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Development of an automated PD-L1 immunohistochemistry (IHC) assay for non-small cell lung cancer.

Phillips T, Simmons P, Inzunza HD, Cogswell J, Novotny J, Taylor C, Zhang X - Appl. Immunohistochem. Mol. Morphol. (2015)

Bottom Line: Nivolumab, a fully human IgG4 programmed death 1 (PD-1) immune checkpoint inhibitor antibody, developed by Bristol-Myers Squibb Inc., has activity across non-small cell lung cancer (NSCLC) histologies and is Food and Drug Administration approved for treatment of metastatic squamous NSCLC with progression on or after platinum-based chemotherapy.PD-L1 has been investigated as a potential biomarker to predict clinical response to nivolumab in clinical settings.The specificity of 28-8 for PD-L1 was demonstrated by antigen competition and genetic deletion of PD-L1 in tumor cell lines.

View Article: PubMed Central - PubMed

Affiliation: *Dako North America Inc., Carpinteria ‡Keck School of Medicine of the University of Southern California, Los Angeles, CA †Bristol-Myers Squibb Inc., Princeton, NJ.

ABSTRACT
Nivolumab, a fully human IgG4 programmed death 1 (PD-1) immune checkpoint inhibitor antibody, developed by Bristol-Myers Squibb Inc., has activity across non-small cell lung cancer (NSCLC) histologies and is Food and Drug Administration approved for treatment of metastatic squamous NSCLC with progression on or after platinum-based chemotherapy. PD-L1 has been investigated as a potential biomarker to predict clinical response to nivolumab in clinical settings. We report an automated PD-L1 immunohistochemistry (IHC) assay, which was developed to detect cell surface PD-L1 in formalin-fixed paraffin-embedded human tumor tissue specimens using Dako's Autostainer Link 48. The primary antibody for this assay is a rabbit monoclonal anti-human PD-L1 antibody, clone 28-8. The specificity of 28-8 for PD-L1 was demonstrated by antigen competition and genetic deletion of PD-L1 in tumor cell lines. The specificity of the PD-L1 IHC assay was further evaluated in a collection of 30 normal human tissues. The PD-L1 IHC assay was optimized for high sensitivity and precision in routine application. A pathology scoring and interpretation method specific to nivolumab clinical studies was adopted for the assay. The analytical performance of the assay was validated for application in the determination of PD-L1 status in human NSCLC specimens. The clinical application of the assay and scoring method was further validated in 3 Clinical Laboratory Improvement Amendments certified labs. The assay is currently being investigated in a variety of clinical studies for use as an in vitro diagnostic to select and stratify patients for treatment with the anti-PD-1 therapeutic antibody, nivolumab.

No MeSH data available.


Related in: MedlinePlus

Photomicrographs (×40) of parental and PD-L1 knock-out (KO)-stable FFPE cell pellets stained by the PD-L1 IHC assay. FFPE indicates formalin-fixed paraffin-embedded; IHC, immunohistochemistry; NCR, negative control reagent; PD-L1, programmed cell death 1 ligand 1.
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Figure 3: Photomicrographs (×40) of parental and PD-L1 knock-out (KO)-stable FFPE cell pellets stained by the PD-L1 IHC assay. FFPE indicates formalin-fixed paraffin-embedded; IHC, immunohistochemistry; NCR, negative control reagent; PD-L1, programmed cell death 1 ligand 1.

Mentions: To demonstrate the specificity of the PD-L1 IHC assay in detecting endogenous PD-L1 protein expressed in tumor cells, FFPE cell pellets of the parental and knock-out cell lines were stained by the PD-L1 IHC assay. Human ovarian clear cell carcinoma tumor cell line ES-2 and human lung adenocarcinoma tumor cell line L2987 showed high level of PD-L1 expression by FACS analysis. PD-L1 gene expression was genetically disrupted and knocked out by a genomic editing approach as described in the Materials and methods section. The knock-out of the PD-L1 expression in the 2 cell lines was verified by direct DNA deep sequencing and FACS analysis of the cells (see the Materials and methods section). Membrane staining of PD-L1 was not detectable in the 100% knock-out cell pellets. Membrane staining of PD-L1 in the parental cell lines was abolished by the addition of PD-L1 antigen to the protein-rich antibody diluent (see the Materials and methods section, Fig. 3). Because the knock-out cells were selected by cell cloning, it is likely that a low percentage of incomplete knock-out cells were present that were below the detection limit of FACS and batch sequencing. Very faint blush cytoplasmic staining was observed in some knock-out cells, and is thus nonspecific for PD-L1. To demonstrate that PD-L1 antigen specifically blocked binding of PD-L1 antibody clone 28-8 to PD-L1, IHC testing with a pair of broadly expressed control epithelial biomarkers CK8/CK18 was performed with and without competition by PD-L1 antigen. The addition of PD-L1 antigen to the CK8/CK18 cocktail did not block the IHC staining of CK8 and CK18, further supporting the specificity of PD-L1 antibody 28-8 in detecting PD-L1 by the PD-L1 IHC assay (Supplemental Figs. 2A, B, Supplemental Digital Content 2, http://links.lww.com/AIMM/A93).


