PD-L1 Immunohistochemistry Assays Research Articles

DRG2 as a Biomarker to Enhance the Predictive Efficacy of PD-L1 Immunohistochemistry Assays.

PD-L1 immunohistochemistry (IHC) assays are used as a companion diagnostic for immunotherapy with immune checkpoint inhibitors (ICIs). However, despite the association between PD-L1 expression and clinical benefit from ICIs, the PD-L1 IHC assay is not sufficiently accurate in predicting response to ICIs; some patients with high PD-L1 expression do not respond to ICIs. Recently, researchers provided insights into why some patients with high PD-L1 expression fail to respond to ICIs. They discovered that DRG2 is a critical regulator of PD-L1 endosomal trafficking in cancer cells, which is essential for the proper localization of PD-L1 on the cell surface. Although DRG2-depleted cells express high levels of PD-L1 and are PD-L1 IHC-positive, the PD-L1 sequestered in early endosomes does not respond to ICIs. Therefore, a companion diagnostic combining DRG2 expression with a PD-L1 IHC assay may improve the therapeutic response to PD-1/PD-L1 ICIs.

Abstract CT053: Exploration of potential biomarkers correlated with efficacy of ociperlimab (anti-TIGIT) plus tislelizumab (anti-PD-1) in 1L PD-L1+ non-small cell lung cancer (NSCLC)

Abstract Background: PD-L1 expression was associated with anti-immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) + anti-PD-(L)1 treatment, in which a high PD-L1 subgroup showed improved efficacy. We investigated if anti-TIGIT mechanism of action (MOA)-related markers were associated with the efficacy of ociperlimab + tislelizumab in Cohort 3 (1L PD-L1+ NSCLC) of the phase 1/1b AdvanTIG-105 trial (NCT04047862) and evaluated a potential patient-enrichment strategy based on tumor tissue gene expression profile (GEP). Methods: Tumor tissue GEP was tested using TruSeq RNA Access technology. Ventana SP263 PD-L1 immunohistochemistry (IHC) assay was used to evaluate PD-L1 expression. Median progression-free survival (mPFS) by investigator was calculated descriptively by Kaplan-Meier methodology. 95% confidence intervals for mPFS were generated using the Brookmeyer method. The primary inferential PFS comparison used unstratified log-rank test with 2-sided descriptive P-values. Results: At data cutoff (Feb 2, 2023), 24 of 45 patients had GEP results. Anti-TIGIT MOA-related genes and signatures correlated with ociperlimab + tislelizumab treatment response. Patients with high (H) vs low (L) expression of TIGIT, CD226, CCR8, or a tumor-associated macrophage (TAM) signature had significantly longer mPFS (Table). Dual biomarkers combining both anti-PD-L1 (PD-L1 IHC) and one of the anti-TIGIT MOA-related factors (TIGIT, CCR8, TAM signature GEP) identified subgroups of PD-L1 H + TIGIT MOA-related factor H patients with improved PFS vs other subgroups (Table). A highly overlapped PD-L1 H + TIGIT H + CCR8 H + TAM signature H patient population was observed in dual biomarker analyses. Conclusions: Anti-TIGIT MOA-related genes and signatures correlated with efficacy in ociperlimab + tislelizumab-treated 1L PD-L1+ NSCLC. Combining anti-TIGIT MOA-related factors with PD-L1 expression identified a subgroup of patients with improved efficacy. TABLE 1. NAND Table. Efficacy Analyses in Patient Subgroups TIGITa CD226b CCR8b TAMb Subgroup H L H L H L H L n 8 16 12 12 12 12 12 12 mPFS, months (95% CI) NR (2.6, NR) 5.26 (2.07, 11.86) NR (4.21, NR) 4.68 (1.41, 15.05) 15.21 (2.6, NR) 4.7 (1.71, 7.16) 15.21 (4.21, NR) 4.17 (1.71, 5.45) PFS, P-value 0.0326 0.0327 0.0131 0.0153 PD-L1c + TIGITb PD-L1c + CCR8b PD-L1c + TAMb Subgroup TIGITHPD-L1H TIGITHPD-L1L TIGITLPD-L1H TIGITL PD-L1L CCR8HPD-L1H CCR8HPD-L1L CCR8LPD-L1H CCR8L PD-L1L TAMHPD-L1H TAMHPD-L1L TAMLPD-L1H TAML PD-L1L n 8 4 8 4 11 1 5 7 9 3 7 5 mPFS, months (95% CI) NR (1.41,NR) 4.76 (2.6,NR) 8.62 (2.07,NR) 2.94 (1.25,NR) NR (1.41,NR) 2.6 (NR,NR) 5.19 (2.07,NR) 4.21 (1.25,7.16) 23.89 (1.41,NR) 5.32 (4.21,NR) 5.29 (2.07,NR) 2.6 (1.25,NR) Cutoff: aTop 1/3; bMedian; cTC≥25% Citation Format: Se Hyun Kim, Jiayuan Zhang, Wei Tan, Han Yan, Tian Tian, Hao Zheng, Ziqi Zhou, Ruihua Wang, Yun Zhang, Zhirong Shen, Hye Ryun Kim, Diansheng Zhong, Shun Lu. Exploration of potential biomarkers correlated with efficacy of ociperlimab (anti-TIGIT) plus tislelizumab (anti-PD-1) in 1L PD-L1+ non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT053.

