Hypermethylated circulating DNA detection using picoliter droplet-based PCR in colorectal cancer.

2016 ◽  
Vol 34 (4_suppl) ◽  
pp. 622-622
Author(s):  
Geraldine Perkins ◽  
Sonia Garrigou ◽  
Fanny Garlan ◽  
Corinne Normand ◽  
Audrey Didelot ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Methylation Status ◽  
Surrogate Marker ◽  
Tumor Evolution ◽  
Specific Gene ◽  
Tumor Type ◽  
Plasma Samples ◽  
Tumor Dna

622 Background: Circulating tumor DNA (ctDNA) is thoroughly investigated as a surrogate biomarker of tumor follow-up, in different cancer types, such as colorectal cancer (CRC). Droplet-based digital PCR (ddPCR) is a highly sensitive and also quantitative method for detection of very low amount of ctDNA. Since many different genes can be mutated within a specific tumor type and also wide mutation spectrum can occur within a specific gene, procedures for ctDNA monitoring can be time consuming and need to be improved for a routinely use. To overcome these drawbacks, we characterized the methylation status of 3 genes frequently hypermethylated in CRC to identify universal markers for tumor follow-up. Methods: The characterization of the methylated status of the WIF, NPY and PENK genes in the tumor DNA was performed in 56 CRC of different stages and 45 corresponding plasma samples using droplet-based dPCR, after DNA bisulfite conversion. A two-panels assay (with albumin as a reference) was developed. Methylation level of these 3 genes in tumor tissues was compared to corresponding normal tissues (n = 22) and plasma samples (MetctDNA). To validate, plasma samples of additional 91 patients were analyzed for the presence of ctDNA both by the characterization of KRAS, BRAF, TP53 and PIK3CA mutations (MutctDNA) and of MetctDNA, at various stages of their follow-up, and 9 of them had MetctDNA assessment during treatment follow-up. Results: All tumor samples were positive for WIF1 and/or NPY markers. Hypermethylation of these two genes was significantly higher in tumor tissue compared to normal, independently of the tumor stage (p < 0.0001). MetctDNA could be detected in 75% of metastatic CRC patients and 24% of localized CRC patients (stage 1 to 3). MetctDNA and MutctDNA fractions were strongly correlated (R2 > 0.9, p < 0.0001). During follow-up, MetctDNA levels changes allowed monitoring of tumor evolution in different stages CRC patients. Conclusions: These results indicate that determination of MetctDNA by droplet-based dPCR can reach same efficiency than MutctDNA for ctDNA assessment, using only 2 markers, and thus could be considered as a universal surrogate marker of tumor follow-up in CRC patients.

scholarly journals A Study of Hypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker

2016 ◽  
Vol 62 (8) ◽  
pp. 1129-1139 ◽  
Author(s):  
Sonia Garrigou ◽  
Geraldine Perkins ◽  
Fanny Garlan ◽  
Corinne Normand ◽  
Audrey Didelot ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Genetic Material ◽  
Surrogate Marker ◽  
Digital Pcr ◽  
Tumor Stage ◽  
Circulating Tumor Dna ◽  
Tumor Evolution ◽  
Highly Sensitive ◽  
Tumor Dna

Abstract BACKGROUND Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

TFAP2E methylation status and prognosis of patients with radically resected colorectal cancer.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3628-3628
Author(s):  
Seong Joon Park ◽  
Seung-mi Kim ◽  
Yong Sang Hong ◽  
Jae-Lyun Lee ◽  
Jeong-Eun Kim ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Methylation Status ◽  
Stage I ◽  
Gene Encoding ◽  
Free Survival ◽  
Factors Affecting ◽  
Distal Location ◽  
Resected Stage ◽  
Significant Factors

