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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.

Objectives To develop a stochastic model relating measurement uncertainty, including reproducibility, to clinical accuracy, as demonstrated by the receiver operating characteristic curve. Methods A model is developed based on the symmetric case of the well-known binormal distribution. The overall distribution is partitioned further into analytical and biological components based on assumptions derived from the Cotlove criterion. Explicit mathematical solutions are derived and further verified by Monte Carlo analyses. Results The model demonstrates that tests with analytical error that conforms to the classic Cotlove criterion can achieve receiver operating characteristic curves with areas under the curve of 0.68 to 0.76 and Youden indices of 0.26 to 0.38 but have overall agreement for duplicate measurements of only 80% to 82%. Furthermore, the analytically accurate agreement is only 75% to 78%, and the clinically accurate agreement is only 50% to 60%. Conclusions The model suggests that assays may have reasonable clinical accuracy despite having reproducibility of less than 85%. Imperfect assays can substantially improve medical decision-making. The findings must be interpreted with caution given the binormal assumptions, but such assumptions are often useful as a first approximation. Practicing pathologists should feel comfortable performing semiquantitative assays shown to have a strong biological association with clinical outcome.

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.

PURPOSE Biomarkers that can predict response to anti–programmed cell death 1 (PD-1) therapy across multiple tumor types include a T-cell–inflamed gene-expression profile (GEP), programmed death ligand 1 (PD-L1) expression, and tumor mutational burden (TMB). Associations between these biomarkers and the clinical efficacy of pembrolizumab were evaluated in a clinical trial that encompassed 20 cohorts of patients with advanced solid tumors. METHODS KEYNOTE-028 ( ClinicalTrials.gov identifier: NCT02054806) is a nonrandomized, phase Ib trial that enrolled 475 patients with PD-L1–positive advanced solid tumors who were treated with pembrolizumab 10 mg/kg every 2 weeks for 2 years or until confirmed disease progression or unacceptable toxicity occurred. The primary end point was objective response rate (ORR; by RECIST v1.1, investigator review). Secondary end points included safety, progression-free survival (PFS), and overall survival (OS). Relationships between T-cell–inflamed GEP, PD-L1 expression, and TMB and antitumor activity were exploratory end points. RESULTS ORRs (with 95% CIs) ranged from 0% (0.0% to 14.2%) in pancreatic cancer to 33% (15.6% to 55.3%) in small-cell lung cancer. Across cohorts, median (95% CI) PFS ranged from 1.7 months (1.5 to 2.9 months) to 6.8 months (1.9 to 14.1 months) in pancreatic and thyroid cancers, respectively, and median OS from 3.9 months (2.8 to 5.5 months) to 21.1 months (9.1 to 22.4 months) in vulvar and carcinoid tumors, respectively. Higher response rates and longer PFS were demonstrated in tumors with higher T-cell–inflamed GEP, PD-L1 expression, and/or TMB. Correlations of TMB with GEP and PD-L1 were low. Response patterns indicate that patients with tumors that had high levels of both TMB and inflammatory markers (GEP or PD-L1) represent a population with the highest likelihood of response. Safety was similar and consistent with prior pembrolizumab reports. CONCLUSION A T-cell–-inflamed GEP, PD-L1 expression, and TMB predicted response to pembrolizumab in multiple tumor types. These biomarkers (alone/in combination) may help identify patients who have a higher likelihood of response to anti–PD-1 therapies across a broad spectrum of cancers.

Pembrolizumab, an anti-programmed death 1 monoclonal antibody, underwent a rapid clinical development program for the treatment of patients with malignancies in parallel to the development of a companion diagnostic for detecting programmed death ligand 1 (PD-L1) expression, which was considered to be a likely biomarker for response and cancer outcome. When investigating outcomes in patients with non-small cell lung cancer (NSCLC) treated with pembrolizumab, PD-L1 tumor proportion score (TPS) was initially identified as a marker of clinical efficacy, with a cutoff of ≥ 50%. This threshold was later reduced to TPS ≥ 1%, which demonstrated that pembrolizumab offered a clinical benefit in a broader population of patients with PD-L1-expressing tumors. As new evidence emerged, it also became clear that immune cell PD-L1 expression has an important role in predicting tumor response in patients treated with pembrolizumab; the combined positive score (CPS), which accounts for both tumor and immune cell PD-L1 expression, was then applied in clinical studies for indications outside of NSCLC. This review summarizes the outcomes of landmark studies in the KEYNOTE clinical development program that investigated the efficacy of pembrolizumab in patients with a variety of malignancies, the relative outcomes when patients were stratified by PD-L1 status, and how these observations have influenced the approved indications for pembrolizumab.

Background PD-L1 is expressed on both tumor and immune cells; however, the mechanism by which PD-L1 modulates the adaptive immune response on tumor versus immune cells may differ. Additionally, the prevalence of PD-L1 expression and the partitioning between tumor and immune compartments varies by tumor type. While PD-L1 expression on tumor or immune cells can be scored separately, the PD-L1 combined positive score (CPS) captures both tumor and immune cell expression in one aggregate score. We performed a retrospective, exploratory analysis of the effectiveness of CPS as an enrichment biomarker across several studies of pembrolizumab monotherapy in patients with multiple tumor types. Methods PD-L1 expression was assessed using PD-L1 IHC 22C3 pharmDx. Expression was measured using CPS (defined as the number of PD-L1–staining cells [tumor cells, lymphocytes, macrophages] divided by the total number of tumor cells, multiplied by 100) in tumor samples from single-arm (KEYNOTE-052 [UC], KEYNOTE-059 cohort 1 [G/GEJ], KEYNOTE-086 [TNBC], KEYNOTE-158 [cervical; SCLC], KEYNOTE-180 [EC], KEYNOTE-224 [HCC], KEYNOTE-427 [RCC]) and randomized (KEYNOTE-040 [HNSCC], KEYNOTE-045 [UC], KEYNOTE-061 [G/GEJ], KEYNOTE-119 [TNBC], KEYNOTE-240 [HCC]) pembrolizumab studies. Data were pooled across tumor types for pembrolizumab and for standard-of-care (in controlled studies), and then estimates of response rate, prevalence, and receiver operating characteristics (ROC) analysis were performed over various CPS cutpoints. CPS distribution by response, tumor type, and line of therapy were also assessed. Results There were 3769 treated patients with available PD-L1 CPS (pembrolizumab, n=2678; standard-of-care, n=1091). The area under the ROC curve for ORR was 0.63 (95% CI, 0.61–0.66) for pembrolizumab and 0.48 (95% CI, 0.43–0.53) for standard-of-care when a positive association was evaluated between CPS and ORR (figure 1); individual cutpoints of 1, 10, 20, and 50 were examined (table 1). Figure 2 shows a boxplot of CPS distribution for response in pembrolizumab-treated patients. Conclusions This retrospective, exploratory pan-tumor analysis demonstrates that CPS is an effective scoring method for measuring PD-L1 expression and can be used as a predictive biomarker to identify patients likely to respond to pembrolizumab monotherapy. CPS demonstrated enrichment of response to pembrolizumab monotherapy across most, but not all, tumor types, including some tumor types for which efficacy favors pembrolizumab regardless of PD-L1 expression, and for which a companion diagnostic is therefore not needed. In the randomized studies, CPS did not show a consistent association with ORR for standard-of-care therapy.