Longitudinal analysis of PD-L1 expression in patients with relapsed NSCLC

BackgroundThe use and approval of immune checkpoint inhibitors for the treatment of non-small cell lung cancer (NSCLC) depends on PD-L1 expression in the tumor tissue. Nevertheless, PD-L1 often fails to...

Comparing Three Different Anti–Programmed Death-Ligand 1 Antibodies in Immunohistochemical Evaluation of Combined Chemoimmunotherapy Response in Patients With NSCLC: A Prospective Study

IntroductionMultiple programmed death-ligand 1 (PD-L1) immunohistochemistry assays performed using different antibodies including DAKO 22C3, DAKO 28-8, and Ventana SP142 PD-L1—predictive markers for response to various immune checkpoint inhibitors in NSCLC—have been approved in several countries. The differences in multiple PD-L1 immunohistochemistry assay results in predicting the therapeutic response to combined chemoimmunotherapy in patients with NSCLC remain unclear. MethodsIn this multicenter prospective observational study, we monitored 70 patients with advanced NSCLC treated with combined chemoimmunotherapy at 10 institutions in Japan. The expression of PD-L1 in pretreatment tumors was evaluated using the 22C3, 28-8, and SP142 assays in all patients. ResultsThe PD-L1 level in tumor cells determined using the 22C3 assay was the highest among the three assays performed with different antibodies. According to the 22C3 assay results, the PD-L1 tumor proportion score greater than or equal to 50% group had a significantly longer progression-free survival period than the PD-L1 tumor proportion score less than 50% group. Nevertheless, the other assays did not reveal remarkable differences in the objective response rate or progression-free survival. ConclusionsIn our study, PD-L1 expression determined using the 22C3 assay was more correlated with the therapeutic response of patients with NSCLC treated with combined chemoimmunotherapy than that determined using the 28-8 and SP142 assays. Therefore, the 22C3 assay may be useful for clinical decision-making for patients with NSCLC treated with combined chemoimmunotherapy. Trial registration number: UMIN 000043958.

Clinical significance of serum-derived exosomal PD-L1 expression in patients with advanced pancreatic cancer

BackgroundInteractions between the programmed cell death receptor 1 (PD-1) and its ligand (PD-L1) lead to immune evasion in various tumors and are associated with poor prognosis in patients with pancreatic cancer; however, the roles of PD-L1-containing exosomes in pancreatic cancer is poorly understood. Here, we investigated the correlation between circulating exosomal PD-L1 (exoPD-L1) and PD-L1 expression in tumor tissue, and survival outcomes in patients with advanced PDAC.MethodsExosomes were derived from pre-treatment serum samples isolated using ExoQuick kit from 77 patients with advanced pancreatic cancer. Exosomal PD-L1 (exoPD-L1) was detected by enzyme-linked immunosorbent assay, and matched tumor tissues PD-L1 expression were evaluated by PD-L1 immunohistochemistry (22C3) assay, described with combined positive score. Cutoff value of exoPD-L1 for survival was assessed with receiver operating characteristic curve analysis. Kaplan-Meier analysis was performed to obtain median overall survival (OS), and hazard ratio was estimated using a stratified Cox regression model.ResultsThe median exoPD-L1 serum concentration was 0.16 pg/mg, with undetected levels in seven patients. ExoPD-L1 levels were significantly higher in patients with systemic disease than in those with locally advanced disease (p = 0.023). There was a significantly higher proportion of elevated exoPD-L1 levels in patients with positive PD-L1 expression compared to patients with negative PD-L1 expression (p = 0.001). Patients were classified into groups with low and high exoPD-L1 levels using ROC curve-derived cutoffs (0.165 pg/mg; area under the curve, 0.617; p = 0.078). At a median follow-up of 8.39 months, the median OS was 13.2 (95% CI, 8.17–18.3) and 6.36 months (95% CI, 3.27–9.45) in the low and high exoPD-L1 groups, respectively (HR = 0.61; 95% CI, 0.35–1.04; p = 0.059). ExoPD-L1 levels did not affect the proportion of CD8+CD69+ effector cytotoxic T cells in either of the groups (p = 0.166).ConclusionsThe serum-derived exoPD-L1 levels were higher in metastatic pancreatic cancer than locally advanced disease. Collectively, higher serum exoPD-L1 levels in patients with advanced pancreatic cancer suggested worse survival outcomes and may have clinical implications.

