Tumor heterogeneity may impact immunohistochemical (IHC) interpretation, thus potentially affecting decision making by treating oncologists for cancer patient management. Programmed cell death ligand-1 (PD-L1) IHC 22C3 pharmDx is a companion diagnostic used as an aid in identifying patient eligibility for treatment with pembrolizumab (KEYTRUDA). This study aims to investigate tumor heterogeneity impact on IHC staining when evaluating PD-L1 expression using PD-L1 IHC 22C3 pharmDx. The effect of tumor heterogeneity was evaluated based on the PD-L1 diagnostic status of PD-L1 IHC 22C3 pharmDx stained tumor tissue sections at relevant diagnostic cutoffs for non–small cell lung carcinoma, gastric or gastroesophageal junction adenocarcinoma, urothelial carcinoma, head and neck squamous cell carcinoma, esophageal cancer and triple negative breast cancer. Overall agreement for the PD-L1 diagnostic status was assessed for each tumor type within a given specimen block (Intra-Block), between specimen blocks from the same surgical resection (Intra-Case), and between intrapatient primary and metastatic specimens. Intrablock and intracase point estimates were above 75%, and primary versus metastatic point estimates were above 50%. The results suggest that PD-L1 expression is consistent across cut sections through a minimum of 150 µm within a tissue block and between blocks from the same surgical resection and is significantly maintained across primary and metastatic blocks from the same patient despite changes to the tissue microenvironment. These data provide confidence for histopathologists and oncologists that evaluation of PD-L1 expression at clinically relevant cutoffs is reproducible among different assessments (or samplings) of a single tumor specimen.
Background: Pembrolizumab monotherapy did not significantly improve overall survival (OS) as second- or third-line treatment for metastatic TNBC vs chemotherapy in the randomized, open-label, phase 3 KEYNOTE-119 study (NCT02555657; N = 622). However, the benefit of pembrolizumab compared with chemotherapy appeared to be greater with increasing PD-L1 expression as quantified by combined positive score (CPS; defined as the number of PD-L1–staining cells [tumor cells, lymphocytes, macrophages] divided by the total number of viable tumor cells, multiplied by 100). In the current exploratory analysis, we aimed to determine whether expression of PD-L1 on tumor cells contributes to the value of PD-L1 as a predictive biomarker in metastatic TNBC.Methods: Patients with centrally confirmed TNBC and 1 or 2 prior systemic treatments for metastatic disease were enrolled in KEYNOTE-119. Patients were randomly assigned 1:1 to pembrolizumab 200 mg Q3W or investigator’s choice of single-agent chemotherapy (capecitabine, eribulin, gemcitabine, or vinorelbine). PD-L1 expression in tumor samples was assessed using PD-L1 IHC 22C3 pharmDx and quantified per tumor proportion score (TPS; defined as the percentage of PD-L1–expressing tumor cells [partial or complete membrane staining] relative to total number of tumor cells) and CPS. Quantitative immune cell density (QID) was defined as CPS minus TPS. QID isolates immune cells but may be truncated when TPS is high. The ability of each scoring method (TPS, CPS, and QID) to predict objective response rate (ORR) with pembrolizumab, including receiver operating characteristics (ROC) analysis, and OS hazard ratios (HRs; pembrolizumab vs chemotherapy) was evaluated.Results: Tumor samples were available for 601 patients (pembrolizumab, 309; chemotherapy, 292) in KEYNOTE-119. ORR was 9.7% (30/309) with pembrolizumab and 11.3% (33/292) with chemotherapy when PD-L1 expression status was not considered. In the pembrolizumab arm, the area under the ROC curve (AUROC; 95% CI) was 0.69 (0.58-0.80) for tumor samples scored for CPS, 0.66 (0.55-0.77) for QID, and 0.55 (0.46-0.64) for TPS. ROC analysis is shown in the Table. At each cutoff, QID had lower estimated sensitivity (ie, missed responders) and a lower Youden Index compared with CPS. The number of missed responders (of 30 total in the pembrolizumab arm) for QID relative to CPS were 2, 5, 5, 6, 2, and 2 at cutoffs of 1, 10, 20, 30, 40, and 50, respectively. Across all practical cutoffs, the OS HR tended to be slightly smaller for CPS than QID. At cutoffs corresponding to the upper percentiles of 10, 20, 40, and 60, OS HRs were 0.497, 0.658, 0.758, and 0.850, respectively, for CPS vs 0.572, 0.712, 0.814, and 0.863, respectively, for QID. QID appeared to be orthogonal to TPS (r = -0.03 for all 