3628 Background: Transcription factor AP-2ε, a member of the AP-2 family has extensively studied in many cancers. Recently, it has been suggested that the gene encoding AP-2ε (TFAP2E) is involved in the development of colorectal cancer (CRC) and is also associated with clinical outcomes of the patients with CRCs. Therefore, we have investigated the clinical significance of TFAP2E in CRC patients who underwent curative resections. Methods: A single-institution cohort of 248 patients with curatively resected, stage I/II/III CRCs between March and December, 2004 were included, and the analyses were performed in 193 patients whose tumors were available for TFAP2E methylation status Results: One hundred twelve patients (58%) showed TFAP2E hypermethylation, which was significantly more common in CRCs with distal location, low pathologic T stage (T1/T2) and stage I. After a median follow-up duration of 86.3 months, the patients with TFAP2E hypermethylation had a trend for better survival outcome in terms of relapse-free survival (RFS) and overall survival (OS) (TFAP2E hypermethylation vs. hypomethylation; 5-year RFS rate 90% vs. 80%, p=0.063; 6-year OS rate 88% vs. 80%, p=0.083). Multivariate analysis showed pathologic nodal stage and TFAP2E methylation status were independent prognostic factors affecting both RFS and OS, which also remained significant factors in the subgroup analysis including 154 patients with stage II/III CRCs who had received adjuvant chemotherapy. Conclusions: TFAP2E hypermethylation was associated with better clinical outcome and may be considered as an independent prognostic factor in the patients with curatively resected CRC.

Role of enterocyte-specific gene polymorphisms in adjuvant treatment for stage III colorectal cancer.

2019 ◽  
Vol 37 (4_suppl) ◽  
pp. 550-550
Author(s):  
Mitsukuni Suenaga ◽  
Shu Cao ◽  
Wu Zhang ◽  
Satoshi Matsusaka ◽  
Satoshi Okazaki ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Survival Rate ◽  
Clinical Significance ◽  
Adjuvant Treatment ◽  
Gene Polymorphisms ◽  
Multivariable Analysis ◽  
Stage Iii ◽  
Specific Gene ◽  
Discovery Cohort

550 Background: Enterocyte subtype of the Colorectal Cancer (CRC) Assigner classifier is known as favorable to oxaliplatin-based adjuvant treatment for stage III CRC. We previously reported potential predictive value of single nucleotide polymorphisms (SNPs) in enterocyte-related genes in metastatic CRC (Suenaga, ASCO2018). In this study, we examined clinical significance of MS4A12 and CDX2 SNPs in adjuvant treatment (AT) for Stage III CRC. Methods: 350 patients with Stage III CRC were included in this study: 274 received AT (discovery cohort: median age = 62, median follow-up = 59.9 months) and 76 received surgery alone (control: median age = 75, median follow-up = 58.0 months). 68 and 206 patients received FOLFOX and oral fluoriopyrimidine, respectively. SNPs were analyzed by PCR-based direct sequencing. Disease-free survival and overall survival (OS) were analyzed using Kaplan-Meier curves, log-rank test, and Cox proportional hazards regression. Results: In discovery cohort, the G/G variant in MS4A12 rs4939378 was associated with lower 5-y survival rate than any A allele in uni- and multi-variate analyses (70% vs 90%, univariate: HR 2.29, 95% CI: 1.03-5.06, P = 0.035; multivariable: HR 2.58, 95% CI: 1.15-5.76, P = 0.021). Patients with the G/G variant in CDX2 rs3812863 had better OS than those with any A, though not significant in multivariable analysis (5 y-survival rate: 95% vs 82%, univariate: HR 0.34, 95% CI: 0.12-0.97, P = 0.034; multivariable: HR 0.39, 95% CI: 0.13-1.11, P = 0.078). There was no significance in the control, and significant interaction was observed between MS4A12 genotypes and groups (interaction P = 0.007). In addition, there was no interaction between MS4A12 rs4939378 and FOLFOX vs oral fluoropyrimidine. Conclusions: Our findings suggest that MS4A12 and CDX2 gene polymorphisms may predict outcome in patients with Stage III CRC. However, clinical significance of the SNPs for oxaliplatin seems to differ depending on tumor stage. Further research and validation study are warranted to explore the association of the SNPs with carcinogenesis or cancer progression.