PD-L1 immunohistochemistry assay optimization to provide more comprehensive pathological information in classic Hodgkin lymphoma

Overexpression of PD-L1 can be a predictive marker for anti-PD-1 therapeutic efficacy in classic Hodgkin lymphoma (CHL); however, harmonization of different IHC assays remains to be accomplished, and interpretations of PD-L1 immunostaining results remain controversial in CHL. In this study, we sought to optimize the PD-L1 immunohistochemistry (IHC) assay in CHL. All tests were performed on a tumour tissue microarray established from 54 CHL cases. Three IHC antibodies (405.9A11, SP142, 22C3) for detecting PD-L1 expression were compared semi quantitatively with the RNAscope assay (No. 310035, ACD), and the difference in the expression in background immune cells (ICs) between assays and the associations of expression levels with densities of TILs/TAMs were also analysed. 405.9A11 demonstrated best specificity in HRS cells and best sensitivity in ICs. Positive expression of PD-L1 was more frequent in ICs (85.2%) than in HRS cells (48.1%). Different subgroups of background ICs, including tumour-associated macrophages (TAMs), were assessed and scored for CD4, CD8, FOXP3, and CD163 expression. PD-L1 expression on ICs was the factor most associated with the density of TAMs. 405.9A11 provided the most convincing PD-L1 expression results. Pathologists should report PD-L1 expression in a combined manner, including both the status of HRS cells and the percentage of PD-L1-positive ICs.

Comparison of PD-L1 tumor cell expression with 22C3, 28-8, and SP142 IHC assays across multiple tumor types

BackgroundMultiple PD-L1 immunohistochemistry (IHC) assays, including DAKO 22C3, DAKO 28-8, and Ventana SP142 PD-L1 IHC assays, have been approved by the Food and Drug Administration as a companion diagnostic (CDx)...

PD-L1 evaluation in the gastrointestinal tract: from biological rationale to its clinical application.

Immune-checkpoint inhibitors targeting the PD-1/PD-L1 axis have brought significant clinical benefit in many solid cancer types, including gastrointestinal malignancies. However, it has been estimated that only 20-40% of patients respond to treatment. The pattern of expression and potential predictive value of PD-L1 as an immunohistochemical biomarker has been extensively studied in gastrointestinal neoplasms. Until now, its predictive value has been demonstrated, and is currently in use only in upper gastrointestinal malignancies (gastroesophageal adenocarcinoma and esophageal squamous cell carcinoma).In this Review, we describe the technical aspects and challenges related to PD-L1 immunohistochemical assays, the current role of PD-L1 as a biomarker in clinical practice and we outline the main studies and clinical trials analyzing the prognostic and predictive value of PD-L1 in gastrointestinal cancers.

Blood exosome PD-L1 is associated with PD-L1 expression measured by immunohistochemistry, and lymph node metastasis in lung cancer

BackgroundPrevious observations illustrated that programmed cell death ligand 1 in exosomes (Exo-PD-L1) may lead to immunosuppression. This study proposed to investigate the significance of Exo-PD-L1 and the results of PD-L1 immunohistochemistry (IHC) assay in the clinical diagnosis and treatment of lung cancer. Methods29 lung cancer patients were enrolled. Exosomes were extracted from the blood of patients and purified, and the extracts were identified by Western blot and transmission electron microscope. Next, the levels of Exo-PD-L1 and PD-L1 in tumor tissue were evaluated by enzyme-linked immunosorbent assay (ELISA) and IHC, respectively. The correlation between Exo-PD-L1, IHC PD-L1 status and pathological features of patients was analyzed by applying Chi-square test. After immune checkpoint inhibitor (ICI) treatment, the objective response rate (ORR) was calculated, and drug response prediction in lung cancer patients by using Exo-PD-L1 alone, IHC PD-L1 alone, and their combined detection were analyzed. ResultsThis study confirmed that lung cancer patients had much expression of PD-L1 in blood exosomes and that Exo-PD-L1 level was associated with IHC PD-L1 status. The expression level of Exo-PD-L1 was evidently related to the positive lymph node metastasis of lung cancer patients, while IHC PD-L1 status was not correlated with clinicopathological features of patients. Moreover, Exo-PD-L1 and IHC PD-L1 alone or their combined detection could be utilized to predict the efficacy of ICI therapy in lung cancer patients. ConclusionThe correlation between Exo-PD-L1 and IHC PD-L1 status was indicated, and Exo-PD-L1 could assist in determining the suitable lung cancer patients suitable for ICI therapy using IHC PD-L1. This study provides references for the application of Exo-PD-L1 as an effective predictor of ICI therapy.

