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Multiplex Immunohistochemistry Analysis

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Standard histology was performed on all biopsies. Monochromatic IHC staining with antibodies to CK20 (KS20.8; Dako), CD8 (C8/144B; Dako), CD4 (SP35; Cell Marque), CM2B4 (MCPyV T-antigen, sc-136172; Santa Cruz Biotechnology), MHC class I (EMR8-5; MBL), PD-L1 (E1L3N; Cell Signaling Technology), and FoxP3 (14-5773-82; eBioscience) was performed. Hematoxylin and eosin- and IHC-stained slides were reviewed for evidence of tumor and necrosis prior to performing further biomarker analyses. For multispectral IHC staining, FFPE specimens were deparaffinized and rehydrated, subjected to heat-induced antigen retrieval, and stained as described previously (37 (link)). Staining was performed using the following antibodies: PD-1 (EPR4877; Abcam), PD-L1 (SP142; Spring Bio), CD4 (RBT-CD4; BioSB), CD8 (C8/144B; Dako), and CD68 (PG-M1; Dako). Slides were imaged with a Vectra Automated Quantitative Pathology Imaging System (Perkin Elmer). Images were analyzed using inForm Software (Perkin Elmer) and were evaluated by a pathologist.

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Bhatia S., Longino N.V., Miller N.J., Kulikauskas R., Iyer J.G., Ibrani D., Blom A., Byrd D.R., Parvathaneni U., Twitty C.G., Campbell J.S., Le M.H., Gargosky S., Pierce R.H., Heller R., Daud A.I, & Nghiem P. (2019). Intratumoral Delivery of Plasmid IL12 Via Electroporation Leads to Regression of Injected and Noninjected Tumors in Merkel Cell Carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(3), 598-607.

Publication 2019

PD-L1 (E1L3N; FoxP3 EPR4877 RBT-CD4 PG-M1 Vectra Automated Quantitative Pathology Imaging System inForm Software

Antibodies Antigen Biomarker Biopsies Cell Ck20 Eosin Hematoxylin Mcpyv Mhc class i Necrosis Pathologist Pd l1 T antigen Tumor Vectra

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Variable analysis

independent variables

Monochromatic IHC staining with antibodies to CK20 (KS20.8; Dako), CD8 (C8/144B; Dako), CD4 (SP35; Cell Marque), CM2B4 (MCPyV T-antigen, sc-136172; Santa Cruz Biotechnology), MHC class I (EMR8-5; MBL), PD-L1 (E1L3N; Cell Signaling Technology), and FoxP3 (14-5773-82; eBioscience)
Multispectral IHC staining with antibodies to PD-1 (EPR4877; Abcam), PD-L1 (SP142; Spring Bio), CD4 (RBT-CD4; BioSB), CD8 (C8/144B; Dako), and CD68 (PG-M1; Dako)

dependent variables

Evidence of tumor and necrosis

control variables

Standard histology performed on all biopsies
FFPE specimens subjected to heat-induced antigen retrieval

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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