601 observations; r = -0.04 after eliminating 7 potentially truncated values).Conclusions: Trends estimated using KN119 suggest that tumor cell expression is an important component of PD-L1 as a predictive biomarker of pembrolizumab efficacy in metastatic TNBC. In this exploratory analysis, when immune cells alone were used to measure PD-L1 expression, a meaningful number of responders was missed and OS benefit trended toward higher HR estimates. Tumor and immune cell PD-L1 expression may represent distinct (presumably negative modulatory) mechanisms. Table. ROC AnalysisCutoffCPS SensCPS SpecCPS YICPS PrevQID SensQID SpecQID YIQID PrevTPS SensTPS SpecTPS YITPS Prev010011001100110.8330.3660.1990.6540.7670.4160.1820.6020.3000.7890.0890.220100.5670.7170.2840.3110.4000.8140.2140.2070.2000.8920.0920.117200.5000.8490.3490.1840.3330.9250.2580.1000.1670.9320.0990.078300.3670.8920.2590.1330.1670.9570.1240.0550.1000.9430.0430.061400.2000.9350.1350.0780.1330.9860.1190.0260.0670.9530.0200.049500.2000.9570.1570.0580.1330.9930.1260.0190.0670.9680.0340.036 Citation Format: Eric P. Winer, Oleg Lipatov, Seock-Ah Im, Anthony Goncalves, Eva Muñoz-Couselo, Keun Seok Lee, Zbigniew Nowecki, Peter Schmid, Kenji Tamura, Laura Testa, Isabell Witzel, Shoichiro Ohtani, Stephanie Hund, Karina Kulangara, Vassiliki Karantza, Jaime A. Mejia, Junshui Ma, Petar Jelinic, Lingkang Huang, Kenneth Emancipator, Javier Cortes. Contribution of tumor and immune cells to PD-L1 as a predictive biomarker in triple-negative breast cancer (TNBC): Analysis from KEYNOTE-119 [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD14-04.
BackgroundThe Combined Positive Score (CPS)1 algorithm includes tumor and immune cells for determination of Programed Death-Ligand 1 (PD-L1) protein expression in tumor tissues and has been analytically and clinically validated for use with PD-L1 IHC 22C3 pharmDx across multiple indications and cutoffs.2 PD-L1 22C3 IHC pharmDx is a qualitative immunohistochemical assay using anti-PD-L1, Clone 22C3 to detect PD-L1 in formalin-fixed, paraffin-embedded (FFPE) tumor tissues using Autostainer Link 48. PD-L1 IHC 22C3 pharmDx is FDA-approved as an aid in identifying patients for treatment with KEYTRUDA® for six tumor indications at clinically validated CPS diagnostic cutoffs2: gastric or gastroesophageal junction (GC/GEJ) adenocarcinoma (CPS ≥ 1), cervical cancer (CPS ≥ 1), urothelial carcinoma (CPS ≥ 10), head and neck squamous cell carcinoma (HNSCC) (CPS ≥ 1), esophageal squamous cell carcinoma (ESCC) (CPS ≥ 10)3, and triple-negative breast cancer (TNBC) (CPS ≥ 10).MethodsPrecision of PD-L1 IHC 22C3 pharmDx using CPS was assessed for all six indications at the corresponding clinically validated diagnostic cutoffs and at additional exploratory cutoffs under normal, day-to-day testing conditions. Precision testing included Combined Precision (inter-instrument/operator/run (day)), Intra-Run Repeatability, and Observer (inter-/intra-) Scoring Reproducibility studies. FFPE specimens were stained with PD-L1 IHC 22C3 pharmDx and scored using CPS as described in the package insert.2 Four CPS cutoffs were evaluated: CPS ≥ 1 (GC/GEJ, urothelial carcinoma, ESCC, cervical cancer, HNSCC, TNBC), CPS ≥ 10 (GC/GEJ, urothelial carcinoma, ESCC, TNBC), CPS ≥ 20 (HNSCC), and CPS ≥ 50 (HNSCC). Data were analyzed using negative percent agreement (NPA), positive percent agreement (PPA), and overall agreement (OA) with two-sided 95% percentile bootstrap confidence intervals (CIs) based on PD-L1 binary status at the applicable cutoff(s). For each study, data from each CPS cutoff-indication pair were individually analyzed. Meta-analyses were also performed by pooling data from all indications per (i) study and cutoff, and (ii) per study for all tested cutoffs.ResultsNearly all agreement analyses (142/144) for each CPS cutoff-indication pair showed NPA/PPA/OA point estimates (PE) ≥ 90% and CI lower bounds (CILB) ≥ 85%. Meta-analyses showed PE ≥ 90% for NPA/PPA/OA and CILB ≥ 85% per study and cutoff, and per study for all tested cutoffs. Discordant comparisons accounted for <5% of total comparisons performed for each study type.ConclusionsCPS used with PD-L1 IHC 22C3 pharmDx provides precise evaluation of PD-L1 expression across multiple tumor indications and cutoffs under normal, day-to-day testing conditions.AcknowledgementsWe thank the IUSCC Cancer Center at Indiana University School of Medicine, for the use of the Tissue Procurement and Distribution Core, which provided Dako North America, Inc. service.The data and biospecimens used in this project were provided by US Biolab (Gaithersburg, MD, USA), Sofia Bio LLC (New York, NY, USA), Contract Research Ltd (Charlestown, Nevis), and Centre Hospitalier Universitaire (CHU) de Nice (Nice, France) with appropriate ethics approval and through Trans-Hit Biomarkers Inc. 