Background PD-L1 IHC 22C3 pharmDx uses Tumor Proportion Score (TPS) and Combined Positive Score (CPS) scoring algorithms for the immunohistochemical (IHC) evaluation of PD-L1 protein expression in human cancer tissues; both algorithms include PD-L1 staining tumor cells (TC) in scoring and CPS also includes scoring of PD-L1 staining mononuclear inflammatory cells to aid in the identification of patients for treatment with pembrolizumab (KEYTRUDA®) using indication-specific diagnostic cut-offs. This study evaluated contribution of TC in determining specimen diagnostic status based on the CPS scoring algorithm by looking into four tumor indications approved for use with KEYTRUDA®: gastric or gastroesophageal junction (GEJ) adenocarcinoma (GC/GEJ), urothelial carcinoma (UC), head and neck squamous cell carcinoma (HNSCC), and esophageal squamous cell carcinoma (ESCC). Detection of specimens expressing PD-L1 is significantly dependent on the PD-L1 staining TC component. Methods A retrospective analysis was done looking at Dako’s internal tumor bank of the mentioned indications that were all stained with PD-L1 IHC 22C3 pharmDx and scored using the TPS, CPS and Quantitative Immune Cell Density (QID) methods described in figure 1. Statistical analysis encompassed looking at the scores generated that met the following criteria: CPS>0, TPS>0 and CPS≠TPS and then evaluating the percentage of those samples that changed from positive to negative diagnostic status upon removal of the TC component from the scoring. Results A noticeable downward trend was observed in all four indications in the total number of positives with the removal of the TC component. Table 1 aptly captures this by showing the number of specimens for each indication that had changed from positive to negative around each indication’s diagnostic cut-off(s). The three indications that showed the highest percentages of diagnostic status change were HNSCC (CPS ≥20) with a remarkable 83.3% (130) followed by UC (CPS ≥10) at 46.3% (57) and ESCC (CPS ≥10) at 36.6% (45) of the specimens reclassified as negative. Conclusions PD-L1 IHC 22C3 pharmDx (Dako, USA) stains both TC and immune cells. Removal of the PD-L1 staining TC from the CPS algorithm reduces the number of specimens scored as positive for each indication’s respective diagnostic cut-off(s). Scoring only IC reduces the number of specimens scored as positive for each indication’s respective cutoff.

Tumor proportion score (TPS) and combined positive score ([CPS] includes immune cells), 2 methods for scoring programmed death ligand 1 (PD-L1) expression, have been used in clinical trials investigating the immune checkpoint inhibitor pembrolizumab in head and neck squamous cell carcinoma (HNSCC). These trials resulted in regulatory approval for pembrolizumab in the first- and second-line setting outside the United States. We performed a post hoc analysis of the KEYNOTE-040 study (NCT02252042) to determine whether CPS is a practical and suitable alternative scoring method to TPS. In KEYNOTE-040, patients with metastatic HNSCC received pembrolizumab or investigator choice of standard of care (SOC). The relative utility and equivalence of CPS ≥ 50 and TPS ≥ 50% for defining PD-L1 expression status in patients with HNSCC and comparability of scoring methods by tandem receiver operating characteristic (ROC) analysis were analyzed. The cutoff for each method was also evaluated. CPS ≥ 50 appeared equivalent to TPS ≥ 50% for predicting objective response rate (ORR), overall survival, and progression-free survival. ORR for pembrolizumab versus SOC was 26.2 versus 8.5% for TPS ≥ 50%, 28.1 versus 7.7% for CPS ≥ 50, 10.6 versus 11.6% for TPS

Context.— Regulatory approval of pembrolizumab for treatment of gastric and gastroesophageal junction (G/GEJ) adenocarcinoma required a reproducible scoring method for use of programmed death ligand-1 (PD-L1) protein expression as a companion diagnostic to identify likely responders to therapy. Objective.— To develop an immunohistochemical scoring algorithm that includes PD-L1 expression for tumor and immune cells, that is, the combined positive score. Design.— Four previously treated tumor types in the KEYNOTE-012 and KEYNOTE-028 studies were analyzed descriptively with a version of the PD-L1 immunohistochemical 22C3 pharmDx assay labeled for investigational use only to determine the relative importance of PD-L1 expression in tumor versus immune cells as a biomarker for pembrolizumab response. A combined positive score was developed as a novel scoring method and was compared with the tumor proportion score in cohort 1 from the KEYNOTE-059 study (G/GEJ cancer). External reproducibility was assessed. Results.— Per combined positive score cutoff of 1 or more, the prevalence of PD-L1 expression in patients with G/GEJ cancer was 57.6% (148 of 257 patients), with reasonable enrichment of responses (odds ratio, 2.8). Per tumor proportion score cutoff of 1% or more, prevalence was 12.5% (32 of 257 patients), with minimal enrichment (odds ratio, 1.4). External reproducibility assessments demonstrated interpathologist overall agreement of 96.6% (591 of 612; 95% CI, 94.0%–98.7%) and intrapathologist overall agreement of 97.2% (595 of 612; 95% CI, 95.3%–98.9%). Conclusions.— Combined positive score is a robust, reproducible PD-L1 scoring method that predicts response to pembrolizumab in patients with G/GEJ cancer. This novel scoring method supported US Food and Drug Administration approval of pembrolizumab as third-line therapy for G/GEJ cancer and has facilitated investigation in other indications.

Purpose Data are limited on programmed death ligand 1 (PD-L1) expression in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). Materials and methods We retrospectively evaluated the relationship between PD-L1 expression and recurrence-free survival (RFS) and overall survival in 319 patients with EGFR-mutant NSCLC who were treated at Samsung Medical Center from 2006 to 2014. Membranous PD-L1 expression on tumor cells was measured using the PD-L1 IHC 22C3 pharmDx antibody and reported as tumor proportion score (TPS). Kaplan-Meier methods, log-rank test, and Cox proportional hazards models were used for survival analysis. Results All patients had ≥1 EGFR mutation-54% in exon 19 and 39% in exon 21. Overall, 51% of patients had PD-L1-positive tumors. The prevalence of PD-L1 positivity was higher among patients with stages II-IV versus stage I disease (64% vs. 44%) and among patients with other EGFR mutations (75%) than with L858R mutation (39%) or exon 19 deletion (52%). PD-L1 positivity was associated with shorter RFS, with an adjusted hazard ratio of 1.52 (95% confidence interval [CI], 0.81 to 2.84; median, 18 months) for the PD-L1 TPS ≥ 50% group, 1.51 (95% CI, 1.02 to 2.21; median, 31 months) for the PD-L1 TPS 1%-49% group, and 1.51 (95% CI, 1.05 to 2.18) for the combined PD-L1-positive groups (TPS ≥ 1%) compared with the PD-L1-negative group (median, 35 months). Conclusion PD-L1 expression is associated with disease stage and type of EGFR mutation. PD-L1 positivity might be associated with worse RFS among patients with surgically treated EGFR-mutant NSCLC.

