Patients and specimens
The formalin-fixed paraffin-embedded (FFPE) tissues used for study were collected from 116 patients with ovarian cancer who underwent curative surgery from 2010 to 2018. The patients received adjuvant chemotherapy according to their tumor stage. The Disease-free survival (DFS) was defined as the length of time for which patient survived after curative resection without any positive radiological imaging test or death from any cause. Overall survival (OS) was evaluated from the date of surgical resection of the primary tumor to the date of death or the last follow-up. This study was approved by the Ethical Board of the Institutional Review Board of Shanghai Outdo Biotech Company. All the patients provided written informed consent before any study-related procedures.
Immunohistochemistry (IHC) and immunofluorescence protocol for ovarian cancer
Ovarian cancer tissue microarrays were prepared semi-automatically with the basic protocols include [1] mark donor blocks showing the tissue of interest that corresponds with the H&E-stained slide, and organize for arrays; [2] punch blank recipient blocks of appropriate size for the array (punches with a diameter of 0.6–1 mm); [3] use the arrayer to insert the tissue cores in the recipient arrayer, putting them in the punches of the recipient block according to a predesigned map, under mild vacuum suction and ensuring that the cores lie in the same plane at the cut surface, in accordance with the arrayer manufacturer’s guidelines; [4] cut with meticulous care on a rotary microtome. Then, 5-µm sections from tissue microarrays were baked at 63°C for 1 hour. The sections were then de-paraffinized in xylene, rehydrated using a gradient of ethanol concentrations, boiled in 1 mM Tris-EDTA buffer with a high-pressure cooker (PH-070, Yiheng Company, Shanghai, China) for 3 minutes to retrieve antigens, blocked with 3% hydrogen peroxide for 10 minutes to inhibit activities of endogenous peroxidases and incubated with 10% goat non-immune serum for 20 minutes to reduce non-specific staining. After that, the sections were incubated with rabbit anti-TOX monoclonal antibody (1:500 dilution; ab155768, Abcam, Cambridge, UK), anti-Ki67 monoclonal antibody( 1:200 dilution; ab16667, Abcam, Cambridge, UK), anti-EGFR monoclonal antibody ( 1:200 dilution; ab32077, Abcam, Cambridge, UK) and anti-PD-L1 monoclonal antibody (1:250 dilution; ab213524, Abcam, Cambridge, UK) at 4°C overnight, then incubated with biotin-labeled secondary antibody (Ultrasensitive SP IHC kit, FuZhou MXB Biotechnology, China) at room temperature for 10 minutes, followed by incubation with HRP-conjugated streptavidin (Ultrasensitive SP IHC kit, FuZhou MXB Biotechnology, China) at room temperature for another 10 minutes. Color development was performed with DAB Substrate Kit (Dako, Glostrup, Denmark). Finally, the sections were counterstained with hematoxylin, dehydrated, cleared, and mounted. Multiplexed fluorescent IHC was performed using Opal 7-color Manual IHC Kit (NEL801001KT,PerkinElmer, USA) and VECTASHIELD® HardSet Antifade Mounting Medium (H-1400, Vector Labs, CA,USA) according to the instructions by the manufactures. Cell nuclei were counterstained with 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). The multiplexed fluorescence-labelled images were analyzed with automated imaging systems (Vectra Polaris, PerkinElmer, USA; TissueFAXS Spectra, Tissue Gnostics, Austria).
IHC staining evaluation of TOX, Ki67, EGFR, PD-L1 expressions
Whole tumor slides were randomly reviewed by two independent pathologists based on the intensity and the proportion of positively stained cells. Both reviewers were blinded until two reviews on two separate days were completed. Stain intensity was evaluated with a 4-grading system: 0 = negative, 1 = weak, 2 = moderate and 3 = strong. The percentage of positive cells were scored as follows: 0 for no cell stained, 1 for 1 − 25% of cells stained, 2 for 26–50% of cells stained, 3 for 51–75% of cells stained and 4 for more than 75% of cells stained. Scores for intensity and percentage of positive cells were multiplied. In order to make sure that the stain intensity should be moderate or strong and the percentage of TOX positive cells was larger than 50%, score 8 was determined as the cut off value. Scores < 8 was used to define tumors with low TOX expression and scores ≥ 8 with high TOX expression.
Statistical analysis
Statistical analyses were performed using SPSS Statistics version 28.0.1 (IBM, Armonk, NY, USA) and Prism version 9.0 (GraphPad, San Diego, CA, USA). The comparison of TOX expression between cancerous tissue and adjacent non-cancerous tissue was tested by Mann-Whitney U test. The correlations between TOX expression and clinical parameters were tested using Fisher’s exact test or Pearson’s chi-square test, as appropriate. DFS and OS were evaluated using the Kaplan-Meier method, and log-rank test was used to compare the difference between groups. For the analysis of DFS, data for patients who are alive and had no disease or who were lost to follow-up were censored at the time of the last assessment. For analysis of OS, data for patients who are alive or who were lost to follow-up were censored at the time of the last assessment. All results were considered significant when p* < 0.05, p**<0.01, p***<0.001, p****<0.0001.
