Genetic Characterization of Cancer of Unknown Primary Using Liquid Biopsy Approaches

Abstract

Cancers of unknown primary (CUPs) comprise a heterogeneous group of rare metastatic tumors whose primary site cannot be identified after extensive clinical-pathological investigations. CUP patients are generally treated with empirical chemotherapy and have dismal prognosis. As recently reported, CUP genome presents potentially druggable alterations for which targeted therapies could be proposed. The paucity of tumor tissue, as well as the difficult DNA testing and the lack of dedicated panels for target gene sequencing are further relevant limitations. Here, we propose that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) could be used to identify actionable mutations in CUP patients. Blood was longitudinally collected from two CUP patients. CTCs were isolated with CELLSEARCH^® and DEPArray^TM NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with <i>Ampli</i>1^TM kits. Circulating cell-free DNA (ccfDNA), purified from plasma at different time points, was tested for tumor mutations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology. In parallel, FFPE tumor tissue was analyzed with three different assays: FoundationOne CDx assay, DEPArray LibPrep and OncoSeek Panel, and the SureSelect custom panel. These approaches identified the same mutations, when the gene was covered by the panel, with the exception of an insertion in <i>APC</i> gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. <i>FGFR2</i> and <i>CCNE1</i> gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in <i>ARID1A</i> gene (p.R1276^∗) was detected in the tumor tissue and ccfDNAs. The alterations were validated by Droplet Digital PCR in all ccfDNA samples collected during tumor evolution. CTCs from a second patient presented a pattern of recurrent amplifications in <i>ASPM</i> and <i>SEPT9</i> genes and loss of <i>FANCC</i>. The 92-gene custom panel identified 16 non-synonymous somatic alterations in ccfDNA, including a deletion (I1485Rfs^∗19) and a somatic mutation (p. A1487V) in <i>ARID1A</i> gene and a point mutation in <i>FGFR2</i> gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy testing in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes.