Background: Comprehensive genomic profiling (CGP) has gradually gained acceptance in clinical pratice to empower precision oncology in recent years. A single assay could provide a broad picture of genetic and genomic alterations in the tumor, which then aids physicians in disease diagnosis, prognosis and treatment selection. This study demonstrated in-depth analytical validation of K-4CARE, a tumor CGP assay. Methods: The assay utilized a panel of 473 cancer- relevant genes with a total length of 1.7 Mb. We first used commercial reference materials to evaluate performance criteria including limit of detection (LOD), concordance, sensitivity, specificity and precision, to detect single nucleotide variants (SNVs), small insertion/deletions (Indels), copy number alterations (CNAs), genomic rearrangement or fusion. Microsatellite instability (MSI) and tumor mutational burden (TMB) were assessed using both reference materials and 83 clinical tumor samples from 10 cancer types. Results: At LOD of 5% for SNVs and Indels, the assay had sensitivity and specificity of 99.96% and 100%, respectively. Genomic fusions and amplifications were detected with specificity of 100%; sensitivity of 94% and 100% respectively. Detection of germline variants, including both SNVs and Indels, also achieved sensitivity and specificity of 100%. TMB measurement showed 100% concordance with reference materials; and in clinical samples, the correlation coefficient between whole-exome sequencing and targeted panel sequencing was 97%. MSI analysis benchmarked against polymerase-chain reaction showed high accuracy with sensitivity of 94%

and specificity of 100%. Conclusions: K-4CARE assay provides comprehensive and reliable genomic information that fulfills all guideline-based biomarker testing for both targeted therapies and immunotherapies.