IHC staining was performed on representative surgical tissue sections from FFPE tissue blocks of LUSC patients using anti–human PD-L1 antibodies: E1J2J, Cell Signaling Technology; 28-8, Abcam; 22C3, Dako; SP142, Spring Bioscience; anti–human CD8 antibodies (C8/144B; Nichirei Bioscience); anti–human PD-1 antibodies (NAT105; Abcam); anti–human β2M antibodies (A0072; Dako); and anti–human HLA class I-A/B/C antibodies (EMR8-5; Hokudo). The detection of immunostaining was performed using Histofine Simple Stain MAX-PO (Nichirei Bioscience) or BOND polymer Refine Detection kit (Leica Biosystems). Some of the sections were stained with the PD-L1 22C3 pharmDx kit (Dako). PD-L1 positivity was defined as membranous or cytoplasmic staining in at least 1% of tumor cells. The staining results of β2M and HLA class I-A/B/C of tumor cells were categorized as negative (0%), focally positive (<50%), or positive (≥50%).
Pd l1 22c3 pharmdx kit
Manufactured by Agilent Technologies
Sourced in United States
The PD-L1 22C3 pharmDx kit is a qualitative immunohistochemistry assay designed to detect the PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) human tissue specimens. The kit utilizes the Agilent 22C3 monoclonal antibody to identify PD-L1 expression in tumor cells and immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Autostainer link 48, by Agilent Technologies (3 mentions)
E1j2j, by Cell Signaling Technology (1 mentions)
A0072, by Agilent Technologies (1 mentions)
Histofine simple stain max po, by Nichirei Biosciences (1 mentions)
Nat105, by Abcam (1 mentions)
Bond polymer refine detection kit, by Leica (1 mentions)
Link 48, by Agilent Technologies (1 mentions)
Pharmdx kit, by Agilent Technologies (1 mentions)
Optiview dab ihc detection kit, by Roche (1 mentions)
Benchmark xt, by Roche (1 mentions)
8 protocols using pd l1 22c3 pharmdx kit
1
PD-L1, CD8, and HLA Expression in LUSC
2
PD-L1 Immunohistochemical Staining Protocol
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Programmed death-ligand 1 (PD-L1) staining of 4 μm thick tissue sections was performed using the US Food and Drug Administration-approved PD-L1 22C3 pharmDx Kit (Dako North America Inc., Carpinteria, CA, USA) on the Dako Autostainer Link 48, as per the manufacturer’s instructions. The slides were assigned depending on the percentage of positive cells divided by the number of fields, to calculate the average value for each individual case, defined at 200× magnification. The PD-L1 combined positive score was calculated as the ratio of PD-L1-positive cells (tumor or immune cells) to the total number of tumor cells × 100 and classified as positive (≥1) or negative (<1).
3
Evaluation of PD-L1 Expression in FFPE Tissue
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The expression of PD-L1 protein was evaluated by immunohistochemistry (IHC) performed on 4-µm formalin-fixed paraffin-embedded (FFPE) tissue sections using a Dako PD-L1 22C3 pharmDx kit (Dako, Carpinteria, CA). PD-L1 protein expression was determined by using the tumor proportion score (TPS), and the cutoffs for low and high expressions were 1 and 50%, respectively (14 (link)).
4
PD-L1 Immunohistochemistry Evaluation Protocol
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Immunohistochemistry (IHC) for PD-L1 was performed at the Section of Anatomic Pathology and Histology of Perugia University, Italy, using the PD-L1 22C3 pharmDx kit (Dako North America Inc., Carpinteria, CA, USA) on the Dako Autostainer Link 48 according to the manufacturer’s instructions. Unstained tissue sections 4-m-thick were prepared from the most representative area of each case. At least 100 viable tumor cells were required for a valid interpretation of PD-L1 staining. Slides were counterstained with Mayer’s hematoxylin. Results were evaluated with known positive and negative tissue controls. The percentage of PD-L1 expression in the invasive tumor cells was calculated as the number of viable invasive carcinoma cells showing membranous staining of any intensity divided by the total number of viable invasive carcinoma cells.
