Circulating cell-free DNA in plasma of melanoma patients: Qualitative and quantitative considerations

Abstract

Keywords:Cell-free DNA DNA integrity index qPCR Melanoma DNA integrity in blood is an emerging biomarker in cancer. Here we report a real time PCR approach for the absolute quantification of four amplicons of 67, 180, 306 and 476 bp in cutaneous melanoma. Three different integrity indexes (180/67, 306/67 and 476/67 ratios) were tested for their ability to reflect differences in plasma cell-free DNA (cfDNA) fragmentation in 79 patients affected by cutaneous melanoma and 34 healthy subjects. All the three integrity indexes showed higher values in melanoma patients in comparison with healthy subjects. According to ROC curve analysis, the ratio 180/67 is the most suitable index to be used in cancer patient selection, even if the combination of the 3 indexes gives the best performance in terms of clinical sensitivity. The most represented fragments in plasma of melanoma patients are those comprised between 181 and 307 bp, while in healthy subjects there is a prevalence of shorter fragments (67-180 bp). In conclusion, DNA integrity indexes can be considered suitable parameters for monitoring cfDNA fragmentation in melanoma patients.

CNAPS in Therapy Monitoring

“…Indeed, Wang et al [85] reported a higher cfDNA integrity in cancer patients using 400 bp and 100 bp DNA amplicons. These results were confirmed with different amplicon sizes in breast [86], colorectal [87,88], esophageal [89], prostate [90], head and neck [91], nasopharyngeal cancer [92], melanoma [93] and acute leukemias [94] while cfDNA integrity was not elevated in other studies on prostate [95], lung [96,97] and breast cancer [98]. In addition, a few studies also found some prognostic relevance of cfDNA integrity for bladder [99], prostate [100], breast [86] and nasopharyngeal cancer [92].…”

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

“…All reactions were performed in triplicate. Conditions for the qPCR reaction amplifying APP were 1× HotStar Taq buffer (Qiagen, Courtaboeuf, France) supplemented with 1.6 mM MgCl 2 , 0.2 mM of each dNTP, 0.08 U of HotStar Taq polymerase, 0.2 mM of forward and reverse primers, 0.1 nM of probe (with 5′-FAM and BBQ-3′), and 0.250 ng of ccfDNA in a 25 μL volume [10,11]. PCR cycling conditions included an initial denaturation step for 10 min at 95°C, followed by 55 cycles of 15 s at 95°C, 60 s at 60°C, and 60 s at 72°C, followed by a melting curve at 20 acquisition per degree from 65 to 95°C and a final cooling at 40°C.…”

“…The size distribution of ccfDNA was evaluated using two previously reported methods: two qPCR assays used to amplify the APP gene (67 and 180 bp) [10,11] and two semiquantitative PCR amplifications (400 and 800 bp) of the TP53 gene, which were subsequently analyzed by gel electrophoresis [12]. An integrity index of ccfDNA was calculated using C P180 /C P67 (C P =cycle threshold of the qPCR assay) for APP and I 800 /I 400 (I=intensity of the gel band) for TP53.…”

“…The isolated ccfDNA was quantified using two newly developed quantitative real-time PCR (qPCR) assays targeting repetitive genomic elements (Kpn, DHFRP2) as well as a fluorometric assay. Finally, the integrity index was calculated using either qPCR assays to amplify the APP gene or gel electrophoresis to amplify products of the TP53 gene [10][11][12].…”

Extracellular Nucleic Acids and Cancer

“…100 bp, to work reliably [294]. This is a very important point since it is known that a reduction in amplicon size results in an increase amount of cfDNA [295,296]. Inhibition of PCR by various inhibitors is also an important point for consideration.…”