The graph reports ddPCR outputs of mutated fractional abundance (p

The graph reports ddPCR outputs of mutated fractional abundance (p.R1276X) (A) and in amplification (B) during sufferers follow-up at three different time points. Click here for additional data file.(528K, TIF) Supplementary Physique 4Patient #95 single-CTCs images and CNA profiles. assay and copy number determination assay by Droplet Digital PCR. The graph reports ddPCR outputs of mutated fractional large quantity (p.R1276X) (A) and in amplification (B) during patients follow-up at three different time points. Image_3.TIF (528K) GUID:?E0EFEF3D-0CD2-4E13-A233-58BCFB6610C4 Supplementary Physique 4: Patient #95 single-CTCs images and CNA profiles. Cell gallery acquired by DEPArray, showing single fluorescent channels and overlays, and bright field (BF) images for 10 CTCs, paired with the corresponding CNA profile. Image_4.TIF (2.6M) GUID:?CC2BE04E-0C94-4A38-81A9-7F49C08F7B3B Supplementary Table 1: List of genes and regions included in SureSelect custom panel. Table_1.XLSX (13K) GUID:?8A9AB909-DA9E-4C42-BC0C-041585A2A5C5 Data Availability StatementThe datasets presented in this article are not readily available because patients have consented to the use of their individual genetic data for biomedical research, but not for unlimited public data release. Requests to access the datasets should be directed to corresponding author. Abstract Cancers of unknown main (CUPs) comprise a heterogeneous group of rare metastatic tumors whose main site cannot be recognized after considerable clinicalCpathological 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 hard DNA screening 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 utilized for single-cell genomic characterization with gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. and gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic PCDH8 variant in gene (p.R1276?) was detected in the tumor tissue and ccfDNAs. The alterations were validated Finasteride by Droplet Digital PCR in all ccfDNA Finasteride samples collected during tumor development. CTCs from a second patient offered a pattern of recurrent amplifications in and genes and loss of gene and a point mutation in gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy screening in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes. (Zhao et al., 2019) and disease modeling (Drapkin et al., 2018) and drug screening (Yu et al., 2014). Size-based or antigen-based technologies for CTC isolation and/or enumeration have been developed in the past 10 years, each one presenting advantages and limitations (Yu et al., 2011). CUP patients are usually diagnosed with an advanced metastatic disease; therefore, they are likely to have a high quantity of CTCs and CTC clusters in the blood circulation. Given the CUP undifferentiated status and variable presentation, it is yet to demonstrate whether CUP CTCs could be isolated using tumor antigen selection (Komine et al., 2014). In this study, we explored liquid biopsy, specifically ctDNA- and CTC-based applications, as approaches to detect CUP druggable Finasteride mutations. We compared two methods to isolate CTCs, one antigen-based, size-agnostic (CELLSEARCH, Menarini Silicon Biosystems) and another antigen-agnostic, size-based (Parsortix, ANGLE plc). CTCs and ctDNA were detectable in the blood of CUP patients and Finasteride analyzed for genomic alterations, which were further compared with genomic alterations recognized in tumor biopsy. Materials and Methods Sample Collection Two patients (Pt#71 and Pt#95) with a diagnosis of malignancy of unknown origin (CUP) were recruited at Bologna University or college Hospital, Italy. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee Center Emilia-Romagna RegionItaly (protocol 130/2016/U/Tess). Patients provided written informed consent. Metastatic tissue from lymph node (Pt#71) and ampulla of Vater (Pt#95) was formalin-fixed and paraffin-embedded (FFPE) and utilized for tumor DNA collection. For Pt#71, blood sampling was performed at three different time points: (A) at diagnosis (August 2018), (B) during FOLFOX-4 treatment (stable disease, November 2018), and (C) at disease progression (May 2019). For Pt#95, blood sampling was performed at diagnosis. Plasma separation was performed centrifugation at 1,900 for 10 min at 4C. A variable number (= 2C5) of plasma aliquots (1 ml) for each patient was collected and stored at C80C prior to isolation of circulating cell-free DNA (ccfDNA). PBMCs were isolated from peripheral blood of Pt#95 using Ficoll-Paque Plus (17-1440-02, GE Healthcare, Chicago, IL, United States). Briefly, after plasma depletion, an equal volume of PBS was added to the remaining blood in EDTA tubes. Following, 4 ml of diluted blood was stratified on 3 ml of Ficoll-Paque Plus and centrifuged at 400 for 30 min at room temperature in a.