Quantification of circulating cell-free DNA was believed to have the potential for the diagnosis of ovarian cancer. But in spite of a few encouraging findings, generally the method is diagnostically imperfect with regard to a number of important parameters [7]. Perhaps, a ratio of long (> 180 bp) to short cfDNA fragments would be a more reliable biomarker of cancer. Longer DNA fragments are assumed to originate from tumor necrosis, changing the ratio of various cfDNA molecules [8].

Using quantitative PCR to amplify variously sized DNA fragments, a few researchers demonstrated that longer fragments make a considerable contribution to the total amount of cfDNA molecules in cancer patients [9, 10]. It was shown that in patients responsive to treatment the proportion of longer fragments decreases during therapy, whereas stably high levels of cfDNA are indicative of poor treatment outcome [9]. However, when amplicon length and tumor-specific somatic mutations were analyzed in another study, it was discovered that tumor cfDNA is shorter [11]. An assumption could be made that a degree of tumor cfDNA fragmentation depends on cancer type and predominant type of cell death.

In this work we describe a multiplex quantitative PCR assay that we designed, validated and used to study the degree of fragmentation of tumor cfDNA in patients with ovarian carcinoma.

METHODS

Our study was conducted in the patients of Blokhin Russian Cancer Research Center. The main group comprised 14 patients with stages III and IV ovarian serous adenocarcinoma, aged 64.3 ± 5.6 years (mean age was 64 years). The control group included 16 seemingly healthy women without cancer aged 60.8 ± 8.2 years (mean age was 60.5 years). The study was approved by the ethics committee of the Center (Protocol No. 3 dated May 16, 2016). Participants’ data were anonymised.

DNA was isolated from the blood plasma of the participants. Blood samples of healthy women were collected during routine medical check-ups. Blood was collected into 6 ml EDTA-containing vacutainer tubes. The samples were then centrifuged at 2,000 g for 10 min; plasma was transferred to new tubes and then centrifuged at 2,000 g for 10 min. Centrifuged plasma aliquots were transferred to new tubes and stored at –70 °С. The aliquots were thawed immediately before DNA isolation performed using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Netherlands).

Designing primers and fluorescent probes for Alu and hLINE-1

Oligo primers and a fluorescent probe for Alu amplification were selected based on the alignment of consensus sequences of the most common Alu families, namely Alu Sx, Alu Sp and Alu Y [12] (fig. 1).

For hLINE-1amplification primer sequences were borrowed from [6]. The design of a fluorescent probe was our own, created in UGENE v1.14 [13] and OligoAnalyzer v1.0.3 (Teemu Kuulasmaa, Finland). To ensure multiplexing, the fluorescent probes for Alu and hLINE-1 contained different dyes. Primer and probe sequences for Alu and hLINE-1 amplification, as well as amplicon lengths, are shown in tab. 1.

Detection of cfDNA in blood plasma by real-time PCR

Real-time PCR was performed in the CFX96 Thermal Cycler (Bio-Rad, USA). The volume of the reaction mixture was 25 μL and contained 65 mM Tris-HCl (pH 8.9), 24 mM ammonium sulfate, 3.5 mM magnesium chloride, 0.05 % Tween-20, 0.2 mM dNTP, 0.3 Mm of each primer, 0.1 mM of each fluorescent probe (tab. 1) and 1 unit of thermally stable modified Taq polymerase (Interlabservice, Russia). The PCR protocol was as follows: 15-min initial denaturation at 96 °C followed by 45 cycles of 10 sec at 96 °C and 30 sec at 58 °C. The optimal annealing temperature was determined empirically.