Quantitative comparison of PD-L1 IHC assays against NIST standard reference material 1934

Companion diagnostic immunohistochemistry (IHC) tests are developed and performed without incorporating the tools and principles of laboratory metrology. Basic analytic assay parameters such as lower limit of detection (LOD) and dynamic range are unknown to both assay developers and end users. We solved this problem by developing completely new tools for IHC—calibrators with units of measure traceable to National Institute of Standards & Technology (NIST) Standard Reference Material (SRM) 1934. In this study, we demonstrate the clinical impact and opportunity for incorporating these changes into PD-L1 testing. Forty-one laboratories in North America and Europe were surveyed with newly-developed PD-L1 calibrators. The survey sampled a broad representation of commercial and laboratory-developed tests (LDTs). Using the PD-L1 calibrators, we quantified analytic test parameters that were previously only inferred indirectly after large clinical studies. The data show that the four FDA-cleared PD-L1 assays represent three different levels of analytic sensitivity. The new analytic sensitivity data explain why some patients' tissue samples were positive by one assay and negative by another. The outcome depends on the assay's lower LOD. Also, why previous attempts to harmonize certain PD-L1 assays were unsuccessful; the assays' dynamic ranges were too disparate and did not overlap. PD-L1 assay calibration also clarifies the exact performance characteristics of LDTs relative to FDA-cleared commercial assays. Some LDTs' analytic response curves are indistinguishable from their predicate FDA-cleared assay. IHC assay calibration represents an important transition for companion diagnostic testing. The new tools will improve patient treatment stratification, test harmonization, and foster accuracy as tests transition from clinical trials to broad clinical use.

Changes in PD-L1 tumor proportion score are associated with CD274 gene (encoding PD-L1) copy number variation in non-small cell lung cancer.

9029 Background: PD-L1 tumor proportion score (TPS) is often used to determine eligibility for first line therapy with immune checkpoint inhibitors (ICIs) in advanced non-small cell lung cancer (NSCLC). However, PD-L1 expression can vary over time and between tumor sites, potentially leading to inaccurate patient stratification. Therefore, it is critical to understand the clinicopathologic and genomic factors that are associated with PD-L1 changes in NSCLC. Methods: Clinicopathologic and genomic data were collected from patients with NSCLC and quantitative PD-L1 immunohistochemistry (IHC) on at least two different biopsies. NSCLC biopsies were categorized as PD-L1 negative, low, and high if they had a PD-L1 TPS < 1%, 1-49%, and ≥50%, respectively. Intrapatient changes in PD-L1 TPS between samples (DPD-L1) were defined as follows: major decrease (decrease in PD-L1 TPS from ≥50% to < 50% or from ≥1% to < 1%), major increase (increase in PD-L1 TPS from < 1% to ≥1% or < 50% to ≥50%), and non-major change (all other cases). Next-generation sequencing (NGS) was used to evaluate copy number (CN) variations at the CD274 locus, which encodes PD-L1. Wilcoxon and Kruskal-Wallis rank sum tests were used to analyze continuous variables and Fisher’s exact test was used to analyze categorical variables. Results: Among 250 patients with NSCLC with PD-L1 IHC assays performed on at least two distinct tissue samples, PD-L1 TPS of the first biopsy was < 1% in 104 (41.6%), 1-49% in 80 (32.0%), and ≥50% in 66 (26.4%) samples, for a median PD-L1 TPS of 2% (range: 0% to 100%). When intrapatient DPD-L1 was examined, there were major decreases and major increases in PD-L1 TPS in 49 (19.6%) and 65 (26.0%) cases, respectively, and non-major changes were observed in the remaining 136 samples (54.4%), with a median DPD-L1 of 0% (range: -90% to +90%). Baseline PD-L1 TPS and DPD-L1 were not significantly affected by histology, smoking status, sex, or treatment. Among 219 NSCLC samples that underwent tissue NGS and had full CN data available, the median PD-L1 TPS differed significantly based on CD274 CN: PD-L1 TPS 1% with single copy deletion vs. 5% with copy neutral vs. 42.5% with low amplification vs. 97.5% with high amplification (p < 0.01). Among 56 patients with paired PD-L1 TPS and NGS on both samples, there was a significant difference in median DPD-L1 according to CD274 CN change: DPD-L1 TPS -49% with acquired CD274 CN loss vs. 0% with no major change in CD274 CN vs. +1.75% with acquired CD274 CN gain (p = 0.01). Conclusions: PD-L1 TPS varies within the same patient in almost half of NSCLC cases, with few clinicopathologic correlates of change in expression. Variation in PD-L1 TPS correlates with changes in CD274 CN across biopsies. These findings suggest a genomic correlate to predict PD-L1 TPS across samples, as well as a potential complementary method in determining in ICI initiation.

Pan-cancer analysis of CD274 (PD-L1) mutations in 314,631 patient samples and subset correlation with PD-L1 protein expression.