BackgroundPD-L1 IHC 22C3 pharmDx is an FDA-approved companion diagnostic for pembrolizumab across multiple tumor types designed for use on the Autostainer Link 48 (AL48). Many pathology laboratories do not have access to the AL48 and therefore do not use PD-L1 IHC 22C3 pharmDx but instead assess PD-L1 using laboratory-developed tests (LDTs) on the Ventana BenchMark platform. We compared our PD-L1 22C3 antibody-based LDT on the BenchMark XT platform with PD-L1 IHC 22C3 pharmDx using cervical cancer (CC), head and neck squamous cell carcinoma (HNSCC), urothelial carcinoma (UC), esophageal SCC (ESCC), and triple-negative breast cancer (TNBC) samples.MethodsTumor specimens from patients with CC, HNSCC, UC, ESCC, and TNBC were stained with the 22C3 antibody, scored using the LDT on the BenchMark XT as previously described,1 and compared with PD-L1 IHC 22C3 pharmDx scored by a trained pathologist, who measured PD-L1 with the use of combined positive score (CPS) and standard cutoffs (HNSCC and CC, ≥1; UC, ESCC, and TNBC, ≥10). Agreement in PD-L1 CPS as determined using the LDT and the PD-L1 IHC 22C3 pharmDx was evaluated.Results423 samples with CC (n = 77), HNSCC (n = 126), UC (n = 121), ESCC (n = 80), and TNBC (n = 19) were evaluated in this study. The pan-tumor (CC, HNSCC, UC, and ESCC) intraclass correlation coefficient (ICC) of PD-L1 CPS as a continuous variable was 0.95 (95% CI, 0.94%–0.96%); Spearman correlations were 0.95. ICC (95% CI) was 0.92 (0.88–0.95) for CC, 0.97 (0.96–0.98) for HNSCC, 0.95 (0.92–0.97) for UC, and 0.92 (0.88–0.95) for ESCC; Spearman correlation was 0.93, 0.96, 0.92, and 0.89, respectively. The overall percentage agreement at the respective CPS cutoff was 96% (CC), 96% (HNSCC), 96% (UC), and 90% (ESCC). Staining patterns by 22C3 LDT and PD-L1 IHC 22C3 pharmDx were also very similar in our TNBC pilot study; however, correlation was not calculated because of the small sample numbers.ConclusionsThe PD-L1 22C3 antibody-based LDT on the BenchMark XT demonstrated high concordance with PD-L1 IHC 22C3 pharmDx. These findings suggest the comparability of PD-L1 IHC 22C3 pharmDx with an LDT based on the 22C3 antibody across several tumor types. Further validation for TNBC is ongoing to confirm the data from the pilot run.ReferenceNeuman T, et al. J Thorac Oncol. 2016;11:1863–1868.

Background: PD-L1 IHC 22C3 pharmDx is an FDA-approved companion diagnostic to pembrolizumab across multiple tumor types designed for use on the Autostainer Link 48 (AL48). Many pathology laboratories do not have access to the AL48 and therefore do not use PD-L1 IHC 22C3 pharmDx to assess PD-L1 status. As a result, these laboratories often assess PD-L1 using laboratory-developed tests (LDTs) on the Ventana BenchMark platform. We compared our PD-L1 22C3 antibody-based LDT on the BenchMark XT platform with PD-L1 IHC 22C3 pharmDx. Data using head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma (UC) samples alone have previously been presented (Vainer GW et al. 31st European Congress of Pathology; September 7-11, 2019). In the current study, we performed a pan-tumor analysis that also included samples from patients with esophageal SCC (ESCC). Methods: The LDT used for this analysis has been described (Neuman T et al. J Thorac Oncol. 2016;11:1863-1868). Tumor specimens from patients with HNSCC, UC, and ESCC were stained with the 22C3 antibody, scored using the LDT on the BenchMark XT platform, and compared with PD-L1 IHC 22C3 pharmDx by one trained pathologist. PD-L1 was measured using the combined positive score (CPS) by the pathologist using standard cutoffs (HNSCC, ≥1; UC and ESCC, ≥10). After PD-L1 status was determined using the LDT and PD-L1 IHC 22C3 pharmDx, the agreement between both assays was measured. Results: Samples from 327 patients with HNSCC (n = 126), UC (n = 121), and ESCC (n = 80) were evaluated for this study. The pan-tumor intraclass correlation coefficient (ICC) of PD-L1 CPS as a continuous variable was 0.96 (95% CI, 0.95-0.97); Spearman correlation was 0.95. Among patients with ESCC, ICC was 0.92 (95% CI, 0.88-0.95) and Spearman correlation was 0.89. The clinical interpretation of PD-L1 status (CPS ≥10) for ESCC samples using the 22C3 antibody LDT on the BenchMark XT platform and PD-L1 IHC 22C3 pharmDx resulted in a negative percentage agreement of 92% (95% CI, 80-97), a positive percentage agreement of 88% (95% CI, 74-95), and an overall percentage agreement of 90% (95% CI, 81-95). Conclusions: The 22C3 antibody-based LDT on the BenchMark XT platform demonstrated high concordance with the FDA-approved PD-L1 IHC 22C3 pharmDx across tumor types and in patients with ESCC alone. These findings suggest the comparability of PD-L1 IHC 22C3 pharmDx with an LDT based on the 22C3 antibody across several tumor types. This analysis will be expanded to include additional indications for inclusion in the presentation. Citation Format: Gilad W. Vainer, Ghadeer Zatara, Lingkang Huang, Kenneth Emancipator, Shanthy Nuti. Analytical comparison of a PD-L1 22C3 antibody LDT protocol on the BenchMark XT and PD-L1 IHC 22C3 pharmDx: Analysis of pan-tumor and esophageal cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 503.

Background Pembrolizumab improved survival as first- and second-line therapy compared with chemotherapy in patients with highly programmed death ligand 1 (PD-L1) expressing advanced non-small cell lung cancer (NSCLC). We report the long-term safety and clinical activity of pembrolizumab as first-line therapy for patients with advanced NSCLC and the correlation between PD-L1 expression and efficacy. Patients and methods In the open-label phase 1b KEYNOTE-001 trial, treatment-naive patients with advanced NSCLC whose tumors expressed PD-L1 (≥1% staining, assessed using a prototype assay) were randomly assigned to intravenous pembrolizumab 2 or 10 mg/kg every 3 (Q3W) or 2 (Q2W) weeks. Response was assessed per central RECIST v1.1 every 9 weeks in all patients who received ≥1 pembrolizumab dose. Using pre-treatment tumor tissue, a clinical assay quantified the percentage of tumor cells expressing PD-L1 as tumor proportion score (TPS). Results Between 1 March 2013 and 18 September 2015, 101 patients received pembrolizumab 2 mg/kg Q3W (n = 6), 10 mg/kg Q3W (n = 49), or 10 mg/kg Q2W (n = 46). Of these, 27 (26.7%) had TPS ≥50%, 52 (51.5%) had TPS 1%-49%, and 12 (11.9%) had TPS