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. Tissue samples supplied by BioIVT (Hicksville, NY, USA).Trial RegistrationN/AReferencesCPS = (# PD-L1 staining cells (tumor cells, lymphotcytes, macrophages))/(Total # viable tumor cells )×100PD-L1 IHC 22C3 pharmDx [Instructions for Use]. Available at: www.agilent.com/library/eifu. Code SK006. Accessed July 2, 2021ESCC was analytically validated as a subtype of esophageal cancer [2].Ethics ApprovalN/AConsentN/A
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: The Tumor Proportion Score (TPS) and Combined Positive Score (CPS) scoring algorithms are used in conjunction with PD-L1 IHC 22C3 pharmDx for the immunohistochemical evaluation of PD-L1 in certain human cancer tissues; both algorithms include PD-L1 staining tumor cells (TC) and have been correlated with response to pembrolizumab therapy (KEYTRUDA®) at specific expression levels in specific tumor indications. This study aims to evaluate whether PD-L1 expression on TC could be important in the evaluation of TNBC with PD-L1 IHC 22C3 pharmDx and the impact on reaching a diagnostic cut-off, as current PD-L1 testing (SP142) for TNBC relies solely on scoring immune cells (IC). TNBC clinical data from KEYNOTE-119 (ClinicalTrials.gov, NCT02555657) and Agilent’s internal TNBC tumor bank data were evaluated by TPS, CPS, and Quantitative Immune Cell Density (QID) to determine the significance of including PD-L1 expressing TC in the scoring algorithms. PD-L1 IHC 22C3 pharmDx is not currently approved for use with TNBC specimens.Methods: TNBC specimens from clinical trial KEYNOTE-119 and Agilent’s internal tumor bank were stained with PD-L1 IHC 22C3 pharmDx and scored using the TPS, CPS, and QID algorithms. The TPS algorithm is defined as the number of PD-L1 staining tumor cells divided by the total number of viable TC, multiplied by 100. The CPS algorithm includes TC and IC and is defined as the number of PD-L1 staining cells (TC, lymphocytes and macrophages) divided by the total number of viable TC, multiplied by 100. In addition to TPS and CPS, QID was also calculated to quantify the contribution from PD-L1 expressing IC. QID is defined as the CPS minus the TPS (QID = CPS - TPS). The data was analyzed to show the percent of specimens that fell below the diagnostic PD-L1 cut-offs of ≥1 and ≥10 when evaluated using QID as compared to CPS. Both scores measure countable PD-L1 staining cells per 100 viable tumor cells. Results: A total of 120 PD-L1 IHC 22C3 pharmDx stained TNBC specimens from Agilent’s internal tumor bank were analyzed. When evaluated using CPS, 71 specimens were positive for the PD-L1 ≥1 cut-off and 49 were positive for the PD-L1 ≥10 cut-off. When analyzed with QID, 8.5% of specimens fell below the PD-L1 ≥1 cut-off and 26.5% fell below the PD-L1 ≥10 cut-off (Table 1). A total of 964 PD-L1 IHC 22C3 pharmDx stained TNBC specimens from KEYNOTE-119 were analyzed. When evaluated using CPS, 604 specimens were positive for the PD-L1 ≥1 diagnostic cut-off and 284 were positive for the PD-L1 ≥10 diagnostic cut-off. When analyzed with QID, 9.3% of patients fell below the PD-L1 ≥1 diagnostic cut-off and 33.5% fell below the PD-L1 ≥10 diagnostic cut-off (Table 1). Conclusion: PD-L1 IHC 22C3 pharmDx stains both TC and IC in TNBC. Removal of the PD-L1 staining TC from the CPS algorithm (QID score) reduces the number of specimens scored as positive for the PD-L1 ≥1 and PD-L1 ≥10 diagnostic cut-offs. This data highlights that a significant portion of TC express PD-L1 in TNBC and inclusion of TC in the scoring algorithm should be considered when evaluating the diagnostic status of a specimen. Table 1. Agilent Tumor Bank and KEYNOTE -119: CPS and QIDDiagnostic Cut-OffCPS Positive SpecimensQID (CPS – TPS) Positive SpecimensSpecimens Flipped at Diagnostic Cut-offDifference (%)Agilent Tumor BankPD-L1 ≥ 1716568.5PD-L1 ≥ 1049361326.5KEYNOTE-119PD-L1 ≥ 1604548569.3PD-L1 ≥ 102841899533.5 Citation Format: Tiffany Evans, Stephanie Hund, Darlene Krohn, Kenneth Emancipator, Jonathon Juco, Bryce Portier, Siena Tabuena-Frolli, Karina Kulangara. Significance of PD-L1 expressing tumor cells in the combined positive score with triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-15.
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