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Purpose: 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). ESR1 epigenetic silencing potentially affects response to endocrine treatment. We evaluated ESR1 methylation in CTCs and paired plasma ctDNA. We evaluated ESR1 methylation in CTCs and paired plasma ctDNA as a potential biomarker for response to everolimus/exemestane treatment.Experimental Design: A highly sensitive and specific real-time MSP assay for ESR1 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. ESR1 methylation status was investigated in CTCs isolated from serial peripheral blood samples of 19 patients with ER+/HER2− advanced breast cancer receiving everolimus/exemestane.Results: ESR1 methylation was detected in: (i) 25/65 (38.5%) FFPEs, (ii) EpCAM+ CTC fractions: 26/112 (23.3%) patients and 1/30 (3.3%) HD, and (iii) plasma ctDNA: 8/108 (7.4%) patients and 1/30 (3.3%) HD. ESR1 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, ESR1 methylation was observed in 10/36 (27.8%) CTC-positive samples, and was associated with lack of response to treatment (P = 0.023, Fisher exact test).Conclusions: We report for the first time the detection of ESR1 methylation in CTCs and a high concordance with paired plasma ctDNA. ESR1 methylation in CTCs was associated with lack of response to everolimus/exemestane regimen. ESR1 methylation should be further evaluated as a potential liquid biopsy-based biomarker. Clin Cancer Res; 24(6); 1500–10. ©2017 AACR.

Background Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa). Methods In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining. Results All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection. Conclusions In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs.

Highly sensitive methodologies for SARS-CoV-2 detection are essential for the control of COVID-19 pandemic. We developed and analytically validated a highly sensitive and specific five-plex one-step RT-ddPCR assay for SARS-CoV-2. We first designed in-silico novel primers and probes for the simultaneous absolute quantification of three different regions of the nucleoprotein (

BackgroundPD-L1, an immune checkpoint protein, is an important biomarker for monitoring cancer patients during the administration of cancer immunotherapy. Droplet digital PCR (ddPCR), is a highly sensitive and accurate tool for the quantification of cancer biomarkers in liquid biopsy. We report the development and analytical validation of a novel duplex RT-ddPCR assay for the simultaneous quantification of PD-L1 and hypoxanthine phosphoribosyltransferase (HPRT) (used as reference gene) transcripts in circulating tumor cells (CTCs).MethodsRT-ddPCR experimental conditions were first optimized and the assay was analytically validated using synthetic standards and the BB49 and SCC47 cancer cell lines. The developed assay was further applied in 71 peripheral blood (PB) samples from head and neck squamous cell carcinoma (HNSCC) patients and 20 PB samples from healthy donors. PD-L1 and HPRT transcripts were quantified in cDNAs derived from CTCs isolated by a size-dependent microfluidic device. The developed RT-ddPCR assay was directly compared to RT-qPCR using 71 identical patient cDNA samples.ResultsAnalytical sensitivity was 0.64 copies/μL, while estimation of intra- and interassay variation revealed a high reproducibility (within-run CV%:4.7–23%; between-run CV%:13%). Using the developed RT-ddPCR assay 33/71(46.5%) HNSCC patients’ samples were found positive for PD-L1 expression in CTCs, while by using RT-qPCR fewer samples (23/71, 32.4%) were positive (concordance: 55/71, 77.5%).ConclusionsThe developed RT-ddPCR assay for PD-L1 in CTCs is highly sensitive, specific, and reproducible; additionally, it offers improved diagnostic sensitivity over RT-qPCR. The clinical utility of the assay should be prospectively evaluated for the real-time monitoring of CTCs of cancer patients under immunotherapy.

Over the last decade, liquid biopsy has gained much attention as a powerful tool in personalized medicine since it enables monitoring cancer evolution and follow-up of cancer patients in real time. Through minimally invasive procedures, liquid biopsy provides important information through the analysis of circulating tumour cells (CTCs) and circulating tumour-derived material, such as circulating tumour DNA (ctDNA), circulating miRNAs (cfmiRNAs) and extracellular vehicles (EVs). CTC analysis has already had an important impact on the prognosis, detection of minimal residual disease (MRD), treatment selection and monitoring of cancer patients. Numerous clinical trials nowadays include a liquid biopsy arm. CTC analysis is now an exponentially expanding field in almost all types of solid cancers. Functional studies, mainly based on CTC-derived cell-lines and CTC-derived explants (CDx), provide important insights into the metastatic process. The purpose of this review is to summarize the latest findings on the clinical significance of CTCs for the management of cancer patients, covering the last four years. This review focuses on providing a comprehensive overview of CTC analysis in breast, prostate and non-small-cell lung cancer. The unique potential of CTC single-cell analysis for understanding metastasis biology, and the importance of quality control and standardization of methodologies used in this field, is also discussed.

Objectives Chemoradiation can induce immunogenic (ICD) or tolerogenic cell death. ICD relies on the generation of damage-associated molecular patterns which can stimulate toll-like receptors (TLRs). We sought to determine whether we can predict responses to chemoradiation by measuring surrogate biomarkers of ICD in a cohort of patients with locally advanced (LA) head and neck squamous cell carcinoma (HNSCC). Materials and Methods In a cohort of 113 LA HNSCC pts we evaluated expression of TLR4, TLR7 and TLR9 in the EpCAM + circulating tumor cell (CTC) fraction at baseline and after cisplatin chemoradiation. We also quantified changes in chemokines CXCL10, CXCL16 and IL-2R in the serum. Results Seventy three patients had evaluable specimens. Among cases with biomarker assessment at baseline and post treatment, 36.8% had an increase in CXCL10 levels (p = 0.022), 73.7% had an increase in CXCL16 levels (p = 0.002) and 63.8% had an increase in IL2Ra levels (p = 0.032) with treatment. 52.0% of evaluable cases at baseline and post-treatment had an increase in TLR4 levels (p = 0.996), 42.9% had an increase in TLR7 levels (p = 0.042) and 27.7% had increase in TLR9 levels (p = 0.011) with treatment. CXCL10 levels at baseline were significantly associated with PFS and OS (p = 0.010 and p = 0.032, respectively). Conclusions Our results suggest that chemoradiation leads to quantifiable effects in surrogate markers of ICD. These effects may inform trials combining chemoradiation with immune checkpoint inhibitors. In addition, CXCL10 has prognostic effect in pts treated with chemoradiation.

