PD-L1 expression as predictor of immunotherapy eligibility in penile squamous cell carcinoma patients

Reagent used to stain PD-L1 in this IHC staining was MD21R clone (Medaysis CA), ready to use (rabbit PD-L1 antibody)

PD-L1 antibody is diluted with ratio 1 uL PD-L1: 100 uL IHC diluent and then mixed for 5 minutes.

Pretreatment was done with EDTA pH8.0, 15 minutes using a Pressure Cooker

0h 15m 0s

OR

1h 0m 0s 95°C to 99°C

Deparaffination and rehydration were done by using Bond Dewax Solution for 3 times, alcohol for 3 times, and Bond Wash Solution for 3 times.

We retrieved the antigen by using Bond Epitope Retrieval Solution I for 4 times

We mixed the solution with PD-L1 (Medaysis) primary antibody for 1 hour.

The detection was done by using post primary procedure for 8 minutes, Bond Wash Solution for two times, and polymer for eight minutes. We used DAB mixture for staining and hematoxylin for counterstaining.

0h 8m 0s post primary procedure

Bond Wash Solution Two times

0h 8m 0s polymer

PD-LI expression was assessed on cytoplasm and/or tumor cell membranes and TILs. The positive control used was the placenta.

PD-LI expression is considered positive if it has a score of +2 or +3 and is considered negative if it has a score of +1 and 0. Intensity measurement then given a score of 0-3 (0= none; 1= weak; 2= moderate; and 3= strong).

The percentage of stained cells was assessed on a scale of 0-100%.

Percentage of stained tumor cells A= percentage of tumor cells intensity in 3+ {3 x (%Cells 3+)}, B= percentage of tumor cells intensity in 2+ {2 x (%Cells 2+)}, C= percentage of tumor cells intensity in 1+ {1 x (%Cells 1+)}

Semi-quantitative assessment refers to research by Chovanec et al. who used histoscore (H-score)

H-Score = {1 x (%cells 1+) + 2x (%cells 2+) + 3x (%cells 3+)}

Total value obtained ranges from 0 to 300, thus PD-L1 expression is categorized into “1” for negative (0 – 99) and “2” for positive (100 – 300).