2605 Background: The effects of non-amplification short variant (SV) mutations in CD274 (PD-L1) on PD-L1 protein expression and immune checkpoint inhibitor (ICPI) therapy are unknown. Here, we present a retrospective analysis of CD274 mutations detected by comprehensive genomic profiling (CGP) and correlate these results with tumor-cell PD-L1 immunohistochemistry (IHC)-based expression assessment to better understand the relationship between mutations and protein expression of PD-L1. Methods: FoundationOne CGP was performed on hybridization-captured, adaptor ligation-based libraries using DNA and/or RNA extracted from 314,631 tumor samples that were sequenced for up to 406 cancer related genes and select gene rearrangements. PD-L1 IHC was performed on a subset of cases (n = 213) using the DAKO 22C3 PD-L1 IHC assay and scored with the tumor proportion score (TPS). Results: Overall, the prevalence of CD274 SV mutations was low (0.3%, 1,081/314,631) with 577 unique variants. The most common CD274 SV mutations were R260H (n = 51), R260C (n = 18), R125Q (n = 12), C272fs*13 (n = 11), R86W (n = 10), and R113H (n = 10). The prevalence of CD274 mutations varied depending on tumor type with diffuse large B-cell lymphoma (1.9%, 19/997), cutaneous squamous cell carcinoma (1.6%, 14/868), endometrial adenocarcinoma (1.0%, 36/3740), unknown primary melanoma (0.9%, 33/3679), and cutaneous melanoma (0.8%, 32/3874) having the highest frequency of mutations. Ultraviolet exposure was likely a mechanism for CD274 SV mutations in cutaneous tumors with high frequencies of ultraviolet mutational signatures (cutaneous squamous cell carcinoma [84.6%, 11/13], cutaneous melanoma [93.8%, 30/32], and unknown primary melanoma [100%, 32/32]), and microsatellite instability (MSI) was likely a mechanism for development of CD274 mutations in non-serous endometrial adenocarcinoma. Of the R260H cases concurrently tested with PD-L1 IHC, most (81.8%, 9/11) had no PD-L1 expression, which contrasts to the five E237K cases where most (80%, 4/5) had PD-L1 expression. This difference in protein expression of these two mutations was significantly different (p = 0.036). It was notable that nearly all samples (88.9%, 16/18) with a clonal truncating variant (nonsense or frame shift indel) and PD-L1 testing showed a PD-L1 TPS score ≤1, whereas three of four samples with sub-clonal truncating variants had TPS scores ≥5. Conclusions: We defined the landscape of CD274 mutations in a large cohort of tumor types that can be used as a reference for examining CD274 mutations as potential resistance biomarkers for ICPI. Furthermore, we presented novel data on the correlation of CD274 mutations and PD-L1 protein expression, providing important new information on the potential functionality of these mutations and can serve as a basis for future research.

Comparison of molecular signatures in small cell lung cancer with or without homologous recombination associated gene mutations.

e20580 Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer with high mortality. Recently, Immune checkpoint inhibitors (ICIs) have been shown to have the potential to improve the prognosis of SCLC, but little is known about immunotherapeutic biomarkers. Homologous recombination deficiency (HRD) is demonstrated to be a response predictor to immunotherapies in gynecologic cancers, while limited studies were reported in small cell lung cancer. Herein, we analyze the mutational pattern of HRR related genes in a Chinese SCLC cohort and further analyze the relationship between HRR-gene mutations and tumor mutational burden. Methods: Target gene sequencing (543 genes) was performed in 133 Genecast cohort with small cell lung cancer. PD-L1 expression were evaluated for 90 among 133 patients using the SP142 PD-L1 immunohistochemistry assay. Results: Among 133 patients, 47 (35.3%) had HRR-gene mutations. ATM (8.3%), NBN (4.5%) and BRCA2 (4.5%) were the top 3 mutated HRR-gene in the cohort，followed by ATR (3.8%), BARD1 (3.8%), BRCA1 (3.8%), PALB2 (3.8%), RAD50 (3.8%), CHEK2 (3.0%), BLM (3.0%), BRIP1(2.3%), CHEK1(1.5%), RAD52(1.5%), and MRE11A (0.8%). Pathogenic somatic and germline mutations of HRR genes were identified in 11 (11/47, 23.4%) and 3 (3/47, 6.4%) patients, respectively. 1 (1/47, 2.1%) patient carried both germline and somatic variants. Genomic landscape revealed that TP53 and RB1 were commonly mutated genes in SCLC cohort. Mutations in KMT2D, AR and RTK-RAS pathway occurred more frequently in the HRR-Mut group, compared with the wildtype ones. Furthermore, we found that mutations in HRR-gene were associated with high TMB (Wilcoxon, p = 0.048), and patients with high TMB (≥median) showed a higher proportion of positive PD-L1 expression in 90 SCLC patients. Conclusions: Our data indicated that genomic alterations associated with HRR-genes have a positive correlation with high TMB, and detection of HRR-gene mutation status probably could help identify patients who might benefit from immune checkpoint blockade therapy. Keywords: Small cell lung cancer, Homologous recombination deficiency, Immunotherapy.