Introduction The Blueprint Programmed Death Ligand 1 (PD-L1) Immunohistochemistry (IHC) Assay Comparison Project is an industrial-academic collaborative partnership to provide information on the analytical and clinical comparability of four PD-L1 IHC assays used in clinical trials. Methods A total of 39 NSCLC tumors were stained with four PD-L1 IHC assays (22C3, 28-8, SP142, and SP263), as used in the clinical trials. Three experts in interpreting their respective assays independently evaluated the percentages of tumor and immune cells staining positive at any intensity. Clinical diagnostic performance was assessed through comparisons of patient classification above and below a selected expression cutoff and by agreement using various combinations of assays and cutoffs. Results Analytical comparison demonstrated that the percentage of PD-L1–stained tumor cells was comparable when the 22C3, 28-8, and SP263 assays were used, whereas the SP142 assay exhibited fewer stained tumor cells overall. The variability of immune cell staining across the four assays appears to be higher than for tumor cell staining. Of the 38 cases, 19 (50.0%) were classified above and five (13%) were classified below the selected cutoffs of all assays. For 14 of the 38 cases (37%), a different PD-L1 classification would be made depending on which assay/scoring system was used. Conclusions The Blueprint PD-L1 IHC Assay Comparison Project revealed that three of the four assays were closely aligned on tumor cell staining whereas the fourth showed consistently fewer tumor cells stained. All of the assays demonstrated immune cell staining, but with greater variability than with tumor cell staining. By comparing assays and cutoffs, the study indicated that despite similar analytical performance of PD-L1 expression for three assays, interchanging assays and cutoffs would lead to "misclassification" of PD-L1 status for some patients. More data are required to inform on the use of alternative staining assays upon which to read different specific therapy-related PD-L1 cutoffs.

Background Safety and efficacy of pembrolizumab, a humanized programmed death 1 monoclonal antibody, was assessed in KEYNOTE-028, a multicohort, phase Ib trial for patients with programmed death ligand 1 (PD-L1)-positive advanced solid tumors. We report results for the cohort of patients with advanced anal carcinoma. Patients and methods Patients with PD-L1-positive tumors (≥1%) received intravenous pembrolizumab 10 mg/kg once every 2 weeks for up to 2 years or until confirmed progression or unacceptable toxicity. Response was assessed every 8 weeks for the first 6 months and every 12 weeks thereafter per Response Evaluation Criteria In Solid Tumors, version 1.1. Primary endpoints were safety and overall response rate per investigator review. Secondary endpoints included progression-free survival, overall survival, and response duration. Data cutoff date was 1 July 2015. Results Of the 43 patients with advanced anal carcinoma evaluable for PD-L1 expression, 32 (74%) had PD-L1-positive tumors as assessed with the 22C3 prototype assay, of whom 25 were enrolled between April and September 2014. Sixteen patients (64%) experienced treatment-related adverse events; the most common ones were diarrhea and fatigue in four patients (16%) each and nausea in three patients (12%). There were no treatment-related deaths or discontinuations as of the data cutoff date. Among the 24 patients with squamous cell carcinoma histology, four had confirmed partial response, for an overall response rate of 17% [95% confidence interval (CI), 5%–37%) and 10 (42%) had confirmed stable disease, for a disease control rate of 58%. One additional patient with non-squamous histology had confirmed stable disease. Conclusion In this population of patients with PD-L1-positive advanced squamous cell anal carcinoma, pembrolizumab demonstrated a manageable safety profile and encouraging antitumor activity. These data support further study of pembrolizumab for this patient population. ClinicalTrials.gov NCT02054806.

Background In KEYNOTE-010, pembrolizumab versus docetaxel improved overall survival (OS) in patients with programmed death-1 protein (PD)-L1-positive advanced non-small-cell lung cancer (NSCLC). A prespecified exploratory analysis compared outcomes in patients based on PD-L1 expression in archival versus newly collected tumor samples using recently updated survival data. Patients and methods PD-L1 was assessed centrally by immunohistochemistry (22C3 antibody) in archival or newly collected tumor samples. Patients received pembrolizumab 2 or 10 mg/kg Q3W or docetaxel 75 mg/m2 Q3W for 24 months or until progression/intolerable toxicity/other reason. Response was assessed by RECIST v1.1 every 9 weeks, survival every 2 months. Primary end points were OS and progression-free survival (PFS) in tumor proportion score (TPS) ≥50% and ≥1%; pembrolizumab doses were pooled in this analysis. Results At date cut-off of 24 March 2017, median follow-up was 31 months (range 23–41) representing 18 additional months of follow-up from the primary analysis. Pembrolizumab versus docetaxel continued to improve OS in patients with previously treated, PD-L1-expressing advanced NSCLC; hazard ratio (HR) was 0.66 [95% confidence interval (CI): 0.57, 0.77]. Of 1033 patients analyzed, 455(44%) were enrolled based on archival samples and 578 (56%) on newly collected tumor samples. Approximately 40% of archival samples and 45% of newly collected tumor samples were PD-L1 TPS ≥50%. For TPS ≥50%, the OS HRs were 0.64 (95% CI: 0.45, 0.91) and 0.40 (95% CI: 0.28, 0.56) for archival and newly collected samples, respectively. In patients with TPS ≥1%, OS HRs were 0.74 (95% CI: 0.59, 0.93) and 0.59 (95% CI: 0.48, 0.73) for archival and newly collected samples, respectively. In TPS ≥50%, PFS HRs were similar across archival [0.63 (95% CI: 0.45, 0.89)] and newly collected samples [0.53 (95% CI: 0.38, 0.72)]. In patients with TPS ≥1%, PFS HRs were similar across archival [0.82 (95% CI: 0.66, 1.02)] and newly collected samples [0.83 (95% CI: 0.68, 1.02)]. Conclusion Pembrolizumab continued to improve OS over docetaxel in intention to treat population and in subsets of patients with newly collected and archival samples. Trial registration ClinicalTrials.gov: NCT01905657.

There is a broad appreciation for the lack of standard operating procedures in the collection, processing, shipping, and storage of tissue samples obtained during clinical trials. This is intensified in the context of clinical sample collection that may include many different sites around the world. This chapter highlights major preanalytic concerns that may impact immunohistochemical biomarker analyses in clinical trials, focusing on key decisions to be made regarding what types and categories of samples to collect. Sample collection logistics for clinical trials are described, ranging from sample collection using a central lab-provided kit through centralized testing of lab biomarker analysis. In addition, sample consent, sample tracking, and companion diagnostic considerations are highlighted in the light of the complexity of performing clinical trial sample analyses.

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.

