Background PD-L1 22C3 IHC pharmDx (SK006) is a qualitative immunohistochemical (IHC) assay using anti-PD-L1, Clone 22C3 to detect PD-L1 in formalin-fixed, paraffin-embedded (FFPE) tumor tissues using the Autostainer Link 48. PD-L1 expression is determined by Combined Positive Score (CPS), which is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. 1 SK006 has been analytically validated using CPS across multiple tumor indications and diagnostic cutoffs and is used as an aid in identifying patients for treatment with KEYTRUDA ® . A previous publication presents the analytical performance of the following indications: gastric or gastroesophageal junction (GC/GEJ) adenocarcinoma (CPS ! 1), cervical cancer (CPS ! 1), head and neck squamous cell carcinoma (HNSCC) (CPS ! 1), esophageal cancer (EC/ESCC) (CPS ! 10), and triple-negative breast cancer (TNBC) (CPS ! 10). 2 This study evaluated the analytical performance of the SK006 assay for an additional five individual tumor indications (ovarian carcinoma (OC), prostate cancer (PC), colorectal carcinoma (CRC), renal cell carcinoma (RCC), biliary tract adeno cancer (BTAC)) and a group of 11 rare tumor indications in a basket trial (BT). 3 Two CPS cutoffs were evaluated: CPS ! 1 (OC, PC, CRC, RCC, BTAC and BT [3]) and CPS ! 10 (OC, CRC and BTAC). Methods Combined precision measured inter-instrument, -operator, -day and -lot variation. Intra-run measured repeatability and Inter/Intra-Observer measured scoring reproducibility. Negative percent agreement (NPA), positive percent agreement (PPA), and overall agreement (OA) with two-sided 95% bootstrap confidence intervals (CIs) were used for data analysis, based on the PD-L1 binary status at the evaluated cutoffs.
BackgroundThe COVID-19 pandemic brought a host of new challenges, including the immediate need for digital solutions addressing the lack of remote options available to pathologists in the field of immunohistochemistry (IHC)-based companion diagnostics for Programmed Death-Ligand 1 (PD-L1) expression evaluation in tumor tissues. Agilent Technologies, Inc. investigated concordance of PD-L1 expression results recorded by trained pathologists between stained glass slides and digital whole slide images (WSIs). Formalin-fixed, paraffin-embedded (FFPE) specimens of eleven tumor indications (table 1) were evaluated in this study. Specimens were stained using the qualitative IHC assay PD-L1 IHC 22C3 pharmDx on Autostainer Link 48 and scored using TPS (Tumor Proportion Score) or CPS (Combined Positive Score) algorithms at six validated cutoffs.1 The objective was to demonstrate equivalency between digital WSI and microscope glass slide scoring.MethodsThree Agilent-certified pathologists evaluated specimen PD-L1 expression level (positive/negative) using CPS and/or TPS at relevant cutoff(s) for each indication (table 1) using two scoring modalities for the same specimen sets: 1) light microscope, and, 2) digital monitor (WSI) with a minimum 14-day washout period between glass slide and WSI reads. WSIs were generated using Leica’s Aperio AT2 scanner and evaluated using Aperio ImageScope software (figure 1) on appropriate monitors (table 2). Concordance between specimen glass slide (reference condition) and WSI PD-L1 expression results was assessed per cutoff on pooled data from all applicable indications using negative percent agreement (NPA), positive percent agreement (PPA) and overall agreement (OA) with 95% two-sided percentile bootstrap confidence intervals (CI); the acceptance criteria for equivalency at each cutoff were set at CI lower-bounds (CILBs) ≥85%. Discordant comparisons with respect to specimen screening data generated prior to inclusion in the study were also analyzed where applicable.ResultsNPA/PPA/OA CILBs for the CPS ≥1, CPS ≥10, TPS ≥1%, and TPS ≥50% cutoffs were ≥85% (table 3). NPA and OA CILBs at CPS ≥20 and CPS ≥50 were ≥85%; PPA CILBs were 83.2% and 84.2%, respectively. Discordant comparisons analysis for CPS ≥20 and CPS ≥50 suggested that WSI is not more prone to discordances in PD-L1 expression level than glass slide scoring when compared to specimen screening data (tables 4 and 5).Abstract 36 Table 1Algorithm-cutoff pairs testedAbstract 36 Figure 1Digital WSI of a triple-negative breast carcinoma (TNBC) specimen stained with PD-L1 IHC 22C3 pharmDx primary antibody and viewed on Aperio ImageScope software with corresponding H&E and NCR WSIs for use as aids in the interpretation of PD-L1 staining. **Tissue sample supplied by BioIVT (Hicksville, NY, USA)Abstract 36 Table 2Minimum computer monitor requirements for viewing WSIs on Aperio ImageScopeAbstract 36 Table 3Glass slide vs. digital WSI NPA/PPA/OA results summary for the six algorithm-cutoff pairs testedAbstract 36 Table 4Number of PD-L1 expression level discordances in HNSCC CPS ≥20 study for predefined negative, near-cutoff (NCO) negative, NCO positive, and positive categories based on specimen screening data assigned by one or more Agilent pathologists prior to the studyAbstract 36 Table 5Number of PD-L1 expression level discordances in HNSCC CPS ≥50 study for predefined negative, NCO negative, NCO positive and positive categories based on specimen screening data assigned by one or more Agilent