Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma

BACKGROUND: Diffuse anaplastic Wilms tumor (DAWT) is a rare, high-risk subtype that is often missed on diagnostic needle biopsy. Somatic mutations in TP53 are associated with the development of anaplasia and with poorer survival, particularly in advanced-stage disease. Early identification of DAWT harboring TP53 abnormalities could improve risk stratification of initial therapy and monitoring for recurrence. METHODS: Droplet digital polymerase chain reaction (ddPCR) was used to evaluate 21 samples from 4 patients with DAWT. For each patient, we assessed TP53 status in frozen tumor, matched germline DNA, and circulating tumor DNA (ctDNA) from plasma, serum, and urine collected throughout treatment. RESULTS: Mutant TP53 was detectable in ctDNA from plasma and serum in all patients. We did not detect variant TP53 in the same volume (200 μl) of urine. One patient displayed heterogeneity of TP53 in the tumor despite both histological sections displaying anaplasia. Concentration of ctDNA from plasma/serum taken prenephrectomy varied significantly between patients, ranging from 0.44 (0.05-0.90) to 125.25 (109.75-140.25) copies/μl. We observed variation in ctDNA throughout treatment, and in all but one patient, ctDNA levels fell significantly following nephrectomy. CONCLUSION: We demonstrate for the first time that ddPCR is an effective method for detection of mutant TP53 in ctDNA from children with DAWT even when there is intratumoral somatic heterogeneity. This should be further explored in a larger cohort of patients, as early detection of circulating variant TP53 may have significant clinical impact on future risk stratification and surveillance.

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“…Results are consistent with a previous study and demonstrate comparable ctDNA yields (Supplementary Figure 2) [17].…”

Section: Resultssupporting

confidence: 92%

Somatic TP53 Mutations Are Detectable in Circulating Tumor DNA from Children with Anaplastic Wilms Tumors

Treger

Chagtai

Butcher

et al. 2018

Translational Oncology

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“…We chose these specific urine kits because the Norgen blood plasma cfDNA isolation kits showed the highest yields in a benchmark [7] and the PerkinElmer plasma cfDNA kit yields in our lab were even higher (data not shown).…”

mentioning

confidence: 99%

Quantifying Cell Free DNA in Urine: Comparison Between Commercial kits, Impact of Gender and Inter-Individual Variation

et al. 2018

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DNA can enter the blood circulation from living cells by extracellular vesicles or at cell death, and pass into urine through the kidney barrier. Urine can be collected non-invasively, making it an interesting source of cell-free DNA (cfDNA) for research studies and ultimately for clinical diagnostics. However, there is currently a lack of data on the quantity and variability of cfDNA in urine. Here, we benchmark two commercial urine cfDNA isolation kits with respect to the quantity of DNA, the labor time, and cost. The results show distinctive differences between each kit. Furthermore, the cfDNA amount from the same probands varied strongly from day to day and may be higher in female samples than in male samples (p = 0.003).

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“…There are various methods available for circulating cell-free DNA quantification, including real-time PCR [567891011121314151618212223242526], fluorescence spectrophotometry [4172023242528], ultraviolet spectrophotometry [22], capillary electrophoresis [2930], mass spectrometry [3031], surface plasmon resonance [3233], and electrochemical luminescence [34]. However, because DNA extraction inevitably leads to DNA loss, all of these methods have thus far been used for scientific research only and have not been applied in clinical laboratories.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, several protocols have been developed for DNA extraction. However, the inevitable and various loss of circulating cell-free DNA during extraction may have a significant impact on the outcome [212223242526]. …”

Section: Introductionmentioning

confidence: 99%

Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA

et al. 2017

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BackgroundCirculating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application.MethodsWe designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients.ResultsCompared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R2=0.916, P<0.0001).ConclusionsThis duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.

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Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma

Cited by 92 publications

References 21 publications

Somatic TP53 Mutations Are Detectable in Circulating Tumor DNA from Children with Anaplastic Wilms Tumors

Somatic TP53 Mutations Are Detectable in Circulating Tumor DNA from Children with Anaplastic Wilms Tumors

Quantifying Cell Free DNA in Urine: Comparison Between Commercial kits, Impact of Gender and Inter-Individual Variation

Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA

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