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Phenoptics Overview & Case Studies

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Posters

49th Society for Neuroscience's Annual Meeting (sFN) Poster Summary

  • Poster Title: Overcoming challenges in neuroscience: Multiplex immunofluorescence and autofluorescence isolation in brain tissue using multispectral imaging
    Synopsis: In this poster, we discuss the expansion of our Phenoptics™ technology to the neuroscience field in order to highlight its ability to overcome some of the challenges specific to imaging and analyzing brain tissue. This proof of concept study was completed using a 10um thick section of human brain tissue, manually stained with our OPAL Polaris 5plex, 6-color fluorophores and imaged on the Vectra Polaris to acquire a whole-slide multispectral image (22.9mm x 46.5mm). Whole slide images and autofluorescence signal were viewed using the Phenochart viewing software. Further analysis was conducted using inForm and PhenoptrReports software to segment tissue (including automated segmentation of tissue imperfections or folds to be excluded from analysis, as well as glass and healthy tissue), cell segmentation and phenotyping (including complex phenotypes) for characterization of human brain tissue with and without chronic traumatic encephalopathy (CTE). In addition, we highlighted the capability of inForm analysis software for pixel-based intensity measurements and signal colocalization and quality reports to evaluate the contribution of AF background signal to the different imaging channels and impact of isolation of this channel for improved signal-to-noise ratios, allowing for increased confidence in measured outcomes.

DPA Poster Summaries

Background: In today’s digital pathology realm, introduction of immunofluorescence-based immunohistochemistry (IHC) has allowed for labeling of different targets of interest, as well as greater flexibility, speed, and quality of data collection. However, conventional fluorescence IHC is currently limited to studying 3-4 markers of interest and is often negatively affected by tissue autofluorescence. Our multispectral imaging (MSI) Phenoptics™ platform overcomes these confounds by not only expanding upon the number of markers of interest that can be studied, but through spectral unmixing is able to remove tissue autofluorescence. Until recently, MSI has been limited to analyzing select fields of view and often has long acquisition times.

  • Poster Title: Whole-Slide Multispectral Imaging: Workflows and Applications
    Synopsis: In this poster, we discuss the advancements to our Phenoptics™ technology, wherein we demonstrate a novel, high-throughput method using our new Opal™ Polaris 6-plex, 7-color fluorophores to acquire a whole-slide multispectral image (1 x 1.5cm tissue) in ~6 minutes on the Vectra Polaris. Annotated regions for analysis were selected in Phenochart using the upgraded viewer that allows for a live unmixing preview of all the fluorophores and autofluorescence, including autofluorescence removal. Further analysis was conducted using inForm and Phenoptr to identify cell phenotypes, locations, and spatial density in order to measure heterogeneity and immune response in the tumor microenvironment.
  • Poster Title: Overcoming Limitations of Conventional Fluorescence Slide Scanning with Multispectral Approaches
    Synopsis: In this poster, we validate our novel, high-throughput whole slide MSI acquisition workflow by comparing it to conventional whole slide fluorescence imaging and field-based MSI (all optimized for Opal™ fluorophores).  Our whole slide workflow displays enhanced autofluorescence unmixing and isolation, via improved limits of detection and increased dynamic range, while reducing crosstalk and improving signal accuracy, known limiting factors in obtaining usable imagery and quantifiable data in immunofluorescence IHC.

SITC Poster Summaries

Background: Development of clinically relevant immuno-oncology (IO) therapies is dependent upon translational biomarker research. Our current Phenoptics™ platform offers a comprehensive workflow consisting of multiplex fluorescence immunohistochemistry staining, multispectral imaging, and tissue analysis, in which multiple markers of interest can be stained on one tissue sample. This technology allows for cell-to-cell interactions to be observed, as well as revealing the underlying biology occurring within the tumor microenvironment.

