Immunohistochemical Analysis of FFPE Samples

Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples were stained for CK17 (Anti-Cytokeratin 17, Rabbit Monoclonal, clone EP1623, dilution 1:100, ab109725, Abcam, Cambridge, United Kingdom), PD-L1 (clone 22C3 for the discovery cohort and clone E1L33N for the validation cohort, respectively), and p16 (E6H4). Both a rabbit monoclonal and polyclonal CK17 antibody (ab109725 and ab53707, respectively) underwent extensive validation using CK17 highly expressing human SCC tissue and CK17 negative tonsil tissue as well as CK17 wild-type and knock-out mouse tissue (as previously described in [22 (link)]). Ultimately, clone ab109725 was selected based on the highest specificity for CK17. HPV status was determined based on p16 status for oropharyngeal tumors [24 ] and high-risk HPV RNA in situ hybridization for non-oropharyngeal tumors [25 (link)]. Detailed protocols are available in Supplemental Data S1.

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Lozar T., Laklouk I., Golfinos A.E., Gavrielatou N., Xu J., Flynn C., Keske A., Yu M., Bruce J.Y., Wang W., Grasic Kuhar C., Bailey H.H., Harari P.M., Dinh H.Q., Rimm D.L., Hu R., Lambert P.F, & Fitzpatrick M.B. (2023). Stress Keratin 17 Is a Predictive Biomarker Inversely Associated with Response to Immune Check-Point Blockade in Head and Neck Squamous Cell Carcinomas and Beyond. Cancers, 15(19), 4905.

Publication 2023

ab109725

Antibody Clone Cytokeratin 17 Dilution Formalin Human In situ hybridization Knock out mouse Oropharyngeal tumors Paraffin Pd l1 Rabbit Tissue Tonsil

Corresponding Organization : University of Wisconsin–Madison

Other organizations : University of Ljubljana, Yale University, Institute of Oncology Ljubljana

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Variable analysis

independent variables

CK17 antibody (clone ab109725)
PD-L1 antibody (clone 22C3 for discovery cohort, clone E1L33N for validation cohort)
P16 antibody (E6H4)

dependent variables

CK17 expression
PD-L1 expression
P16 expression
HPV status

control variables

Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples
CK17 highly expressing human SCC tissue
CK17 negative tonsil tissue
CK17 wild-type and knock-out mouse tissue

positive controls

CK17 highly expressing human SCC tissue
CK17 wild-type mouse tissue

negative controls

CK17 negative tonsil tissue
CK17 knock-out mouse tissue

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