DISCOVERY Proximity detection is a powerful technique for demonstrating the presence of 2 unique targets in direct proximity of each other in tissue sections. It allows visualization of interacting proteins in distance of 40 nm. Recently this technique became available on the Roche DISCOVERY ULTRA staining platform. With the DISCOVERY Proximity detection you can not only detect the presence/absence of a protein, but also to determine its functional states; i.e., whether it carries post-translational modifications or is present in complex with other proteins, in which cells it is expressed, and in which cell compartments the different states of the protein are present. By having the possibility on the DISCOVERY ULTRA system to add chromogenic IHC multiplexing you can create the advantage of visualizing which cells are undergoing protein interaction. In our recent publication we investigated the interaction between PD1/PDL1 where we studied tumour draining lymph nodes of melanomas and the primary tumours with proximity and chromogenic IHC multiplexing. Drugs targeting the PD1/PDL1 pathway have revolutionized the treatment of multiple cancer types including non-small cell lung cancer, renal cancer, and melanoma with a subset of patients experiencing durable responses. PD1/PDL1 blocking antibodies are believed to act primarily in the tumour microenvironment, by reinvigorating exhausted T cells and thereby reviving pre-existing anti-tumour immunity. In this presentation, we will review this technology by setting up PD1/PDL1 Proximity assay in combination with chromogenic IHC multiplexing with CD8, CD68 and CD11c.

Learning Objectives:

1. Obtain knowledge on how to set up the Proximity detection on Roche DISCOVERY ULTRA Platform

2. Obtain knowledge on how to add chromogenic IHC multiplexing to Proximity detection

3. Understand PD-1/PD-L1 interactions in Tumor Draining Lymph Nodes (TDLN) but not tumor correlate with prognosis in melanoma

Ensuring that clinical tests are not affected by tumor heterogeneity requires a sampling methodology that captures the genomic, proteomic, and cellular diversity of entire solid tumors. However, today the vast majority of surgically removed solid tumor tissue is considered surgical waste and incinerated, or bio-banked as formalin fixed blocks. In fact, in a clinical survey we show that standard molecular tests use only 0.0005% of the initial tumor volume. Other fields outside of medicine such as the mining industry and political polling have solved the challenge of heterogeneity through the statistically powered process of representative sampling. We have applied the principle of representative sampling to solid tumors by homogenizing residual fixed tumor tissue not taken for pathology into a well-mixed solution across multiple tumor types including breast, NSCLC, and colon adenocarcinoma. Representative sampling followed by NGS (Rep-Seq) allows for clear discernment of clonal vs. sub-clonal variants and detects more total variants compared to single biopsies. The homogenized representative sample can be further processed to enable flow cytometry and a comprehensive quantification of infiltrating immune cells, activated fibroblasts, and the percentage of cancer cells within the tumor. Representative sampling of tumors through homogenization of fixed, unused tumor tissue enables researchers and clinicians to generate data sets that are not biased by spatial heterogeneity, and to assess the genomic and cellular diversity of solid tumors.

Three-dimensional (3D) cell culture spheroids and aggregates are preferred over monolayer cell culture due to their architectural and functional similarity to solid tumors. To study expression profiles of key tumor biomarkers, physiologically relevant preclinical models of non-small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line‐derived 3D spheroids and aggregates were utilized. We performed multiplex immunofluorescence (mIF) staining, whole slide scanning and image analysis to enable simultaneous detection and quantification of multiple biomarkers on a single formalin-fixed paraffin embedded section.
We describe the workflow optimized in our lab at Roche Tissue Diagnostics to establish mIF assay for 3D spheroids and aggregates sections. Multiplex staining of 3D sections were performed on Roche DISCOVERY Ultra automated staining instruments with Roche reagent and fluorescent dyes. Biomarker panels were designed to examine the expression of frequently studies key markers of two cancer subtypes. The slides were scanned and multispectral images captured in Vectra 3 (Akoya Biosciences) fitted with filters compatible to the dyes. Quantitative assessment of the expressed biomarkers was performed through image analysis.

Immunohistochemistry (IHC) has been an indispensable tool for daily pathology practice. Multiplex designs have further facilitated its utility with improved efficacy. Several techniques of multiplex IHC are available; one of which is the Discovery Ultra system of Ventana. It allows combination of AP and HRP based chromogens on sequential runs. In this presentation, we will demonstrate our experience with Discovery Ultra system and our pentaplex design as well as a triplex design with new Beta-product HRP based chromogens.

