We demonstrate that detection of CTCs overexpressing PD-L1 is feasible and may provide important prognostic information in HNSCC. Our results suggest that adjuvant PD1 inhibitors deserve evaluation in HNSCC patients in whom PD-L1(+) CTCs are detected at the end of curative treatment.

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Liquid biopsy, based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides non-invasive real-time monitoring of tumor evolution and therapeutic efficacy. We performed for the first time a direct comparison study on gene expression and DNA methylation markers in CTCs and paired plasma-derived exosomes and evaluated their prognostic significance in metastatic castration resistant prostate cancer. This prospective liquid biopsy (LB) study was based on a group of 62 metastatic castration resistant prostate cancer (mCRPC) patients and 10 healthy donors (HD) as controls. Identical blood draws were used to: a) enumerate CTC and tumor-derived extracellular vesicles (tdEVs) using CellSearch (CS) and b) analyze CTCs and paired plasma-derived exosomes at the gene expression and DNA methylation level. CTCs were enumerated using CellSearch in 57/62 patients, with values ranging from 5 to 854 cells/7.5mLPB. Our results revealed for the first time a significantly higher positivity of gene expression markers (CK-8, CK-18, TWIST1, PSMA, AR-FL, AR-V7, AR-567 and PD-L1 mRNA) in EpCAM-positive CTCs compared to plasma-derived exosomes. GSTP1, RASSF1A and SCHLAFEN were methylated both in CTC and exosomes. In CTCs, Kaplan–Meier analysis revealed that CK-19(p = 0.009), PSMA(p = 0.001), TWIST1(p = 0.001) expression and GSTP1(p = 0.001) methylation were correlated with OS, while in exosomes GSTP1 (p = 0.007) and RASSF1A (p = 0.001) methylation was correlated with OS. Our direct comparison study of CTCs and exosomes at gene expression and DNA methylation level, revealed for the first time a significantly higher positivity in EpCAM-positive CTCs compared to plasma-derived exosomes. Future perspective of this study should be the evaluation of clinical utility of molecular biomarkers in CTCs and exosomes on independent multicentric cohorts with mCRPC patients.

Lung cancer is a leading cause of cancer death, and late diagnosis is one of the most important reasons for the high mortality rate. microRNAs (miRNAs) are key players in gene regulation and therefore in tumorigenesis. As far as lung carcinogenesis is concerned, miRNAs open novel fields in biomarker research, in diagnosis, and in therapy. In this review we focus on miR-21 in lung cancer and especially on how miR-21 is involved 1) as a biomarker in response or resistance to therapy or 2) as a therapeutic target.

Standardization of preanalytical conditions and implementation of quality control steps is extremely important for reliable liquid biopsy analysis, and a prerequisite for routine applications in the clinic.

ANGLE) and an epcAM-dependent positive immune-magnetic isolation procedure were applied in parallel, using 10 mL PB from 50 HNSCC patients and 18 healthy donors. Total RNA was isolated from enriched CTCs and Rt-qpcR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M (reference gene). Real time methylation specific PCR (MSP) was used to study the methylation status of RASSF1A and MLL3 genes. In identical blood draws, the label-free size-dependent CTC-isolation system was superior in terms of sensitivity when compared to the epcAM-dependent ctc enrichment, since a significantly higher percentage of identical PB samples was found positive at the gene expression and DNA methylation level, while the specificity was not affected. Our results indicate that future studies focused on the evaluation of clinical utility of ctc molecular characterization in HnScc should be based on size-dependent enrichment approaches.Liquid biopsy provides a valuable source of biomarkers on prognosis and response to treatment of cancer patients 1 and has recently shown a significant potential even for early cancer diagnosis and screening 2 . Isolation of circulating tumor cells (CTCs) from peripheral blood (PB) and their further downstream molecular characterization at the DNA, RNA and protein level is very important for reliable liquid biopsy analysis 3 . However, the identification and molecular characterization of CTCs is very challenging since these cells are extremely rare, and the amount of available sample for analysis in most cases is very limited 1,3 .A variety of molecular assays have been developed for CTCs detection and molecular characterization. Molecular assays are based on total RNA isolation from CTCs and subsequent mRNA quantification of specific genes, and gDNA isolation for mutation analysis and DNA methylation studies 4 . CTC molecular characterization at the gene expression level has the potential to elucidate the critical signaling pathways involved in metastasis biology and even improve patient management. We have shown many years ago that the detection of CK-19 expression in CTCs has prognostic significance in both early and metastatic breast cancer 5-7 . Beyond gene expression, DNA methylation analysis in CTCs has a high potential to provide novel epigenetic biomarkers for diagnosis, prognosis, risk assessment, and disease monitoring in many types of cancer 4 . Based on this, we selected www.nature.com/scientificreports www.nature.com/scientificreports/ gDNA isolation from CTCs. gDNA was extracted from CTCs using the TRIZOL-LS reagent (ThermoFisher Scientific, USA) as previously described. Isolated gDNA 44 was dissolved in 30 μL of 8 mmol/L NaOH. Sodium Bisulfite (SB) treatment. gDNA samples were treated with SB, to convert all non-methylated cytosines to uracil, while methylated cytosines were not converted, using the EZ DNA Methylation Gold Kit (ZYMO Research, USA). SB-treated DNA was stored at −70 °C until use. In each SB reaction, dH 2 O and 100% methylated DNA were included as ...

BACKGROUND Circulating tumor cells (CTCs) and microRNAs (miRNAs) are important in liquid biopsies in which peripheral blood is used to characterize the evolution of solid tumors. We evaluated the expression levels of miR-21, miR-146a, miR-200c, and miR-210 in CTCs of breast cancer patients with verified metastasis and compared their expression levels in corresponding plasma and primary tumors. METHODS Expression levels of the miRNAs were quantified by quantitative reverse transcription PCR (RT-qPCR) in (a) 89 primary breast tumors and 30 noncancerous breast tissues and (b) CTCs and corresponding plasma of 55 patients with metastatic breast cancer and 20 healthy donors. For 30 of these patients, CTCs, corresponding plasma, and primary tumor tissues were available. RESULTS In formalin-fixed, paraffin-embedded tissues, these miRNAs were differentially expressed between primary breast tumors and noncancerous breast tissues. miR-21 (P < 0.001) and miR-146a (P = 0.001) were overexpressed, whereas miR-200c (P = 0.004) and miR-210 (P = 0.002) were underexpressed. In multivariate analysis, miR-146a overexpression was significantly [hazard ratio 2.969 (1.231–7.157), P = 0.015] associated with progression-free survival. In peripheral blood, all miRNAs studied were overexpressed in both CTC and corresponding plasma. There was a significant association between miR-21 expression levels in CTCs and plasma for 36 of 55 samples (P = 0.008). In plasma, ROC curve analysis revealed that miR-21, miR-146a, and miR-210 could discriminate patients from healthy individuals. CONCLUSIONS Metastasis-related miRNAs are overexpressed in CTCs and corresponding plasma; miR-21 expression levels highly correlate in CTCs and plasma; and miR-21, miR-146a, and miR-210 are valuable plasma biomarkers for discriminating patients from healthy individuals.

Androgen-receptor splice variant 7 (AR-V7) is a highly promising liquid biopsy predictive biomarker showing primary or acquired resistance to novel androgen receptor signaling inhibitors in metastatic castration resistant prostate cancer (mCRPC).