Formalin-fixed paraffin-embedded tumor samples were stained with PD-L1 antibody (clone 22C3) on the Dako automated staining platform following the manufacturer’s standard protocol (Dako 22C3 PharmDx Assay, Dako Autostainer Link 48, Agilent, Santa Clara, CA, USA). All the areas in each tissue section were evaluated for the PD-L1 expression. Tissue sections evaluated to have moderate to strong membrane staining in at least 5% of the tumor cells are considered to be positive for PD-L1 overexpression, while tissue sections with an absence or detection of staining in less than 5% of the cells were considered to be negative [33 (link), 34 (link)]. PD-L1 expression was expressed as tumor proportion score, with scores of 0–5% as negative, between 5 and 49% as low expression and ≥50% as high expression level. All the slides were scored for PD-L1 membrane staining by two independent pathologists.
Dako 22c3 pharmdx assay
Manufactured by Agilent Technologies
Sourced in United States
The Dako 22C3 PharmDx Assay is a qualitative immunohistochemical assay used to detect the PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue samples. The assay utilizes the Dako 22C3 antibody to identify the presence of the PD-L1 protein on the surface of tumor cells.
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4 protocols using dako 22c3 pharmdx assay
1
Immunohistochemical Analysis of PD-L1 Expression
2
Wnt5b and PD-L1 Immunohistochemistry
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Experimental procedures were performed as previous studies [13] . We incubated tissue sections with Wnt5b antibody (1:50, Abcam, ab94914). The intensity of Wnt5b staining was scored as following: 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The percentage scores of Wnt5b staining were assigned as following: 1 (1%-25%), 2 (26%-50%), 3 (51%-75%), and 4 (76%-100%). The above two scores were multiplied to give a final score: 0 to 12. The scores of ≥4 were defined as Wnt5b overexpression (positive expression); the scores between 1 and 4 were defined as weak expression (negative expression). The informations of PD-L1 staining were in Supplemental materials and methods. PD-L1 staining was performed by using the Dako automated staining platform according to the manufacturer's standard steps (Dako 22C3 PharmDx Assay, Dako Autostainer Link 48, Agilent, Santa Clara, CA, USA). We determined the PD-L1 expression in tumor cells by using Tumor Proportion Score (TPS). TPS was calculated as the percentage of at least 100 viable tumor cells with complete or partial membrane staining. The percentage of ≥50% was defined as PD-L1 positive expression; and that of＜50% was defined as PD-L1 positive expression.
3
PD-L1 IHC Assay for Melanoma
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All PD-L1 IHC testing were performed using the DAKO 22C3 PharmDx assay (Agilent, Santa Clara, CA) following the manufacturer’s instructions in a Clinical Laboratory Improvement Amendments (CLIA)-certified and College of American Pathologists (CAP)-accredited reference laboratory (Foundation Medicine, Morrisville, NC). A positive PD-L1 IHC produces a brown stain that can be obscured by heavy melanin pigmentation, rendering it impossible to distinguish positive from negative results. Whenever this technical problem resulted in uninterpretable IHC, it was documented, allowing us to identify HPMel cases. As noted above, cases with interpretable IHC were designated LPMel.
4
Immunohistochemical Assessment of PD-L1 Expression
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Immunohistochemical staining of PD-L1 expression was performed using the Dako 22C3 pharmDx assay (Agilent Technologies, Carpinteria, USA) on tissue sections of primary tumor specimens removed by palliative gastrectomy for all patients. PD-L1 expression was evaluated in both tumor and immune cells by two pathologists. The combined positive score (CPS) was calculated by dividing the number of PD-L1 stained cells (tumor cells and immune cells) by the total number of viable tumor cells and by multiplying the value by 100. The PD-L1 CPS is currently recognized as a cost-effective screening tool that is easy to apply clinically [28 (link)]. The PD-L1 negative was defined as a CPS < 1, and high PD-L1 expression was defined as a CPS ≥ 10.
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