Abstract Background: Eosinophils are innate immune granulocytes that migrate to areas of inflammation to combat against infection and disease. Best known for their detrimental role in asthma and allergic disorders, there is growing interest in the involvement of eosinophils in cancer. Eosinophils are routinely observed in the tumor microenvironment (TME) and, depending on the cancer type, have been shown to drive other immune cells to either suppress or promote tumor growth - In colorectal cancer (CRC), eosinophil infiltration into the TME has been linked to a favorable prognosis. However, the behavior of eosinophils and their effect on associated immune mediators in the TME remains poorly understood. Currently, eosinophils are primarily identified from H&E stained tissue sections based on morphological features by a pathologist, but it can be challenging to reliably and efficiently identify all eosinophils based on morphology alone. Here, we present a novel image analysis workflow that established an AI-based cell classifier which can accurately quantify eosinophils in H&E stained CRC tissue sections by leveraging biomarker staining of eosinophils using multiplex immunofluorescence (mIF) imaging in conjunction with the morphological characteristics of eosinophils observed by H&E. Methods: CRC tissue sections were labeled with a 5plex panel of eosinophil-specific markers and imaged by mIF using the PhenoImager HT platform (Akoya). Biomarker fluorescence was then quenched, sections stained by H&E, and reimaged using the PhenoImager. mIF and H&E images were then imported into the HALO® platform for algorithm development. Halo AI cell classification of eosinophils was trained on the morphological features of eosinophil staining in H&E images, guided by eosinophil-specific labeling in the 5plex mIF images. H&E staining was also performed on a serial section of each specimen. Morphological identification of eosinophils in H&E images was performed by a pathologist. Results: Correlation analysis was performed to evaluate the relationship, per high power field (HPF), between manual eosinophil counts by a pathologist and AI algorithm derived eosinophils counts. The results showed the two methods are highly correlated, demonstrating reliable algorithm performance. Conclusion: The AI-based eosinophil detection algorithm established here enables high-throughput analysis and quantification of eosinophils from H&E stained CRC tissue specimens and facilitates morphology diagnosis. Citation Format: Arezoo Hanifi, Elizabeth Blain, James Hargrove, Jeff Lock, Nam Tran, Vladislav Chizhevsky, Qingyan Au. Development of an AI-based algorithm to quantify eosinophils in H&E images from colorectal cancer (CRC) tissue sections guided by biomarker staining using multiplex immunofluorescence imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2352.