scholarly journals Tumor DNA Mutations From Intraparenchymal Brain Metastases Are Detectable in CSF

2021 ◽  
pp. 163-172
Author(s):  
Stephanie Kim Cheok ◽  
Azeet Narayan ◽  
Anna Arnal-Estape ◽  
Scott Gettinger ◽  
Sarah B. Goldberg ◽  
...  
Keyword(s):  
Brain Metastases ◽  
Cohort Analysis ◽  
Surrogate Marker ◽  
Normal Pressure ◽  
Tumor Evolution ◽  
Leptomeningeal Disease ◽  
Dna Mutations ◽  
Sequencing Method ◽  
Cancer Types ◽  
Tumor Dna

PURPOSE Discordant responses between brain metastases and extracranial tumors can arise from branched tumor evolution, underscoring the importance of profiling mutations to optimize therapy. However, the morbidity of brain biopsies limits their use. We investigated whether cell-free DNA (cfDNA) in CSF could serve as an effective surrogate marker for genomic profiling of intraparenchymal (IP) brain metastases. METHODS CSF and blood were collected simultaneously from patients with progressive brain metastases undergoing a craniotomy or lumbar puncture. Mutations in both biofluids were measured using an error-suppressed deep sequencing method previously published by our group. Forty-three regions of 24 cancer-associated genes were assayed. RESULTS This study enrolled 14 patients with either IP brain metastases (n = 12) or cytology-positive leptomeningeal disease (LMD, n = 2) and two controls with normal pressure hydrocephalus. Primary cancer types were lung, melanoma, renal cell, and colorectal. cfDNA was measurable in all sixteen samples of CSF. Cancer-associated mutations were found in the CSF of ten patients (eight with IP [67%] and two with LMD [100%]) and plasma of five patients (five with IP [42%] and none with LMD). All patients with plasma cfDNA had extracranial tumors. Among the five patients in the cohort who also had mutation data from time-matched brain metastasis tissue, four patients (80%) had matching mutations detected in CSF and brain, whereas only one patient (20%) had matching mutations detected in plasma and brain. CONCLUSION The detection of mutational DNA in CSF is not restricted to LMD and was found in two thirds of patients with IP brain metastases in our cohort. Analysis of CSF can be a viable alternative to biopsy for detection of somatic mutations in brain metastases.

scholarly journals DNA methylation markers detected in blood, stool, urine, and tissue in colorectal cancer: a systematic review of paired samples

2020 ◽  
Author(s):  
Eivor Alette Laugsand ◽  
Siv Sellæg Brenne ◽  
Frank Skorpen
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Data Extraction ◽  
Meta Analysis ◽  
Methylation Status ◽  
High Sensitivity ◽  
Liquid Biopsies ◽  
Non Invasive ◽  
Paired Samples

Abstract Purpose Methylated cell-free DNA in liquid biopsies are promising non-invasive biomarkers for colorectal cancer (CRC). Optimal markers would have high sensitivity and specificity for early detection of CRC and could be detected in more than one type of material from the patient. We systematically reviewed the literature on DNA methylation markers of colorectal cancer, detected in more than one type of material, regarding their potential as contributors to a panel for screening and follow-up of CRC. Methods The databases MEDLINE, Web of Science, and Embase were systematically searched. Data extraction and review was performed by two authors independently. Agreement between methylation status in tissue and other materials (blood/stool/urine) was analyzed using the McNemar test and Cohen’s kappa. Results From the 51 included studies, we identified seven single markers with sensitivity ≥ 75% and specificity ≥ 90% for CRC. We also identified one promising plasma panel and two stool panels. The correspondence of methylation status was evaluated as very good for four markers, but only marginal for most of the other markers investigated (12 of 21). Conclusion The included studies reported only some of the variables and markers of interest and included few patients. Hence, a meta-analysis was not possible at this point. Larger, prospective studies must be designed to study the discordant detection of markers in tissue and liquid biopsies. When reporting their findings, such studies should use a standardized format.

