Investigating novel oncogenic pathways using liquid biopsy in cancers of unknown primary

Cancer of unknown primary (CUP) is a metastatic malignancy with an indeterminate origin, accounting for 2–5% of all cancer diagnoses. In this study, we employed a 92-gene CUP-specific targeted panel to analyze liquid biopsies from 39 CUP patients, examining circulating cell-free DNA (ccfDNA) and matching germline DNA (gDNA) samples. Somatic mutations were identified in 78 genes, with frequent alterations in KMT2C, ARID1A, MDC1, TP53, and KRAS. Actionable mutations were found in 21 genes, providing potential targets for molecularly guided therapies. Clonal hematopoiesis of indeterminate potential (CHIP) was detected in PBMC of 17 patients. Additionally, pathogenic germline mutations in NTRK1 and MITF were observed in two patients. CUPs represent a challenge with no existing in vitro or in vivo models reported in the literature. We present the first established two CUP cell lines characterized using a custom targeted sequencing panel. The cell lines were developed from two patients #55 and #96both growing in suspension. The FGFR2 amplification that we previously described [1] was confirmed via ddPCR copy number assay and fluorescence in situ hybridization (FISH), revealing its presence as extrachromosomal DNA (ecDNA). Small RNA-sequencing and RNA-sequencing profiling demonstrated differential regulation of the miR-200 family and epithelial-mesenchymal transition (EMT)-associated pathways. Further analyses focused on tumor microenvironment modulation by characterizing extracellular vesicles (EVs) released by CUP cell lines. Functional coculture experiments with THP-1 cells demonstrated selective transfer of FGFR2 and microRNAs(miRNA) via EVs. Collectively, this study highlights the potential of liquid biopsy as a valuable tool for molecular profiling in CUPs and underscores the importance of paired ccfDNA/gDNA analysis to differentiate somatic mutations from germline and CHIP-associated variants. Also, these findings provide the first comprehensive molecular and functional characterization of CUP models, revealing EV-mediated molecular communication as a critical mechanism by which CUP cells modulate their microenvironment.