Incorporation of the TSA Technology available from Ventana’s AmpHQ kit allows for improved signal and reduced background in IHC staining at NovoVita Histopath Laboratory. This presentation will describe how the technology is used and share examples of its use.

The development of many different chromogens for light microscopy gives us the opportunity to explore different usage of multiplex immunohistochemistry (IHC) for clinical diagnostic purposes as well as for research. The main advantage over the immunofluorescence (IF) multiplex technology is that a special microscope or a dark room for review of the slide is not required and the pathologist can easily recognize the morphology of the specimen evaluated, giving instantly a multi-parameter semi-quantitative assessment. Also by using, image analysis and artificial intelligence (AI) algorithms, an accurate spatial and cellular distribution of the cellular components of interest can be achieved. The disadvantage is that we still have limited number of chromogens to use, and the co-expression of colors in a single cellular compartment, if more than two, is requiring image analysis algorithms.
In this presentation you will see examples of potential clinical usage of light microscopy chromogenic multiplex IHC for diagnostic purposes. This has applications to surgical pathology as well as cytology specimens; In other words how you can replace IHC-panels of different antibodies with Multiplex IHC; or how to combine in-situ-hybridization (ISH) with protein expression on the same slide. This presentation will also demonstrate the use of Multiplex chromogenic IHC in the research setting, with examples of evaluation of tumor micro-environment with quantitative and spatial distribution of different cellular components.
This work was performed on the Discovery BechMark Ultra and partially supported by Roche Tissue Diagnostics. The Image Analysis Algorithms were custom created for the Department of Pathology, Thomas Jefferson Universiry hospital with the VisioPharm Image Analysis software.

Learning Objectives:
1. The use of chromogenic multiplex IHC in research
2. The future of chromogenic multiplex IHC in Diagnostic Cytopathology
3. The future of chromogenic multiplex IHC in Diagnostic Surgical Pathology

Ensuring that clinical tests are not affected by tumor heterogeneity requires a sampling methodology that captures the genomic, proteomic, and cellular diversity of entire solid tumors. However, today the vast majority of surgically removed solid tumor tissue is considered surgical waste and incinerated, or bio-banked as formalin fixed blocks. In fact, in a clinical survey we show that standard molecular tests use only 0.0005% of the initial tumor volume. Other fields outside of medicine such as the mining industry and political polling have solved the challenge of heterogeneity through the statistically powered process of representative sampling. We have applied the principle of representative sampling to solid tumors by homogenizing residual fixed tumor tissue not taken for pathology into a well-mixed solution across multiple tumor types including breast, NSCLC, and colon adenocarcinoma. Representative sampling followed by NGS (Rep-Seq) allows for clear discernment of clonal vs. sub-clonal variants and detects more total variants compared to single biopsies. The homogenized representative sample can be further processed to enable flow cytometry and a comprehensive quantification of infiltrating immune cells, activated fibroblasts, and the percentage of cancer cells within the tumor. Representative sampling of tumors through homogenization of fixed, unused tumor tissue enables researchers and clinicians to generate data sets that are not biased by spatial heterogeneity, and to assess the genomic and cellular diversity of solid tumors.

Three-dimensional (3D) cell culture spheroids and aggregates are preferred over monolayer cell culture due to their architectural and functional similarity to solid tumors. To study expression profiles of key tumor biomarkers, physiologically relevant preclinical models of non-small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line‐derived 3D spheroids and aggregates were utilized. We performed multiplex immunofluorescence (mIF) staining, whole slide scanning and image analysis to enable simultaneous detection and quantification of multiple biomarkers on a single formalin-fixed paraffin embedded section.
We describe the workflow optimized in our lab at Roche Tissue Diagnostics to establish mIF assay for 3D spheroids and aggregates sections. Multiplex staining of 3D sections were performed on Roche DISCOVERY Ultra automated staining instruments with Roche reagent and fluorescent dyes. Biomarker panels were designed to examine the expression of frequently studies key markers of two cancer subtypes. The slides were scanned and multispectral images captured in Vectra 3 (Akoya Biosciences) fitted with filters compatible to the dyes. Quantitative assessment of the expressed biomarkers was performed through image analysis.

Immunohistochemistry (IHC) has been an indispensable tool for daily pathology practice. Multiplex designs have further facilitated its utility with improved efficacy. Several techniques of multiplex IHC are available; one of which is the Discovery Ultra system of Ventana. It allows combination of AP and HRP based chromogens on sequential runs. In this presentation, we will demonstrate our experience with Discovery Ultra system and our pentaplex design as well as a triplex design with new Beta-product HRP based chromogens.