The association between programmed death ligand 1 (PD-L1) expression and survival among melanoma patients treated with chemotherapy is unclear.1-5 We retrospectively characterized PD-L1 levels in patients with melanoma treated at Aarhus University Hospital in Denmark (2001-2012) using a prototype immunohistochemistry assay (QualTek Molecular Laboratories, Goleta, California, USA) and investigated the association between PD-L1 and overall survival (OS) in patients who received chemotherapy or immunotherapy (interleukin-2, interferon, ipilimumab). None of the patients received pembrolizumab or nivolumab. This article is protected by copyright. All rights reserved.

Background Predictive biomarkers of patients likely to benefit from anti–programmed death 1 inhibitor therapy have clinical relevance. We examined whether line of therapy or tumour programmed death ligand 1 (PD-L1) expression affects the efficacy and safety of pembrolizumab, compared with ipilimumab, in advanced melanoma. Methods Of 834 patients enrolled in the randomised, open-label phase III KEYNOTE-006 study, 833 were included in this analysis. Patients were randomly assigned 1:1:1 to receive pembrolizumab 10 mg/kg every 2 or 3 weeks (for 24 months) or ipilimumab 3 mg/kg every 3 weeks (for four doses) until disease progression/intolerable toxicity. This analysis evaluated progression-free survival (PFS), overall survival (OS) and objective response rate (ORR). Data cut-off: 03 November 2016. Results Of the patients, 60.3% were male, 65.9% were treatment naive and 80.6% had PD-L1–positive tumours (median follow-up was 33.9 months). Twenty-four–month survival rates were higher with pembrolizumab than with ipilimumab in treatment-naive (PFS 31.0% versus 14.6%; OS 58.0% versus 44.7%) and previously treated patients (PFS 25.7% versus 11.3%; OS 49.2% versus 37.9%). Twenty-four–month survival rates were higher with pembrolizumab than with ipilimumab in patients with PD-L1–positive tumours (PFS 33.2% versus 13.1%; OS 58.4% versus 45.0%) and similar in PD-L1–negative tumours (PFS 14.9% versus NR [no data at 24 months for a PFS estimate]; OS 43.6% versus 31.8%). Safety of pembrolizumab by subgroup was consistent with previous reports. Conclusions Findings support pembrolizumab monotherapy as standard of care in patients with advanced melanoma, regardless of first- or second-line therapy or PD-L1 status. ClinicalTrials.gov identifier NCT01866319 .

Background Two techniques are available for scoring programmed death ligand 1 (PD-L1) expression: tumor proportion score (TPS), which measures PD-L1 in tumor cells, and combined positive score (CPS), which measures PD-L1 in tumor and immune cells. The European Medicines Agency approved pembrolizumab for the second-line treatment of patients with HNSCC whose tumors express PD-L1 based on a TPS ≥50%. To determine whether CPS can be used interchangeably with TPS to evaluate patients with HNSCC for second-line therapy, a post hoc analysis from the open-label phase 3 KEYNOTE-040 (NCT02252042) trial was conducted. Methods Patients with platinum-refractory HNSCC received pembrolizumab (200 mg Q3W) or standard of care (SOC; chemotherapy or cetuximab). Tumor samples were collected during screening (up to 42 days before baseline) and were evaluated by TPS and CPS. Scoring methods were evaluated for efficacy outcomes, including objective response rate (RECIST v1.1), progression-free survival, and overall survival. Results Of 495 patients enrolled, 475 were assessed for PD-L1 expression per TPS and CPS. Efficacy results based on TPS and CPS cutpoints are summarized in the table. Concordance between TPS and CPS cutpoints was 77% at 1, 91% at 20, and 95% at 50. Conclusions At lower expression levels, CPS detects a larger fraction of responders than TPS while maintaining similar survival results. At higher expression levels, CPS ≥50 can be used interchangeably with TPS ≥50%. Thus, CPS is a valid scoring method for determining PD-L1 status in patients with HNSCC being evaluated for second-line treatment. Efficacy results demonstrating treatment benefit with pembrolizumab compared with SOC were similar regardless of scoring method used. Clinical trial identification NCT02252042. Editorial acknowledgement Holly C. Cappelli, PhD, and Dana Francis, PhD, of the ApotheCom pembrolizumab team (Yardley, PA, USA), funded by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. Legal entity responsible for the study Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. Funding Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. Disclosure E. Cohen: Advisory / Consultancy: Amgen, AstraZeneca, Bayer, BMS, Incyte, MSD, Merck. K. Harrington: Research grant / Funding (institution), For trial conduct: MSD, AstraZeneca, Amgen, BMS, Boehringer-Ingelheim, Merck-Serono, Pfizer, Replimune, Vyriad; Advisory / Consultancy: MSD, AstraZeneca, Amgen, BMS, Boehringer-Ingelheim, Merck-Serono, Pfizer, Replimune, Vyriad; Speaker Bureau / Expert testimony: MSD, AstraZeneca, Amgen, BMS, Merck-Serono; Honoraria (self): MSD, AstraZeneca, Amgen, BMS, Boehringer-Ingelheim, Merck-Serono, Pfizer, Replimune, Vyriad. D. Soulieres: Research grant / Funding (institution), Trial conduct: MSD; Advisory / Consultancy: Merck, MSD; Speaker Bureau / Expert testimony: Merck, MSD. C. Le Tourneau: Advisory / Consultancy: MSD, BMS, Merck Serono, Amgen, Nanobiotix, AstraZeneca, Roche. L.F. Licitra: Honoraria (self): Eisai, BMS, MSD, Merck–Serono, Boehringer Ingelheim, Novartis, AstraZeneca, Roche, Bayer, Debiopharm, Sobi, Kura Oncology, Health & Life srl, Ipsen Innovation, Immuno-Oncology Hub, Incyte Biosciences Italy srl, Doxa Pharma srl, Amgen and Nanobiotics Sa.; Advisory / Consultancy, Research grant / Funding (institution): Bayer, GSK, MSD, Kura Oncology, Ipsen, Health & Life srl, Merck–Serono, Doxa Pharma srl.; Research grant / Funding (institution): Eisai, MSD, Merck–Serono, Boehringer Ingelheim, Novartis, AstraZeneca, Roche, BMS, Celgene International, Exelixis inc, Hoffmann-La roche ltd, IRX Therapeutics inc, Medpace inc and Pfizer.; Travel / Accommodation / Expenses: Merck–Serono, BMS, MSD, Debiopharm, Sobi, Bayer, Stilema, AccMed, Aiocc, Aiom. B. Burtness: Research grant / Funding (institution), For trial conduct: MSD, Boehringer-Ingelheim; Honoraria (self), For steering committee: MSD; Honoraria (self), Travel / Accommodation / Expenses, For steering committee and presentation of trial results: Boehringer-Ingelheim; Advisory / Consultancy: Amgen, Alligator Biosciences, Aduro; Honoraria (self), Advisory / Consultancy: Bayer, AstraZeneca, Cue Biosciences, BMS, Genentech/Roche; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Celgene, Debiopharm, Maverick Therapeutics, GSK; Honoraria (self), Data Safety Monitoring Commttee: VentiRx; Honoraria (self), Travel / Accommodation / Expenses, Data Safety Monitoring Committ: IDDI. T. Bal: Shareholder / Stockholder / Stock options, Full / Part-time employment: Merck & Co., Inc. J. Juco: Full / Part-time employment: Merck & Co., Inc.; Shareholder / Stockholder / Stock options: Merck & Co., Inc., Regeneron, Illumina. R.F. Swaby: Shareholder / Stockholder / Stock options, Full / Part-time employment: Merck & Co., Inc. K. Emancipator: Full / Part-time employment: Merck & Co., Inc.; Shareholder / Stockholder / Stock options: Merck & Co., Inc., Johnson & Johnson, Bayer AG, Celgene; Full / Part-time employment, Spouse: Celgene. All other authors have declared no conflicts of interest.