Abstract Background: Programmed death-1/programmed death ligand 1 (PD-[L]1) inhibitors are approved for use in a range of cancers. PD-L1 expression in the tumor microenvironment, assessed with an FDA-approved PD-L1 immunohistochemistry (IHC) diagnostic assay such as the Dako PD-L1 IHC 28-8 and 22C3 pharmDx or Ventana PD-L1 SP142 and SP263 assays, is associated with improved PD-(L)1 inhibitor treatment outcomes in some tumor types, including breast cancer (BC). In March 2019, the FDA approved atezolizumab + nab-paclitaxel for the treatment of patients with advanced triple-negative BC and immune cell (IC) PD-L1 expression ≥ 1% using the SP142 assay. Here, we investigated test utilization, test turnaround time (TAT), PD-L1 expression prevalence by assay and biopsy location, and analytical concordance between assays in real-world BC samples. Design: The study included samples from patients with BC that were tested for PD-L1 expression between Oct 2015 and Sep 2019 at NeoGenomics Laboratories, a US national reference laboratory. Patient characteristics from Symphony Healthcare Solutions were matched to PD-L1 test results using unique identifiers. Test volume and TAT were assessed for the 28-8, 22C3, SP142, and SP263 assays. PD-L1 expression was determined by trained pathologists using the 28-8, 22C3, or SP142 assays. Results for the 28-8 assay for the entire study period and for the 22C3 assay until Dec 2018 were reported as the percentage of tumor cells (% TC) with PD-L1 expression. From Jan 2019 onwards, 22C3 assay results were reported as a combined positive score (CPS). All SP142 assay results were reported as the percentage of ICs (% IC) with PD-L1 expression. Analytical concordance between assays was assessed in patients with matched samples (biopsies from the same site and collected on the same date). BioStat Solutions performed statistical analyses. Results: 2955 PD-L1 tests were performed on samples from 2508 patients with BC. The volume of PD-L1 tests on BC samples increased > 100-fold over the study period. Mean TAT was < 5 days for all 4 assays pooled. Table 1 shows PD-L1 expression prevalence in patients with a 28-8, 22C3, or SP142 test result. Median PD-L1 expression did not differ between primary tumors and metastatic sites. In matched samples, overall percentage agreement (OPA) between the 28-8 (TC ≥ 1%) and 22C3 (CPS ≥ 1) assays was 94%, and OPA between the 22C3 (CPS ≥ 1) and SP142 (IC ≥ 1%) assays was 64% (Table 2). Analytical concordance between the 28-8 and 22C3 assays for % TC scoring in matched samples from 27 patients was high (Kendall’s tau = 0.997 [95% CI, 0.883-1.000]). Conclusion: Mean PD-L1 test TAT for BC samples remained < 5 days across all tests despite a large increase in test volume over the study period. Prevalence of PD-L1 expression ≥ 1(%) was higher with the CPS and % IC algorithms than the % TC algorithm, although differences could be due to multiple confounding factors. Despite a small sample size, analytical concordance between the 28-8 and 22C3 assays in matched samples was high. These data provide real-world context for the PD-L1 testing landscape in BC. Table 1. Prevalence of PD-L1 expression in patients with BCPD-L1 expressiona28-8 and 22C3b22C3cSP142% TC, n (%)CPS, n (%)% IC, n (%)(N = 608)(N = 609)(N = 1080)<1(%)390 (64)253 (42)367 (34)≥1(%)218 (36)356 (58)713 (66)All patients had a single test result or ≥ 2 identical results. aThe CPS algorithm is reported on a scale of 0-100, not as a percentage; bSamples tested with the 22C3 assay between Q4 2015 and Q4 2018 were scored using the % TC algorithm; cSamples tested with the 22C3 assay between Q1 2019 and Q4 2019 were scored using the CPS algorithm. Table 2. Agreement between assays on matched samples from patients with BCAgreement between 28-8 (TC ≥ 1%) and 22C3 (CPS ≥ 1) (N = 18)a28-8 as reference22C3 as referenceOPA (n/N)94 (17/18)PPA (n/N)100 (6/6)86 (6/7)NPA (n/N)92 (11/12)100 (11/11)Agreement between 22C3 (CPS ≥ 1) and SP142 (IC ≥ 1%) (N = 33)b22C3 as referenceSP142 as referenceOPA (n/N)64 (21/33)PPA (n/N)86 (12/14)55 (12/22)NPA (n/N)47 (9/19)82 (9/11)aData are presented for 28-8 and 22C3 tests on matched samples with 22C3 tests performed between Q1 2019 and Q4 2019; bData are presented for 22C3 and SP142 tests on matched samples with 22C3 tests performed between Q1 2019 and Q4 2019. N, total number of samples; n, number of samples with the same results with the 2 tests; NPA, negative percentage agreement; PPA, positive percentage agreement. Citation Format: Shreya Mitra, Emily A. Prince, James Pratt, James Novotny, Jr, Vladislav Chizhevsky, Josette William Ragheb, David Huron. Real-world PD-L1 test utilization and analytical concordance of the PD-L1 IHC 28-8 and 22C3 assays in patients with 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 PS4-18.

