p16 IHC on formalin-fixed, paraffin-embedded (FFPE) tumor tissue was performed as part of the routine diagnostics process (Supplementary Table 1). Additional IHC was performed on FFPE tissue using standard protocols for the automated platforms Dako PT Link for Heat Induced Epitope Retrieval and Dako Autostainer 48S Link (Agilent, Santa Clara, CA, USA) with staining of tumor-infiltrating lymphocytes using anti-CD8 antibodies (Anti-Human CD8, Clone C8, 144B, Dako, concentration: 1:100, Agilent, Santa Clara, CA, USA). An unsuccessful immunohistochemical approach for HPV16 E2 and E7 was performed with following antibodies [Anti-HPV16 E2, NBP2-53115, NovusBio (no literature), Centennial, CO, USA; Anti-HPV16 E7, ab20191, Abcam, Cambridge, UK (32 ); Anti-HPV16 E7 (716–325), sc-51951, Santa Cruz Biotechnology, Dallas, TX, USA (33 (link))]. Different concentrations and antigen retrieval techniques revealed still unspecific staining.
Frequency and density of CD8 positive T cells were determined as previously described (34 (link), 35 (link)). One case was excluded in the further IHC analysis due to non-specific IHC staining.
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