BackgroundWe assessed the efficacy and safety of programmed cell death 1 (PD-1) inhibition with pembrolizumab in patients with advanced non–small-cell lung cancer enrolled in a phase 1 study. We also sought to define and validate an expression level of the PD-1 ligand 1 (PD-L1) that is associated with the likelihood of clinical benefit. MethodsWe assigned 495 patients receiving pembrolizumab (at a dose of either 2 mg or 10 mg per kilogram of body weight every 3 weeks or 10 mg per kilogram every 2 weeks) to either a training group (182 patients) or a validation group (313 patients). We assessed PD-L1 expression in tumor samples using immunohistochemical analysis, with results reported as the percentage of neoplastic cells with staining for membranous PD-L1 (proportion score). Response was assessed every 9 weeks by central review. ResultsCommon side effects that were attributed to pembrolizumab were fatigue, pruritus, and decreased appetite, with no clear difference according to dose or schedule. Among all ...

Introduction: The PD-1 receptor-ligand pathway can be used by tumors to evade immune surveillance, thereby allowing neoplastic growth. Pembrolizumab is a highly selective, humanized IgG4/kappa isotype mAb designed to block PD-1 interaction with its ligands PD-L1 and PD-L2, thereby reactivating the immune system to eradicate tumors. Methods: This is a multi-center, non-randomized trial of single agent MK-3475 treatment given intravenously at 10 mg/kg every 2 weeks, in patients with recurrent/metastatic triple-negative (ER, PR, and HER2 negative) breast cancer (TNBC). PD-L1 expression in tumor or stroma was required for study entry. PD-L1 status was determined by immunohistochemical analysis of patient’s tumor tissues using the Merck proprietary 22C3 antibody. Primary objectives of this study were to determine the safety, tolerability, and anti-tumor activity of MK-3475 in patients with PD-L1 positive, advanced TNBC. Secondary objectives included assessments of progression-free survival, overall survival, and response duration. Adverse events (AEs) reported in any patient receiving at least 1 dose of study treatment were monitored and graded using NCI CTCAE v. 4.0. Radiographic imaging was obtained every 8 weeks and evaluated by both investigator and an independent radiologist to assess clinical responses as defined by RECIST 1.1. This study (Clinicaltrials.gov: NCT01848834) is being conducted in conformance with Good Clinical Practices. Results: A total of 32 female patients with a median age of 50.5 years (range 29 – 72 years) with PD-L1 positive, recurrent/metastatic TNBC were enrolled in the study. Most of these patients had received and progressed on multiple lines of therapy for advanced disease. A preliminary analysis of data collected as of 23May2014 indicates that 5 patients (15.6%) experienced at least one drug-related serious adverse event (SAE); each of 4 patients experienced one of the following: Grade 3 anemia, headache, aseptic meningitis or pyrexia, and a fifth patient experienced disseminated intravascular coagulation (DIC) with thrombocytopenia and decreased blood fibrinogen. The patient who experienced DIC died. Preliminary analysis of data collected from investigators as of 23May2014 indicates that no patient had a complete response, 16.1% of patients had a partial response, 9.7% had stable disease, and 64.5% had progressive disease. As of 23May2014, all but one of the responders, in addition to three patients with stable disease, remain on treatment. Conclusion: This is the first report of clinical activity of an immune checkpoint inhibitor in TNBC. The preliminary results from this study suggest that single agent MK-3475 is a well-tolerated and effective treatment with significant therapeutic activity in a subset of heavily pre-treated patients with recurrent/metastatic triple-negative breast cancer. Citation Format: Rita Nanda, Laura Q Chow, E Claire Dees, Raanan Berger, Shilpa Gupta, Ravit Geva, Lajos Pusztai, Marisa Dolled-Filhart, Kenneth Emancipator, Edward J Gonzalez, Jennifer Houp, Kumudu Pathiraja, Vassiliki Karantza, Robert Iannone, Christine K Gause, Johnathan D Cheng, Laurence Buisseret. A phase Ib study of pembrolizumab (MK-3475) in patients with advanced triple-negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S1-09.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecological system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

BACKGROUND: Programmed death ligand 1 (PD-L1) expression may predict response to anti-programmed death 1 (anti-PD-1) or anti-PD-L1 treatment. There is limited information on changes in PD-L1 expression over time in patients with non-small cell lung cancer (NSCLC).PATIENTS AND METHODS: Eligible patients with NSCLC who received surgery or underwent biopsy at Samsung Medical Center, Seoul, Republic of Korea, and Aarhus University Hospital, Aarhus, Denmark, between February 2004 and April 2012 were included. PD-L1 expression in paired tumor tissue samples from the same patients at different dates and lesions was measured using a laboratory-developed prototype immunohistochemistry assay (22C3 antibody). PD-L1 positivity was defined as tumor cell membrane positivity in ≥ 1% of tumor cells (proportion score). Concordance of PD-L1 expression was analyzed by treating proportion score as categoric or continuous variables.RESULTS: Ninety-one patients were included in the analysis. The median interval between the 2 tumor collection dates was 20 months, with 91% of paired samples collected > 3 months apart. The concordance rate for PD-L1 classification between paired samples was 67% (95% confidence interval, 57%-77%). When treating the immunohistochemistry proportional score as a continuous variable, a significant correlation of PD-L1 expression was observed between the paired samples (Pearson correlation coefficient, 0.61; P < .001).CONCLUSION: There are good correlations of PD-L1 expression from paired NSCLC samples. For patients whose PD-L1 status is negative, it may be valuable to obtain additional tissue samples for retesting PD-L1 expression when anti-PD-1 immunotherapy is considered.