Background/aim Effective targeted therapies for triple-negative breast cancer (TNBC) are limited. In a subset of TNBC, androgen receptor (AR) plays an important role, while the human proviral integration site for Moloney murine leukemia virus-1 (PIM1) overexpression is also implicated. PIM1 kinases phosphorylate AR, thus regulating its transcriptional activity, regardless of the presence or not of androgens. We evaluated the expression of AR and PIM1 and their prognostic significance in TNBC. Materials and methods AR and PIM1 transcripts were quantified by quantitative reverse transcription polymerase chain reaction in formalin-fixed paraffin-embedded tumor from 141 patients with TNBC. Results AR was expressed in 38.3%, PIM1 in 10.6%, while co-expression of AR and PIM1 was detected in 7/141 cases (5.0%). No prognostic significance of AR or PIM1 was reached for overall or disease-free survival. Conclusion Co-expression of AR and PIM1 exists in only in a small percentage of patients with TNBC. The implications of this finding in the therapeutic management of patients with TNBC should be investigated in larger patient cohorts.

Background In metastatic breast cancer (MBC) the molecular characterization of Circulating Tumor Cells (CTCs) provides a unique tool to understand metastasis-biology and therapy-resistance. We evaluated the prognostic significance of gene expression in EpCAM(+) CTCs in 46 MBC patients based on a long follow-up. Methods We selected a panel consisting of stem cell markers (CD24, CD44, ALDH1), the mesenchymal marker TWIST1, receptors (ESR1, PGR, HER2, EGFR) and the epithelial marker CK-19. Singleplex RT-qPCR was used for TWIST1 and CK-19 and multiplex RT-qPCR for a) CD24, CD44, ALDH1, HPRT and b) ESR1, PR, HER2, EGFR. A group of 19 healthy donors (HD) was used as control. Results Univariate (p=0.001) and multivariate analysis (p=0.002) revealed the prognostic value of combined gene expression of CK-19(+), CD44high/CD24-/low, ALDH1high/CD24-/low and HER2 over-expression for overall survival (OS). The Kaplan–Meier estimates of OS were significantly different in patients positive for CK-19 (p = 0.028), CD44high/CD24-/low (p = 0.002), ALDH1high/CD24-/low (p = 0.007) and HER2-positive (p = 0.022). Conclusions Our results indicate that combined gene expression analysis in EpCAM(+) CTCs provides prognostic information in MBC but need to be further confirmed in a prospective study, including a larger and well-defined patient cohort.

Background We sought to determine the prognostic role of indoleamine 2,3-dioxygenase 1 (IDO1) by evaluating IDO1 expression in circulating tumour cells (CTCs) at baseline and after completion of chemoradiotherapy in patients with locally advanced (LA) head and neck squamous cell carcinoma (HNSCC) treated with curative intent. Methods In a prospective cohort of 113 patients with LA HNSCC, we evaluated expression of IDO1 in the EpCAM+ CTC fraction at baseline and after cisplatin chemoradiation. The prognostic value of combined programmed cell death ligand-1 (PDL-1) and IDO1 expression was assessed. Results IDO1 was significantly overexpressed at baseline compared with the post-treatment counterparts (p=0.007). IDO1 messenger RNA (mRNA) expression at baseline was associated with better survival in terms of progression-free survival (PFS) (HR=0.19, p=0.017). Post-treatment IDO1 mRNA levels were correlated with unfavourable prognosis in terms of overall survival (OS) (HR=3.27, p=0.008). Patients with combined decreased expression levels of PDL-1 and IDO1 after treatment exhibited superior PFS (p=0.043) and OS (p=0.021). Conclusions Our results strongly suggest that IDO1 mRNA expression is an independent prognostic factor for clinical outcome. Our study provides useful information for future trials combining chemoradiation with immune checkpoint inhibitors and IDO1 inhibitors.

Liquid biopsy, based on the molecular information extracted from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), offers the possibility to characterize the evolution of a solid tumor in real time and is highly important for diagnostic and therapeutic purposes. The aim of the present study was the development and validation of a novel liquid bead array methodology for the molecular characterization of CTCs and its application in breast cancer. In the present study we developed and evaluated a multiplex polymerase chain reaction (PCR)-coupled liquid bead array (MLBA) assay for studying simultaneously the expression of 14 genes in CTCs. The 14-gene MLBA assay is characterized by high analytical specificity, sensitivity, and reproducibility. The analytical performance of the 14-gene MLBA assay was compared with a commercially available test (AdnaTest BreastCancer, Qiagen, Germany) and our previously described multiplex quantitative reverse transcription PCR (RT-qPCR) assays. The developed assay has the potential to be further expanded in order to include up to 100 gene targets. The assay is highly specific for each target gene and is not affected by the numerous primers and probes used for multiplexing; hence, it constitutes a sample-, cost-, and time-saving analysis.