Incorporation of the TSA Technology available from Ventana’s AmpHQ kit allows for improved signal and reduced background in IHC staining at NovoVita Histopath Laboratory. This presentation will describe how the technology is used and share examples of its use.

The development of many different chromogens for light microscopy gives us the opportunity to explore different usage of multiplex immunohistochemistry (IHC) for clinical diagnostic purposes as well as for research. The main advantage over the immunofluorescence (IF) multiplex technology is that a special microscope or a dark room for review of the slide is not required and the pathologist can easily recognize the morphology of the specimen evaluated, giving instantly a multi-parameter semi-quantitative assessment. Also by using, image analysis and artificial intelligence (AI) algorithms, an accurate spatial and cellular distribution of the cellular components of interest can be achieved. The disadvantage is that we still have limited number of chromogens to use, and the co-expression of colors in a single cellular compartment, if more than two, is requiring image analysis algorithms.
In this presentation you will see examples of potential clinical usage of light microscopy chromogenic multiplex IHC for diagnostic purposes. This has applications to surgical pathology as well as cytology specimens; In other words how you can replace IHC-panels of different antibodies with Multiplex IHC; or how to combine in-situ-hybridization (ISH) with protein expression on the same slide. This presentation will also demonstrate the use of Multiplex chromogenic IHC in the research setting, with examples of evaluation of tumor micro-environment with quantitative and spatial distribution of different cellular components.
This work was performed on the Discovery BechMark Ultra and partially supported by Roche Tissue Diagnostics. The Image Analysis Algorithms were custom created for the Department of Pathology, Thomas Jefferson Universiry hospital with the VisioPharm Image Analysis software.

Learning Objectives:
1. The use of chromogenic multiplex IHC in research
2. The future of chromogenic multiplex IHC in Diagnostic Cytopathology
3. The future of chromogenic multiplex IHC in Diagnostic Surgical Pathology

Multiplexing IHC is a technique that is increasing in demand as more and more researchers and physicians are being tasked with doing more with less. With multiplexing, you have the advantage to view different targets and their levels of expression in relation to one another. In this presentation, we will review the different technologies and techniques that are currently available to assist with developing a multiplex assay. After a brief overview of the technologies, we will apply some of those technologies and walk through an example of optimizing a multiplex assay. Although digital analysis of multiplexed slides can be an important factor in the interpretation of a multiplexed assay, this presentation will focus on and emphasize the staining aspects of the assay optimization.

Learning Objectives: 

1. Understand the utility and methods for multiplexing

2. Understand the principles behind multiplex assay development

Immunohistochemistry (IHC) is used to determine spatial relationships where we can identify the localization of target proteins in specific regions and cells of a tissue.  IHC has been the gold standard to analyze protein expression analysis in tissues; however, the IHC assay may not always work as not all antibodies are robust reagents for detection of target proteins.  For that reason, we have utilized several orthogonal assays to analyze localization of proteins and mRNA of targets within cells where robust antibody reagents are not available or specificity is in question. I will briefly describe several effective techniques that have helped my pathobiology team in the method development of assays that are used for target validation and characterization of human diseases and animal disease models.

Learning Objectives:

1. The presentation will provide the audience the many tools available for the method development of antibodies by utilizing orthogonal methods to ensure specificity of the IHC assay or equivalent histology-related assay

2. The presentation will help you discern how to resolve common problems encountered in immunohistochemical staining

Cancer cells can acquire immune resistance resulting in a loss of immunosurveillance. Tumor cells can upregulate PDL-1 which binds to PD-1. This complex inhibits CD8 Tumor Infiltrating cells ( TILs) response to tumor antigens inducing immune blockade. Anti-blockade immunotherapy blocks the blockade and restores immunosurveillance (self-healing).This presentation emphasizes clinical examples of immunotherapy with both an individual patient with malignant melanoma and population-based examples including triple negative breast cancer.. The cases demonstrate the importance of intact tissue chemistry, in particular multi-analyte immunohistochemistry to inform treatment.. It emphasizes the future need to add more markers and cancer types to the testing repertoire and the need to go beyond diagnosis to prediction and monitoring of immunotherapy responses.

Learning Objectives:

1. Understand the concepts of tumor immunosurveillance, immune blockade, and immune restoration via immunotherapy

2. Understand the pivotal role of the diagnostic laboratory in informing immunotherapy by using intact multi-analyte immunohistochemistry.