CHAPTER 3 70 nih.gov/clinvar/), The Clinical Knowledgebase (CKB, https://ckb.jax.org/), Cancer Genome Interpreter (CGI, https://www.cancergenomeinterpreter.org/home), the Catalog of Somatic Mutations in Cancer (COSMIC, https://cancer.sanger.ac.uk/cosmic), OncoKB (https://www.oncokb.org/), Varsome (https:// varsome.com/). The three first categories were taken into consideration and included known activating hotspot mutations for the oncogenes, and missense, nonsense, frameshift and splice site mutations for the included TSGs. Intronic mutations were excluded with exception of splice site sequences. To determine whether sufficient DNA molecules were sequenced to reliably exclude mutation, a cumulative binomial distribution was used for calculating the required unique read depths, above a certain mutant allele frequency with a certainty of >95%.6 These required read depts were assessed in the context of estimated tumor percentage cells by microscopy. Immunohistochemical staining For p53 staining, antigen retrieval (30 minutes, pH 6·7) and blocking of endogenous peroxidase with hydrogen peroxide was performed. Subsequently, slides were incubated with p53 antibody (clone DO-7 + BP53-12, dilution 1:600). Powervision+ Poly-HRP was used and visualization was accomplished by using PowerVision DAB substrate solution (Leica Biosystems, Buffalo Grove, IL, US). Counterstaining was performed with hematoxylin, slides were dehydrated and mounted. For PMS2 and MSH6 staining, antigen retrieval with EnVision FLEX High pH Target Retrieval Solution, and blocking of endogenous peroxidase with hydrogen peroxide was performed. After, slides were incubated with anti-MSH6 (clone EPR3945 1:400, Abcam, Cambridge, UK) or anti-PMS2 (clone A16-4 dilution 1:20, BD Biosciences , San Jose, CA). Incubation was performed with EnVision FLEX and visualized with High pH visualization system. Counterstaining was performed with hematoxylin, slides were dehydrated and mounted.
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