Citations

Multiplex immunofluorescence and immunohistochemistry (mIF/IHC) are increasingly employed antibody-based technologies that use tissue sparingly and facilitate the detection of co-localized or neighboring biomarkers. Specifically, these platforms enable spatial analyses of the tumor microenvironment as well as extended applications, for example, describing normal tissue anatomy, autoimmunity, infectious diseases, etc. mIF/IHC has greatly enhanced biomarker discovery efforts, and a growing number of studies suggest superiority to traditional IHC. Standardization of staining approaches, reporting of image analysis strategies and resultant data is critical for facilitating cross-study comparisons, validation, deployment, and generalization of findings. To address this challenge, The Society for Immunotherapy of Cancer (SITC) previously published two articles providing best practice guidelines for mIF/IHC staining, image analysis, and data sharing. Here, SITC convened stakeholders to develop the third article in the series, a consensus checklist for scientific reporting of mIF/IHC data to support and complement the best practice guidelines. The checklist includes critical components of mIF/IHC applications to be defined within publications such as detailed descriptions of analytical validation; image acquisition, selection, and registration methods; and cell clustering and spatial analysis strategies, amongst others. Such information will help with data reproducibility and comparison across studies towards future drug and assay development.

Classic Hodgkin Lymphoma (cHL) is characterized by the presence of abnormal large cells known as Hodgkin Reed-Sternberg (HRS) cells. Curative intent chemotherapy has a 90% response rate, but for refractory patients, current immunotherapies remain insufficient. Ongoing research aims to enhance therapy and patient outcomes by focusing on the tumor microenvironment (TME), a complex environment that includes diverse non-malignant cells, including immune cells, which constitute a critical component of the TME. Utilizing multiplex analysis to comprehend these intricate interactions can pave the way for identifying novel strategies to combat cancer more effectively. CD30-targeted therapies and PD1/PDL1 inhibitors are under investigation clinically and could potentially benefit from adding checkpoint markers such as TIM-3 to enhance patient outcomes by addressing T-cell exhaustion. Such combination strategies will require assays and analysis strategies that can describe both spatial and multi-marker data. Multiplex immunofluorescence (mIF) is a valuable tool for analyzing spatial biology, patient selection, mechanism of action, pharmacodynamics and biomarker assays predictive of response. Describing the relationship between T cell exhaustion and response in cHL may guide the development of targeted treatments aimed at restoring the immune system's ability to recognize and eliminate cancer cells. Twenty-five to thirty percent of cHL patients with advanced stages do not respond to standard therapies. To understand the mechanisms of resistance, we developed a mIF panel (CD4, CD8, CD30, PD-1, PD-L1, and TIM3) for formalin-fixed and paraffin-embedded cHL tissues on the OPAL Tyramide Signal Amplification system and the Leica Bond RX. A set of commercially sourced cHL whole tissue samples (n=5) were used to develop and validate the panel, with qualitative evaluations by a Hematopathologist affirming that the mIF and 3, 3'-diaminobenzidine (DAB) staining yielded comparable results. Subsequently, a cohort of cHL samples (n=34) were stained and underwent exploratory image (Halo) and data (Microsoft Excel and R Studio) analyses. The image analysis strategy includes cell segmentation and phenotyping for all markers in the panel, identifying individual cell populations and multi-marker co-expressing cell populations. Additionally, spatial analysis describes the relationships between key phenotypes in the TME. The quantity of exhausted T cells, their average distance to HRS cells and the average count of exhausted T cells within different proximities of HRS cells will be presented. The development of this mIF assay for cHL demonstrates its consistent application at a mid-scale level across a diverse patient population with varying medical backgrounds and treatment histories. This approach provides a deeper understanding of immune response diversity and cellular composition within cHL. Future deployment of the panel against clinical trial samples with outcomes and responses will yield further insights.