Purpose Data are limited on programmed death ligand 1 (PD-L1) expression in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). Materials and Methods We retrospectively evaluated the relationship between PD-L1 expression and recurrence-free survival (RFS) and overall survival in 319 patients with EGFR-mutant NSCLC who were treated at Samsung Medical Center from 2006 to 2014. Membranous PD-L1 expression on tumor cells was measured using the PD-L1 IHC 22C3 pharmDx antibody and reported as tumor proportion score (TPS). Kaplan-Meier methods, log-rank test, and Cox proportional hazards models were used for survival analysis. Results All patients had ≥1 EGFR mutation—54% in exon 19 and 39% in exon 21. Overall, 51% of patients had PD-L1–positive tumors. The prevalence of PD-L1 positivity was higher among patients with stages II-IV versus stage I disease (64% vs. 44%) and among patients with other EGFR mutations (75%) than with L858R mutation (39%) or exon 19 deletion (52%). PD-L1 positivity was associated with shorter RFS, with an adjusted hazard ratio of 1.52 (95% confidence interval [CI], 0.81 to 2.84; median, 18 months) for the PD-L1 TPS ≥ 50% group, 1.51 (95% CI, 1.02 to 2.21; median, 31 months) for the PD-L1 TPS 1%-49% group, and 1.51 (95% CI, 1.05 to 2.18) for the combined PD-L1–positive groups (TPS ≥ 1%) compared with the PD-L1–negative group (median, 35 months). Conclusion PD-L1 expression is associated with disease stage and type of EGFR mutation. PD-L1 positivity might be associated with worse RFS among patients with surgically treated EGFR-mutant NSCLC.

Purpose Expression of programmed death-ligand 1 (PD-L1) is a potential predictive marker for response and outcome after treatment with anti–programmed death 1 (PD-1). This study explored the relationship between anti–PD-1 activity and PD-L1 expression in patients with advanced melanoma who were treated with pembrolizumab in the phase Ib KEYNOTE-001 study (clinical trial information: NCT01295827). Patients and Methods Six hundred fifty-five patients received pembrolizumab10 mg/kg once every 2 weeks or once every 3 weeks, or 2 mg/kg once every 3 weeks. Tumor response was assessed every 12 weeks per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 by independent central review. Primary outcome was objective response rate. Secondary outcomes included progression-free survival (PFS) and overall survival (OS). Membranous PD-L1 expression in tumor and tumor-associated immune cells was assessed by a clinical trial immunohistochemistry assay (22C3 antibody) and scored on a unique melanoma (MEL) scale of 0 to 5 by one of three pathologists who were blinded to clinical outcome; a score ≥ 2 (membranous staining in ≥ 1% of cells) was considered positive. Results Of 451 patients with evaluable PD-L1 expression, 344 (76%) had PD-L1–positive tumors. Demographic and staging variables were equally distributed among PD-L1–positive and –negative patients. An association between higher MEL score and higher response rate and longer PFS (hazard ratio, 0.76; 95% CI, 0.71 to 0.82) and OS (hazard ratio, 0.76; 95% CI, 0.69 to 0.83) was observed ( P < .001 for each). Objective response rate was 8%, 12%, 22%, 43%, 57%, and 53% for MEL 0, 1, 2, 3, 4, and 5, respectively. Conclusion PD-L1 expression in pretreatment tumor biopsy samples was correlated with response rate, PFS, and OS; however, patients with PD-L1–negative tumors may also achieve durable responses.

Context.—Programmed death ligand-1 (PD-L1) expression by tumors may enable them to avoid immunosurveillance.Objective.—To develop a PD-L1 immunohistochemical assay using the 22C3 anti–PD-L1 murine monoclonal antibody on the Dako platform as a possible companion diagnostic for pembrolizumab in patients with non–small cell lung cancer.Design.—Tumor samples from 146 patients with non–small cell lung cancer treated with pembrolizumab in KEYNOTE-001 and for whom response data were available were scored according to their staining intensity by a single pathologist using 4 methods: percentage of tumor cells staining at any intensity (PS1), moderate/strong intensity (PS2), strong intensity (PS3), and H-score (PS1 + PS2 + PS3). The cutoff score for predicting response to pembrolizumab was determined using receiver operating characteristic analysis. Progression-free and overall survival were assessed in patients with measurable disease per Response Evaluation Criteria in Solid Tumors, version 1.1 (n = 146).Results.—The 4 scoring methods assessed performed similarly; PS1 with a 50% cutoff score is the simplest and easiest method to implement in practice. Response to pembrolizumab was observed in 19 of 44 patients (43%) with a PS1 score of 50% or higher and 8 of 102 patients (8%) with PS1 lower than 50% (odds ratio, 8.93). Median progression-free and overall survival was 4.0 months and not yet reached, respectively, for patients with a PS1 of 50% or higher, and 2.1 and 6.1 months, respectively, for those with PS1 lower than 50%.Conclusion.—The PD-L1 immunohistochemical assay shows the potential for enrichment of trial populations and as a companion diagnostic tool in non–small cell lung cancer.

A companion diagnostic assay was codeveloped by Dako for pembrolizumab non-small-cell lung cancer clinical trials to detect PD-L1 expression by immunohistochemistry (IHC). This automated IHC assay has been analytically verified and validated using Dako's autostainer Link 48 and 22C3 mouse anti-PD-L1 monoclonal antibody to detect the PD-L1 expression in formalin-fixed paraffin-embedded human tumor tissue specimens. The PD-L1 22C3 IHC assay was optimized for high sensitivity and specificity. Repeatability and reproducibility studies were conducted at Dako and at 3 Clinical Laboratory Improvement Amendments certified laboratories during assay development. The studies included: intersite and intrasite, interobserver and intraobserver, interinstrument, interoperator, interday, and interlot, and intraday and intrarun. All precision studies performed at Dako and external laboratories achieved >85% point-estimate agreements for all 3 agreement types (negative, positive, and overall). A clinical cutoff (tumor proportion score ≥50%) of PD-L1 expression was determined and evaluated through a phase 1 clinical trial (KEYNOTE-001) for advanced non-small-cell lung cancer patients treated with pembrolizumab. The treatment effect of pembrolizumab in the 61 subjects who had a tumor PD-L1 of tumor proportion score ≥50% was substantial, with an overall response rate of 41% (95% confidence interval, 28.6-54.3) as compared with 20.6% (95% confidence interval, 15.5-26.5) observed in the 223 subjects irrespective of PD-L1 status. PD-L1 IHC 22C3 pharmDx is a sensitive, precise, and robust companion diagnostic assay, which will facilitate safe and effective use for pembrolizumab in cancer patients.

Immunohistochemistry (IHC) is an essential tool for visualization and assessment of protein expression in sections of tissue in the context of sample morphology. IHC is utilized in preclinical applications to determine target engagement and pharmacodynamics, as well as proof of concept studies. However, transitioning an assay from the preclinical stage to implementation for human clinical trial sample analyses can be challenging for a number of reasons. For example, correlation of preclinical results on surrogate tissues may not be indicative of the final results in humans and assay conditions and reagents may need to be changed for optimal use on human formalin-fixed paraffin-embedded (FFPE) tissue. In clinical trials, IHC can be deployed to address different types of biomarker needs, such as for prospective patient enrollment where the assay validated to stringent clinical laboratory standards, or as a research assay used solely to generate observational data. Using a chromogen-based oncology IHC biomarker analyses as an example, this chapter will cover a range of considerations for implementation of IHC assays in clinical trials, from sample collection considerations through assay validation and implementation.