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). We present for the first time the expression pattern of AR-FL, AR-V7, and AR-567es at a quantitative level in circulating tumor cells (CTCs) and paired plasma-derived extracellular vesicles in mCRPC. We first developed and analytically validated a novel multiplex RT-qPCR assay for AR full length (AR-FL), AR-V7, AR-567es and AR-total. We then quantified the expression levels of AR-splice variants, CK-19 (epithelial marker) and B2M (reference gene) in EpCAM+ CTCs, and paired plasma-derived extracellular vesicles isolated from peripheral blood (20 mL) of 62 mCRPC patients and 10 healthy donors. CTCs were enumerated using the FDA-cleared CellSearch® system. In CTCs AR-FL was detected in 64/69 (92.3%), AR-V7 in 34/69 (49.3%), AR-567es in 16/69 (23.2%) and AR-total in 62/69 (89.9%). In 52 out of 69 samples, paired plasma-derived extracellular vesicles were analyzed. AR-FL was detected in 40/52 (76.9%), AR-V7 in 4/52 (7.7%), AR-567 in 2/52 (3.8%) and AR total in 39/52 (75.0%). In all cases AR splice variants were expressed in higher levels in CTCs than in paired extracellular vesicles, while AR-V7 was detected in higher percentages than in AR-567es. Using CellSearch®, CTCs were detected in 52/69 (75.4%) mCRPC patient samples; 27/52 (51.9%) of these samples were CTC+/AR-V7+ and 14/52 (26.9%) were CTC+/AR-567es+, while 7/17 (41.2%) were CTC-/AR-V7+ and 2/17 (11.8%) were CTC-/AR-567es+. Our results reveal for the first time a remarkable heterogeneity in the expression levels of AR-FL, AR-V7 and AR-567es in EpCAM+ CTCs and paired extracellular vesicles between individual mCRPC patients. The clinical significance of this finding will be further investigated in a large patient cohort with respect to therapy response.

We directly compared two different approaches used for Circulating Tumor Cell (CTC) isolation, a size-dependent microfluidic system versus an EpCAM-dependent positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in Head and Neck Squamous Cell Carcinoma (HNSCC). A size-dependent microfluidic device (Parsortix, 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.

International audience; Background: Successful application of programmed death 1 (PD1) checkpoint inhibitors in the clinic may ultimately benefit from appropriate patient selection based upon predictive biomarkers. Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while predictive biomarkers for response to PD1 checkpoint inhibitors are lacking. We sought to assess whether overexpression of PD-L1 in CTCs could be detected at baseline and at different timepoints during treatment in a prospective cohort of head and neck squamous cell carcinoma (HNSCC) patients and used to predict clinical outcome after treatment with curative intent. Patients and methods: We developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA expression in EpCAM(+) CTCs. In a prospective cohort of 113 locally advanced HNSCC patients treated with curative intent we evaluated PD-L1 expression in the EpCAM(+) CTC fraction at baseline, after 2 cycles of induction chemotherapy (week 6) and at the end of concurrent chemoradiotherapy (week 15). Results: PD-L1 overexpression was found in 24/94 (25.5%) patients at baseline, 8/34 (23.5%) after induction chemotherapy and 12/54 (22.2%) patients at the end of treatment. Patients with CTCs overexpressing PD-L1 at end of treatment had shorter progression-free survival (P = 0.001) and overall survival (P \textless 0.001). Multivariate analysis revealed that PD-L1 overexpression at end of treatment was independent prognostic factor for progression-free survival and overall survival. The absence of PD-L1 overexpression at the end of treatment was strongly associated with complete response with an odds ratio = 16.00 (95% CI = 2.76-92.72, P = 0.002). Conclusions: 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.

(1) Background: The aim of the study was to evaluate the prognostic significance of EMT-associated (TWIST1) and stem-cell (SC) transcript (CD24, CD44, ALDH1) quantification in EpCAM+ circulating tumor cells (CTCs) of early breast cancer patients. (2) Methods: 100 early stage breast cancer patients and 19 healthy donors were enrolled in the study. CD24, CD44, and ALDH1 transcripts of EpCAM+ cells were quantified using a novel highly sensitive and specific quadraplex RT-qPCR, while TWIST1 transcripts were quantified by single RT-qPCR. All patients were followed up for more than 5 years. (3) Results: A significant positive correlation between overexpression of TWIST1 and CD24&minus, /low/CD44high profile was found. Kaplan&ndash, Meier analysis revealed that the ER/PR-negative (HR-) patients and those patients with more than 3 positive lymph nodes that overexpressed TWIST1 in EpCAM+ cells had a significant lower DFI (log rank test, p &lt, 0.001, p &lt, 0.001) and OS (log rank test, p = 0.006, p &lt, 0.001). Univariate and multivariate analysis also revealed the prognostic value of TWIST1 overexpression and CD24&minus, /low/CD44high and CD24&minus, /low/ALDH1high profile for both DFI and OS. (4) Conclusions: Detection of TWIST1 overexpression and stem-cell (CD24, CD44, ALDH1) transcripts in EpCAM+ CTCs provides prognostic information in early stage breast cancer patients.

ObjectivesHuman papillomavirus-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) is increasing in incidence. Although HPV+ OPSCC has favorable prognosis, 10 to 25% of HPV+ OPSCCs eventually recur. We sought to evaluate the feasibility of detection of HPV16 E6/E7 expression in Circulating Tumor Cells (CTCs) and its utility as a prognostic tool in HPV16-associated OPSCC.Materials and methodsWe developed a highly sensitive RT-qPCR assay for HPV mRNA expression in EpCAM(+) CTCs. In 22 patients with early stage and locally advanced OPSCC we evaluated HPV16 E6/E7 expression in the EpCAM(+) CTC fraction at baseline and at the end of concurrent chemoradiotherapy. HPV status in pre-therapy formalin-fixed paraffin-embedded (FFPE) tumor biopsies was assessed by p16 immunohistochemistry and polymerase chain reaction (PCR) and double positives were subjected to Real-time qPCR assay for detection of HPV16, 18 and 31 types.ResultsFourteen of 22 OPSCC (63.6%) were HPV DNA+/p16+. Among HPV+/p16+ patients, 10 patients (71.4%) were HPV16 DNA+. HPV16 E6/E7(+) CTCs were detected in 3 of 10 patients (30%) at baseline and 4 of 9 patients (44.4%) at the end-of-treatment, all of which were p16+/HPV16 DNA+. Survival analysis showed a significantly higher risk for disease relapse (p = 0.001) and death (p = 0.005) in patients with HPV16 E6/E7(+) baseline CTCs.ConclusionDetection of HPV E6/E7(+) CTCs might be a useful noninvasive test in liquid biopsy samples for determination of a clinically relevant HPV infection in HPV+ OPSCC. Combined interpretation of HPV E6/E7(+) CTCs with UICC staging data may lead to alteration of risk definition of patient subsets, with improved risk discrimination in early-stage disease.