Abstract Background: Programmed death-1/programmed death ligand 1 (PD-1/PD-L1) inhibitors are approved in a range of tumor types, including non-small cell lung cancer, with PD-L1 immunohistochemistry (IHC) diagnostic assays approved to inform treatment in some settings. There is evidence that PD-L1 expression can vary between primary tumors and metastatic sites, but the relationship remains unclear. In this real-world study, we compared PD-L1 expression between matched primary tumor and metastatic site biopsies in patients with lung cancer. Methods: NeoGenomics Laboratories Inc (Fort Myers, FL), a US national reference laboratory, provided results for PD-L1 tests performed on samples from 21,224 patients with lung cancer between Oct 2015 and Mar 2018. Test results were linked to clinical characteristics provided by Symphony Healthcare Solutions using unique identifiers. PD-L1 tests were performed using the Dako PD-L1 IHC 28-8 or 22C3 pharmDx assays according to the manufacturers' protocols at the time. The percentage of tumor cells (TCs) expressing PD-L1 was determined by trained pathologists. Patients were included in the analysis if they had matched biopsies from a primary lung tumor and a metastatic site that were collected in any order within a 3-month period, and if both samples were tested with the same PD-L1 assay ≤ 3 months apart. Patients were excluded if they received treatment between biopsies or had > 2 biopsies. Statistical analysis was performed by BioStat Solutions Inc. Results: In total, 121 patients had matched primary and metastatic biopsy samples, with sites biopsied in any order; a subgroup of 59 patients had their second biopsy obtained after the PD-L1 test result for the first biopsy was reported. Matched biopsy pairs showed modest concordance (Kendall's tau 0.43 [95% CI, 0.33–0.54]; Spearman's correlation 0.56 [95% CI, 0.42–0.67]). Overall percentage agreement was 69–80% (Cohen's kappa 0.34–0.53) across a range of PD-L1 expression cutoffs (1%, 5%, 10%, 25%, and 50% of TCs). Identical PD-L1 expression was observed in 26% of matched biopsy pairs; 44% of sample pairs had a < 5% difference and 35% of sample pairs had a > 20% difference in PD-L1 expression scores between primary and metastatic sites. PD-L1 expression in primary tumor and metastatic sites was heterogeneous, with no clear trends across biopsy sites. In the subgroup of 59 patients whose second biopsy was obtained after the test result for their first biopsy was reported, 50% of patients (15/30) with PD-L1 expression on < 1% of TCs in their first biopsy had PD-L1 expression on ≥ 1% of TCs in their second biopsy. Conclusion: This real-world study suggests that agreement of PD-L1 expression between matched primary and metastatic biopsy sites is low, further highlighting PD-L1 expression heterogeneity in lung cancer. Variation in PD-L1 expression between biopsy sites may affect treatment decisions relating to PD-1/PD-L1 inhibitors. Citation Format: Emily A. Prince, Vladislav Chizhevsky, Josette William Ragheb, James L. Pratt, Dimple Pandya, David Huron. Comparison of PD-L1 expression in primary and metastatic lung cancer biopsies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2004.

AimsProgrammed death-1/programmed death ligand 1 (PD-1/PD-L1) inhibitor therapy is accompanied by companion or complementary PD-L1 testing in some tumour types. We investigated utilisation of the Dako PD-L1 IHC 28-8 and 22C3 pharmDx assays and the Ventana PD-L1 (SP142) assay and evaluated concordance between the 28-8 and 22C3 assays in a real-world cohort of patients tested at a single US national reference laboratory.MethodsNeoGenomics Laboratories performed PD-L1 testing on tumour samples between October 2015 and March 2018. PD-L1 test results were matched with patient characteristics using unique identifiers. Concordance between the 28-8 and 22C3 assays was evaluated in matched tumour samples. Data were evaluated across multiple tumour types and in subgroups of patients with lung cancer, melanoma, squamous cell carcinoma of the head and neck, and urothelial carcinoma.Results62 180 individual PD-L1 tests were conducted on samples from 55 652 patients. PD-L1 test volume increased ~10-fold over the period evaluated. Test failure rates were typically low, and test turnaround time (TAT) ranged between 2 and 4 days. Concordance between the 28-8 and 22C3 assays was strong in the overall population and across tumour type subgroups (Kendall’s tau correlations of 0.94 and 0.92–0.98, respectively).ConclusionsTest failure rates for PD-L1 tests were low and TAT remained reasonable despite marked increases in test volume. Concordance was high between the 28-8 and 22C3 assays across a range of tumour types and biopsy locations. These findings add to the literature showing high concordance between the 28-8 and 22C3 assays.