Development of a Companion Diagnostic PD-L1 Immunohistochemistry Assay for Pembrolizumab Therapy in Head and Neck Squamous Cell Carcinoma

Objectives: FDA approval of PD-L1 IHC 22C3 pharmDx for use as an aid in identifying head and neck squamous cell carcinoma (HNSCC) patients for treatment with pembrolizumab was based on the results of rigorous analytical and clinical validation testing. Methods: For the HNSCC indication, the device was validated at Agilent Technologies on the performance of sensitivity and precision using the Combined Positive Score (CPS) ≥ 1 and CPS ≥ 20 cutoffs; external validation studies were performed at three external laboratories. CPS ≥ 1 and CPS ≥ 20 cutoffs were evaluated in KEYNOTE-048, a phase 3 clinical trial. Results: Analytical validation studies supporting the companion diagnostic indication (CPS ≥ 1) achieved point estimates of > 85% for negative, positive, and overall percent agreement. Clinical validation studies show that HNSCC patients treated with pembrolizumab as a single agent had an overall survival (OS) of 12.3 months at CPS ≥ 1 (95% CI, 10.8-14.9) compared with patients receiving cetuximab, platinum, and 5-fluorouracil (CPS ≥ 1 OS of 10.3 months (95% CI, 9.0-11.5)). Conclusion: Analytical and clinical validation studies demonstrate that PD-L1 IHC 22C3 pharmDx is a precise companion diagnostic assay, allowing for selection of eligible HNSCC patients for treatment with pembrolizumab.

PD-L1 as a biomarker of response to immune-checkpoint inhibitors.

Immune-checkpoint inhibitors targeting PD-1 or PD-L1 have already substantially improved the outcomes of patients with many types of cancer, although only 20-40% of patients derive benefit from these new therapies. PD-L1, quantified using immunohistochemistry assays, is currently the most widely validated, used and accepted biomarker to guide the selection of patients to receive anti-PD-1 or anti-PD-L1 antibodies. However, many challenges remain in the clinical use of these assays, including the necessity of using different companion diagnostic assays for specific agents, high levels of inter-assay variability in terms of both performance and cut-off points, and a lack of prospective comparisons of how PD-L1+ disease diagnosed using each assay relates to clinical outcomes. In this Review, we describe the current role of PD-L1 immunohistochemistry assays used to inform the selection of patients to receive anti-PD-1 or anti-PD-L1 antibodies, we discuss the various technical and clinical challenges associated with these assays, including regulatory issues, and we provide some perspective on how to optimize PD-L1 as a selection biomarker for the future treatment of patients with solid tumours.

Harmonization of Molecular Testing for Non-Small Cell Lung Cancer: Emphasis on PD-L1.

Comprehensive molecular testing plays a critical role in the choice of treatment for non-small lung cell cancer (NSCLC). The analysis of druggable alterations in EGFR, BRAF, MET, KRAS, ALK, ROS1, RET and NTRK1/2/3 genes is more or less standardized and can be achieved using a single diagnostic platform, e.g., next generation sequencing (NGS) or polymerase chain reaction (PCR). In contrast to above targets, PD-L1 testing requires the use of immunohistochemistry (IHC). There are multiple PD-L1 IHC assays, which utilize distinct antibodies and detection systems. These PD-L1 tests are tailored to distinct drugs, often rely on different thresholds and scoring guidelines, and are characterized by incomplete inter-laboratory and inter-observer reproducibility. Several studies evaluated the performance of PD-L1 RNA expression tests, as PCR-based RNA analysis is compatible with other NSCLC molecular testing platforms, can be performed in a semi-automated manner, and has a potential for proper standardization. These investigations revealed a correlation between PD-L1 protein and RNA expression; however, there were NSCLCs demonstrating decent amounts of PD-L1 transcript in the absence of PD-L1 IHC staining. Clinical studies are required to evaluate, which of the two PD-L1 testing approaches, i.e., RNA or protein expression measurement, has a better predictive value.

Economic evaluation of the SP142 versus 22C3 PD-L1 assays in the treatment of atezolizumab plus nab-paclitaxel for patients with advanced triple negative breast cancer in the Brazilian private healthcare system

Objective: The aim of the study was to demonstrate the economic impact of two PD-L1 immunohistochemistry (IHC) assays, SP142 versus 22C3, in the treatment with atezolizumab plus nab-paclitaxel in patients with advanced triple negative breast cancer (aTNBC) in the Brazilian private healthcare system (BPHS). Methods: The study performed two analyses: one per patient and other of the potential population projected for the BPHS (budget impact analysis). Data of progression-free survival and overall survival were extracted from a post hoc analysis of the IMpassion130 trial to develop a partitioned-survival model to simulate the economic impact of the treatment with atezolizumab plus nab-paclitaxel guided by the SP142 and 22C3 assays on patients with aTNBC. The analyses included only direct costs that were based on CBHPM (Classificação Brasileira Hierarquizada de Procedimentos Médicos) and CMED (Câmara de Regulação do Mercado de Medicamentos) PF18% tables. A univariate sensitivity analysis was performed with the parameters varying ± 20%. Results: The study has demonstrated that the SP142 assay has the potential to save –BRL 179,730 with the treatment of atezolizumab plus nab-paclitaxel per patient with aTNBC in five years. Conclusion: The SP142 assay can optimize the use of atezolizumab plus nab-paclitaxel avoiding its prescription in patients who will not have a significant clinical improvement.

