Abstract 6467: Specificity and sensitivity assessment of Xenium in situ platform in multiple human carcinomas for clinical studies

Abstract Spatial transcriptomics is advancing at a rapid pace since being named the Nature ‘Method of the Year’ in 2020. The 10x Genomics Visium and Xenium platform and assays are now providing sub cellular resolution and mapping transcripts to the tissue morphology to study complex biology from whole transcriptomics to precisely targeted gene panels. These assays are of great significance to clinical research in precision medicine and biomarker discovery. However, third party validations on reproducibility, orthogonal concordance, specificity and sensitivity raise questions that are critical to their use. At BioChain Institute Inc. (BioChain), we are running a series of tests on Xenium V1 as well as Visium Cytassist V2 chemistry, using multiple human carcinoma FFPE tissues to address the specificity and sensitivity questions in regard to these platforms. FFPE tissues from BioChain’s repository were screened for quality assessment by our pathologist and molecular scientists for tumor content above 30% as well as the RNA quality. Four carcinoma tissues from Breast, Colon, Lung, and Kidney with high DV200 scores were selected. An array was constructed with these tissues to fit the Xenium slide’s imageable area. Two serial sections were used for the Xenium V1 run per 10x Genomics’ protocol. The Off-the-shelf Human Multi-Tissue and Cancer Panel was used for targeting 377 genes. After the Xenium run completion, these slides were used for H&E staining and imaging as well as Visium V2 assay. The H&E images were annotated by the pathologist and integrated with Xenium images using Xenium Explorer.The data was sub-sampled for the different regions per pathologist’s annotations of stroma, tumor and immune cell regions. Data from these regions were compared between the serial sections from Xenium run as well as the Visium run for specificity and sensitivity. Sensitivity of Xenium data versus the visium data was also compared for these specific regions.We have previously demonstrated reproducibility of Xenium data between serial sections prepared by different operators as well as concordance of the two assays run on the same section. With this study we are zooming into specificity and sensitivity via pathologist annotations of the morphology and the gene expression data across multiple carcinomas. For example, in the breast cancer sample cluster 10 in the Xenium data corresponded to the immune region as annotated by the pathologist. This cluster was noted to have high expression of immune cell infiltration marker genes such as CD3E, CD8A, CD2, CCL5, PTPRC, etc. Our findings show the sensitivity and specificity of Xenium and Visium assays to delve further into the heterogeneity of tumor microenvironment and patient stratification for precision medicine and biomarker discovery studies. Citation Format: Jinghao Tian, Tong Lu, Elim Cheung, Lutong Zhang, Vidyodhaya Sundaram, Rikita Gakhar. Specificity and sensitivity assessment of Xenium in situ platform in multiple human carcinomas for clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6467.