447 Background: Programmed death-1/programmed death ligand 1 (PD-1/PD-L1) inhibitors are FDA-approved for urothelial carcinoma (UC) among other tumor types. PD-L1 immunohistochemistry (IHC) testing for UC has evolved with several diagnostic approvals. As of August 2018, the Dako PD-L1 IHC 22C3 pharmDx is a companion diagnostic, while the Dako PD-L1 IHC 28-8 pharmDx and the Ventana PD-L1 (SP263) assays are complementary diagnostics. The Ventana PD-L1 (SP142) assay has complementary status for platinum-treated patients with UC but gained companion status in July 2018 for cisplatin-ineligible patients with PD-L1-expressing UC. Here, we assess PD-L1 testing with the 22C3, 28-8, and SP142 assays on real-world UC samples. Methods: Analyses on 55,652 tumor samples from Symphony Health Solutions were performed between October 2015 and March 2018. PD-L1 results were linked to clinical characteristics using unique identifiers. PD-L1 testing on UC samples was performed at NeoGenomics Laboratories, Inc. based on the manufacturer’s protocols at the time. Results from the Dako assays were reported as % of tumor cells with PD-L1 expression. Test failure (TF) was defined as the absence of adequate sample with measurable PD-L1. Turnaround time (TAT) was defined as the time from sample receipt by the laboratory to test-report delivery. Paired 28-8 and 22C3 testing was performed on 13 samples. Results: 251 confirmed PD-L1 IHC tests (0.4% of tests conducted for the entire dataset) were performed on 223 UC samples, 98.2% of which had quantifiable PD-L1 expression. Mean TAT was 3.0 (1.0–4.2) days and the TF rate was 2.0% for all tests. The 22C3, 28-8, and SP142 assays were used in 52.2%, 9.6%, and 38.3% of tests, respectively. Paired testing with the 22C3 and 28-8 assays was highly correlated (Spearman’s r = 0.94; n = 13). Further analyses of UC PD-L1 prevalence will be presented. Conclusions: Based on the total tests conducted by this US national reference laboratory, PD-L1 testing was not commonly requested for UC during the analysis period. However, PD-L1 tests that were conducted displayed high success rates and reasonable TAT. This is the first comparison of the 22C3 and 28-8 assays on real-world UC samples, adding to the literature on PD-L1 testing concordance.

Targeting the programmed death-1/programmed death ligand 1 (PD-1/PD-L1) pathway has improved clinical outcomes, expediting the US FDA approval of 5 agents as treatment for several tumor types. PD-L1 immunohistochemistry (IHC) diagnostic assays have been developed to guide treatment with anti–PD-1/PD-L1 agents. The Dako PD-L1 IHC 22C3 pharmDx and Ventana PD-L1 (SP142) assays are FDA-approved diagnostics for non-small cell lung cancer (NSCLC) as a companion to pembrolizumab and complementary to atezolizumab, respectively. The Dako PD-L1 IHC 28-8 pharmDx is approved for nonsquamous NSCLC, complementary to nivolumab. We sought to overcome barriers to PD-L1 testing by characterizing the use of 22C3, 28-8, and SP142 PD-L1 IHC assays on real-world lung cancer samples.

Background: A number of programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) diagnostic (Dx) tests have been approved by the FDA to guide treatment (Tx) with programmed death-1/PD-L1 inhibitors. Here, we evaluate the utilization of the Dako PD-L1 IHC 28-8 and 22C3 pharmDx and Ventana PD-L1 (SP142) assays on real-world samples across multiple tumor types, characterize PD-L1 testing practices among physicians who order a PD-L1 test, and investigate how PD-L1 test results impact physicians’ Tx decision-making and use of immuno-oncology (I-O) Tx.Methods: 55,652 samples with clinical characteristics were provided by Symphony Health Solutions. NeoGenomics Laboratories, Inc assessed PD-L1 expression between October 2015–April 2018, according to manufacturers’ protocols at time of study. Clinical characteristics were matched to PD-L1 test results using unique identifiers for the 4714 patients (pts) whose diagnoses and treatment could be determined.Results: Across tumor types, 56% of pts received a test prior to first-line (1L) Tx. Most lung cancer (57%) or melanoma (MEL; 65%) pts had a PD-L1 test prior to 1L Tx, while most squamous cell carcinoma of the head and neck (70%) or urothelial carcinoma (73%) pts had a PD-L1 test after Tx initiation, in line with drug and complementary Dx approvals for these tumor types during the testing period. The percentage of lung cancer pts whose PD-L1 expression was tested prior to 1L Tx rose from 32% during Q4 2015–Q3 2016 to 63% during Q4 2016–Q1 2018, in line with FDA approval of a companion PD-L1 IHC Dx for non-small cell lung cancer. Regardless of PD-L1 test used, most lung cancer (73%) or MEL (93%) pts received I-O Tx, defined as nivolumab (NIVO; + ipilimumab [IPI] for MEL only), pembrolizumab (pembro) ± chemotherapy, atezolizumab, or IPI alone. Moreover, the majority of lung cancer pts who had ≥1% PD-L1 expression or MEL regardless of PD-L1 expression received 1L I-O Tx irrespective of the PD-L1 test used. Analysis of PD-L1 expression, Dx test used, and Tx received showed that, for MEL pts tested with 22C3, 28%, 17%, and 0% of pts received NIVO or IPI, while 28%, 43%, and 43% received NIVO+IPI, at the expression cutoffs of 0%, 1–49%, and ≥50%, respectively. Lung cancer pts with 1–49% PD-L1 expression, as determined by a 22C3 test, received NIVO or pembro monotherapy at similar rates (12–18%). Of those who had ≥50% PD-L1 expression, as determined by the 28-8 test only, 52% received pembro monotherapy vs other Tx.Conclusions: Since FDA approval of the first PD-L1 IHC Dx in 2015, analyses of PD-L1 testing practices and physicians’ Tx decision behavior at a US national reference laboratory have shown that physicians’ adoption of PD-L1 testing is responsive to FDA approvals and that most testing for lung cancer and MEL occurs before 1L Tx initiation. In lung cancer or MEL pts who had a PD-L1 test result available, Tx decisions do not appear to be tied to the specific intended use of the PD-L1 assay that was ordered.Citation Format: Gabriel S. Krigsfeld, Emily Prince, Kim Zerba, Vladislav Chizhevsky, Josette William Ragheb, James White. Real-world utilization of PD-L1 IHC testing and results across multiple tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3993A.

