# A New Tool to Decrease Interobserver Variability in Biomarker Annotation in Solid Tumor Tissue for Spatial Transcriptomic Analysis

## TL;DR

A new MATLAB tool reduces variability in biomarker annotation for spatial transcriptomic analysis of solid tumors.

## Contribution

A MATLAB-based tool was developed to standardize γH2AX annotation in spatial transcriptomics, reducing interobserver variability.

## Key findings

- Manual annotation of γH2AX showed significant interobserver variability (Kappa = 0.345).
- The MATLAB tool enabled reproducible annotation of γH2AX-positive spots with user-defined parameters.
- Reproducible gene expression analysis was achieved using the new tool.

## Abstract

Integrating spatial transcriptomic data with immunofluorescence image data is challenging using existing tools due to their differences in spatial resolution. Immunofluorescence provides information about protein expression at the cellular or subcellular level, whereas spatial transcriptomic platforms typically rely on multicellular “spots” for RNA profiling. Our study coupled spatial transcriptomics of irradiated glioblastoma tissues with immunofluorescence for γH2AX, a marker of DNA damage within the nuclei of cells. We then compared gene expression in γH2AX-positive and negative regions within the tissue. There was significant interobserver variability in manual annotation of γH2AX positivity in multicellular spots by three different researchers (Kappa statistic = 0.345), despite all of them being familiar with γH2AX immunofluorescence and having predefined imaging parameters for annotation. This variability led to different researchers nominating different genes as being associated with DNA repair. To overcome this problem, we have developed a new tool using MATLAB. This tool performs “spot”-wise image analysis and uses researcher-defined parameters such as immunofluorescent marker intensity threshold and number of positive cells to annotate the “spots” as γH2AX positive or negative. The tissue with the most variability in manual annotation was annotated reproducibly by our MATLAB tool, leading to reproducible downstream analysis.