DNA methylation profiling from circulating tumor DNA for early-detection of colorectal cancer.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e15076-e15076
Author(s):  
Wei Zhang ◽  
Jinke Sui ◽  
Xianrui Wu ◽  
Fuao Cao ◽  
Guanyu Yu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Early Detection ◽  
Methylation Status ◽  
Stage I ◽  
Support Vector ◽  
Plasma Samples ◽  
Neoplastic Progression ◽  
Healthy Individuals ◽  
Early Stage Disease

e15076 Background: Colorectal cancer (CRC) develops as a result of neoplastic progression, which often takes decades, providing a window for early detection. Unfortunately, there has been little success in developing blood-based screening method due to the low amount of ctDNA present in the circulation, especially in patients with early stage disease. The role of aberrant DNA methylation, occurring very early in tumorigenesis, has been well elucidated. In this prospective study, we evaluated the potentiality of DNA methylation status obtained from ctDNA as an early detection method. Methods: Panel Design: Methylation data of tumor samples (12 types, n = 4,772), adjacent normal (8 types, n = 411), and normal white blood cells (n = 656) from TCGA and GSE were compared. Differentially methylated sites were extracted using modified wald-test with an adjusted p-value < 0.05 and fold-change > 2. Our panel covers 80,672 CpG sites, spanning 1.05Mb of human genome. We performed targeted bisulfite sequencing on plasma samples of 67 (stage I: 13, II:29, III: 23, IV: 2) Chinese CRC patients and 144 healthy individuals to construct a model for deriving markers that are differentially methylated and their associated weight. The model was validated in 2 independent cohorts. Results: We constructed a model using a support vector machine (SVM)-based machine learning classifier based on top 4,000 differentially methylated regions (DMRs) selected by random forest between tumor and normal plasma samples. Subsequently, 5-fold cross-validation with 100-time repeats were performed to gain a robust estimation of model performance, achieving a sensitivity of 91%, specificity of 98% and area under curve (AUC) of 98.6%. The model was subsequently validated in 2 independent cohorts: one consisted of 57 stage I-III CRC patients and 74 healthy individuals and another one with 47 stage IV patients and the same 74 healthy individuals. The model yielded a sensitivity of 83% and 95% for the early and late stage cohorts, respectively. A specificity of 95% was obtained for both cohorts. Conclusions: Our findings demonstrated the potential of profiling DNA methylation, which can effectively distinguish cancerous from healthy, for the purpose of screening. This method has potential to serve as a supplementary or alternative approach in early detection.

scholarly journals Serial Circulating Tumor DNA Mutational Status in Patients with KRAS-Mutant Metastatic Colorectal Cancer from the Phase 3 AIO KRK0207 Trial

2020 ◽  
Vol 66 (12) ◽  
pp. 1510-1520
Author(s):  
Smiths S Lueong ◽  
Andreas Herbst ◽  
Sven-Thorsten Liffers ◽  
Nicola Bielefeld ◽  
Peter A Horn ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Metastatic Colorectal Cancer ◽  
Treatment Initiation ◽  
Multivariable Analysis ◽  
Circulating Tumor Dna ◽  
Post Treatment ◽  
First Line ◽  
Phase 3 ◽  
Tumor Dna