151 Background: A number of programmed death ligand 1 (PD-L1) IHC diagnostic tests have been approved by the FDA to guide treatment with programmed death-1/PD-L1 inhibitors. We evaluated PD-L1 assay utilization and concordance across all tumor types, including melanoma (MEL) and squamous cell carcinoma of the head and neck (SCCHN), using real-world cancer samples tested at a single reference laboratory in the United States. Methods: 55,652 samples with clinical characteristics were provided by Symphony Health Solutions. NeoGenomics Laboratories, Inc assessed PD-L1 expression using the Dako PD-L1 IHC 28-8 or 22C3 pharmDx, or Ventana PD-L1 SP142 assay tests between October 2015 and April 2018, according to the manufacturers’ protocols at the time of the study. Clinical characteristics were matched to PD-L1 test results using unique identifiers. Data were analyzed by BioStat Solutions, Inc. Results: 61,568 tests from 55,652 patients were included. Across all 61,568 tests, 88.8% were carried out with 22C3, 9.8% with 28-8, and 1.4% with SP142. The 28-8 and 22C3 pharmDx assays showed high analytical concordance in all 3113 matched samples with PD-L1 expression data for both assays (OPA = 96.2%, Spearman’s r = 0.96; Table). The failure rate across all tests was 3.5%, and 96.6% of patients had a quantifiable test result. Average test turnaround time was 3.1 days, with a 9.1-fold increase in total number of tests performed between Q4 2015 and Q1 2018 (from 1163 to 10,544). Subgroup analyses for 678 patients (1.2%) with MEL and 270 patients (0.5%) with SCCHN will be presented. Conclusions: Despite an increased demand for PD-L1 testing in the US, most patients received a result in 2–4 days. These real-world analytical data support the potential interchangeability of the Dako PD-L1 IHC 28-8 and 22C3 pharmDx assays in clinical practice for assessing tumor cell membrane PD-L1 expression across tumor types, and provide context on the evolution and adoption of PD-L1 testing for patients with cancer. [Table: see text]

Immune checkpoint inhibitors targeting the programmed death-1/programmed death ligand 1 (PD-1/PD-L1) pathway have improved clinical outcomes in patients with cancer. Testing for PD-L1 with immunohistochemistry (IHC) is performed to inform therapy decisions. Among the FDA-approved PD-L1 IHC diagnostic assays, the Dako PD-L1 IHC 22C3 pharmDx assay is approved as a companion diagnostic to pembrolizumab in non-small cell lung cancer (NSCLC) and the Dako PD-L1 IHC 28-8 pharmDx assay as a complementary diagnostic to nivolumab for nonsquamous NSCLC. Previous studies have compared the 2 assays in NSCLC and urothelial carcinoma. Here, we present a comparison of the 28-8 and 22C3 assays on the largest real-world dataset of lung cancer samples reported to date.

