BACKGROUND:Circulating tumor cells (CTCs) are associated with prognosis in a variety of human cancers and have been proposed as a liquid biopsy for follow-up examinations. We show that tumor suppressor and metastasis suppressor genes are epigenetically silenced in CTCs isolated from peripheral blood of breast cancer patients.

INTRODUCTION Detection of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in the peripheral blood of patients with solid tumors has been widely studied for the early detection of metastatic spread. We evaluated whether there was an association between the origin of cfDNA and CTCs. We investigated whether SRY (sex determining region Y)-box 17 (SOX17) promoter methylation in CTCs was associated with the methylation pattern of this gene in matched cfDNA isolated from plasma of patients with breast cancer. METHODS We examined SOX17 methylation in 79 primary breast tumors, in 114 paired samples of DNA isolated from CTCs and cfDNA, and in 60 healthy individuals. Isolated DNA was modified by sodium bisulfite and subjected to methylation specific PCR. RESULTS The SOX17 promoter was methylated in 68 (86.0%) of 79 of primary breast tumors. In CTCs, SOX17 was methylated in 19 (34.5%) of 55 patients with early breast cancer, 27 (45.8%) of 59 patients with metastatic cancer, and 1 (4.3%) of 23 healthy individuals, whereas in matched cfDNA SOX17 was methylated in 19 (34.5%) of 55, 24 (40.7%) of 59, and 1 (2.0%) of 49 of these same groups, respectively. There was a significant correlation between SOX17 methylation in cfDNA and CTCs in patients with early breast cancer (P = 0.008), but not in patients with verified metastasis (P = 0.283). CONCLUSIONS The SOX17 promoter is highly methylated in primary breast tumors, in CTCs isolated from patients with breast cancer, and in corresponding cfDNA samples. Our findings indicate a direct connection between the presence of CTCs and cfDNA in patients with operable breast cancer, after surgical removal of the primary tumor.

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..

Background:Breast-cancer metastasis suppressor 1 (BRMS1) gene encodes for a predominantly nuclear protein that differentially regulates the expression of multiple genes, leading to suppression of metastasis without blocking orthotropic tumour growth. The aim of the present study was to evaluate for the first time the prognostic significance of BRMS1 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of non-small cell lung cancer (NSCLC) patients. Towards this goal, we examined the methylation status of BRMS1 promoter in NSCLC tissues, matched adjacent non-cancerous tissues and corresponding cfDNA as well as in an independent cohort of patients with advanced NSCLC and healthy individuals.Methods:Methylation of BRMS1 promoter was examined in 57 NSCLC tumours and adjacent non-cancerous tissues, in cfDNA isolated from 48 corresponding plasma samples, in cfDNA isolated from plasma of 74 patients with advanced NSCLC and 24 healthy individuals.Results:The BRMS1 promoter was highly methylated both in operable NSCLC primary tissues (59.6%) and in corresponding cfDNA (47.9%) but not in cfDNA from healthy individuals (0%), while it was also highly methylated in cfDNA from advanced NSCLC patients (63.5%). In operable NSCLC, Kaplan–Meier estimates were significantly different in favour of patients with non-methylated BRMS1 promoter in cfDNA, concerning both disease-free interval (DFI) (P=0.048) and overall survival (OS) (P=0.007). In advanced NSCLC, OS was significantly different in favour of patients with non-methylated BRMS1 promoter in their cfDNA (P=0.003). Multivariate analysis confirmed that BRMS1 promoter methylation has a statistical significant influence both on operable NSCLC patients' DFI time and OS and on advanced NSCLC patients' PFS and OS.Conclusions:Methylation of BRMS1 promoter in cfDNA isolated from plasma of NSCLC patients provides important prognostic information and merits to be further evaluated as a circulating tumour biomarker.

Our results show that SOX17 promoter is highly methylated in primary tumors and in corresponding plasma samples both in operable and advanced NSCLC. In the advanced setting, SOX17 promoter methylation in plasma ctDNA has a statistical significant influence on NSCLC patient's survival time. Detection of SOX17 promoter methylation in plasma provides prognostic information and merits to be further evaluated as a circulating tumor biomarker in patients with operable and advanced NSCLC.

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Breast Cancer Metastasis Suppressor-1 differentially regulates expression of multiple genes, leading to metastasis suppression without affecting orthotopic tumor growth. We evaluated BRMS1 promoter methylation as prognostic biomarker in primary breast tumors and studied BRMS1 promoter methylation in a subset of corresponding Circulating Tumor Cells (CTC) for the first time. We analyzed 118 formalin-fixed paraffin embedded samples: 5 pairs of breast tumors and adjacent non-cancerous tissues, 14 non-cancerous tissues, 10 benign fibroadenomas, and 84 primary breast tumors. Peripheral blood mononuclear cells from 39/84 of these patients were fixed in cytospins. BRMS1 methylation status was investigated in all FFPE and cytospin stained CTC using methylation specific PCR. BRMS1 expression in cytospins was examined by double-immunofluorescence using anti-BRMS1 and pancytokeratin A45-B/B3 antibodies. BRMS1 promoter methylation was not observed in noncancerous breast tissues (0%), and benign fibroadenomas (0%), while it was observed in 36.9% of primary breast tumors. BRMS1 promoter methylation in primary tumors was associated with reduced disease-free interval (P=0.009) while a trend towards a reduced overall survival was also observed (P=0.071). 13/39 cytospin samples (33.3%) were positive for the presence of CTC and the total number of the detected CTC was 41. Most CTC (80.5%) were negative for BRMS1 or maintained low expression, implying that BRMS1 is down regulated in these cells. BRMS1 promoter methylation was observed in 5/39 (12.8%) samples. BRMS1 promoter methylation in primary breast tumors provides prognostic information for DFS. BRMS1 expression in CTC was highly heterogeneous, between patients and even in the same patient.

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.