Abstract Background We assessed the usefulness of circulating tumor DNA (ctDNA) pre- or post-treatment initiation for outcome prediction and treatment monitoring in metastatic colorectal cancer (mCRC). Methods Droplet digital PCR was used to measure absolute mutant V-Ki-ras2 Kirsten rat sarcoma viral oncogene ((mut)KRAS) ctDNA concentrations in 214 healthy controls (plasma and sera) and in 151 tissue-based mutKRAS positive patients with mCRC from the prospective multicenter phase 3 trial AIO KRK0207. Serial mutKRAS ctDNA was analyzed prior to and 2–3 weeks after first-line chemotherapy initiation with fluoropyrimidine, oxaliplatin, and bevacizumab in patients with mCRC and correlated with clinical parameters. Results mut KRAS ctDNA was detected in 74.8% (113/151) of patients at baseline and in 59.6% (90/151) at follow-up. mutKRAS ctDNA at baseline and follow-up was associated with poor overall survival (OS) (hazard ratio [HR] =1.88, 95% confidence interval [CI] 1.20–2.95; HR = 2.15, 95% CI 1.47–3.15) and progression-free survival (PFS) (HR = 2.53, 95% CI 1.44–4.46; HR = 1.90, 95% CI 1.23–2.95), respectively. mutKRAS ctDNA clearance at follow-up conferred better disease control (P = 0.0075), better OS (log-rank P = 0.0018), and PFS (log-rank P = 0.0018). Measurable positive mutKRAS ctDNA at follow-up was the strongest and most significant independent prognostic factor on OS in multivariable analysis (HR = 2.31, 95% CI 1.40–3.25). Conclusions Serial analysis of circulating mutKRAS concentrations in mCRC has prognostic value. Post treatment mutKRAS concentrations 2 weeks after treatment initiation were associated with therapeutic response in multivariable analysis and may be an early response predictor in patients receiving first-line combination chemotherapy. Clinicaltrialsgov Identifier NCT00973609.

scholarly journals BRAF Mutation Status in Circulating Tumor DNA from Patients with Metastatic Colorectal Cancer: Extended Mutation Analysis from the AGEO RASANC Study

Cancers ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 998 ◽  
Author(s):  
Leo Mas ◽  
Jean-Baptiste Bachet ◽  
Valerie Taly ◽  
Olivier Bouché ◽  
Julien Taieb ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Liver Metastasis ◽  
Metastatic Colorectal Cancer ◽  
Mutation Analysis ◽  
Braf Mutation ◽  
Circulating Tumor Dna ◽  
Kappa Coefficient ◽  
Plasma Samples ◽  
Mutation Status ◽  
Tumor Dna

In patients with metastatic colorectal cancer (mCRC), RAS and BRAF mutations are currently determined by tumor sample analysis. Here, we report BRAF mutation status analysis in paired tumor tissue and plasma samples of mCRC patients included in the AGEO RASANC prospective cohort study. Four hundred and twenty-five patients were enrolled. Plasma samples were analyzed by next-generation sequencing (NGS). When no mutation was identified, we used two methylated specific biomarkers (digital droplet PCR) to determine the presence or absence of circulating tumor DNA (ctDNA). Patients with conclusive ctDNA results were defined as those with at least one mutation or one methylated biomarker. The kappa coefficient and accuracy were 0.79 (95% CI: 0.67–0.91) and 97.3% (95% CI: 95.2–98.6%) between the BRAF status in plasma and tissue for patients with available paired samples (n = 405), and 0.89 (95% CI: 0.80–0.99) and 98.5% (95% CI: 96.4–99.5%) for those with conclusive ctDNA (n = 323). The absence of liver metastasis was the main factor associated to inconclusive ctDNA results. In patients with liver metastasis, the kappa coefficient was 0.91 (95% CI, 0.81–1.00) and accuracy was 98.6% (95% CI, 96.5–99.6%). We demonstrate satisfying concordance between tissue and plasma BRAF mutation detection, especially in patients with liver metastasis, arguing for plasma ctDNA testing for routine BRAF mutation analysis in these patients.

Sign in / Sign up

Export Citation Format

Share Document