Abstract
Gene expression studies are fundamental in molecular biology, offering insights into development, disease progression, and therapeutic targets. To address the need for precise analysis of large datasets, we developed THRESHOLD, a novel tool that introduces the concept of gene saturation. Unlike traditional methods focused on absolute or binary expression levels, THRESHOLD quantifies the consistency of gene expression across patients, revealing co-regulation patterns critical for understanding disease mechanisms and stratifying patients by molecular signatures. The tool offers several features, including user-defined parameters, statistical comparisons, and interactive data visualization. THRESHOLD has uncovered compelling insights into disease progression using TCGA cancer datasets. For instance, bladder urothelial carcinoma demonstrated increasing upregulated gene saturation in progressive cancer stages (P < .00001). Moreover, THRESHOLD identified heightened gene saturation in patients with earlier onset of prostate adenocarcinoma (P < .0001) and revealed a critical fusion transcript, SLC45A2-AMACR, implicated in prostate adenocarcinoma progression, recurrence, and metastasis. Additionally, novel biomarkers and potential candidates for drug therapies were identified through protein-protein interaction networks and functional analyses of saturation data in colon adenocarcinoma and breast invasive carcinoma. THRESHOLD offers a new approach for studying gene expression dynamics and patient stratification. The tool is publicly available at Zenodo: https://zenodo.org/records/15287195.