Abstract A07: Assessment of concordance among 22C3, SP263, and SP142 immunohistochemistry assay for PD-L1 expression in non-small cell lung cancer

Abstract Background: PD-L1 expression, as determined by immunohistochemistry (IHC), is the most commonly use predictive biomarker for anti-PD-1 and anti-PD-L1 agents in currently available in clinical practice. However, multiple antibodies have been utilized in IHC assays and interpretation of their results has not been standardized. Recently, several studies have compared different antibodies and IHC systems for PD-L1 tests. We aimed to evaluate the similarity and concordance between three validated, commercially available PD-L1 IHC assays for non-small cell lung cancer (Dako 22C3, Ventana SP263, and Ventana SP142). Methods: We evaluated 219 samples from patients with non-small cell lung cancer at the Daegu Catholic University Medical Center between June 2017 and July 2018. Each tumor sample was stained with PD-L1 IHC 22C3, SP263, and SP142. PD-L1 expression was scored at different cut-off levels for each IHC antibodies (22C3 assay: <1%, 1-49%, and ≥50%, SP263: <10%, ≥10 and <25%, and ≥25%, SP142: <1%, 1-4%, 5-49%, and ≥50%). Results: Among the 219 samples, three different antibodies showed well correlation for PD-L1 expression (22C3 vs. SP263; r=0.896, p<0.001, 22C3 vs. SP142; r=0.750, p<0.001, SP263 vs. SP142; r=0.728, p<0.001). 22C3 IHC assay produced the similar results with SP263 IHC assay (Kappa value=0.510, p<0.001). However, 22C3 vs. SP263 and SP263 vs. SP142 showed poor concordance (Kappa value=0.171, p=0.004, and Kappa value=0.240, p<0.001, respectively). Conclusions: 22C3, SP263, and SP142 IHC assays showed good correlation for PD-L1 expression. 22C3 IHC assay showed good concordance with SP263, but SP142. Citation Format: Chi Young Jung, Eun Jin Kim, Woo Jung Sung. Assessment of concordance among 22C3, SP263, and SP142 immunohistochemistry assay for PD-L1 expression in non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A07.

Agreement between PDL1 immunohistochemistry assays and polymerase chain reaction in non-small cell lung cancer: CLOVER comparison study

The goal of the CLOVER study was to perform a pairwise comparison of four tests based on the same patient population with non-small cell lung cancer (NSCLC): three validated PDL1 immunohistochemistry (IHC) assays (Ventana SP142, Ventana SP263, Dako 22C3) and one PCR test. Four hundred seventy-three NSCLC samples were obtained from a biobank and were stained using PDL1 IHC assays. Four trained pathologists independently evaluated the percentage of tumor cells (TC) and immune cells (IC) that stained positive at any intensity. PDL1 transcripts were quantified in 437 patients by a standard Taqman RT-PCR assay using SDHA as a reference gene. A concordance analysis was performed to assess (1) the correlation of TC and IC between different assays and (2) the predictive properties of one test for another. “High” RNA expression was detected in 187 of 437 (43%) patients. The percentage of PDL1-positive cells (≥1%) was higher among the IC than the TC in all IHC three assays. The Pearson correlation coefficients (PCC) for TC were 0.71, 0.87, and 0.75 between 22C3/SP142, 22C3/SP263, and SP263/SP142, respectively. The PCC for IC were 0.45, 0.61, and 0.68 for the same pairs. A low correlation was observed between the PCR test and each of the three IHC assays; however, if a patient tested low/negative by PCR, then they were likely to test negative by any single IHC test with a high probability (92–99%). Among patients who tested positive by PCR, only 9–45% tested positive by IHC assays. There was excellent positive and negative agreement (>91%) between 22C3 and SP263 staining using the recommended individual cutoffs for first-line treatment. PCR RNA expression analysis is not equivalent to IHC. However, this method may have some potential for the identification of PDL1-negative tumors. 22C3 could be considered as a substitute for SP263 in first-line treatment.