Background Liquid biopsy is based on minimally invasive blood tests and has the potential to characterize the evolution of a solid tumor in real time, by extracting molecular information from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Epigenetic silencing of tumor and metastasis suppressor genes plays a key role in survival and metastatic potential of cancer cells. Our group was the first to show the presence of epigenetic alterations in CTCs. Methods We present the development and analytical validation of a highly specific and sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) for the simultaneous detection of the methylation status of three tumor and metastasis suppressor genes (CST6, SOX17 and BRMS1 ) in liquid biopsy material (CTCs, corresponding ctDNA) and paired primary breast tumors. Results In the EpCAM-positive CTCs fraction we observed methylation of: a) CST6, in 11/30(37%) and 11/30(37%), b) BRMS1 in 8/30(27%) and 11/30(37%) c) SOX17 in 8/30(27%) and 13/30(43%) early breast cancer patients and patients with verified metastasis respectively. In ctDNA we observed methylation of: a) CST6, in 5/30(17%) and 10/31(32%), b) BRMS1 in 8/30 (27%) and 8/31 (26%) c) SOX17 in 5/30(17%) and 13/31(42%) early breast cancer patients and patients with verified metastasis respectively. Conclusions Our results indicate a high cancerous load at the epigenetic level in EpCAM-positive CTCs fractions and corresponding ctDNA in breast cancer. The main principle of the developed methodology has the potential to be extended in a large number of gene-targets and be applied in many types of cancer.

Simple Summary “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. Our results revealed for the first time a significantly higher positivity of all markers in EpCAM-positive CTCs compared to plasma-derived exosomes. We report that in EpCAM-positive CTCs, CK-19, PSMA, TWIST1 expression and GSTP1 methylation are significantly correlated with worse overall survival (OS), while in exosomes, CK-8 expression and GSTP1 and RASSF1A methylation status were significantly correlated with a lower OS. We also enumerated CTC and tumor-derived extracellular vesicles (tdEVs) using CellSearch (CS) and found a correlation between the CTC and tumor-derived extracellular vesicles (tdEVs) enumeration values. Abstract 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/7.5 mL PB. 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.

PIM-1 is an oncogene involved in cell cycle progression, cell growth, cell survival and therapy resistance, activated in many types of cancer, and is now considered as a very promising target for cancer therapy. We report for the first time that PIM-1 is overexpressed in circulating tumor cells (CTCs) from metastatic castration-resistant prostate cancer patients (mCRPC). We first developed and validated a highly sensitive RT-qPCR assay for quantification of PIM-1 transcripts. We further applied this assay to study PIM-1 expression in EpCAM(+) CTC fraction isolated from 64 peripheral blood samples of 50 mCRPC patients. CTC enumeration in all samples was performed using the FDA-cleared CellSearch&reg, system. PIM-1 overexpression was detected in 24/64 (37.5%) cases, while in 20/24 (83.3%) cases that were positive for PIM-1 expression, at least one CTC/7.5 mL PB was detected in the CellSearch&reg, Our data indicate that PIM-1 overexpression is observed at high frequency in CTCs from mCRPC patients and this finding, in combination with androgen receptor splice variant 7 (AR-V7) expression in CTCs, suggest its potential role as a very promising target for cancer therapy. We strongly believe that PIM-1 overexpression in EpCAM(+) CTC fraction merits to be further evaluated and validated as a non-invasive circulating tumor biomarker in a large and well-defined patient cohort with mCRPC.

BACKGROUND Liquid biopsy provides important information for the prognosis and treatment of cancer patients. In this study, we evaluated the effects of preanalytical conditions on gene expression and DNA methylation analyses in liquid biopsies. METHODS We tested the stability of circulating tumor cell (CTC) messenger RNA by spiking MCF-7 cells in healthy donor peripheral blood (PB) drawn into 6 collection-tube types with various storage conditions. CTCs were enriched based on epithelial cell adhesion molecule positivity, and RNA was isolated followed by cDNA synthesis. Gene expression was quantified using RT-quantitative PCR for CK19 and B2M. We evaluated the stability of DNA methylation in plasma under different storage conditions by spiking DNA isolated from MCF-7 cells in healthy donor plasma. Two commercially available sodium bisulfite (SB)-conversion kits were compared, in combination with whole genome amplification (WGA), to evaluate the stability of SB-converted DNA. SB-converted DNA samples were analyzed by real-time methylation-specific PCR (MSP) for ACTB, SOX17, and BRMS1. Quality control was assessed using Levey–Jennings graphs. RESULTS RNA-based analysis in CTCs is severely impeded by the preservatives used in many PB collection tubes (except for EDTA), as well as by time to analysis. Plasma and SB-converted DNA samples are stable and can be used safely for MSP when kept at −80 °C. Downstream WGA of SB-converted DNA compensated for the limited amount of available sample in liquid biopsies. CONCLUSIONS 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.

