Cell-free circulating nucleic acids in cancer and pregnancy

Pamela Pinzani, Francesca Salvianti, Serena Vinci, Roberta Cascella, Claudio Santucci, Sara Zaccara, Mario Pazzagli, Claudio Orlando, Department of Clinical Physiopathology, University of Florence, Italy

Circulating nucleic acids (CNA) are present in the blood of humans and other vertebrates. During the last ten years researchers actively studied cell-free nucleic acids present in plasma or serum with great expectations as potential biomarkers for cancer and other pathologic conditions. Our interest was focused on cutaneous melanoma and, among pregnancy pathologies, preeclampsia (PE) and fetal growth restriction (FGR). In the compartment of plasma CNA, it is possible to evaluate either the total DNA plasma concentration or alternatively the concentration of particular DNA sequences characterized by specific genetic or epigenetic changes. In the proposed clinical areas, we tried to optimize a multi-parametric approach for the simultaneous evaluation of abnormal increase of genetic and epigenetic variants to increase our diagnostic potency. In particular, BRAF V600E is a mutation occurring with high frequency in melanoma and detectable in CNA of the same patients. Methylation of RASSF1A promoter is one of the epigenetic changes identified in many cancer types including melanoma. Moreover, the RASSF1A promoter is methylated in placenta and unmethylated in maternal blood cells, thus representing a DNA marker independent from fetal gender to be used as a tool to monitor pregnancy outcome. We developed real time methods for the quantification and characterization of cell-free plasma DNA. In particular we evaluated the following parameters in human plasma: i. total DNA concentration; ii. DNA integrity index; iii. density of RASSF1A methylation; iv. abundance of BRAF mutated alleles. All assays were based on the initial real time detection of the amiloyd precursor protein gene (APP) as a single copy gene. Quantitative evaluation of RASSF1A methylated sequences was based on methylation-sensitiverestriction enzyme digestion to selectively remove the unmethylated derived RASSF1A sequences. These method avoids the bisulfite treatment, which is a highly variable, cumbersome and labor-intensive procedure. For BRAF V600E we developed an allele specific Taqman–based real-time PCR assay, that allows the sensitive, accurate and reliable measurement of the variant DNA in plasma providing a non-invasive tool to support tumor diagnosis. As regard to pregnancy, the results showed, a statistically significant increase of both fetal and total DNA in overt PE and FGR, with unvaried proportion between the two, confirming that these pathologies involve both mother and fetus. As regard to cancer, the analyzed parameters showed a significant increase in patients affected by melanoma compared to controls. Our data suggest the simultaneous determination of several circulating biomarkers which significantly increases the diagnostic sensitivity in cutaneous melanoma. Consequently, we propose a diagnostic approach based on the sequential determination of APP, BRAF, RASSF1A.

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