Summary Background Expression of PD-L1 has been shown to be upregulated in some patients with gastric cancer. As part of the phase 1b KEYNOTE-012 study, we aimed to assess the safety and activity of the anti-PD-1 antibody pembrolizumab in patients with PD-L1-positive recurrent or metastatic adenocarcinoma of the stomach or gastro-oesophageal junction. Methods This study was a multicentre, open-label, phase 1b trial done at 13 cancer research centres in the USA, Israel, Japan, South Korea, and Taiwan. We enrolled patients with PD-L1-positive recurrent or metastatic adenocarcinoma of the stomach or gastro-oesophageal junction. Patients received intravenous pembrolizumab at 10 mg/kg once every 2 weeks for 24 months or until progression or unacceptable toxic effects occurred. Response was assessed every 8 weeks in accordance with Response Evaluation Criteria in Solid Tumors version 1.1. The primary objectives were safety in patients who received at least one dose of pembrolizumab and the proportion of patients achieving overall responses in patients who received at least one pembrolizumab dose and who either had a post-baseline scan or who discontinued therapy because of clinical disease progression or a treatment-related adverse event before the first post-baseline scan. The study is registered with ClinicalTrials.gov, number NCT01848834, and is ongoing but no longer enrolling patients. Findings From Oct 23, 2013, to May 5, 2014, 39 patients were enrolled. 36 were evaluable for response by central assessment. Eight (22%, 95% CI 10–39) patients were judged to have had an overall response at central review; all responses were partial. All 39 patients were included in the safety analyses. Five (13%) patients had a total of six grade 3 or 4 treatment-related adverse events, consisting of two cases of grade 3 fatigue, one case each of grade 3 pemphigoid, grade 3 hypothyroidism, and grade 3 peripheral sensory neuropathy, and one case of grade 4 pneumonitis. No treatment-related deaths occurred. Interpretation In this population of patients with recurrent or metastatic PD-L1-positive gastric cancer, pembrolizumab had a manageable toxicity profile and promising antitumour activity, warranting further study in phase 2 and 3 trials. Funding Merck & Co.

Purpose We explored the association between different molecular biomarkers and PD-L1 mRNA and protein expression in Asian patients with lung adenocarcinoma. Experimental design PD-L1 protein expression level was evaluated using a prototype immunohistochemistry (IHC) assay with the 22C3 antibody in tumor samples from 157 patients with lung adenocarcinoma from Samsung Medical Center. Other biomarkers, including PD-L1, PD-L2 and IFN-γ mRNA expression detected using the Affymetrix array, EGFR and KRAS mutation detected using the Sequenom platform, ALK fusion detected using the NanoString system and tumor mutation burden (TMB) calculated as total number of non-synonymous single nucleotide substitution from Illumina HiSeq 2000 sequencing platform were provided as part of the Asian Cancer Research Group collaboration. Spearman correlation test, t test, Fisher exact test, and multivariate regression models were utilized to test the association between different biomarkers and PD-L1 mRNA/protein expression. PD-L1 IHC tumor proportion score was analyzed as a continuous or categorical variable, where PD-L1 strong and weak positivity were defined to be traceable to the 1% and 50% cutoffs used in the clinical trial version of the assay. Results The median age was 61 years (range, 20-84), 50% were female, 40% were smokers, and 77% had stage I/II disease. There was strong correlation between PD-L1 mRNA and protein expression (N = 79, Spearman R = 0.762, P < 0.0001). Absence of EGFR mutation was associated with higher PD-L1 expression (for mRNA, N = 166, t test P = 0.023; for protein, N = 83, Fisher test P = 0.051), and presence of ALK fusion was associated with higher PD-L1 expression (for mRNA, N = 229, t test P = 0.001; for protein, N = 144, Fisher test P = 0.002). Similar associations between EGFR mutation, ALK fusion, and PD-L1 mRNA and protein expression were observed after adjusting for age, sex, smoking status, and disease stage. No association was found between PD-L1 expression and KRAS mutation (for mRNA, N = 140, t test P = 0.305; for protein, N = 78, Fisher test P = 0.243). mRNA expression of PD-L2, the other PD-1 ligand, was highly correlated with PD-L1 expression (for mRNA, N = 253, Spearman R = 0.650, P < 0.0001; for protein, N = 79, Spearman R = 0.444, P < 0.0001). As evidence for IFN-γ-inducible biology, IFN-γ mRNA expression was significantly correlated with PD-L1 expression (for mRNA, N = 253, Spearman R = 0.57, P < 0.0001; for protein, N = 79, Spearman R = 0.44, P < 0.0001). TMB was not significantly correlated with PD-L1 expression (for mRNA, N = 31, Spearman R = 0.092, P = 0.62; for protein, N = 8, Spearman R = 0.19, P = 0.65), which suggested independent predictive ability for anti-PD-1 therapy. Conclusion Our study provides evidence that DNA and RNA features, including PD-L1 mRNA, lack of EGFR mutation, and ALK fusion are associated with PD-L1 protein expression for Asian lung adenocarcinoma patients. Citation Format: Xiaoqiao Liu, Jinseon Lee, Soo Jung Lee, Jong Ho Cho, Jong-Mu Sun, Lu Zhang, Yunfei Pei, Lan Chen, Marisa Dolled-Filhart, Kenneth Emancipator, Jared Lunceford, Ronghua Chen, Wei Zhou, Jhingook Kim. Molecular biomarker study of programmed death receptor ligand 1 (PD-L1) in Korean patients with lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4213.

4065Background: TPS, the percentage of viable tumor cells with partial or complete membrane staining at any intensity, has been invaluable for assessing PD-L1 expression in non–small cell lung canc...

The aim of our analysis was to evaluate the prognostic effect of programmed cell death ligand-1 (PD-L1) expression in patients with non-small cell lung cancer (NSCLC).PD-L1 expression among 1070 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. Data were analyzed using Cox proportional hazard models adjusting for age, sex, smoking status, histologic type, stage, and performance status.Sixty-eight patients (6%) were strongly PD-L1 positive and 410 (38%) were weakly PD-L1 positive. A significantly higher prevalence of PD-L1 positivity was observed among patients with squamous cell carcinoma and among stage IIIB and IV patients. PD-L1 expression may be associated with poorer overall survival, with an adjusted hazard ratio of 1.56 (95% confidence interval [CI]: 1.08-2.26, p = 0.02) for strong PD-L1 positivity, 1.18 (95% CI: 0.96-1.46; p = 0.12) for weak PD-L1 positivity, and 1.23 (95% CI: 1.00-1.51; p = 0.05) for the combined strongly and weakly positive groups compared with PD-L1 negativity. Negative prognostic effect of PD-L1 expression was not statistically significant after adjustment for postoperative chemotherapy or radiotherapy. Similar results were observed for progression-free survival. Among stage I patients, the disease recurrence rate was higher in the PD-L1-positive versus in the PD-L1-negative group (48% versus 27%, p0.001), with an adjusted hazard ratio for disease-free survival of 2.01 (95% CI, 1.08-3.73; p = 0.03) for strong PD-L1 positivity and 1.57 (95% CI, 1.17-2.11; p = 0.003) for weak PD-L1 positivity compared with PD-L1 negativity.Tumor PD-L1 expression may be associated with poor prognosis in patients with NSCLC, although its significance weakens when postoperative therapy is considered.