Msh2 clone g219 1129

Immunohistochemical staining was performed on TMAs with the Ventana Benchmark XT platform. The following panel of antibodies was used: anti-MLH1 (clone G168-728, Roche), -PMS2 (clone EPR3947, Roche), -MSH2 (clone G219-1129, Roche), -MSH6 (clone 44, Roche), and -ARID1A (clone D2A8U, Roche). According to the published criteria of abnormal expression of MMR and ARID1A [15 , 23 ], both markers were interpreted as loss of expression, whereas no tumor cells were stained.
We chose the recommended guidelines that the immunohistochemistry results be reported in a binary manner, either positive (indicating intact mismatch repair, showing intact nuclear expression in tumor cells) or negative (indicating deficient mismatch repair, showing nuclear expression completely lost in tumor cells) [24 , 25 ] Nuclei of lymphocytes and ovarian stromal cells served as positive internal controls. All staining results were reviewed by the above 2 pathologists. Because immunohistochemical examination was performed using a tissue microarray, when deficient mismatch repair (dMMR) was observed, the immunohistochemistry procedure was repeated on whole-slide sections to avoid heterogeneous expression, such as for MSH6 [26 ]. Ultimately, MMR protein restaining was performed in 51 instances involving 27 of 176 cases; ARID1A restaining was performed in 15 instances involving 3 cases of dMMR among 135 cases.

Tissue microarray (TMA) techniques were used to make paraffin blocks for immunohistochemical slides using a TMA Master 3D Histech manual tissue arrayer. From each paraffin block containing invasive adenocarcinoma tissue, two cylindrical cores measuring 2 mm were taken from random tumor tissue sites. All cores were collected in a recipient TMA paraffin block. Each recipient block contained 20 samples from 10 cases. For immunohistochemistry, four µm-thick tissue sections were stained in a Ventana BenchMark ULTRA auto-stainer (Ventana Medical Systems, Tucson, Arizona). Monoclonal antibodies against SATB2 (CellMarque, EP281, 1:200), CK7 (clone OV-TL, BioSB, 1:500), PD-L1 (clone SP263, Roche, diagnostic kit), MutS homolog 2 (MSH2, clone G219-1129, Roche, ready to use), postmeiotic segregation 2 (PMS2, clone A16-4, Roche, ready to use), MutS homolog 6 (MSH6, clone 44, Roche, ready to use), and MutL homolog 1 (MLH1, clone M1, Roche, ready to use) were used. The positive reactions were visualized using the Ultraview Detection System (Ventana Medical Systems); slides were counterstained with hematoxylin. Stained slides were dehydrated and covered in a xylene-based mounting medium.