  • Poster Title: A fully optimized end-to-end solution for I/O multiplex immunofluorescence staining using Opal Polaris 7-Color PD1/PD-L1 Panel Kits for lung cancer and melanoma
    Synopsis: Our new MOTiF™ PD1/PD-L1 Panel Kits were developed to address the need for a reproducible, simple, and standardized multiplex immunofluorescent assay that can assess spatial relationships between biomarkers. In this study, our automated MOTiF™ workflow was validated on lung cancer and melanoma FFPE samples. The kits integrate with a pre-configured whole slide multispectral imaging and analysis workflow (on the Vectra® Polaris™ and InForm® software) to provide the only validated end-to-end solution for quantitative data for translational immuno-oncology research.
  • Poster Title: Applying multispectral unmixing and spatial analyses to explore tumor heterogeneity with a pre-optimized 7-color immuno-oncology workflow
    Synopsis: We characterized the tumor heterogeneity of lung cancer samples using our new end-to-end translational workflow based on the Phenoptics™ platform. The MOTiF™ PD1/PD-L1 Panel Kits include six clinically relevant antibodies and their Opal fluorophores. In combination with pre-defined parameters for multispectral scanning on the Vectra® Polaris™ and a pre-configured algorithm for the inForm® software, MOTiF™ provides an end-to-end solution from staining to analysis. The heterogeneity of spatial interactions was demonstrated among different lung cancer samples using tissue microarrays (TMA). Multispectral imaging (on Vectra® Polaris™) was shown to improve sensitivity of detection through spectral unmixing, and the full workflow from staining, imaging, unmixing, and spatial analysis provides a robust platform for analysis of the tumor microenvironment.
  • Poster Title: Advances in Multiplex Fluorescence Immunohistochemistry: 9-color imaging; Whole Slide Multispectral
    Synopsis: In this poster, we describe advancements made to our Phenoptics™ platform, including two new Opal™ fluorophores, Opal Polaris 480 and Polaris 780, allowing for 8-plex, 9-color multispectral imaging when acquired on the Vectra Polaris®. Analysis with inForm® software was able to identify cell more than 20 phenotypes of interest relevant to immune-oncology and categorize their respective densities within tumor and stroma. Additional analyses included Ki67+ proliferation and PD-1/ PD-L1 proximity.
  • Poster Title: A Fully-Automated Multiplex Fluorescence IHC Assay with Whole Slide Multispectral Imaging on Mouse Tissue: Phenoptics™ Quantitative Pathology Solutions Translational Workflow
    Synopsis: In this poster, we expand beyond human IO tissue research into animal models in order to create a fully translational platform. We discuss our study in which we utilized our Phenoptics™ platform, along with our newly developed Opal™ anti-rabbit HRP secondary antibodies, to analyze formal-fixed paraffin-embedded serial sections of mouse breast cancer.

AACR Poster Summaries

Background: The utilization of multiplex immunofluorescence in immuno-oncology continues to grow at a rapid pace. However, given the innate complexities that can encompass assay development and implementation, it is important to develop standardized methodologies that can be employed across different sites to improve confidence in data gathered and achieve consistent intra- and inter-assay reproducibility. In addition, as the number of markers of interest that can be analyzed on a single tissue section continues to increase, the amount of data collected grows exponentially as well. As such, it is necessary to develop new tools to visualize this information, particularly in regards to spatial relationships of distinct cell populations and how they relate to the invasive margins of the tumor versus the stroma.

  • Poster Title: Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE Study): Reproducibility assessment of an automated multiplexed immunofluorescence slide staining, imaging, and analysis workflow
    Synopsis: This poster, encompassing data gathered in a joint collaboration involving six institutions, discusses the development and validation of an automated 6-plex, 7-color assay assessing inter- and intra-site reproducibility, with emphasis on the PD-1/ PD-L1 axis and %PD-L1 expression by immune cells. Using the Leica BOND RX autostainer and the Phenoptics™ workflow, a 7-color multiplex immunofluorescence panel (PD-L1, PD-1, CD8, CD68, FoxP3, Cytokeratin, and DAPI) was optimized and stained on serial sections of tonsil and lung TMA at each site. Images were acquired using the Vectra Polaris® and analyzed using inForm®. There was strong reproducibility amongst all the sites, with a coefficient of variation of 10% for tonsil samples, and inter-site concordance for lung TMAs of tumor cell and immune cell densities averaging R 2 =0.86, and %PD-L1 averaging R 2 =0.81. Ultimately, the implementation of these methodologies may help to reduce data variability and increase translational confidence in using this technology for clinical trials.
  • Poster Title: Quantifying tumor heterogeneity and mapping complex immune cell interactions with high-throughput, 7-color multispectral slide scans
    Synopsis: In this poster, we build upon our previous Phenoptics™ platform advancements and demonstrate that, using our Opal™ Polaris 6-plex, 7-color fluorophores and the Vectra Polaris, spatial measurements can now be revealed using this high-throughput, translational workflow, including distance between nearest cell neighbors to the extent of the invasive margin. Tissue sections from lung, melanoma, colorectal, and lymphoma were stained for the key immune markers of interest, CD8, FoxP3, PD-1, PD-L1, CD68, plus respective tumor markers and DAPI counterstain. Data was analyzed with inForm® and R software using the phenoptr and phenoptrReports packages. Our spatial tools revealed hot spots of immune activity and overall density differences of individual markers of interest, which were then used to compare and categorize the properties of different tumors. These findings were not observed using conventional 3- and 4-color slide scans. As such, these spatial analysis tools for 7-color whole slide multispectral scans provide researchers with the opportunity to discover and validate biomarkers that capture the full scope of the immune-tumor interactions, which can help to better understand patient responses and drug mechanisms of action.