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The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell‐free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell‐free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (next‐generation sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high‐quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained. However, the choice of blood collection tubes is a critical pre‐analytical variable., We assessed whether a combined analysis of circulating tumor cells, circulating tumor DNA, and microRNA in total plasma or extracellular vesicles from one blood sample is feasible. We show that high‐quality data from a single blood tube can be obtained in melanoma patients. However, the choice of the tube affects the outcome of the analysis, underlining the importance of pre‐analytical factors.

Liquid biopsies are emerging noninvasive diagnostic tools in cancer. MLL3 histone methyltransferase is a transcriptional regulator encoded by the tumor suppressor KMT2C gene. We evaluated the prognostic significance of KMT2C epigenetic changes in plasma of NSCLC patients. The detection of KMT2C promoter methylation in cfDNA predicts poor outcomes in NSCLC and merits further evaluation as a circulating epigenetic biomarker., MLL3 histone methyltransferase, encoded by the KMT2C gene, is a tumor suppressor that has an essential role in cell‐type‐specific gene expression. We evaluated the prognostic significance of KMT2C promoter methylation as a circulating epigenetic biomarker in plasma cell‐free DNA (cfDNA) in non‐small cell lung cancer (NSCLC). We examined the methylation status of KMT2C promoter using a novel highly specific and sensitive real‐time methylation‐specific PCR (MSP) assay in (a) operable NSCLC: 48 fresh‐frozen NSCLC tissues, their corresponding adjacent non‐neoplastic tissues, and 48 matched plasma samples; (b) metastatic NSCLC: 91 plasma samples; and (c) 60 plasma samples from healthy donors (HD). KMT2C promoter methylation in plasma cfDNA was detected in 7/48 (14.6%) patients with operable and in 18/91 (19.8%) patients with advanced NSCLC but in none (0/60, 0%) of the plasma samples from HD. In operable NSCLC, in corresponding adjacent non‐neoplastic tissue samples, KMT2C promoter methylation was detected in 3/48 (6.3%) cases. Moreover, in operable NSCLC, KMT2C promoter methylation in plasma cfDNA was related to reduced disease‐free survival (ΗR = 0.239; P = 0.001) and worse overall survival (OS; HR = 0.342, P = 0.023). In metastatic NSCLC, KMT2C promoter methylation in plasma cfDNA was related to worse progression‐free survival (PFS; HR = 0.431; P = 0.005) and worse OS (HR = 0.306; P

Liquid biopsy analysis, mainly based on circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides an extremely powerful tool for the molecular profiling of cancer patients in real time. In this study, we directly compared PIK3CA hotspot mutations (E545K, H1047R) in EpCAM‐positive CTCs and paired plasma‐ctDNA in breast cancer (BrCa). PIK3CA hotspot mutations in CTCs and ctDNA were analyzed using our previously developed highly sensitive (0.05%), specific, and validated assay in plasma‐ctDNA from 77 early and 73 metastatic BrCa patients and 40 healthy donors. We further analyzed and directly compared PIK3CA hotspot mutations in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma‐ctDNA, in 56 cases of early and 27 cases of metastatic breast cancer, and 16 corresponding primary tumors. In plasma‐ctDNA,PIK3CA hotspot mutations were identified in 30/77(39.0%) early and 35/73(47.9%) metastatic BrCa cases; none (0/40, 0%) of the healthy donors’ plasma‐ctDNA samples were positive. Our direct comparison study in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma‐ctDNA from the same blood draws has shown a lack of concordance in early BrCa (27/56, 48.2%), while the concordance in the metastatic setting was higher (18/27, 66.6%). Our results were validated by ddPCR methodology, and the concordance between our assay and ddPCR for PIK3CA E545K hotspot mutation was 30/37 (81.1%). In many cases, PIK3CA hotspot mutations were detected in samples found to be negative for CTCs in CellSearch®. Our data demonstrated for the first time that (a) PIK3CA hotspot mutations are present at high frequencies in CTCs isolated from CellSearch® cartridges and paired plasma‐ctDNA both in early and metastatic BrCa, (b) the detection and concordance of PIK3CA hotspot mutations between plasma‐ctDNA and CTCs are higher in the metastatic setting, (c) PIK3CA mutational status significantly changes after therapeutic intervention, and (d) PIK3CA mutation detection in CTCs and plasma‐ctDNA provides complementary information., Our direct comparison study on the presence of PIK3CA hotspot mutations (E545K, H1047R) in EpCAM‐positive CTCs and paired plasma‐ctDNA demonstrated for the first time that PIK3CA mutations are present at high frequencies in both CTCs and paired plasma‐ctDNA and that there is a high concordance in the metastatic setting. PIK3CA mutational status significantly changes after therapeutic intervention.

A variety of molecular assays have been developed for CTCs detection and molecular characterization. Molecular assays are based on the nucleic acid analysis in CTCs and are based on total RNA isolation and subsequent mRNA quantification of specific genes, or isolation of genomic DNA that can be for DNA methylation studies and mutation analysis. This review is mainly focused on gene expression and methylation studies in CTCs in various types of cancer.

Papillary renal cell carcinoma (pRCC) is the second most common RCC subtype and can be further classified as type 1 (pRCC1) or 2 (pRCC2). There is currently minimal understanding of pRCC1 pathogenesis, and treatment decisions are mostly empirical. The aim of this study was to identify biological pathways that are involved in pRCC1 pathogenesis using an integrated genomic approach. By microarray analysis, we identified a number of significantly dysregulated genes and microRNAs (miRNAs) that were unique to pRCC1. Integrated bioinformatics analyses showed enrichment of the focal adhesion and extracellular matrix (ECM) pathways. We experimentally validated that many members of these pathways are dysregulated in pRCC1. We identified and experimentally validated the downregulation of miR-199a-3p in pRCC1. Using cell line models, we showed that miR-199a-3p plays an important role in pRCC1 pathogenesis. Gain of function experiments showed that miR-199a-3p overexpression significantly decreased cell proliferation (p = 0.013). We also provide evidence that miR-199a-3p regulates the expression of genes linked to the focal adhesion and ECM pathways, such as caveolin 2 (CAV2), integrin beta 8 (ITGB8), MET proto-oncogene and mammalian target of rapamycin (MTOR). Using a luciferase reporter assay, we further provide evidence that miR-199a-3p overexpression decreases the expression of MET and MTOR. Using an integrated gene/miRNA approach, we provide evidence linking miRNAs to the focal adhesion and ECM pathways in pRCC1 pathogenesis. This novel information can contribute to the development of effective targeted therapies for pRCC1, for which there is none currently available in the clinic.