Rajarsi Gupta

Introduction: AI assisted computed higher tumor infiltrating lymphocyte% (TIL%) scores from hematoxylin and eosin (H&E) stained whole slide images (WSI) may serve as a useful biomarker for stratifying patients for treatment with immune checkpoint inhibitors. Methods: To test the hypothesis that self-identified African ancestry (AA) vs. European ancestry (EA) was associated with higher TIL% scores, WSI were assembled from three diverse cohorts of formalin-fixed paraffin embedded (FFPE) colon and rectal adenocarcinoma (COAD-READ) tumor tissues: 1.) 242 AA vs. EA WSI downloaded from The Cancer Genome Atlas (TCGA)-COAD-READ database; 2.) 97 independent US self-identified AA vs. EA WSI assembled from three US medical centers; 3.) 48 (of 51) Nigerian WSI assembled from a single Nigerian medical center. There were 33 self-identified AA WSI suitable for analysis in the TCGA cohort and 49 self-identified AA WSI in the US cohort. For the TCGA cohort, 241 whole transcriptome RNA-sequence data were generated from parallel frozen tumor samples collected alongside the FFPE samples. For the US cohort, 87 RNA-seq enriched by exome capture data were generated from the same FFPE blocks as WSI. Results: No difference in median TIL% scores was detected between the combined TCGA and US AA cohorts and the Nigerian cohort. Exploratory multiple regression analysis of the combined TCGA and US cohorts revealed that AA had a higher TIL% score (Estimated difference = 3.01%, 95% CI (0.54%, 5.49%), P-value = 0.0170), while controlling for MMR/MSI status and other covariates. However, this result needs to be interpreted with caution because of differences between the TCGA and US cohorts with respect to representation of self-identified AA. After adjustment for cohort in the model, the association of AA with a higher TIL% score was no longer significant (Estimated difference = 2.05%, 95% CI(-0.59%, 4.70%), P-value = 0.1277). Moderate correlations were detected in the TCGA and US cohorts analyzed separately between computed TIL% scores and CIBERSORTx estimates of lymphocyte and T-cell abundance. Moderate correlations were also detected between TIL% scores and CXCL10 and CCL5 gene expression values. Conclusions: These preliminary results underscore the need for expanding the representation of minority populations in publicly accessible datasets.

Brightfield Multiplex Immunohistochemistry (mIHC) provides simultaneous labeling of multiple protein biomarkers in the same tissue section. It enables the exploration of spatial relationships between the inflammatory microenvironment and tumor cells, and to uncover how tumor cell morphology relates to cancer biomarker expression. Color deconvolution is required to analyze and quantify the different cell phenotype populations present as indicated by the biomarkers. However, this becomes a challenging task as the number of multiplexed stains increase. In this work, we present self-supervised and semi-supervised approaches to mIHC color deconvolution. Our proposed methods are based on deep convolutional autoencoders and learn using innovative reconstruction losses inspired by physics. We show how we can integrate weak annotations and the abundant unlabeled data available to train a model to reliably unmix the multiplexed stains and generate stain segmentation maps. We demonstrate the effectiveness of our proposed methods through experiments on mIHC dataset of 7-plexed IHC images.

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.

Digital pathology has seen a proliferation of deep learning models in recent years, but many models are not readily reusable. To address this challenge, we developed WSInfer: an open-source software ecosystem designed to streamline the sharing and reuse of deep learning models for digital pathology. The increased access to trained models can augment research on the diagnostic, prognostic, and predictive capabilities of digital pathology.

Quantifying tumor‐infiltrating lymphocytes (TILs) in breast cancer tumors is a challenging task for pathologists. With the advent of whole slide imaging that digitizes glass slides, it is possible to apply computational models to quantify TILs for pathologists. Development of computational models requires significant time, expertise, consensus, and investment. To reduce this burden, we are preparing a dataset for developers to validate their models and a proposal to the Medical Device Development Tool (MDDT) program in the Center for Devices and Radiological Health of the U.S. Food and Drug Administration (FDA). If the FDA qualifies the dataset for its submitted context of use, model developers can use it in a regulatory submission within the qualified context of use without additional documentation. Our dataset aims at reducing the regulatory burden placed on developers of models that estimate the density of TILs and will allow head‐to‐head comparison of multiple computational models on the same data. In this paper, we discuss the MDDT preparation and submission process, including the feedback we received from our initial interactions with the FDA and propose how a qualified MDDT validation dataset could be a mechanism for open, fair, and consistent measures of computational model performance. Our experiences will help the community understand what the FDA considers relevant and appropriate (from the perspective of the submitter), at the early stages of the MDDT submission process, for validating stromal TIL density estimation models and other potential computational models. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

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 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.