Pilot study of whole-exome sequencing and single-nucleus RNA sequencing to identify actionable alterations in cancer.

e15078 Background: DNA sequencing employing comprehensive genomic profiling (CGP) or whole-exome sequencing (WES) is widely used to identify targetable mutations and genomic instability phenotypes that guide treatment decisions in cancer. However, these approaches fail to characterize critical features such as cellular composition, tumor and immune cell states, and cell type-specific expression of therapeutic targets. Single-cell transcriptomic approaches have been extensively used in research to address these limitations, but have not yet been translated into clinical practice. Methods: We undertook a pilot study of 13 tumor samples from four cancer types (colorectal, lung, ovarian, pancreatic) using an integrated WES and single-nucleus RNA sequencing (snRNA-seq) approach. Nuclei were extracted from frozen tissue using One Biosciences' protocol optimized to preserve nuclei integrity. A single nuclei suspension was used to prepare both WES and snRNA-seq libraries. WES libraries were sequenced to a median depth of 330X for tumor DNA and 74X for constitutional DNA, while snRNA-seq generated ~600 million reads per sample. Following quality control, molecular features relevant for treatment decisions were extracted and integrated across both modalities. Results: WES identified 11,412 somatic mutations (median 374 per sample). Actionable mutations were detected in 2 patients, including KRAS G12C (targetable with adagrasib or sotorasib in lung cancer) and BRAF V600E (targetable with encorafenib plus cetuximab in colorectal cancer). Two colorectal samples also exhibited mismatch repair deficiency signatures, associated with immunotherapy response. Overall, WES revealed clinically relevant therapeutic opportunities in 3 of 13 patients. SnRNA-seq generated high-quality transcriptomic profiles from 52,302 individual cells (median 4,002 cells per sample; median 1,829 genes per cell), enabling quantitative characterization of lymphoid, myeloid and vascular populations. Analysis of target gene expression for immune checkpoint inhibitors (ICI), antibody–drug conjugates (ADC), and bispecific antibodies (BsAb) suggested additional opportunities in 5 of 13 patients. Integrating WES and snRNA-seq identified potential treatment strategies in 7 of 13 patients. Conclusions: This study demonstrates the feasibility of combining WES with snRNA-seq from a single frozen tumor biopsy. The two modalities provide complementary, clinically actionable insights, identifying opportunities in 3 of 13 patients with WES alone and 7 of 13 patients with the integrated approach. WES primarily guided small-molecule therapy selection based on mutations and DNA repair defects, whereas snRNA-seq facilitated prioritization of biologics, including ICI, ADC, and BsAb. Future efforts will focus on developing an integrated clinical report to support therapeutic decision-making.