Real-time PCR quantification of plasma DNA in non-small cell lung cancer patients and healthy controls

Abstract

IntroductionFree-circulating DNA is present in minute amounts in plasma of healthy individuals, whereas increased levels are found in a number of malignant pathologies including non-small cell lung cancer (NSCLC). The objective of this research was the evaluation of the plasma DNA quantification capacity to distinguish between healthy subjects and non-small cell lung cancer (NSCLC) patients.Materials and methodsPlasma samples were collected prospectively from 16 healthy volunteers and 30 untreated NSCLC patients (I-IIIA). Subsequently, free-circulating DNA extraction and quantitative real-time PCR analysis were performed.ResultsThe values of plasma DNA concentration ranged from 0.9 up to 7.0 ng/ml in healthy individuals and from 1.5 up to 50 ng/ml in NSCLC patients before treatment. Cancer group showed several-fold higher mean free-circulating DNA concentration than that present in healthy subjects (mean 12.00 vs. 2.65 ng/ml; P < 0.001). A greater variability of plasma DNA concentrations was observed in NSCLC patients than in controls (SD 14.50 vs. 2.02, respectively). The area under the ROC curve was 0.87 (95% CI, 0.744 to 0.954, P < 0.001).ConclusionNon-small cell lung cancer is associated with elevated levels of cell-free DNA in plasma with respect to healthy controls. Real-time PCR method proved its utility in effective free-circulating DNA detection and quantification.

Cell-free circulating tumor DNA in plasma/serum of non-small cell lung cancer

“…From Table 1, we can see that almost all studies employed the QIAamp DNA Mini kit or DNA Blood Mini kit to isolate DNA from plasma or serum. Apart from two studies using fluorescence detection method with PicoGreen dsDNA kit [17,24], most of the studies determined the concentration of ctDNA by qPCR [5,6,[8][9][10][18][19][20][21][22][23]. At present, qPCR is regarded as a standard method for DNA quantification, characterized by high accuracy, reproducibility, and time effectiveness.…”

Circulating cell-free DNA: a potential biomarker in lung cancer

“…No consistent relationship has been found between cfDNA levels and histology, age, smoking status, sex [1,37,40,45-47], number of metastatic sites, performance status [48] or pulmonary comorbidities [37]. Pre-and post-operative cfDNA levels were found to be prognostic for recurrence in some [45,49], but not all, studies [18].…”

Optical biosensors to analyze novel biomarkers in oncology

“…In cancer patients, several tumor-dependent alterations allow the concentration of higher amounts of circulating DNA. Circulating DNA can be found at concentrations of 0.5-0.9 ng/ml in healthy individuals, and 1.5-50 ng/ml in lung cancer patients [90]. Interestingly, tumor-derived DNA has been employed to detect specific genetic [73] and epigenetic [91] alterations in cancer patients.…”

“…Devices should be optimized, by properly engineering, to improve performance (reproducibility, sensitivity, detection limit, analysis times) with respect to currently available techniques. In particular, detection limit should range between 1 and 50 ng/ml, according to circulating tumor DNA concentration [90]. 3.…”

“…Histone modification profiling could be particularly useful to predict response to an emerging class of cancer drugs, targeting histone acetylation and DNA methylation [97,98]. 3) Analysis of easily accessible biological fluids: blood samples [90] but also tumor-specific fluids (urine for genito-urinary neoplasms, saliva for head and neck cancer). This would allow for rapid and reproducible epigenetic profiling.…”