5
PD-L1 Expression Analysis by IHC
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For PD-L1 expression analysis, IHC using an automated stainer (Link 48, Dako) was performed on 4μm sections cut from archival FFPE tumor samples from 232 patients diagnosed with non-small cell lung cancer that were retrospectively selected from the QEII Health Sciences Centre. PD-L1 IHC using the PD-L1 22C3 pharmDx kit on the Dako platform (Product number: SK006) was performed according to manufacturer recommendations [17 (link)]. The positive and negative controls were from known PD-L1 IHC positive and negative cases confirmed by IHC testing. The pharmDx kit (Dako) is designed to perform the staining using a linker and a chromogen enhancement reagent. Pre-treatment of the slides including deparaffinization and rehydration was performed using PT Link machine. Next, the specimens were incubated with monoclonal mouse IgG antibody to PD-L1, followed by incubation with a mouse linker and with a ready-to-use Visualization Reagent consisting of Goat secondary antibodies against mouse immunoglobulin and horseradish peroxidase coupled to a dextran polymer backbone. Then, chromogen and chromogen enhancement reagents were added, resulting in a brown color at the site of the antigen-antibody interaction. All slides were cover slipped and visualized with a light microscope.
6
PD-L1 Immunohistochemistry Analysis
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Tumor PD-L1 analysis was performed in the Section of Anatomic Pathology and Histology of Perugia University, Italy. Immunohistochemistry (IHC) for PD-L1 was performed using the PD-L1 22C3 pharmDx kit (Dako North America Inc., Carpinteria, CA, USA) on the Dako Autostainer Link 48, according to the manufacturers’ instructions. Unstained tissue section 4-μm thick were prepared from the most representative area of each case. At least 100 viable tumor cells were required for a valid interpretation of PD-L1 staining. Slides were counterstained with Mayer’s hematoxylin. Results were evaluated with known positive and negative tissue controls. The percentage of PD-L1 expression on invasive tumor cells was calculated as the number of viable invasive carcinoma cells showing membranous staining of any intensity divided by the total number of viable invasive carcinoma cells.
7
Immunotherapy in Advanced NSCLC Patients
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Advanced NSCLC patients receiving a combination of immunotherapy and chemotherapy, or immunotherapy alone as the first- or second-line treatment at the Fudan University Shanghai Cancer Center (FUSCC) from January 2016 to July 2018 were enrolled in this study. All the patients had wild-type epidermal growth factor/anaplastic lymphoma kinase receptor. The patients were enrolled in clinical trials for ICIs, including nivolumab, pembrolizumab, atezolizumab, durvalumab, tremelimumab, and camrelizumab. The ICIs were administrated as per the study protocols, and the therapeutic effects were evaluated as a complete/partial response (CR/PR), stable disease (SD), or progressive disease (PD) according to the response evaluation criteria in solid tumors (RECIST) 1.1. This study was performed in accordance with the principles of the Declaration of Helsinki (as revised in 2013). The Institutional Review Board of Fudan University Shanghai Cancer Center approved the study (Date 2012-12-24; No. 050432-4-1212B), and all patients provided informed consent.
Tumoral PD-L1 expression was tested by immunohistochemistry (PD-L1 22C3 pharmDx kit, Agilent, Santa Clara, CA, USA) in archived tissue samples, when available, to calculate the tumor proportion scores (TPSs). 5 mL of peripheral blood samples were collected before immunotherapy from all the patients.
Tumoral PD-L1 expression was tested by immunohistochemistry (PD-L1 22C3 pharmDx kit, Agilent, Santa Clara, CA, USA) in archived tissue samples, when available, to calculate the tumor proportion scores (TPSs). 5 mL of peripheral blood samples were collected before immunotherapy from all the patients.
8
PD-L1 and MMR Protein Evaluation
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PD-L1 staining was done using the PD-L1 22C3 pharmDx kit on the Link 48 system autostainer (Agilent Technologies, USA) according to the manufacturer's instructions. Combined positive score (CPS) was calculated as the number of PD-L1-expressing tumor and intra-or peri-tumoral inflammatory cells divided by the number of viable tumor cells and then multiplied by 100.
For the assessment of mismatch repair (MMR) status, tissue specimens were stained using MLH1 (Novo, Newcastle, UK), MSH2 (Cell Marque, CA, USA), MSH6 (Cell Marque, CA, USA), and PMS2 (Cell Marque, CA, USA) using a Bench Mark XT automatic immunostaining device with an OptiView DAB IHC Detection Kit (Ventana Medical System). dMMR was defined as the loss of expression of one or more of the MMR proteins (i.e., MLH1, MSH2, MSH6, and PMS2).
For the assessment of mismatch repair (MMR) status, tissue specimens were stained using MLH1 (Novo, Newcastle, UK), MSH2 (Cell Marque, CA, USA), MSH6 (Cell Marque, CA, USA), and PMS2 (Cell Marque, CA, USA) using a Bench Mark XT automatic immunostaining device with an OptiView DAB IHC Detection Kit (Ventana Medical System). dMMR was defined as the loss of expression of one or more of the MMR proteins (i.e., MLH1, MSH2, MSH6, and PMS2).
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