Cancer of unknown primary genomic profiling from cell-free DNA provides insights into CUP biology and vulnerabilities.

Cancer of unknown primary (CUP) is a metastatic malignancy with no identifiable site of origin, accounting for 2-5% of cancer diagnoses. Its marked heterogeneity and the limited availability of tumor tissue pose major challenges to genomic profiling. In this retrospective observational study, we applied a 92-gene CUP-specific targeted sequencing panel to liquid biopsy samples from 39 CUP patients, analyzing circulating cell-free DNA (ccfDNA) together with paired germline DNA (gDNA), when available. Somatic, germline, and CHIP-related variants were classified using predefined variant allele frequency (VAF) thresholds and paired ccfDNA/gDNA comparison. We identified somatic mutations in 78 of 92 genes and 44 clinically relevant variants (Tier I-III), most frequently affecting NF1, KRAS, ARID1A, and PIK3CA. Mutated genes were primarily involved in cell-cycle regulation, receptor tyrosine kinase signaling, and NOTCH pathways. Recurrent genetic alterations were detected in 15 genes, including canonical hotspot mutations in TP53, KRAS, and PIK3CA, 10 of which were shared by three or more patients. Actionable mutations, as defined by current clinical annotation guidelines, were identified in 13 genes, supporting the potential role of molecularly guided therapies in CUP. Likely CHIP-associated variants, defined as mutations in CHIP-associated genes with VAF ≥ 2% in PBMC-derived genomic DNA, were detected in 17 patients. In addition, pathogenic germline variants in MITF, NTRK1, and BAP1 were identified in three patients; notably, the NTRK1 germline variant was accompanied by an independent somatic mutation in the same gene. Overall, these findings support liquid biopsy as a valuable approach for molecular profiling of CUP and highlight the critical importance of paired ccfDNA/gDNA analysis, including CHIP assessment, to accurately distinguish somatic, germline, and hematopoiesis-related variants.