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Cell-free DNA (cfDNA) quantity and quality in plasma has been investigated as a non-invasive biomarker in cancer. Previous studies have demonstrated increased cfDNA amount and length in different types of cancer with respect to healthy controls. The present study aims to test the hypothesis that the presence of longer DNA strands circulating in plasma can be considered a biomarker for tumor presence in thyroid cancer. We adopted a quantitative real-time PCR (qPCR) approach based on the quantification of two amplicons of different length (67 and 180 bp respectively) to evaluate the integrity index 180/67. Cell-free DNA quantity and integrity were higher in patients affected by nodular thyroid diseases than in healthy controls. Importantly, cfDNA integrity index was higher in patients with cytological diagnosis of thyroid carcinoma (Thy4/Thy5) than in subjects with benign nodules (Thy2). Therefore, cfDNA integrity index 180/67 is a suitable parameter for monitoring cfDNA fragmentation in thyroid cancer patients and a promising circulating biomarker in the diagnosis of thyroid nodules., Competing Interests: The authors declare no conflict of interest.

This papers deals with the preanalytical and analytical phase of cell-free DNA analysis, highlighting some criticism on sample collection and extraction. We describe a method to accurately quantify total cfDNA in plasma and our particular approach to the measurement of tumor deriving cfDNA.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: BRAF is the most frequently mutated oncogene in melanoma with BRAF(V600E) mutation accounting for 92% of all BRAF variants. As this event occurs early in melanoma progression, the quantification of BRAF-mutated alleles in plasma may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy., Methods: We propose an assay based on the use of a locked nucleic acid probe and an allele specific primer to measure plasma-circulating BRAF(V600E) concentration in patients affected by cutaneous melanoma (n=55) and non-melanoma skin cancers (n=13) as well as 18 healthy subjects. The assay is highly sensitive and accurate in detecting down to 0.3% of mutated allele in plasma., Results: A significant difference between the control group and invasive melanomas (p<0.01) was evidenced in BRAF(V600E) concentration, either as relative percentage or absolute values. ROC curve indicated that BRAF(V600E) absolute concentration has the maximal diagnostic relevance with 97% sensitivity and 83% specificity. Comparison of the results obtained in plasma with those found in the corresponding tissues indicated an 80% concordance., Conclusions: The allele specific Taqman-based real-time PCR assay allows the sensitive, accurate and reliable measurement of BRAF(V600E) mutated DNA in plasma., (Copyright 2010 Elsevier B.V. All rights reserved.)