Targeting of the PD1/PD-L1 immune checkpoint pathway has rapidly gained acceptance as a therapeutic strategy for a growing number of malignancies. Testing for expression of PD-L1 in tumor cells and immune cells has been used as a companion or complementary test for drugs targeting the PD1/PD-L1 pathway. We evaluated the results of PD-L1 testing in a large reference lab cohort. Using Food and Drug Administration-approved methods and interpretive instructions for each individual test, 62,896 cases were evaluated for PD-L1 using antibody clone 22C3, 28-8, SP142, or SP263. Case data analyzed included test results and information on tumor location and clinical history. No clinical outcome information was available and no attempt was made to correlate PD-L1 results with any other tests performed. The following numbers of cases were evaluated: 22C3 with tumor proportion score [n = 52585], 22C3 with combined positive score [n = 2631], 28-8 [n = 4191], SP142 [n = 850], and SP263 [n = 70]. In 22C3/tumor proportion score cases, the general results were as follows: negative 33.1% (n = 17,405), (low) expression 33.9% (n = 17,822), and high expression 29.5% (n = 15,486). In cases identified as metastatic, the results were as follows: negative 35.9% (n = 1411), (low) expression 30.8% (n = 1211), and high expression 30.7% (n = 1208). We found broad ranges of expression in tumor types with increasing positivity, as adenocarcinomas were reported as poorly differentiated, whereas squamous cell carcinomas showed more positivity as tumors were described as well-differentiated. The results of many individual tumor types were evaluated and showed, in general, high levels of positive expression. Practical challenges and observations of PD-L1 stain results and interpretation are also discussed.

Aims At the time of analysis, two widely used, drug-specific, tumour-cell programmed death ligand 1 (PD-L1) assays were approved by the US Food and Drug Administration for anti-PD-1 therapies: the Dako PD-L1 immunohistochemistry (IHC) 28-8 pharmDx assay and the Dako PD-L1 IHC 22C3 pharmDx assay. Given that the majority of current PD-L1 testing in US clinical practice is performed at commercial reference laboratories, we aimed to evaluate the concordance of the 28-8 and 22C3 assays in a real-world setting. Methods Matched PD-L1 IHC 28-8 and 22C3 results from routine assessment were obtained from 1930 patients, including 412 confirmed to have lung cancer, submitted from hospitals in over 38 US states/territories. Biopsies were stained, reviewed and scored by trained/certified pathologists at a single cancer reference laboratory between 2015 and 2017. Rate of concordance between assay findings was assessed by Bland-Altman analysis; overall per cent agreement (OPA), positive per cent agreement and negative per cent agreement; and Cohen's kappa. Results PD-L1 IHC 28-8 and 22C3 displayed strong correlation across all samples and in samples with a confirmed lung cancer diagnosis irrespective of biopsy site. The OPA was 97%-98% for all samples, depending on the expression level defining PD-L1 positivity. In the Bland-Altman analysis, the mean difference in percentage of tumour cells positively stained for PD-L1 between the paired assay findings was -0.80% for all samples and -0.93% in samples with a confirmed lung cancer diagnosis. Conclusions These data, in conjunction with recent findings, support the analytical concordance of the PD-L1 IHC 28-8 and 22C3 assays for assessing per cent tumour-cell membrane PD-L1 expression.

3022 Background: DNA mismatch repair deficiency (dMMR) can be tested by immunohistochemistry (IHC) or microsatellite instability (MSI). While either IHC and MSI is adequate for establishing Lynch syndrome, the relevance of discordant results in selecting patients for immune checkpoint treatment is unknown. We investigated MSI and IHC in detecting dMMR and correlated with PD-L1 expression. Methods: Community-based practice tissue samples were submitted for PD-L1 expression and dMMR by both IHC and MSI. PD-L1 testing was performed by IHC using clone 22C3, dMMR using IHC against four MMR proteins (MHL1, MSH2, MSH6, and PMS2), and MSI using PCR with five Bethesda markers. Results: Of the 396 cases tested for both PD-L1 and dMMR by IHC, 18 (4.5%) were reported dMMR positive. Of the 610 cases tested for both PD-L1 and dMMR by MSI, 27 (4.4%) were dMMR positive. The dMMR positivity was determined as having at least one MMR protein expressed at ≤ 6%. There was no statistically significant correlation between PD-L1 expression and the presence or absence of dMMR as detected by IHC. In contrast, patients with MSI had significantly higher PD-L1 positive cells when PD-L1 expression is considered as a continuous variable (P = 0.04), and at cut-offs of 5% (P = 0.003) and 10% (P = 0.004). When a cut-off point of 6% for IHC is used, 8.9% of positive cases by MSI were negative (FN) by IHC and 2.6% of MSI negative cases were positive (FP) by IHC. If a 20% cut-off for IHC is used, FP was at 4.4% but FP was at 3.6%, and if a 30% IHC cut-off is used, FP was at 3.1% and FP was at 5.7%. This difference between cut-off points was statistically significant (P = 0.0008 for 20% and P = 0.0001 for 30% cut-off). Conclusions: There is significant correlation between PD-L1 expression and dMMR as detected by MSI, but not by IHC testing. Based on this and the established association between tumor mutation burden and MSI, MSI should be considered the gold standard for dMMR testing for checkpoint blockade therapy consideration. dMMR by IHC cut-off MSI % positive % IHC FP % IHC FN Pos Neg Total by MSI by IHC 6% Pos 267 16 283 32.38 31.27 8.9 2.6 Neg 26 596 622 20% Pos 280 22 302 32.38 33.37 4.4 3.6 Neg 13 590 603 30% Pos 284 35 319 32.38 35.25 3.1 5.7 Neg 9 577 586 Total 293 612 905

