Patients with advanced ovarian cancer who were admitted to the Department of Gynaecology, Cancer Hospital, Chinese Academy of Medical Sciences between March 2018 and September 2018 were included in the study. Serum samples were collected before any antitumor treatment. The inclusion criteria were as follows: patients with stage III or IV epithelial ovarian cancer confirmed by histopathology; patients who could not achieve optimal cytoreductive surgery according to the comprehensive judgement of two gynaecological oncologists using imaging combined with clinical indicators; received neoadjuvant chemotherapy, with a regimen of paclitaxel (175 mg/m2, iv Q3w) combined with carboplatin (AUC = 5, iv Q3w), 2 to 4 courses of treatment, inclusive; PFS less than 6 months (chemoresistant group) or longer than 12 months (chemosensitive group). The exclusion criteria were as follows: mucinous or low-grade serous carcinoma; undergoing primary debulking surgery; PFS 6–12 months. This study was approved by the Ethics Committee of CAMS (approval number 19–218/1796). All subjects signed informed consent forms.
Blood Sample Collection And Specimen Processing
Before the initial treatment, peripheral venous blood (3.5 ml) was collected using biochemical blood collection tubes (BD, USA). Within 2 h of collection, the serum was separated by centrifugation at 4 000 r/min for 10 min. The upper serum was aspirated and transferred to an EP tube and stored at -80°C.
Detection Of Serum Polypeptide Peaks By Maldi-tof-ms
Seven microlitres of weak cation-exchange magnetic bead suspension (Beijing Yixin Bochuang Biotechnology Co., Ltd.), 10 µL of serum and 95 µL of magnetic bead binding buffer were pipetted into a 200 µL sample tube, mixed repeatedly, and allowed to stand for 5 min at room temperature. The sample tube was then placed on the magnetic bead separator for 1 min to allow the magnetic beads to adhere to the wall. The clarified liquid was then aspirated. The sample tube was removed from the magnetic bead separator, and 100 µL of solid-phase extraction polyethylene glycol solution was added, mixed repeatedly, and allowed to stand for 2 min at room temperature. The sample tube was placed on the magnetic bead separator again for 1 min to allow the magnetic beads to adhere to the wall. The clarified liquid was then aspirated. The operation was repeated 2 more times. The sample tube was then removed from the magnetic bead separator, 10 µL of magnetic solid-phase extraction solution was added, and the solution was mixed repeatedly more than 10 times, after which it was placed at room temperature for 5 min. The sample tube was then placed on the magnetic bead separator for 1 min to allow the magnetic beads to adhere to the wall. After the liquid was clear, the liquid was transferred into a clean sample tube and detected with MALDI-TOF-MS (Beijing Yixin Bochuang Biotechnology Co., Ltd.) for mass spectrometry analysis. The primary mass spectrometry collision energy was 65 eV, the scanning range was from 1 000 m/z to 10 000 m/z, and the optimal pulsed ion extraction value was 5 000.00 Da. Each sample point accumulated 500 spectra and was superimposed to analyse the mass spectrum of the tested sample.
Data Processing And Maldi-tof-ms Analysis
The R language MALDIquant package was used to preprocess the original spectra (Fig. 1). The spectral peaks were then sorted by the linear discriminant analysis algorithm in the R language sda program package, and the differential peptide peaks (score > 5) were selected. Sample cluster analysis and principal component analysis were performed on the differential polypeptide peaks between the chemoresistant and chemosensitive groups. The receiver operating characteristic (ROC) curve of each differential polypeptide peak was drawn, and the area under the curve was selected (area under curve, AUC) ≥ 0.80. According to the Youden index, the optimal cut-off value of the differential polypeptide peaks was determined, and the differential polypeptide peaks with a sensitivity > 70% and specificity > 90% for predicting chemotherapy efficacy were further screened.
Lc‒ms/ms Detection And Identification Of The Components Of The Differential Peptide Peaks
A 20 µL sample was separated on a nanolitre flow high-performance liquid chromatography system (EASY-nLC 1000, Thermo Fisher Scientific, USA). Liquid phase A was 0.1% formic acid acetonitrile aqueous solution, phase B was 0.1% formic acid acetonitrile aqueous solution, the flow rate was 200 nl/min, the chromatographic spray needle was a GlassTip glass spray needle, and the quartz electrospray needle was an FS360-20-10-N-20-C12. The samples were separated by liquid chromatography and analysed by a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA). The ion source voltage was 3.5 kV, the analysis time was 120 min, the detection method was positive ion, and the precursor ion scanning range was 300–2000 m/z. Twenty fragment spectra were collected after each full scan; the resolution of the first-stage mass analyser was 70,000 and that of the second-stage mass analyser was 17,500. The parent ion mass tolerance was 10 ppm, and the fragment ion mass tolerance was 0.02 Da. The fixed modification was carbamidomethyl, and the variable modification was oxidation. Peak 8.5 was used to analyse the obtained spectra, which were imported into the SEQUEST retrieval program and finally queried in the Bioworks database to retrieve the corresponding possible proteins.
The R language pheatmap program package was used to draw the heatmap of differential peptide peaks in the chemoresistant and chemosensitive groups. The R language ggpubr program package was used to draw box plots of differential polypeptide expression, and PAST 3 software was used to draw the main component analysis plot. The Wilcoxon nonparametric test and ROC curve analysis were performed using SPSS 24.0. The inspection level was α = 0.05.