Abstract PD1-07: Exploratory analytical harmonization of PD-L1 immunohistochemistry assays in advanced triple-negative breast cancer: A retrospective substudy of IMpassion130

Abstract Background: In the Phase 3 IMpassion130 (NCT02425891) trial in metastatic triple-negative breast cancer (mTNBC), first-line atezolizumab + nab-paclitaxel (A+nP) significantly improved PFS vs placebo + nab-paclitaxel (P+nP) in the intent-to-treat (ITT) and PD-L1+ (PD-L1-stained immune cells [IC] ≥ 1% of the tumor area by VENTANA PD-L1 SP142 assay) populations. SP142 is currently the only validated assay for selecting patients who may derive benefit with A+nP. In post-hoc analyses of IMpassion130 (Rugo, ESMO 2019, submitted), PD-L1 status was also evaluated by Dako 22C3 and VENTANA SP263 assays. The SP142+ population (IC ≥ 1%; 46% prevalence) was captured within the 22C3 (CPS ≥ 1, 81% prevalence) and SP263 (IC ≥ 1%, 75% prevalence) subgroups, which identified more patients (pts) with PD-L1+ tumors. A+nP clinical activity was highest in pts identified as PD-L1+ by both SP142 and 22C3/SP263. HRs for clinical activity were lower in pts identified as PD-L1+ by 22C3/SP163, but PD-L1- by SP142. There was no suggestion of clinical benefit in pts identified as PD-L1- by both SP142 and 22C3/SP263. In the current retrospective exploratory analysis, we attempted to harmonize the PD-L1 assays by identifying cutoffs for 22C3 or SP263 that replicate the SP142 IC ≥ 1% population. Methods: Samples from IMpassion130 were assessed by a central laboratory for PD-L1 expression using VENTANA SP142 or SP263 IHC assays or Dako 22C3 IHC assay. Optimal cutoffs for 22C3 and SP263 were identified as those that maximize the analytical concordance (defined as overall percentage agreement; OPA) with the clinically validated SP142 IC 1% cutoff as the reference standard. Association with clinical activity was analyzed in the biomarker-evaluable population (BEP) in tumor samples from pts evaluated by the three PD-L1 assays. Results: In the BEP (n = 614; 68% of ITT), the intraclass correlation index (Spearman r) between SP142 IC and 22C3 CPS or SP263 IC was 0.57 and 0.69, respectively. The model-derived cutoffs with highest OPA (75%) for SP142 IC ≥ 1% were CPS 10 for 22C3 and IC 4% for SP263. Compared with our previous analyses at standard cutoffs (22C3 CPS 1; SP263 IC 1%), model-derived cutoffs resulted in negative percentage agreement increases from 45% to 74% (22C3) and from 34% to 77% (SP263), accompanied by positive percentage agreement reductions from 98% to 74% (22C3) and to 73% (SP263). These data suggest that the SP142 assay may identify a different population from the 22C3 or SP263 assays. For 22C3 at CPS 10, 36% of pts were SP142+/22C3+, but 10% were SP142+/22C3-, and 17% SP142-/22C3+. For SP263 at IC 4%, 34% of pts were SP142+/SP263+, but 12% were SP142+/SP263-, and 12% SP142-/SP263+. See table for prevalences, medians and HR point estimates for PFS and OS with the model-derived cutoffs. Conclusions: The suboptimal OPAs achieved with the model-derived cutoffs indicate that the assays could not be harmonized. Differences in SP142+ vs 22C3+ or SP263+ populations at model-derived cutoffs suggest that SP142, 22C3 and SP263 may not identify the same tumor biology. Additional data are required to understand these differences. Findings from these hypothesis-generating, post-hoc exploratory analyses based on mathematical modeling should be interpreted with caution. Currently, the VENTANA PD-L1 SP142 IHC assay (IC ≥ 1%) is the only clinically validated companion assay to select pts with mTNBC for A+nP treatment. Populationn (%)Median PFS, moPFS HRMedian OS, moOS HRA+nPP+nP(95% CI)A+nPP+nP(95% CI)BEP614 (100)7.45.40.7221.119.20.84(0.61, 0.86)(0.68, 1.03)SP142IC ≥ 1%285 (46)8.34.10.627.317.90.74(0.47, 0.78)(0.54, 1.01)IC < 1%329 (54)5.75.60.8620.820.70.95(0.68, 1.09)(0.72, 1.27)22C3CPS ≥ 10325 (53)7.55.50.712218.70.77(0.56, 0.91)(0.57, 1.03)CPS < 10289 (47)5.85.40.7320.219.40.94(0.57, 0.93)(0.69, 1.26)SP263IC ≥ 4%286 (47)8.75.50.6428.919.60.71(0.49, 0.83)(0.51, 0.98) Citation Format: Hope Rugo, Sherene Loi, Sylvia Adams, Peter Schmid, Andreas Schneeweiss, Carlos H Barrios, Hiroji Iwata, Véronique Diéras, Eric P Winer, Mark M Kockx, Dieter Peeters, Stephen Y Chui, Jennifer C Lin, Anh Nguyen Duc, Guiseppe Viale, Luciana Molinero, Leisha A Emens. Exploratory analytical harmonization of PD-L1 immunohistochemistry assays in advanced triple-negative breast cancer: A retrospective substudy of IMpassion130 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD1-07.