Background: Higher levels of PD-L1 expression at the surface of tumor cells have been associated with increased response to anti-PD-1 therapies (Santabarbara G et al. Ann Transl Med. 2016;4:215; Borghaei H et al. N Engl J Med. 2015;373:1627-1639; Brahmer J et al. N Engl J Med. 2015;373:123-135). The FDA has 2 approved tests associated with the use of these agents. The Dako PD-L1 IHC 22C3 kit is approved as a companion diagnostic for the use of pembrolizumab in the first- and second-line non-small-cell lung cancer (NSCLC) settings, where diagnostic testing is required for its use. The Dako PD-L1 IHC 28-8 kit is approved as a complementary diagnostic for nivolumab therapy in second-line NSCLC, where testing is not required for its use. NeoGenomics Laboratories stained a subset of the samples received for PD-L1 testing with both 22C3 and 28-8 on the same biopsy specimen. This dataset presents an opportunity to explore real-world concordance testing for 22C3 and 28-8 antibodies. Methods: The analysis was performed on a dataset obtained from Symphony Health Solutions describing the PD-L1 biomarker test results and annotations reported by NeoGenomics Laboratories between Oct 7, 2015, and Aug 31, 2016. A total of 5217 biomarker test results were available for 4528 patients afflicted by diverse malignancies. Concordance testing was performed on 556 unique patients that had a single 22C3/28-8 pairing collected from the same biopsy specimen. All staining was performed using the FDA-approved in vitro diagnostic assay using the 22C3 antibody or the 28-8 antibody on the Dako Link 48. Cases were reviewed and scored randomly across multiple pathologists, who received special training and certification in scoring. All statistical analyses were performed in SAS. Results: Paired biopsy specimens stained with both 22C3 and 28-8 displayed high degrees of correlation (Spearman’s correlation co-efficient = 0.97). In addition, Bland-Altman analysis revealed that the mean difference in the percentage of tumor cells positively stained for PD-L1 between the paired 22C3 and 28-8 assay findings was 0.48% [95% prediction limit (-12.41% to 13.37%)]. Across expression levels both antibodies displayed a similar probability of being interpreted as greater than one another (43 of 556 where 28-8>22C3 and 40 of 556 where 22C3>28-8), with no clear direction of a single antibody displaying greater sensitivity. When analyzed at the ≥ 1%, ≥ 5%, ≥ 10%, ≥ 25% and ≥ 50% thresholds, the overall rate of agreement was between 96.8% and 98.2% and associated with a level of agreement (Cohen’s kappa) between 0.92 and 0.96. Conclusion: Both the 22C3 and 28-8 diagnostic assays show strong agreement in a single central laboratory real-world setting. These data, in conjunction with recent findings from analytical comparability studies reported elsewhere, support the potential interchangeability of these assays for diagnostic interpretation. Citation Format: Cory Batenchuk, Maher Albitar, Sucha Sudarsanam, Vladislav Chizhevsky, Chelsea Jin, Virginia A. Burns. A comparative study of PD-L1 IHC 22C3 and 28-8 FDA-approved diagnostic assays in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4015. doi:10.1158/1538-7445.AM2017-4015