Development of an automated PD-L1 immunohistochemistry (IHC) assay for non-small cell lung cancer.

Phillips T, Simmons P, Inzunza HD, Cogswell J, Novotny J, Taylor C, Zhang X - Appl. Immunohistochem. Mol. Morphol. (2015)

Photomicrographs (×40) of parental and PD-L1 knock-out (KO)-stable FFPE cell pellets stained by the PD-L1 IHC assay. FFPE indicates formalin-fixed paraffin-embedded; IHC, immunohistochemistry; NCR, negative control reagent; PD-L1, programmed cell death 1 ligand 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561627&req=5

Figure 3: Photomicrographs (×40) of parental and PD-L1 knock-out (KO)-stable FFPE cell pellets stained by the PD-L1 IHC assay. FFPE indicates formalin-fixed paraffin-embedded; IHC, immunohistochemistry; NCR, negative control reagent; PD-L1, programmed cell death 1 ligand 1.
Mentions: To demonstrate the specificity of the PD-L1 IHC assay in detecting endogenous PD-L1 protein expressed in tumor cells, FFPE cell pellets of the parental and knock-out cell lines were stained by the PD-L1 IHC assay. Human ovarian clear cell carcinoma tumor cell line ES-2 and human lung adenocarcinoma tumor cell line L2987 showed high level of PD-L1 expression by FACS analysis. PD-L1 gene expression was genetically disrupted and knocked out by a genomic editing approach as described in the Materials and methods section. The knock-out of the PD-L1 expression in the 2 cell lines was verified by direct DNA deep sequencing and FACS analysis of the cells (see the Materials and methods section). Membrane staining of PD-L1 was not detectable in the 100% knock-out cell pellets. Membrane staining of PD-L1 in the parental cell lines was abolished by the addition of PD-L1 antigen to the protein-rich antibody diluent (see the Materials and methods section, Fig. 3). Because the knock-out cells were selected by cell cloning, it is likely that a low percentage of incomplete knock-out cells were present that were below the detection limit of FACS and batch sequencing. Very faint blush cytoplasmic staining was observed in some knock-out cells, and is thus nonspecific for PD-L1. To demonstrate that PD-L1 antigen specifically blocked binding of PD-L1 antibody clone 28-8 to PD-L1, IHC testing with a pair of broadly expressed control epithelial biomarkers CK8/CK18 was performed with and without competition by PD-L1 antigen. The addition of PD-L1 antigen to the CK8/CK18 cocktail did not block the IHC staining of CK8 and CK18, further supporting the specificity of PD-L1 antibody 28-8 in detecting PD-L1 by the PD-L1 IHC assay (Supplemental Figs. 2A, B, Supplemental Digital Content 2, http://links.lww.com/AIMM/A93).

Bottom Line: Nivolumab, a fully human IgG4 programmed death 1 (PD-1) immune checkpoint inhibitor antibody, developed by Bristol-Myers Squibb Inc., has activity across non-small cell lung cancer (NSCLC) histologies and is Food and Drug Administration approved for treatment of metastatic squamous NSCLC with progression on or after platinum-based chemotherapy.PD-L1 has been investigated as a potential biomarker to predict clinical response to nivolumab in clinical settings.The specificity of 28-8 for PD-L1 was demonstrated by antigen competition and genetic deletion of PD-L1 in tumor cell lines.

View Article: PubMed Central - PubMed

Affiliation: *Dako North America Inc., Carpinteria ‡Keck School of Medicine of the University of Southern California, Los Angeles, CA †Bristol-Myers Squibb Inc., Princeton, NJ.

ABSTRACT
Nivolumab, a fully human IgG4 programmed death 1 (PD-1) immune checkpoint inhibitor antibody, developed by Bristol-Myers Squibb Inc., has activity across non-small cell lung cancer (NSCLC) histologies and is Food and Drug Administration approved for treatment of metastatic squamous NSCLC with progression on or after platinum-based chemotherapy. PD-L1 has been investigated as a potential biomarker to predict clinical response to nivolumab in clinical settings. We report an automated PD-L1 immunohistochemistry (IHC) assay, which was developed to detect cell surface PD-L1 in formalin-fixed paraffin-embedded human tumor tissue specimens using Dako's Autostainer Link 48. The primary antibody for this assay is a rabbit monoclonal anti-human PD-L1 antibody, clone 28-8. The specificity of 28-8 for PD-L1 was demonstrated by antigen competition and genetic deletion of PD-L1 in tumor cell lines. The specificity of the PD-L1 IHC assay was further evaluated in a collection of 30 normal human tissues. The PD-L1 IHC assay was optimized for high sensitivity and precision in routine application. A pathology scoring and interpretation method specific to nivolumab clinical studies was adopted for the assay. The analytical performance of the assay was validated for application in the determination of PD-L1 status in human NSCLC specimens. The clinical application of the assay and scoring method was further validated in 3 Clinical Laboratory Improvement Amendments certified labs. The assay is currently being investigated in a variety of clinical studies for use as an in vitro diagnostic to select and stratify patients for treatment with the anti-PD-1 therapeutic antibody, nivolumab.

No MeSH data available.


Related in: MedlinePlus