Jeppe Thagaard’s research while affiliated with Visiopharm and other places

Aim Manual detection and scoring of Ki67 hotspots is difficult and prone to variability, limiting its clinical utility. Automated hotspot detection and scoring by digital image analysis (DIA) could improve the assessment of the Ki67 hotspot proliferation index (PI). This study compared the clinical performance of Ki67 hotspot detection and scoring DIA algorithms based on virtual dual staining (VDS) and deep learning (DL) with manual Ki67 hotspot PI assessment. Methods Tissue sections of 135 consecutive invasive breast carcinomas were immunohistochemically stained for Ki67. Two DIA algorithms, based on VDS and DL, automatically determined the Ki67 hotspot PI. For manual assessment; two independent observers detected hotspots and calculated scores using a validated scoring protocol. Results Automated hotspot detection and assessment by VDS and DL could be performed in 73% and 100% of the cases, respectively. Automated hotspot detection by VDS and DL led to higher Ki67 hotspot PIs (mean 39.6% and 38.3%, respectively) compared to manual consensus Ki67 PIs (mean 28.8%). Comparing manual consensus Ki67 PIs with VDS Ki67 PIs revealed substantial correlation ( r = 0.90), while manual consensus versus DL Ki67 PIs demonstrated high correlation ( r = 0.95). Conclusion Automated Ki67 hotspot detection and analysis correlated strongly with manual Ki67 assessment and provided higher PIs compared to manual assessment. The DL‐based algorithm outperformed the VDS‐based algorithm in clinical applicability, because it did not depend on virtual alignment of slides and correlated stronger with manual scores. Use of a DL‐based algorithm may allow clearer Ki67 PI cutoff values, thereby improving the clinical usability of Ki67.

Recent advances in the field of immuno‐oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high‐throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi‐marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The clinical significance of the tumor-immune interaction in breast cancer is now established, and tumor-infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2-negative) breast cancer and HER2-positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state-of-the-art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in-depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple-negative breast cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Modern histologic imaging platforms coupled with machine learning methods have provided new opportunities to map the spatial distribution of immune cells in the tumor microenvironment. However, there exists no standardized method for describing or analyzing spatial immune cell data, and most reported spatial analyses are rudimentary. In this review, we provide an overview of two approaches for reporting and analyzing spatial data (raster versus vector-based). We then provide a compendium of spatial immune cell metrics that have been reported in the literature, summarizing prognostic associations in the context of a variety of cancers. We conclude by discussing two well-described clinical biomarkers, the breast cancer stromal tumor infiltrating lymphocytes score and the colon cancer Immunoscore, and describe investigative opportunities to improve clinical utility of these spatial biomarkers. © 2023 The Pathological Society of Great Britain and Ireland.

The growth in cancer immunotherapy agents requires an understanding of the immune contexture of the tumor microenvironment (TME). Understanding immune contexture requires multiplex staining, imaging, and analysis to obtain multi-marker phenotypes of specific cells and analyze their biodistribution in the TME. Imaging Mass Cytometry™ (IMC) is the method of choice for single-step staining and highplex imaging of FFPE tissues. FFPE tissue is autofluorescent, which limits the utility of immunofluorescence methods. Lung and colorectal tissue (and bone, skin, etc) are highly autofluorescent, and therefore good targets for IMC. However, developments in analysis software for highplex imagery have not kept pace with imaging advances. We present a comprehensive workflow designed specifically for highplex image analysis, covering tissue segmentation, cell segmentation based on IMC DNA images, cellular phenotyping, and spatial analyses. Lung and colorectal tissue sections with a 30-marker IMC panel of structural, tumor, stroma, immune cell, and immune activation markers were imaged (Hyperion+™, Standard BioTools). Highplex image analysis (Phenoplex™, Visiopharm) was performed as a multi-step workflow in a single software package that includes: conversion of IMC images to pyramidal format; easy visualization methods for displaying different marker subsets; a paint-to-train algorithm for tissue segmentation (into tumor, stroma, blood vessels, etc.); deep-learning-based nuclear segmentation pre-trained on IMC DNA channels; cellular phenotyping based on thresholds based on visual assessment of positivity; spatial biodistribution metrics for cell populations; and a flexible set of outputs for downstream analysis. Tissue segmentation was used to divide the tissue into tumor, stromal, and tumor margin regions, and these regions were used to compare the immune contexture through a series of t-SNE images partitioned by spatial region. We demonstrate that a simple analysis workflow can be used for highplex images of different tissue types by users with no programming knowledge. Visualization templates for the marker subsets and the pre-trained IMC nuclear segmentation are reusable. A new tissue segmentation algorithm for each tissue type is required, as are new thresholds for biomarker positivity. Spatial biodistribution metrics, heatmaps and partitioned t-SNE plots were generated for each tissue type with a minimum of work. Highplex IMC imaging of lung and colorectal tumor samples is a simple and effective means of obtaining high-parameter images without interfering autofluorescence. Having a comprehensive workflow for the analysis of this complex data makes obtaining useful results from highplex images more accessible to biologists and immunologists by circumventing the requirement for expert programming for each specific application. Citation Format: Brenna O'Neill, Smriti Kala, Sam Lim, Clinton Hupple, Nina Lane, Rasmus Norre Sorensen, Rasmus A. Lyngby, Alessandro Massaro, Andreas Hussing, Jeppe Thagaard, Johan Dore-Hansen, James Robert Mansfield. A comprehensive guided workflow for highplex imaging, tissue segmentation, and multiplex cellular phenotyping for tumor microenvironment analysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4625.