e21050 Background: BRAF mutation and PD-L1 expression appear independent in melanoma. We have established that PD-L1 clones 22C3 and 28.8 show almost identical results. In this study we correlated between BRAF mutation and PD-L1 expression as detected by 22C3/28.8 and SP142 in melanoma clinical samples. Methods: Melanoma samples were tested for PD-L1 expression and BRAF mutation. IHC testing for PD-L1 22C3 or 28.8 were tested using FDA-approved kits as recommended. Testing with SP142 (Spring Biosciences; LDT) was performed using standard techniques. BRAF testing in combination with SP142 PD-L1 testing was performed using next generation sequencing covering exons 11 and 15. Samples tested with 22C3 and 28.8 clones were tested using FDA-cleared BRAF kits (Cobas, Therascreen) testing V600 only. Results: 68 samples were tested for PD-L1 expression with clone 22C3, 67 with 28.8, and 56 with SP142. There was no overall statistical difference between the three clones in PD-L1 expression (P = 0.41). For combined 22C3 and 28.8, 61 of 135 cases (45%) had PD-L1 expression and 31 (23%) had BRAF mutation detected using FDA kits (V600). There was no statistical correlation between PD-L1 expression and BRAF mutation in this group (P = 0.9) at any cut-off. In cases tested with SP142 clone, BRAF mutation was detected in 31 cases (55%) by NGS, higher than using the FDA kit (P < 0.0001). In V600 mutations by NGS, 14 cases (25%) were positive, similar FDA kits. In SP142 cases, PD-L1 was positive in 45% of cases. There was no correlation between BRAF mutation and PD-L1 using SP142 expression as a continuous variable (P = 0.59) or cut-off points of 5% or 50%. When cut-off of 20% was used, significant inverse correlation with BRAF mutation (P = 0.004) was identified and was maintained if V600 codon only was evaluated. Conclusions: There is no correlation between BRAF mutation and PD-L1 expression as detected using clones 22C3 and 28.8. With SP142, a negative correlation between PD-L1 expression and BRAF mutation is identified with a 20% cut-off. The rate of BRAF mutations practically doubles when NGS is used and exons 11 and 15 are included in the testing, in contrast to FDA approved testing.

T-cell lymphomas (TCLs) are a heterogenous group of diseases that show histologic and immunophenotypic features overlapping with reactive lymphoid proliferations and often require the use of ancillary testing for accurate diagnosis. The oncoprotein, bcl-2, is expressed in various types of lymphoma. At present, expression of this protein is useful for distinguishing several B-cell lymphomas. Although there are some anecdotal reports that the lack of bcl-2 expression by T cells might also be a useful marker for the diagnosis of TCL, there are no focused studies to address this hypothesis. Another antigen with value in TCL diagnosis is programmed death-1 (PD-1), a marker of follicular helper T cells, which has been reported to be sensitive in the detection of angioimmunoblastic TCL and peripheral T-cell lymphoma, unclassified. However, several reports have also shown that PD-1-positive cells may be increased in a number of settings other than TCL, including reactive and atypical lymphadenopathies. Finally, lymphoma cells express a variety of cytokine receptors and signaling molecules that are current or potential targets for immunomodulatory therapy. One such target is the interleukin (IL)-2 receptor (CD25), which is acted on by denileukin diftitox/ONTAK, a recombinant diphtheria toxin-IL-2 fusion protein. Selection of suitable patients for therapy often includes pretreatment assessment of CD25 expression in tumor cells. In order to further assess the diagnostic and therapeutic utility of these antigens, we compared the expression of the CD25, PD-1, and bcl-2 in 119 cases of T-cell non-Hodgkin lymphoma using immunohistochemical techniques applied to routinely processed and paraffin-embedded tissues. We show that lack of expression of bcl-2 was observed in 52% cases of TCL and may aid in identification of neoplastic T-cell populations. In combination, bcl-2, CD25, and PD-1 provide diagnostic utility and may aid in selecting appropriate patients for immunomodulatory therapy.

IgG4-related sclerosing disease encompasses a family of disorders associated with increased numbers of IgG4 plasma cells and mass forming lesions in various tissues. Lymphadenopathy is a common finding, seen in up to 80% of cases. In the largest series of cases to date, we describe histologic, immunohistochemical, special stain and flow cytometric findings in 29 cases of enlarged lymph nodes with increased IgG4 plasma cells. Lymph node biopsies showed all resection specimens; no needle core biopsies of tissue were evaluated. Cases were considered to have increased numbers of IgG4 plasma cells using the histological criteria outlined by Cheuk and Chan (2010): IgG4 plasma cells >50 cells in a high-power field and >40% of IgG-positive plasma cells positive for IgG4. Additionally, increased intrafollicular plasma cells were a common finding. The lymph nodes showed a variety of reactive histological features including follicular hyperplasia, progressive transformation of germinal centers, interfollicular expansions, variable degrees of fibrosis, increased histiocytes and occasionally an appearance similar to that of plasma cell Castleman disease.