Detection of circulating tumor DNA (ctDNA) by digital droplet polymerase chain reaction (dd-PCR) in liquid biopsies

2019 ◽  
pp. 1-15 ◽  
Author(s):  
Sara Valpione ◽  
Luca Campana
Keyword(s):  
Polymerase Chain Reaction ◽  
Circulating Tumor Dna ◽  
Chain Reaction ◽  
Liquid Biopsies ◽  
Polymerase Chain ◽  
Tumor Dna

Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction

2020 ◽  
Vol 58 (4) ◽  
pp. 527-532 ◽  
Author(s):  
Jee-Soo Lee ◽  
Miyoung Kim ◽  
Moon-Woo Seong ◽  
Han-Sung Kim ◽  
Young Kyung Lee ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Circulating Tumor Dna ◽  
Analytical Sensitivity ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Specimen Type ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Ctdna Analysis ◽  
Tumor Dna

AbstractBackgroundChoosing the specimen type is the first step of the pre-analytical process. Previous reports suggested plasma as the optimal specimen for circulating tumor DNA (ctDNA) analysis. However, head-to-head comparisons between plasma and serum using platforms with high analytical sensitivity, such as droplet digital polymerase chain reaction (ddPCR), are limited, and several recent studies have supported the clinical utility of serum-derived ctDNA. This study aimed to compare the DNA profiles isolated from plasma and serum, characterize the effects of the differences between specimens on ctDNA measurement, and determine the major contributors to these differences.MethodsWe isolated cell-free DNA (cfDNA) from 119 matched plasma/serum samples from cancer patients and analyzed the cfDNA profiles by DNA fragment sizing. We then assessed KRAS mutations in ctDNA from matched plasma/serum using ddPCR.ResultsThe amount of large DNA fragments was increased in serum, whereas that of cfDNA fragments (<800 bp) was similar in both specimens. ctDNA was less frequently detected in serum, and the KRAS-mutated fraction in serum was significantly lower than that in plasma. The differences in ctDNA fractions between the two specimen types correlated well with the amount of large DNA fragments and white blood cell and neutrophil counts.ConclusionsOur results provided detailed insights into the differences between plasma and serum using DNA fragment sizing and ddPCR, potentially contributing to ctDNA analysis standardization. Our study also suggested that using plasma minimizes the dilution of tumor-derived DNA and optimizes the sensitivity of ctDNA analysis. So, plasma should be the preferred specimen type.

scholarly journals Analytic and Clinical Validation of an Ultrasensitive, Quantitative Polymerase Chain Reaction Assay for EGFR Mutation Analysis With Circulating Tumor DNA

2017 ◽  
Vol 141 (7) ◽  
pp. 978-984 ◽  
Author(s):  
Xiaowei Wang ◽  
Yunhua Gao ◽  
Bei Wang ◽  
Zhenrong Zhang ◽  
Chaoyang Liang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Mutation Analysis ◽  
Polymerase Chain Reaction Assay ◽  
Egfr Mutation ◽  
Quantitative Polymerase Chain Reaction ◽  
Circulating Tumor Dna ◽  
Egfr Mutations ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tumor Dna

Context.— The mutation analysis of epidermal growth factor receptor (EGFR) has become a common test to guide therapeutic decision making for lung cancer. Molecular testing with circulating tumor DNA in plasma allows diagnosis of mutations when tumor tissue is not available as well as monitoring treatment response with repeat biopsies. Objectives.— To develop a timely and cost-effective assay that can accurately detect EGFR mutations in circulating tumor DNA and to evaluate the analytic and clinical performance of the assay. Design.— Analytic assessment was conducted with a set of reference materials carrying classic EGFR mutations. A recently developed Poisson distribution–based approach was employed to understand the assay sensitivity. Clinical evaluation was performed with 224 pairs of plasma and matched tissues from patients with stage I to IV disease. EGFR mutation rates of 390 consecutive plasma samples processed in the central service laboratory were compared with previously reported prevalence in an Asian population. Results.— Our results suggested that limit of detection for the EGFR quantitative polymerase chain reaction assay was 10 mutation copies, and the lowest detectable copy numbers could be extended to a single-digit level. The clinical sensitivity was 53.3% for all stages combined and 81.4% for late stages, with a high specificity of 100%. Clinical observations showed an overall positive finding rate of 32.5% and 41.4% for stage IV disease, which is consistent with previously reported EGFR mutation prevalence in an Asian population. Conclusions.— Our results supported the clinical utility of the ultrasensitive, quantitative polymerase chain reaction assay for EGFR mutation analysis with circulating tumor DNA.

scholarly journals A rare case of melanoma of the rectum hemorrhoid nodule

2021 ◽  
Vol 55 (1) ◽  
pp. 40-44
Author(s):  
Vladimir Selaković ◽  
Milan Ranisavljević ◽  
Tijana Vasiljević ◽  
Bratislav Stoiljković ◽  
Biljana Kukić
Keyword(s):  
Polymerase Chain Reaction ◽  
Case Report ◽  
Rare Case ◽  
Polymerase Chain Reaction Analysis ◽  
Symptomatic Therapy ◽  
Anorectal Melanoma ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Rare Malignancy ◽  
Tumor Dna

Anorectal melanoma (AM) is an aggressive and very rare malignancy. The aim of this case report is to present a case of anorectal melanoma that occured in hemorrhoidal node after a colorectal surgery. A patient aged 69 years was treated at the Oncology Institute of Vojvodina for histopathological (HP) verified melanoma of the anorectum (S100, HMB45 and Melan A positive) localized in the hemorrhoidal node. Wide local excision was made. Three months after the operation secondary deposits ocurred at the site of the colorectal anasthomosis, stomach, lung and brain. Polymerase chain reaction analysis of tumor DNA found no mutation in the BRAF V600 gene. Five months after the operation the patient is still alive and on supportive and symptomatic therapy. Despite its rarity, AM should always be considered in unusual anorectal lesions first at all in hemorrhoid nodules.

scholarly journals Heterogeneity and Coexistence of T790M and T790 Wild-Type Resistant Subclones Drive Mixed Response to Third-Generation Epidermal Growth Factor Receptor Inhibitors in Lung Cancer

2018 ◽  
pp. 1-15 ◽  
Author(s):  
Zofia Piotrowska ◽  
Mehlika Hazar-Rethinam ◽  
Coleen Rizzo ◽  
Brandon Nadres ◽  
Emily E. Van Seventer ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Growth Factor Receptor ◽  
Third Generation ◽  
Chain Reaction ◽  
Liquid Biopsies ◽  
Epidermal Growth ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Purpose Third-generation epidermal growth factor receptor (EGFR) inhibitors like nazartinib are active against EGFR mutation–positive lung cancers with T790M-mediated acquired resistance to initial anti-EGFR treatment, but some patients have mixed responses. Methods Multiple serial tumor and liquid biopsies were obtained from two patients before, during, and after treatment with nazartinib. Next-generation sequencing and droplet digital polymerase chain reaction were performed to assess heterogeneity and clonal dynamics. Results We observed the simultaneous emergence of T790M-dependent and -independent clones in both patients. Serial plasma droplet digital polymerase chain reaction illustrated shifts in relative clonal abundance in response to various systemic therapies, confirming a molecular basis for the clinical mixed radiographic responses observed. Conclusion Heterogeneous responses to treatment targeting a solitary resistance mechanism can be explained by coexistent tumor subclones harboring distinct genetic signatures. Serial liquid biopsies offer an opportunity to monitor clonal dynamics and the emergence of resistance and may represent a useful tool to guide therapeutic strategies.

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