1. Inclusion and exclusion criteria
Inclusion criteria and exclusion criteria: inclusion criteria: (1) the primary lesion occurred in the ovary; (2) ovarian cancer staging surgery, tumor cell reduction surgery or laparoscopic biopsy was performed; (3) The pathological diagnosis was clear and the pathological specimens were well preserved; (4) FIGO stage was I-IV; (5) the follow-up data were complete; (6) no radiotherapy, chemotherapy and other anti-tumor treatment were received before surgery.
2. Clinical data
1) Group for genetic test: 36 patients who in accordance with the inclusion criteria were enrolled in Beijing Union Medical College Hospital from November 19, 2012 to November 18, 2016, including 24 cases of clear cell carcinoma and 12 cases of high-grade serous carcinoma. All patients were treated by cytoreductive surgery/ovarian cancer staging, and platinum based first-line chemotherapy was performed.
2) Group for Immunohistochemistry: 39 patients who in accordance with the inclusion criteria were enrolled in Beijing Union Medical College Hospital from January 7, 2015 to April 25, 2018. All of the 39 cases were all clear cell carcinoma. All patients were treated by cytoreductive surgery/ovarian cancer staging, and platinum based first-line chemotherapy was performed.
3. NanoString expression analysis
The differential expression level of genes in tumor tissue samples of the patients were analyzed. The requirements of histologic specimen of the selected cases are: effective area >1.5*1.5cm, 3-5 wax rolls with the thickness of 10um, and the number of wax roll increased according to the tissue area of the puncture specimen. The experimental process includes: total RNA extraction, quality control of samples, overnight hybridization, elution and purification of hybrid products, sample plate preparation, sample plate scanning, and results output.
4. Immunohistochemistry
Immunohistochemistry was performed to detect the expression of MLH1, MSH2, MSH6 and PMS2 in tumor tissues: paraffin blocks with samples were taken from tumor tissues and paracancerous tissues of patients, and the tumor cell rich areas (> 50% were tumor cells) and paracancerous tissues were stained with immunohistochemistry. Among them, the ready-to-use MLH1 mouse anti-human monoclonal antibody (ESO5), the ready-to-use MSH2 mouse anti-human monoclonal antibody (FE11), the ready-to-use MSH6 mouse anti-human monoclonal antibody (EP49) and the ready-to-use PMS2 mouse anti-human monoclonal antibody (EP51) were purchased from Zhongshan Company (Beijing). The experiment was performed according to the instructions, the sections were dewaxed and repaired by high pressure thermal repair with 1mmol/L EDTA antigen repair solution (PH8.0). The sections were incubated with 30ml/L H2O2 for 10min and washed by the distilled water. Then soaked by phosphate buffered saline (PBS) for 5min. The sections were incubated with normal rabbit serum working solution for 10min, and then incubated with primary antibodies of MLH1, MSH2, MSH6 and PMS at 37 ℃ for 2h, washed with PBS for 3 times, 3min each time. After that, the sections were incubated with the secondary antibody for 15 min, rinsed with PBS for 3 times, 3 min for each time. Then the sections were incubated with the third antibody for 15ml, and washed with PBS for 3 times, 3 min for each time. At last, diaminobenzidine (DAB) was used for developing, and hematoxylin was used for counterstaining. Then Dehydration, transparent, and sealing was performed for each section. PBS was used as the negative control of primary antibody, and the expression of paracancerous tissue was considered as positive control.
5. Results determination
MLH1, MSH2, MSH6 and PMS2 were all located in the nucleus of tumor cells, with yellow or brown granules as the expression patterns (+), and the nucleus of tumor cells without staining were considered as the deletion (-). The absence of any one type of MLH1, MSH2, MSH6 and PMS2 protein (-) was determined as defective DNA mismatch repair(dMMR), while the expression of all of the MLH1, MSH2, MSH6 and PMS2 proteins were determined as proficient DNA mismatch repair (pMMR) [6, 7]. The evaluation process was independently completed by two senior pathologist using double blind method, and the disputed cases were further submitted to the superior physician for interpretation.
6. Follow up
The follow-up was performed by outpatient reexamination and telephone follow-up. The follow-up lasted from the beginning of the surgery to January 2019. Progression free survival (PFS) was defined as the time from staging or cytoreductive surgery to tumor recurrence, metastasis or death caused by any reason. Overall survival (OS) was defined as the time from staging or cytoreductive surgery to death of patients for any reason.
7. Statistical analysis
SPSS 23.0 software was used for statistical analysis. Chi-square test was performed to compare the enumeration data, and Kaplan-Meier method was performed for survival analysis.