New diagnostic tools can be useful and give clinical benefits, including diagnosis, prognosis, treatment and monitoring of the disease. A new non-invasive method is the study of liquid biopsies, performed in fluids containing cell-free circulating DNA (cfDNA). Analyses with digital PCR technique (dPCR) allows to establish the levels of cfDNA as well as the absolute quantification of mutant alleles with accuracy. This system scatters the sample among twenty thousand wells (microfluids on the chip), where amplification reactions occur independently and are recorded by a reader.
Peripheral blood samples were obtained from breast cancer patients and healthy controls. From each sample, the cfDNAs were extracted from plasma using the MagMAX™ Cell Free DNA Isolation Kit and dPCR was done for quantification of samples. We evaluated two genes, PUM1 and RNaseP. For amplification of fragments, master mix 1X (Applied Biosystems) and PCR detection assays of both genes were combined with 1.5 μl of plasma cfDNA, dispersed in the chips and placed in a ProFlex ™ thermocycler (Applied Biosystems) following the program pre-established by the manufacturer, with additional five cycles. Finally, quantification data were obtained with QuantStudio® 3D AnalysisSuite™ Cloud Software. Comparison of DNA concentration in copies per microliter and other statistical calculations were performed with InfoStat 2015.
Significant differences were found in the values of cfDNA between patients and controls for PUM1 (p = 0.0001) and RNase P (p = 0.0003). These results allowed to establish cut-off points between groups at 78,995 and 51,154 copies/uL, respectively. These values can be considered in the classification of groups for further analysis of others samples. Statistical support for the use of markers in diagnosis was also evaluated using the ROC curve that favors the PUM1 marker, with a sensitivity of 75% and a specificity of 95.2%.
Based on the significant differences found between breast cancer patients and controls, cell free DNA is a good biomarker that can be used in the diagnostic of breast cancer. On the other hand, digital PCR has been established as a good tool to check cfDNA levels from plasma of breast cancer patients.
Clinical trial identification
Legal entity responsible for the study
Programa Nacional para la competitividad y Productividad (Innovate Perú)-No.138-PNICP-PIAP-2015, Universidad de San Martin de Porres, Oncosalud - AUNA
All authors have declared no conflicts of interest.