Aims: Digital image analysis (DIA) is used increasingly as an assisting tool to evaluate biomarkers, including human epidermal growth factor receptor 2 (HER2) in invasive breast cancer (BC). DIA can assist pathologists in HER2 evaluation by presenting quantitative information about the HER2 staining in APP assisted reading (AR). Concurrently the HER2-low category (HER2-1+/2+ without HER2 gene amplification) has gained prominence due to newly developed antibody-drug conjugates. However, major inter- and intraobserver variability have been observed for the entity. The present quality assurance study investigated the concordance between DIA and AR in clinical use, especially concerning the HER2-low category. Methods and results: HER2 IHC in 761 tumors from 727 patients was evaluated in tissue microarray (TMA) cores by DIA (Visiopharm HER2-CONNECTTM ) and AR. Overall concordance between HER2-scores were 73% (n=552, weighted-κ: 0.66), and 88% (n=669, weighted-κ: 0.70), when combining HER2-0/1+. A total of 205 scores were discordant by one category, while four were discordant by two categories. A heterogeneous HER2 pattern was relatively common in the discordant cases and a pitfall in the categorization of HER2-low BC. AR more commonly reassigned a lower HER2 score (from HER2-1+ to HER2-0) within the HER2-low subgroup (n=624) compared with DIA. Conclusions: DIA and AR display moderate agreement with heterogeneous and aberrant staining representing a source of discordance and pitfall in the evaluation of HER2.

Background The growth in cancer immunotherapy agents requires an understanding of the immune contexture of the tumor microenvironment (TME). Imaging Mass Cytometry™ (IMC™) is a powerful tool for the study of complex cellular interactions in the TME and in the discovery of biomarkers that can predict disease outcome or response to therapy. The Hyperion Imaging System (Standard BioTools) utilizes CyTOF® technology to simultaneously assess 40+ protein markers at subcellular resolution without spectral overlap or background autofluorescence, thus providing unprecedented insight into the organization and function of the TME. However, despite the advances in staining and imaging methods, developments in analysis software had not kept pace, as a complete, user-defined workflow in a single software package for the analysis of highplex imaging data was lacking. Methods This study demonstrates a tissue phenotyping workflow in highly autofluorescent lung and colorectal cancer tissues using highplex IMC, which offers the advantage of zero autofluorescence, and hence more reliable results. The data analysis pipeline uses Oncotopix® Discovery (Visiopharm) software for easy, accurate and quantifiable phenotyping. Serial sections of both tissue types were stained with a 40-marker panel comprised of structural, tumor, stroma, and immune cell markers, including immunoregulatory proteins that are targets of immunotherapy. The IMC cell segmentation kit was included in the panel for improved nucleus and plasma membrane demarcation. The analysis pipeline consisted of tissue segmentation (tumor, stroma, necrosis, etc), nuclear detection using a deep-learning algorithm pre-trained on IMC DNA channels, a threshold-based cellular phenotyping step, and spatial analyses. Results In this work, we have shown that analysis of IMC images from lung and colorectal cancer tissues can uncover tissue phenotypic signatures of the TME through the determination of immune cell types found in the vicinity of cancerous cells and their numbers. Moreover, cell counts and tissue phenotypes were highly consistent across the serial sections, demonstrating the power of IMC in generating robust data. Conclusions Overall, this work demonstrates that even for highly autofluorescent tissues, IMC can generate high-quality data, consistent across serial sections, which can be easily and accurately analyzed using a single software package, thus empowering IMC users to be confident in biological interpretation of high-dimensional proteomic data. This study showcases the capability of IMC technology combined with Oncotopix Discovery analysis in classification of cellular components within the TME, which is important for development of systematic digital profiling of the spatial TME.