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 DEPArrayTM NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with Ampli1TM 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 APC gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. FGFR2 and CCNE1 gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in ARID1A 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 ASPM and SEPT9 genes and loss of FANCC. 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 ARID1A gene and a point mutation in FGFR2 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.

An Integrated Bioinformatics Analysis towards the Identification of Diagnostic, Prognostic, and Predictive Key Biomarkers for Urinary Bladder Cancer

“…In previous findings, Annexin A5 ( ANXA5 ) plasma levels were investigated as a potential biomarker for asthma diagnosis [ 109 ], pregnant and non-pregnant subjects [ 110 ], as well as for liver cirrhosis and hepatocellular carcinoma [ 111 ]. Abnormal spindle protein homolog ( ASPM ) was detected in circulating tumor cells through single-cell genomic characterization in cancer patients [ 112 ]. CD34 serves as an essential marker in disease research, as it is routinely used for identifying and isolating human hematopoietic stem/progenitor cells applied in bone marrow transplantation.…”

Synergic activity of FGFR2 and MEK inhibitors in the treatment of FGFR2-amplified cancers of unknown primary

“…When we applied the predictive algorithms to the two tumors, we obtained a site-of-origin prediction: CUP#55 was predicted to be of biliary tract origin (probability of 93%) and CUP#96 of gastrointestinal tract origin (probability of 99%). The tumor tissue, circulating cell-free DNA (ccfDNA), cell line and PDX were tested for genetic alterations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology (Agilent Technologies) as described in (Laprovitera et al, 2021c). The summary of all genetic alterations is reported in Table 1 and 2.…”

“…However, these regimens remain empirical since they are mostly based on results of singlearm phase II clinical trials (Briasoulis et al, 2008;Pentheroudakis et al, 2008;Warner et al, 1998) or small phase III trials (Gross-Goupil et al, 2012;Hainsworth et al, 2015;Huebner et al, 2009). The use of molecular tests based on gene/miRNA expression signatures or methylation profiles to identify the most probable site-of-origin could assist the oncologists in the selection of the best treatment options and potentially improve CUPs outcome (Laprovitera et al, 2021b;Laprovitera et al, 2021c). NCCN guidelines for occult primary tumors recently introduced the recommendation to use NGS genetic testing to guide therapeutic decision (v.2/2023) and suggest 11 different chemotherapy regimens for adenocarcinoma and 9 for squamous histology.…”

“…The use of molecular tests based on gene/miRNA expression signatures or methylation profiles to identify the most probable site-of-origin could assist the oncologists in the selection of the best treatment options and potentially improve CUPs outcome (Laprovitera et al, 2021b; Laprovitera et al, 2021c). NCCN guidelines for occult primary tumors recently introduced the recommendation to use NGS genetic testing to guide therapeutic decision (v.2/2023) and suggest 11 different chemotherapy regimens for adenocarcinoma and 9 for squamous histology.…”

ARID1A in cancer: Friend or foe?

“…Specifically, we longitudinally evaluated the variant allele frequency in circulating cell-free DNA (ccfDNA) samples of a CUP case with ARID1A mutations. The study reports how the fractional abundance of ARID1A mutation (p.R1276_) in ccfDNA decreases during the initial treatment and then increases again during disease worsening, thus suggesting a role in the expansion of the more aggressive subclones ( 78 ).…”