Objective Estrogen receptor, coded by the ESR1 gene, is highly expressed in epithelial ovarian cancer. ESR1 gene is frequently methylated in many types of gynecological malignancies. However, only a few studies attempted to investigate the role of ESR1 methylation and its clinical significance in ovarian cancer so far. The aim of our study was to examine ESR1 methylation status in primary tumors and corresponding circulating tumor DNA of patients with high-grade serous ovarian cancer (HGSC). Methods ESR1 methylation was detected by a highly specific and sensitive real-time methylation-specific PCR assay. Two groups of HGSC samples were analyzed: group A (n = 66 primary tumors) and group B (n = 53 primary tumors and 50 corresponding plasma samples). Results ESR1 was found methylated in both groups of primary tumors: in 32/66 (48.5%) of group A and in 15/53 (28.3%) of group B. 19/50 (38.0%) corresponding plasma samples of group B were also methylated for ESR1. A significant agreement for ESR1 methylation was observed between primary tumors and paired plasma ctDNA samples (P = 0.004). Interestingly, the presence of ESR1 methylation in primary tumor samples of group B was significantly correlated with a better overall survival (P = 0.027) and progression-free survival (P = 0.041). Conclusions We report for the first time the presence of ESR1 methylation in plasma ctDNA of patients with HGSC. The agreement between ESR1 methylation in primary tumors and paired ctDNA is statistically significant. Our results indicate a correlation between the presence of ESR1 methylation and a better clinical outcome in HGSC patients.

INTRODUCTION: Androgen-receptor splice variant 7 (AR-V7)is a highly promising liquid biopsy predictive biomarker indicating primary or acquired resistance to novel androgen receptor signaling inhibitors in metastatic castration resistant prostate cancer (mCRPC). We present for the first time the expression pattern of AR-FL, AR-V7, and AR-567es at a quantitative level in circulating tumor cells (CTCs) and paired plasma-derived exosomes in mCRPC. METHODS: We first developed and analytically validated a novel multiplex RT-qPCR assay for AR full length (AR-FL), AR-V7, AR-567es and AR-total. We then quantified the expression levels of AR-splice variants, CK-19 (epithelial marker) and B2M (reference gene) in EpCAM+ CTCs and paired plasma-derived exosomes isolated from peripheral blood (20mL) of 62 mCRPC patients and 10 healthy donors. RESULTS: In CTCs AR-FL was detected in 57/62(92%),AR-V7 in 32/62(52.0%),AR-567es in15/62(24.2%) and AR-total in 56/62(90.0%). In paired plasma-derived exosomes, AR-FL was detected in 45/62(72.5%), AR-V7 in 4/62(6.5%), AR-567 in 2/62(3.2%) and AR total in 47/62(75.8%).CK-19 expression was detected in 18/62(29.0%) of CTCs and in 28/62(45.2%) of exosomes. In all cases AR splice variants were expressed at higher levels in CTCs than in paired exosomes, while AR-V7 was detected at higher percentages than AR-567es. CONCLUSIONS: Our results reveal for the first time a remarkable heterogeneity on the expression levels of AR-FL, AR-V7 and AR-567es in EpCAM+ CTCs and paired exosomes between individual mCRPC patients. The clinical significance of this finding will be further investigated in large patient cohorts in respect to therapy response. Citation Format: Areti D. Strati, Martha Zavridou, Evangelos Bournakis, Sophia Mastoraki, Evi S. Lianidou. Expression pattern of androgen receptor (AR), splice variant 7 (AR-V7) and splice variant 567 (AR-567) in circulating tumor cells and paired plasma-derived exosomes in metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2290.

Background: Circulating tumor cells (CTCs) heterogeneity is highly affecting the efficiency of their isolation and thus the reliability of downstream analysis. Especially in Head and Neck Squamous Cell Carcinoma (HNSCC) epithelial mesenchymal transition (EMT) is highly affecting CTC isolation and downstream analysis. We directly compared two different approaches used for CTC isolation, a label-independent size-based microfluidic-based system versus an EpCAM-based positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in HNSCC. Methods: Peripheral blood (PB) in EDTA (20mL) was collected from 50 HNSCC patients and 18 healthy donors (HD). A size-based microfluidic device (Parsortix, ANGLE) and an EpCAM-based positive immune-magnetic isolation procedure were applied in parallel, using 10mL PB in each case. 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. Real time methylation specific PCR (MSP) was used to study the methylation status of SOX17, RASSF1A and MLL3 genes in DNAs isolated from the same enriched CTCs. Results: In identical blood draws, the label-free size-based CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-based CTC enrichment, since a significantly higher percentage of identical PB samples was found positive for all genes tested both at the gene expression and DNA methylation level, while the specificity was not affected. Conclusions: In HNSCC CTC molecular characterization at the gene expression and DNA methylation level should be based on a label-free size-based isolation system. Citation Format: Martha Zavridou, Sophia Mastoraki, Areti D. Strati, George Koutsodontis, Apostolos Klinakis, Amanda Psyrri, Evi S. Lianidou. Molecular characterization of circulating tumor cells in head and neck squamous cell carcinoma: Direct comparison of a label-independent size-based microfluidic device with EpCAM-based CTC enrichment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2241.

Objectives We have recently shown that detection of CST6 promoter methylation in primary breast tumors can provide important prognostic information in patients with operable breast cancer and that CST6 promoter is also methylated in Circulating Tumor Cells (CTC). In this study we evaluated the presence of CST6 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of breast cancer patients. Design and methods Our study material consisted of: a) a pilot testing group of 27 patients with stage I-III operable breast cancer, 46 patients with verified metastasis and 37 healthy donors and b) an independent cohort of 123 consecutive stage I-III operable breast cancer patients. Methylated and unmethylated CST6 promoter sequences were detected by using methylation-specific PCR (MSP). CST6 immunohistochemical detection was performed in 20 corresponding primary tumor tissues. Results In the pilot testing group, CST6 promoter was methylated in 8/27 (29.6%) operable breast cancer patients, in 6/46 (13.0%) patients with verified metastasis but none of 37 healthy individuals (0%). In the independent cohort, 49/123 (39.8%) operable breast cancer patients were found positive. During the follow up period, 25/123 (20.3%) patients relapsed and 9/123 (7.3%) died. CST6 was methylated in cfDNA of 13/25 (52%) patients that relapsed and in 3/9 (33.3%) patients that died. Conclusions CST6 promoter is highly methylated in cfDNA of breast cancer patients, but not in healthy individuals. CST6 promoter methylation in cfDNA, should be prospectively validated as a novel plasma tumor biomarker for breast cancer in a large cohort 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.

