.ResultsiSAFE NIPT achieved a 100% analytical sensitivity (95% CI 94.9-100% trisomy 21; 79.4-100% trisomy 18; 73.5-100% trisomy 13) and 100% specificity (95% CI 96.3-100% trisomy 21; 97.6-100% trisomy 18; 97.6-100% trisomy 13). It also achieved a 100% clinical sensitivity and specificity for trisomy 21 detection in the 211 clinical samples (95% CI for sensitivity is 69.1-100%, and 95% CI for specificity is 98.2-100%).ConclusionsThe iSAFE NIPT is a highly multiplexed ddPCR based assay for detection of fetal trisomies from maternal blood. Based on clinical validation, the iSAFE NIPT has high diagnostic sensitivity and specificity. It can be decentralized in routine clinical laboratories, is fast, easy to use and economical comparing to current NIPT.

scholarly journals Reporting Subclonal Immunohistochemical Staining of Mismatch Repair Proteins in Endometrial Carcinoma in the Times of Ever-Changing Guidelines

2021 ◽  
Author(s):  
Ashley Scheiderer ◽  
Courtney Riedinger ◽  
Kristopher Kimball ◽  
Larry Kilgore ◽  
Amila Orucevic
Keyword(s):  
Genetic Testing ◽  
Endometrial Carcinoma ◽  
Mismatch Repair ◽  
Promoter Methylation ◽  
Methylation Status ◽  
Nuclear Staining ◽  
Mlh1 Promoter ◽  
History Of ◽  
The Times

Context.— The current College of American Pathologists reporting guideline for mismatch repair protein (MMRP) immunohistochemistry for Lynch syndrome (LS) screening considers the presence of any positive nuclear staining as intact MMRP expression. This would include tumors with combined areas of subclonal retention and loss of MMRP staining. Objective.— To evaluate the clinical significance of reporting subclonal staining patterns of MMRP immunohistochemistry in endometrial carcinoma. Design.— We retrospectively reviewed 455 consecutive MMRP immunohistochemistry results of endometrial carcinoma in hysterectomy specimens from 2012 through 2017 and identified cases with subclonal MMRP staining. These results were correlated with the patient's personal and family history of LS-associated carcinoma, MLH1 promoter methylation status, and LS genetic testing. Results.— Subclonal staining of MMRP was seen in 48 of 455 cases (10.5%) on review. Thirty cases demonstrated isolated subclonal staining and were reported by pathologists as follows: subclonal (n = 5), complete MMRP loss (n = 4), and intact MMRP (n = 21). Eighteen cases had subclonal staining in combination with complete loss of other MMRP. Cases reported as subclonal or complete MMRP loss had appropriate clinical follow-up. Two of 2 cases with isolated subclonal MSH6 loss tested positive for LS. One of 3 cases with isolated subclonal MLH1/PMS2 loss was negative for MLH1 promoter methylation; LS genetic testing was not performed because of cost. Conclusions.— Our study reveals that LS germline mutation can be detected in endometrial carcinoma patients whose tumors display sole subclonal MMRP staining. Our results stress the importance of reporting subclonal staining patterns to ensure appropriate clinical follow-up.

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