Abstracts J Immunother Cancer: first published as 10.1136/jitc-2020-SITC2020.0041 on 10 December 2020. Downloaded from

2. Banik G, et al. High-dimensional multiplexed immunohistochemical characteriza- therapies is downregulation of CD20.12Development of an – tion of immune contexture in human cancers. Methods Enzymol 2020;635:1 20. assay that provides highly sensitive and accurate detection of 3. Keren L, et al. A structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging. Cell 2018;174:1373–1387. CD20 levels in the tissue context may help to assess whether there is a minimum CD20 threshold associated with response http://dx.doi.org/10.1136/jitc-2020-SITC2020.0039 to rituximab or other CD20-targeted therapies. Here, we describe the development of a novel Quanticell™ assay for sensitive and quantitative detection of CD20 expression in for- 40 THE ORION™ PLATFORM FROM RARECYTE® ENABLES malin-fixed paraffin-embedded (FFPE) biopsy samples from SAME-DAY 21-PLEX FLUORESCENCE TISSUE ANALYSIS NHL patients. Methods A CD20 (Abcam, clone SP32) Quanticell-based assay, Daniel Campton, Jeremy Cooper, Steven Reese, Kyla Teplitz, Jeffrey Werbin, which utilizes Konica Minolta’s novel fluorescent phosphor- Joshua Nordberg*, Eric Kaldjian, Tad George. RarCyte, Seattle, WA, USA integrated dots (PIDs)3 was optimized on a panel of B lym- Background Tissue consists of heterogenous cell types, each phoma cell lines. Flow cytometry was performed to bench- with diverse functions and functional states, arranged spatially mark assay performance. Next, a human B lymphoma tissue in a way that impacts patient health status. Resolving this microarray (TMA, n=39 cores) was stained using DAB-IHC complexity at the subcellular level has historically been chal- to evaluate CD20 expression. Tumor cores (n=10) showing lenged by fluorescence overlap, autofluorescence, a limited CD20highCD19high expression by DAB-IHC and immuno- number of targets that can be simultaneously assessed, and fluorescence (IF)-IHC were selected for further evaluation. low throughput. Orion technology breaks these barriers, pro- Human tonsil tissue was used to assess CD20 assay perform- viding rapid, straightforward, highly multiplexed whole slide ance as a Quanticell singleplex or duplexed with CD19 IF- tissue analysis. The recently announced Orion platform rapidly IHC. The TMA was stained with CD20 Quanticell plus generates high resolution 21-channel images to enable compre- CD19-AF488 to measure CD20 expression on a per cell basis. hensive phenotypic profiling and characterization of tissue To assess sensitivity of CD20 Quanticell detection, a CD19 architecture including micro-anatomy, analysis of tumor hetero- negative non-B cell core was analyzed. CD20 expression deter- geneity and the complex tissue microenvironment. mined by Quanticell was compared to results generated with a Methods FFPE tissue samples are stained with TissuePlex™ commercially available method enabling digital profiling of reagents and whole slides are imaged on the Orion Instru- CD20 in FFPE sections. ment. Resulting 21-channel datasets are reviewed by a pathol- Results Analytical comparison between the Quanticell assay ogist to verify staining specificity. The TissuePlex core panel and flow cytometry on cell lines showed strong concordance includes antibodies against CD45, CD4, FoxP3, CD8A, between the two methods (CD20 Quanticell score versus copyright. CD11b, LAG-3, PD-, CD11c, CD163, CD68, PD-1, Ki-67, CD20 receptor number). The Quanticell method demonstrated CD3d, and CD20. Single marker IHC is used as the gold a broader dynamic range in CD20 expression in the TMA standard to evaluate TissuePlex reagents and instrument spec- samples compared to DAB-IHC. Both the Quanticell and digi- tral isolation performance is evaluated using fluorescent tal protein detection assays appropriately clustered cores into microspheres. CD20low and CD20high categories. Notably, the CD20 Quan- Results We will present the novel Orion spatial biology plat- ticell assay demonstrated the ability to measure CD20 expres- form, provide a technology and workflow overview, demon- sion accurately and precisely over a broader dynamic range strate instrument validation results, and present sample when compared to the digital method. datasets. Conclusions Relative to DAB IHC, the novel CD20 Quanticell Conclusions The Orion platform enables rapid and deep phe- assay provides significantly enhanced detection and quantifica- notypic analysis of tissue samples for high resolution, whole tion of CD20 in FFPE tissue samples. This technology may be http://jitc.bmj.com/ slide sample analysis. useful to assess whether there are critical antigen densities associated with response to CD20-targeting therapies. http://dx.doi.org/10.1136/jitc-2020-SITC2020.0040 Acknowledgements The authors gratefully acknowledge techni- cal assistance from Ankit Gandhi and Marie Zamanis. The authors also thank Sean Gerrin for technical writing review. 41 OPTIMIZATION OF AN ULTRASENSITIVE, QUANTITATIVE Trial Registration N/A IMMUNOASSAY FOR DETECTION OF CD20 IN NON- Ethics Approval N/A on September 24, 2021 by guest. Protected HODGKIN’S LYMPHOMA (NHL) FFPE SAMPLES Consent N/A

1 2 1 1 1 Apollina Goel*, Michael Ross, Jeanette Rheinhardt, Peter Duval, Michael Maker, REFERENCES 3 1 1 2 1 1 Hiroyuki Yokota, Kenneth Bloom, George Abe, Ann Ranger, Joseph Krueger. Invicro, a 1. Johnson NA, Boyle A, Bashashati A, et al. Diffuse large B-cell lymphoma: Konica Minolta Company, Boston, MA, USA; 2Unum Therapeutics Inc., Cambridge, MA, Reduced CD20 expression is associated with an inferior survival. Blood; USA; 3Konica Minolta, Inc., Hino-shi Tokyo, Japan 2009;113:3773. 2. Rasheed AA, Samad A, Raheem A, et al. CD20 expression and effects on out- Background CD20, a membrane B cell marker, is expressed come of relapsed/refractory diffuse large B cell lymphoma after treatment with Rit- uximab. Asian Pac J Cancer Prev 2018; 19: 331 on the majority of mature B cell neoplasms, including diffuse 3. Gonda K, Watanabe M, Tada H, et al. Quantitative diagnostic imaging of cancer large B cell lymphoma and follicular lymphoma. Importantly, tissues by using phosphor-integrated dots with ultra-high brightness. Sci Rep CD20 is the target of rituximab as well as autologous T cell 2017;7:7509. and BiTE® therapies in clinical development. Studies show http://dx.doi.org/10.1136/jitc-2020-SITC2020.0041 that one mechanism of resistance to rituximab-containing

A24 J Immunother Cancer 2020;8(Suppl 3):A1–A559