// Maria Chimonidou 1 , Areti Strati 1 , Nikos Malamos 2 , Sophia Kouneli 2 , Vassilis Georgoulias 3 and Evi Lianidou 1 1 Analysis of Circulating Tumour Cells Laboratory, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, Greece 2 Department of Pathology, Oncology Unit, Helena Venizelou Hospital, Athens, Greece 3 Laboratory of Tumour Cell Biology, Medical School, University of Crete, Heraklion, Greece Correspondence to: Evi Lianidou, email: lianidou@chem.uoa.gr Keywords: liquid biopsy, circulating tumour cells, circulating tumour DNA, breast cancer, methylation specific PCR Received: March 25, 2016 Accepted: March 29, 2017 Published: June 27, 2017 ABSTRACT Circulating Tumour Cells (CTCs) and circulating tumour DNA (ctDNA) represent a non-invasive liquid biopsy approach for the follow-up and therapy management of cancer patients. We evaluated whether DNA methylation status in CTCs and ctDNA is comparable and whether it reflects the status of primary tumours. We compared the methylation status of three genes, SOX17, CST6 and BRMS1 in primary tumours, corresponding CTCs and ctDNA in 153 breast cancer patients and healthy individuals, by using real time methylation specific PCR. We report a clear association between the EpCAM-positive CTC-fraction and ctDNA for SOX17 promoter methylation both for patients with early ( P = 0.001) and metastatic breast cancer ( P = 0.046) but not for CST6 and BRMS1. In early breast cancer, SOX17 promoter methylation in the EpCAM-positive CTC-fraction was associated with CK-19 mRNA expression ( P = 0.006) and worse overall survival (OS) ( P = 0.044). In the metastatic setting SOX17 promoter methylation in ctDNA was highly correlated with CK-19 ( P = 0.04) and worse OS ( Ρ = 0.016). SOX17 methylation status in CTCs and ctDNA was comparable and was associated with CK-19 expression but was not reflecting the status of primary tumours in breast cancer. DNA methylation analysis of SOX17 in CTCs and matched ctDNA provides significant prognostic value.

Blood testing using Circulating Tumor Cells (CTCs) has emerged as one of the hottest fields in cancer diagnosis. Research on CTCs present nowadays a challenge, as these cells are well defined targets for understanding tumour biology and improving cancer treatment. The presence of tumor cells in patient's bone marrow or peripheral blood is an early indicator of metastasis and may signal tumor spread sooner than clinical symptoms appear and imaging results confirm a poor prognosis. CTC enumeration can serve as a "liquid biopsy" and an early marker to assess response to systemic therapy. Definition of biomarkers based on comprehensive characterization of CTCs has a strong potential to be translated to individualized targeted treatments and spare breast cancer patients unnecessary and ineffective therapies but also to reduce the costs for the health system and to downsize the extent and length of clinical studies. In this review, we briefly summarize recent studies on the molecular characterization of circulating tumor cells in breast cancer and discuss challenges and promises of CTCs for individualized cancer treatment.

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. METHODS We obtained peripheral blood from 56 patients with operable breast cancer, 27 patients with verified metastasis, and 23 healthy individuals. We tested DNA extracted from the EpCAM-positive immunomagnetically selected CTC fraction for the presence of methylated and unmethylated CST6, BRMS1, and SOX17 promoter sequences by methylation-specific PCR (MSP). All samples were checked for KRT19 (keratin 19, formerly CK-19) expression by reverse-transcription quantitative PCR. RESULTS In CTCs of patients with operable breast cancer, promoter methylation of CST6 was observed in 17.9%, BRMS1 in 32.1%, and SOX17 in 53.6% of patients. In CTCs of patients with verified metastasis, promoter methylation of CST6 was observed in 37.0%, BRMS1 in 44.4%, and SOX17 in 74.1%. In healthy individuals, promoter methylation of CST6 was observed in 4.3%, BRMS1 in 8.7%, and SOX17 in 4.3%. DNA methylation of these genes for both operable and metastatic breast cancer was significantly different from that of the control population. CONCLUSIONS DNA methylation of tumor suppressor and metastasis suppressor genes is a hallmark of CTCs and confirms their heterogeneity. Our findings add a new dimension to the molecular characterization of CTCs and may underlie the acquisition of malignant properties, including their stem-like phenotype.

Background: The estrogen receptor is highly expressed in epithelial ovarian cancer and represents the main target for endocrine therapy. The ESR1 gene is frequently methylated in many types of gynecological malignancies and previous studies have shown an inverse correlation between ESR1 methylation and gene expression. Few studies attempted to investigate the role of ESR1 methylation in ovarian cancer so far, and the clinical significance of ESR1 methylation status is not as yet clear. Methods: The ESR1 methylation status was examined in primary tumors and corresponding circulating tumor DNA (ctDNA) samples of patients with high-grade serous ovarian cancer. For the detection of methylation we applied a novel highly specific and sensitive real-time methylation specific PCR (real-time MSP) assay. Two groups of primary tumor samples were recruited (training group, n=66 and validation group, n=61), along with the corresponding plasma samples (n=58) for the validation group. ESR1 methylation was also analyzed in a small group of 16 normal fallopian tube samples. Results: ESR1 was found methylated in both groups of primary tumor samples and in the corresponding plasma samples of the validation group. More specifically, ESR1 methylation was detected in 32/66 (48.5%) and 17/61 (27.9%) primary tumor samples of the training and the validation group, respectively, and in 23/58 (39.7%) corresponding plasma samples. A significant agreement between ESR1 methylation in primary tumors and paired ctDNA was observed in 40/56 (71.4%) samples of the validation group (P=0.004, k=0.360). Almost all normal fallopian tube samples (15/16) were found methylated. Interestingly, the presence of ESR1 methylation in the primary tumor samples of the validation group was nearly significantly correlated (P=0.057) with a better overall survival. Conclusions: We detected for the first time ESR1 methylation in ctDNA of patients with high-grade serous ovarian cancer. The agreement between ESR1 methylation in the primary tumors and paired ctDNA is statistically significant. Our results indicate a potential correlation between ESR1 methylation and better overall survival in high-grade serous ovarian cancer patients. Citation Format: Lydia Giannopoulou, Sofia Mastoraki, Areti Strati, Issam Chebouti, Kitty Pavlakis, Sabine Kasimir-Bauer, Evi Lianidou. ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4587.

