Oct 07, 2022

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

DOI
dx.doi.org/10.17504/protocols.io.bp2l69b4klqe/v1
  • 1Universitas Indonesia;
  • 2Universitas Sumatera Utara
  • Muhammad Haritsyah Warli: Department of Urology, Faculty of Medicine, Unviersitas Indonesia - Haji Adam Malik General Hospital;
  • Fauriski Febrian Prapiska: Division of Urology, Department of Surgery, Faculty of Medicine, Universitas Sumatera Utara – Haji Adam Malik General Hospital
  • Ginanda Putra Siregar: Division of Urology, Department of Surgery, Faculty of Medicine, Universitas Sumatera Utara – Haji Adam Malik General Hospital
  • Bungaran Sihombing: Division of Urology, Department of Surgery, Faculty of Medicine, Universitas Sumatera Utara – Haji Adam Malik General Hospital
  • Syah Mirsya Warli: Division of Urology, Department of Surgery, Faculty of Medicine, Universitas Sumatera Utara – Haji Adam Malik General Hospital and Department of Urology, Faculty of Medicine, Universitas Sumatera Utara Hospital – Universitas Sumatera Utara
Muhammad Haritsyah Warli
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bp2l69b4klqe/v1
Protocol Citation: Muhammad Haritsyah Warli, Fauriski Febrian Prapiska, Ginanda Putra Siregar, Bungaran Sihombing, Syah Mirsya Warli 2022. PD-L1 expression as predictor of immunotherapy eligibility in penile squamous cell carcinoma patients. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69b4klqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 05, 2022
Last Modified: October 07, 2022
Protocol Integer ID: 70872
Keywords: PD-L1, Squamous Cell Carcinoma
Abstract
We investigated the correlation between PD-L1 expression and poor prognosis. We examined the correlation between PD-L1 expression and survival of penile SqCC and analyzed the adjusted hazard ratios (HR) of the patients with PD-L1 expression compared to anatomical stage of the tumor.
Materials
-MD21R clone (Medaysis CA)
-Rabbit PD-L1 antibody
-DAB mixture
-Hematoxylin
PD-L1 Antibody Preparation
PD-L1 Antibody Preparation
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.



5m
Pretreatment
Pretreatment
Pretreatment was done with EDTA pH8.0, 15 minutes using a Pressure Cooker

Duration00:15:00 Pressure cooker
OR

Duration01:00:00 30 – 60 minutes using a water bath Temperature95 °C to Temperature99 °C

1h 15m
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
PD-L1 and Sample Mixing and Detection
PD-L1 and Sample Mixing and Detection
We mixed the solution with PD-L1 (Medaysis) primary antibody for 1 hour.
1h
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.

Duration00:08:00 post primary procedure
Bond Wash Solution Two times
Duration00:08:00 polymer


16m
Analysis
Analysis
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).