Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of circulating tumor cells (CTCs) and plasma-circulating tumor DNA (ctDNA). epigenetic silencing potentially affects response to endocrine treatment. We evaluated methylation in CTCs and paired plasma ctDNA. We evaluated methylation in CTCs and paired plasma ctDNA as a potential biomarker for response to everolimus/exemestane treatment. A highly sensitive and specific real-time MSP assay for methylation was developed and validated in (i) 65 primary breast tumors formalin-fixed paraffin-embedded (FFPE), (ii) EpCAM CTC fractions (122 patients and 30 healthy donors; HD), (iii) plasma ctDNA (108 patients and 30HD), and (iv) in CTCs (CellSearch) and in paired plasma ctDNA for 58 patients with breast cancer. methylation status was investigated in CTCs isolated from serial peripheral blood samples of 19 patients with ER/HER2 advanced breast cancer receiving everolimus/exemestane. methylation was detected in: (i) 25/65 (38.5%) FFPEs, (ii) : 26/112 (23.3%) patients and 1/30 (3.3%) HD, and (iii) 8/108 (7.4%) patients and 1/30 (3.3%) HD. methylation was highly concordant in 58 paired DNA samples, isolated from CTCs (CellSearch) and corresponding plasma. In serial peripheral blood samples of patients treated with everolimus/exemestane, methylation was observed in 10/36 (27.8%) CTC-positive samples, and was associated with lack of response to treatment ( = 0.023, Fisher exact test). We report for the first time the detection of methylation in CTCs and a high concordance with paired plasma ctDNA. methylation in CTCs was associated with lack of response to everolimus/exemestane regimen. methylation should be further evaluated as a potential liquid biopsy-based biomarker..

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 ...

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.

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).

No abstract