Introduction: Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of CTCs and plasma-ctDNA. ESR1 epigenetic silencing potentially affects response to endocrine treatment. We evaluated ESR1 methylation in CTCs and paired plasma-ctDNA. We evaluated ESR1 methylation in CTCs and paired plasma-ctDNA as a potential biomarker for response to everolimus/exemestane treatment. Materials and methods: A highly sensitive and specific real-time MSP assay for ESR1 methylation was developed and validated in: a) 65 primary breast tumors (FFPEs), b) EpCAM+ CTC-fractions (122 patients and 30 healthy donors; HD), c) plasma-ctDNA (108 patients and 30HD), d) in CTCs (CellSearch®) and in paired plasma-ctDNA for 58 BrCa patients. ESR1 methylation status was investigated in CTCs isolated from serial peripheral blood samples of 19 patients with ER+/ HER2- advanced BrCa receiving everolimus/exemestane. Results: ESR1 methylation was detected in: a) 25/65(38.5%) FFPEs, b) EpCAM+ CTC-fractions: 26/112(23.3%) patients and 1/30(3.3%) HD, c) plasma-ctDNA: 8/108(7.4%) patients and 1/30(3.3%) HD. ESR1 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, ESR1 methylation was observed in 10/36(27.8%) CTC-positive samples, and was associated with lack of response to treatment (p=0.023 Fisher's Exact Test). Conclusions: We report for the first time the detection of ESR1 methylation in CTCs and a high concordance with paired plasma-ctDNA. ESR1 methylation in CTCs was associated with lack of response to everolimus/exemestane regimen. ESR1 methylation should be further evaluated as a potential liquid biopsy-based biomarker. Citation Format: Sofia Mastoraki, Areti Strati, Eleni Tzanikou, Maria Chimonidou, Eleni Politaki, Alexandra Voutsina, Amanda Psyrri, Vassilis Georgoulias, Evi S. Lianidou. ESR1 methylation: A liquid biopsy-based epigenetic assay for the follow up of patients with metastatic breast cancer receiving endocrine treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4577.

Objectives To evaluate the potential of ellagic acid to inhibit the expression of human telomerase reverse transcriptase ( hTERT ) α+β+ splice variant in MCF-7 breast cancer cells. Design and methods MCF-7 cells were incubated with ellagic acid (10 − 9 M–10 − 5 M) in the absence and in the presence of 17β-estradiol (10 − 8 M), a known inducer of hTERT transcription, and hTERT α+β+ mRNA expression was quantified by real-time RT-PCR. 17β-estradiol and ICI182780, a known estrogen antagonist, served as positive and negative controls respectively. Results Ellagic acid, when alone, increased hTERT α+β+ mRNA while its coexistence with 17β-estradiol reduced significantly the 17β-estradiol-induced increase in hTERT α+β+ mRNA, implicating thus both its estrogenic and anti-estrogenic effects in breast cancer cells. Conclusions The potential of ellagic acid to down-regulate the 17β-estradiol-induced hTERT α+β+ mRNA expression may be a mechanism via which ellagic acid exerts, at least in part, its chemopreventive effects in breast cancer.

Detection of Circulating Tumor Cells (CTC) in peripheral blood can serve as a “liquid biopsy” approach and has thus emerged lately as one of the hottest fields in cancer research. A variety of molecular assays are continuously been developed for CTC detection and molecular characterization. Molecular assays are based on the nucleic acid analysis in CTCs like RT-qPCR, multiplex RT-qPCR, methylation specific PCR, ARMS-PCR, and next-generation sequencing technologies. The main strategies are based on total RNA isolation and subsequent mRNA quantification of specific genes, and isolation of genomic DNA for DNA methylation studies and mutation analysis. Molecular characterization of CTC holds considerable promise for the identification of therapeutic targets and resistance mechanisms in CTCs as well as for the stratification of patients and real-time monitoring of systemic therapies. Quality control and standardization of these methodologies is very important for the incorporation of CTCs into prospective clinical trials testing their clinical utility. This review is mainly focused on the basic principles and clinical applications of molecular assays that are currently used for the detection and molecular characterization of CTCs.

Detection of Circulating Tumor Cells (CTCs) in peripheral blood can serve as a "liquid biopsy" approach and as a source of valuable tumor markers. CTCs are rare, and thus their detection, enumeration and molecular characterization are very challenging. CTCs have the unique characteristic to be non-invasively isolated from blood and used to follow patients over time, since these cells can provide significant information for better understanding tumour biology and tumour cell dissemination. CTCs molecular characterization offers the unique potential to understand better the biology of metastasis and resistance to established therapies and their analysis presents nowadays a promising field for both advanced and early stage patients. In this chapter we focus on the latest findings concerning the clinical relevance of CTC detection and enumeration, and discuss their potential as tumor biomarkers in various types of solid cancers. We also highlight the importance of performing comparison studies between these different methodologies and external quality control systems for establishing CTCs as tumor biomarkers in the routine clinical setting.

