Synuclein-γ (SNCG) expression in ovarian cancer is associated with high-risk clinicopathologic disease

After obtaining IRB approval, the Pathology archive at Roswell Park Cancer Institute, Buffalo, New York was searched for ovarian, fallopian tube, and primary peritoneal cancer cases from 1995 to 2007. A chart review was conducted with extraction of clinical information, including patient’s age at the time of diagnosis, the surgical stage, postoperative treatment. All patients underwent a primary surgical staging surgery, including total abdominal hysterectomy with bilateral salpingo-oophorectomy, with or without pelvic and para-aortic lymph nodes dissection and pelvic washings, depending on tumor stage. Patients were treated according to National Comprehensive Cancer Network guidelines (https://​www.​nccn.​org). Patient’s general information and tumor features are summarized in Table 1.

Tumor grade was assessed using the International Federation of Gynecology and Obstetrics (FIGO) system and tumor stage was assigned based on the 2014 FIGO surgical staging guidelines. All slides were examined by a gynecologic pathologist for confirmation of tumor morphology and tumor grade. The viable tumor tissues and control tissues (fallopian tube) from each case were circled by pathologists. 0.6 mm tissue cores were punched and arrayed into high-density TMA receipting blocks.

Tissues were prepared for analysis, as previously described by Mhawech-Fauceglia et al. [19]. Briefly, high-density TMA blocks were sectioned in 4 μm thickness followed by deparaffinization with xylene, then washed with ethanol. Sections were cooled for 20 min and incubated for 10 min with 3 % H2O2 to quench endogenous peroxidase activity. Blocking was performed using serum-free protein block, Dakocytomation (Carpenteria, CA) for 30 min. Antigen retrieval was done using a citrate-based buffer (pH 6; Bond Epitope Retrieval Solution, Leica Biosystems) and sections were incubated with the SNCG antibody (Abcam) for 1 h at room temperature on the Dako Autostainer Plus. The diaminobenzidine complex was used as a chromogen. Immunostained slides were blindly reviewed and scored by two gynecologic pathologists. Immunostain for SNCG was present in both the nucleus and cytoplasm. Immunoreactivity was semiquantitatively scored in immunointensity of 0 (negative), 1 + (weak), 2+ (moderate) and 3+ (strong) and immunopercentage of <10 %, 10–50 % and >50 %. For the sake of statistical analysis, tumors were grouped as positive (SNCG+) or negative (SNCG-). Tumors were considered SNCG+ if ≥10 % of the tumor epithelial cells were immunoreactive with immunointensity of ≥2. Examples of positive and negative cases are illustrated in Fig. 1a-d.

RNA from benign fallopian tube epithelium and ovarian cancer tissues was reverse transcribed. First-strand cDNA synthesis was performed using 700 ng of RNA and M-MLV reverse transcriptase (Life Technologies). Real time PCR was performed using Taqman reagent in a QuantStudio 5 system and primers to SNCG and the housekeeping gene TBP (Applied Biosystems/Life Technologies). Fold-change values were calculated using TBP as the housekeeping gene.

To test the association between the biomarker and the clinical parameters, Wilcoxon rank-sum test was used to compare the median ages and Fisher’s exact test was used to compare frequencies and percentages for categorical variables. Progression-free survival (PFS), was defined as observed length of time from date of diagnosis to recurrence or censoring at date of last contact. Overall survival (OS) was defined as observed length of life from date of diagnosis to death or censoring at date of last contact. For survival analysis, Kaplan-Meier curves were used to estimate PFS, and OS curves. Curves were compared for tumors with and without SNCG expression using the log-rank test. Univariate cox proportional hazards models were used to estimate hazard ratios for SNCG status for both the OS and PFS outcomes. Multivariate Cox regression was then conducted with SNCG expression as the main predictor and adjustment for clinical factors that were associated with SNCG expression (CA125 level, ascites, debulking status, FIGO grade, tumor stage and histologic subtype). Since several of the clinical covariates were highly associated with each other, sensitivity of the hazard ratio estimates for SNCG was examined after adjustment for each clinical variable in separate models. All reported p values are two sided with P < 0.05 significance. Statistical analyses was performed using SAS v 9.4. Unpaired t-test was used to compare SNCG mRNA levels in benign fallopian tube and ovarian cancer tissues using Graphpad Prism version 6.0 (Graphpad Software, La Jolla, CA, USA).