pathologists prior to the studyConclusionsGlass slide and WSI scoring are equivalent across multiple validated cutoffs and tumor indications tested for PD-L1 expression using PD-L1 IHC 22C3 pharmDx with CPS and/or TPS algorithms and are, thus, considered interchangeable scoring modalities.Acknowledgements< i >We would like to thank our colleagues at Agilent Technologies, Inc. and all of the pathologists involved in study specimen scoring for their valuable contributions to this study. Samples/tissue supplied by Conversant Biologics.Tissue samples supplied by BioIVT (Hicksville, NY, USA)The data and biospecimens used in this project were provided by Centre Hospitalier Universitaire (CHU) de Nice (Nice, France), Contract Research Ltd (Charlestown, Nevis), National BioService LLC (Saint Petersburg, Russia), Sofia Bio LLC (New York, NY, USA), US Biolab (Gaithersburg, MD, USA), Nottingham University Hospitals NHS Trust (Nottingham, UK), Gundersen Medical Foundation Center Biobank (La Crosse, WI, USA), LLC Biomedica CRO (Kyiv, Ukraine), Clinfound Clinical Research Services Pvt Ltd (Idukki, Kerala, India), SageBio LLC (Sharon, MA, USA), GLAS (Winston-Salem, NC, USA), Hospices Civils de Lyon (Lyon, France), IOM Ricera (Viagrande, Italy), Clin-Path Diagnostics (Tempe, AZ, USA), Centre Antoine Lacassagne (CAL; Nice, France), CHU de Bordeaux (Biobank ID: BB-0033–00036; Bordeaux, France) and contributions by clinical personnel from Centre de ressources biologiques, and SELARL DIAG (Nice, France) with appropriate ethics approval and through Trans-Hit Biomarkers Inc. Biological materials were provided by the Ontario Tumour Bank, which is supported by the Ontario Institute for Cancer Research (Toronto, Ontario, Canada) through funding provided by the Government of Ontario.Tissue samples were provided by the Cooperative Human Tissue Network which is funded by the National Cancer Institute. Other investigators may have received specimens from the same subjects.</i >Trial RegistrationN/AReferenceP02893/13 Instructions for Use (IFU) for PD-L1 IHC 22C3 pharmDx Human Cancer (SK00621-4) Package InsertEthics ApprovalN/AConsentN/A
Background In-person pathologist trainings during the COVID-19 pandemic became impossible, necessitating a shift to remote-digital whole slide image (WSI) training. High concordance between WSI and glass slide scores from the same specimens stained with PD-L1 IHC 22C3 pharmDx (SK006) across multiple tumor indications supported the validity of digital training. 1 However, in-person microscope (glass-slide) training versus remote-digital (WSI) training effectiveness must be assessed. Collated testing data on specimens (SK006 stained) spanning multiple indications scored by external pathologists during Agilent led training and testing (T&T) sessions via glass slides were compared to sessions utilizing WSIs. Methods Stained slides (30 unique specimens per tumor indication) were scanned on an Aperio AT2 scanner to generate WSIs for digital T&T. Remote T&T sessions used WebEx and PathcoreScholar's online platform to discuss scoring guidelines and WSI training cases. Subsequently, external pathologists evaluated WSIs in PathcoreScholar for PD-L1 expression using either Tumor Proportion Score (TPS) or Combined Positive Score (CPS) scoring algorithms and interpreted these scores at predefined cutoffs (figure 1). In both glass and WSI scoring test modalities, passing is defined as inter and intra-observer overall agreement (OA) !85%. Training effectiveness pass rates from glass slide data (2018-2020) and WSI data (2021-2022) spanning multiple indications and scoring algorithms were calculated and then compared using the Fisher-Freeman-Halton test, with a significance threshold of 0.05. Only data from initial pathologist tests were included in the pass rate calculation; data from re-tests executed after initial test failure were excluded. Results The differences between pass rates for microscope (glass slide) and digital (WSI) testing were not statistically significant (p-value > 0.05) (tables 1 and 2). Testing pass rates for indications scored with TPS or CPS using microscope glass slide vs digital WSI T&T was not statistically significant (pvalue > 0.05) (table 3). Conclusions No statistically significant differences in pathologist training effectiveness for PD-L1 were observed between remote and in-person trainings across multiple tumor indications, scoring algorithms, and cutoffs. These results demonstrate the effectiveness and equivalency of remote-digital pathologist trainings for evaluation of PD-L1 expression as detected by PD-L1 IHC 22C3 pharmDx in multiple tumor indications when compared to in-person-microscope glass slide T&T. Use of digital training and scoring proficiency testing can provide pathologists around the world with access to high-quality, interactive training from leading experts in PD-L1 expression evaluation. Acknowledgements We would like to thank our colleagues at Agilent Technologies, Inc. and all the pathologists who completed Agilent scoring certification training and testing for their valuable contributions to this study. Tissue samples were provided by the Cooperative Human Tissue Network wh...
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