Detection of Circulating Tumor Cells (CTCs) in peripheral blood can serve as a “liquid biopsy” approach and as a source of valuable tumor markers. CTCs are rare, and thus their detection, enumeration and molecular characterization are very challenging. CTCs have the unique characteristic to be non-invasively isolated from blood and used to follow patients over time, since these cells can provide significant information for better understanding tumour biology and tumour cell dissemination. CTCs molecular characterization offers the unique potential to understand better the biology of metastasis and resistance to established therapies and their analysis presents nowadays a promising field for both advanced and early stage patients. In this chapter we focus on the latest findings concerning the clinical relevance of CTC detection and enumeration, and discuss their potential as tumor biomarkers in various types of solid cancers. We also highlight the importance of performing comparison studies between these different methodologies and external quality control systems for establishing CTCs as tumor biomarkers in the routine clinical setting.

Background: Estrogen receptor (ER) is an important prognostic biomarker in breast cancer. Epigenetic silencing of ESR1 could be of important clinical significance especially for its potential impact on endocrine treatment efficacy. Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of CTCs and ctDNA. Our group has evaluated for the first time epigenetic silencing of tumor and metastasis suppressor genes in CTCs and corresponding ctDNA. In this study, we evaluated for the first time ESR1 methylation in CTCs, paired ctDNA and primary tumors of breast cancer patients. Methods: We developed and validated a highly sensitive and specific real-time MSP assay for ESR1 methylation. We further applied the developed assay in sodium bisulfite (SB) treated DNA samples from: a) FFPEs from 40 patients with operable breast cancer, 25 patients with metastasis, 30 mammoplasties and 15 fibroadenomas, b) EpCAM+ immunomagnetically isolated CTCs fractions, from 74 early breast cancer patients, 48 patients with metastasis and 30 healthy donors, c) CellSearch® cartridges from 36 early breast cancer patients, 22 patients with metastasis, d) ctDNA isolated from plasma of matched samples and 54 healthy donors as a control group. Results: By using this highly specific and sensitive assay (sensitivity 0.1%) we detected methylation of ESR1 in: a) FFPEs: 16/40(40%) early breast cancer patients, 9/25(36%) patients with verified metastasis, 7/30(23.3%) mammoplasties and 5/15(33.3%) fibroadenomas. A statistically significant negative correlation was observed between ESR1 methylation status and ER protein expression (56/65 samples, 86%, p Conclusions: ER expression and ESR1 methylation were found 100% inversely correlated in primary tissues. The EpCAM+ CTC fraction of patients with breast cancer was found methylated for ESR1. Interestingly, ESR1 methylation was detected exclusively in CTC+ samples as analyzed from CellSearch® cartridges but in none of CTC- samples. In paired plasma samples, ESR1 methylation showed a high concordance (p Citation Format: Sophia Mastoraki, Areti Strati, Eleni Tzanikou, Eleni Politaki, George Koutsodontis, Loukas Kaklamanis, Nikolaos Malamos, Amanda Psyrri, Vassilis Georgoulias, Evi Lianidou. ESR1 methylation in circulating tumor cells, ctDNA and primary tumors of breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1730. doi:10.1158/1538-7445.AM2017-1730

Prostate cancer (PC) is the second leading cause of cancer death in men in the US. The aberrant functioning of androgen receptor signaling is the central driving force behind prostatic tumorigenesis and its transition into metastatic castration resistant disease. Hence, androgen deprivation therapy (ADT) is the first line of treatment for PC patients. However, many patients progress becoming resistant to ADT therapy, due to the expression of AR splice variants (AR-Vs), which lack the ligand binding domain and are constitutively active in the nucleus. Expression of the AR splice variant, AR-v7, in circulating tumor cells (CTCs) isolated from the blood of PC patients was correlated with resistance to enzalutamide and abiraterone, which are the next generation AR signaling inhibitors in CRPC. Further, there is evidence that AR-Vs may convey cross-resistance, not only to enzalutamide and abiraterone, but also to taxanes, highlighting that their assessment in the clinic may have clinical utility. We developed a novel, specific and highly sensitive assay to measure mRNA expression of the AR full length (AR-FL) and the splice variants ARv7 and ARv567es, by using Droplet Digital PCR in CTCs isolated from CRPC patients. The analytical specificity of the assay was determined by transfecting cells with plasmids encoding AR-FL, AR-v7 and AR-v567 and showed that each probe detected signal only in cells expressing the respective transcript. No signal was detected against genomic DNA, indicating lack of non-specific binding. Also, the assay detected endogenous expression of AR-FL and AR-v7 in VCAP or 22RV1 cells, while no variant expression was detected in healthy donor blood. The analytical sensitivity of the assay was determined in a series of serial dilution experiments that showed sensitivity down to single cell. We then used this assay to determine the clinical prevalence and expression pattern of each of these variants in CTCs from about 200 mCRPC patient samples and blood from 40 healthy donors. CTCs were enriched by EpCAM- or PSMA-based positive selection or CD45 negative depletion in an antigen-agnostic manner. AR-FL was detected in ~80% of mCRPC samples irrespective of CTC-enrichment technology. AR-v7 was expressed in 65% of the samples in which in CTCs were enriched either by PSMA-positive selection or by negative depletion. In contrast, EpCAM-based CTC enrichment showed lower AR-v7 expression both in terms of expression levels and prevalence. In addition, CTC enrichment following negative depletion showed that 30% of the samples had higher AR-v7 expression levels as compared to AR-FL. This expression pattern was not observed in the samples using EpCAM-based selection. Collectively, these data suggest distinct CTC subpopulations are present in CRPC patient samples, with differential expression of AR-Vs that could have important predictive and prognostic implications. Citation Format: Ada Gjyrezi, Giuseppe Galletti, Areti Strati, Seaho Kim, Evi Lianidou, David M. Nanus, Jun Luo, Emmanuel Antonarakis, Scott T. Tagawa, Andrew Armstrong, Paraskevi Giannakakou. A digital droplet PCR assay for the quantitation of androgen receptor and splice variant expression in CTCs from metastatic castration resistant prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2736. doi:10.1158/1538-7445.AM2017-2736