2.1 Patient cohort: All healthy volunteers and EC patients were recruited at the Regina Elena National Cancer Institute. We collected serum samples from 21 healthy volunteers with no gynecological conditions, and 63 EC patients. Patients included in this study were not previously selected, but randomly chosen and recruited between 2014 and 2017. According with the histologic grade, we analyzed samples from 9 G1, 33 G2 and 21 G3 ECs. Supplementary table 1 depicts clinical-pathological characteristics of patients enrolled in this study.
2.2 Immunohistochemistry: Citrullinated Histone H3 expression was evaluated by immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) tissues using rabbit polyclonal anti-Histone H3 (citrulline R2 + R8 + R17) (Abcam) at the diluition of 1:50 (citrate based pH 6.0 epitope retrieval solution). Immunoreactions were revealed by a streptavidin-biotin enhanced immunoperoxidase technique in an automated autostainer (Bond III, Leica Biosystem, Milan, Italy), according to the manufacturer’s instructions. Immunoreactivity was evaluated by two investigators (LR and MC) and discordant cases were subsequently discussed and agreed upon.
2.3 Immunofluorescence: Immunofluorescence staining to observe the expression of NETosis markers was performed on formalin-fixed paraffin-embedded (FFPE) tissues utilizing the following primary antibodies: rabbit polyclonal anti-Elane (Atlas) at the diluition of 1:50 (citrate based pH 9.0 epitope retrieval solution); chicken polyclonal anti-Histone H2B (Abcam) at the diluition of 1:500 (citrate based pH 9.0 epitope retrieval solution) rabbit polyclonal anti-Histone H3 (citrulline R2 + R8 + R17) (Abcam) at the diluition of 1:50 (citrate based pH 6.0 epitope retrieval solution). The following were used as secondary antibodies: CyTM2 Affinipure Donkey Anti-rabbit (Jackson Immuno Research) at the diluition of 1:400, CYTM3 Affinipure Donkey Anti-chicken (Jackson Immuno Research) at the diluition of 1:400. The colorations were read and interpreted by using a fluorescence confocal microscope. Immunoreactivity was evaluated by two investigators (LR and AG) and discordant cases were subsequently discussed and agreed upon. Thermo Scientific DAPI Nuclear Counterstains was used for nuclear staining.
2.4 Blood processing: Venous blood of cancer patients were obtained before surgery and before the beginning of any treatment. The blood tests were obtained within 24 hours for all patients as routine clinical practice before surgery. NLR, MLR and PLR were defined as the absolute neutrophil count in peripheral blood divided by the absolute lymphocyte count, the absolute count of monocyte divided by the absolute lymphocyte count, and the absolute platelet count in peripheral blood divided by the absolute lymphocyte count, respectively. For serum processing, blood samples were collected in Vacutainer tubes without anticoagulant and processed within 1–4 h. After collection the blood was allowed to clot at room temperature. The blood serum was separated by centrifugation at 1000–2000 x g for 10 min in a refrigerated centrifuge and stored at − 80°C.
2.5 Serum deactivation: 20 µL of each serum sample were mixed with 20 µL of a preparation buffer (2.5% of tween 20, 50 mmol/L Tris-HCl, and 1 mmol/L EDTA). This mixture was digested with proteinase K (20 µg) solution for 50 min (Promega) at 56°C, followed by 5 min of heat deactivation and insolubilization for 10 min at 95°C. After subsequent centrifugation of 10,000×g for 5 min, supernatant was used as a template for each quantitative real-time polymerase chain reaction (qRT-PCR) using SYBR Green Power Up Master Mix (Thermo Scientific) followed by evaluation of the average of CT values from triplicate reactions from Real Time PCR software.
2.6 DNA extraction: Isolation from 1 ml of blood serum was performed with commercial kit for blood DNA according to manufacturer’s specifications from Qiamp Circulating Nucleid Acid kit (QIAGEN). Concentration and purity of the extracted DNA were evaluated by performing a Qubit Fluorometric Quantification.
2.7 Bioanalyzer analysis: DNA was isolated from 1 ml of blood serum with commercial kit for blood DNA according to isolation manufacturer’s specifications from Qiamp Circulating Nucleid Acid kit (QIAGEN). Microfluidic electrophoresis using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips (Agilent technologies Inc., Palo Alto, CA, USA) was performed to assess DNA fragment length for a size range between 35 and 10.380 base pairs (bp) based on manufacturer’s recommended protocol. Bioanalyzer 2100 Expert Software was used for the analysis of electrophoretic runs.
2.8 Measurement of cfDNA levels: SYBR Gold Nucleic Acid Gel Stain (Invitrogen) was diluted first at 1:1000 in dimethyl sulphoxide (DMSO) and then at 1:8 in phosphate-buffered saline (PBS). Ten microliters of DNA solutions or sera were applied to a black 96-well plates. Forty microliters of diluted SYBR Gold were added to each well (final dilution 1:10,000) and fluorescence measured with a 96well fluorometer at an emission wavelength of 535 nm and an excitation wavelength of 485 nm. Serum samples were diluted in PBS fivefold (20%). Assay was performed in triplicate. Standards were prepared with commercial Salmon sperm DNA.
2.9 qPCR: One microliter of deactivated serum was used and both the mtDNA and nDNA content were measured by qPCR using SYBR Green Power Up Master Mix (Applied Biosystems, CA, USA) followed by evaluation of the average CT values from triplicate reactions from Real Time PCR software. Primers were designed and used for relative quantification for mtDNA to nDNA content. Two primer pairs were used for the amplification of two mitochondrial genes: MT-ND1 and mtDNA 16S. One primer set was used for the amplification of the single-copy nuclear gene 36B4. Primers sequences will be: forward ND1: 5’-CCCTAAAACCCGCCACATCT-3’ and reverse ND1: 5’-GAGCGATGGTGAGAGCTAAGGT-3’; forward mtDNA 16S 5’-CAGCCGCTATTAAAGGTTCG-3’ and reverse: mtDNA 16S 5’-CCTGGATTACTCCGGTCTGA-3’; forward 36B4 5’-CAGCAAGTGGGAAGGTGTAATCC-3’ and reverse: 36B4 5’-CCCATTCTATCATCAACGGGTACAA-3’. To determine the mtDNA content relative to nDNA, the following equations were used: ΔCT = (nDNA CT – mtDNA CT). Relative mitochondrial DNA content = 2 × 2ΔCT.
2.10 ELISA assay: To quantify CitH3 in the blood, we employed the citrullinated Histone H3 (Clone 11D3) ELISA Kit (Cayman) following the manufacturer’s recommended protocol.
2.11 Statistics: The examined variables were not normally distributed, as verified by the Shapiro–Wilk test, thus the most suitable non-parametric test was applied to perform comparisons between groups. Receiver operating characteristic (ROC) curves and the relative area under the curve (AUC) were calculated for all continuous variables of interest. Youden’s index was used to maximize the difference between sensitivity and specificity in order to individuate the optimal cut-off for each parameter. Multiple correspondance analysis (MCA), a descriptive/exploratory technique designed to analyze simple two-way and multi-way table, was used to obtain an overview of the potential association among parameters. P-values <0.05 were considered statistically significant. The SPSS (version 21.0) was used for all statistical analyses.