A Comprehensive Molecular Analysis of in Vivo Isolated EpCAM-Positive Circulating Tumor Cells in Breast Cancer

Abstract

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.

Introduction

Liquid biopsy is a minimally invasive tool for real-time monitoring of patients with cancer that provides information about tumor burden, early evidence of recurrence, and treatment efficacy or resistance (1–3). The main advantages of liquid biopsy over conventional tissue biopsy are its minimally invasive nature, real-time profiling of tumors, and a global representation of tumor heterogeneity that reflects the clonal dynamics of tumors (1, 2).

Circulating tumor cells (CTCs) are detected at very low numbers in the blood of most patients with cancer, and their enrichment and downstream molecular characterization require specific technologies with extreme analytical sensitivity and specificity (1, 2, 4). Immunomagnetic separation, based on EpCAM expression, is the most widely used approach to isolate CTCs from patients’ blood. Currently, the only FDA-cleared technology for the isolation and enumeration of CTCs in metastatic breast (5), prostate, and colorectal cancer is the CellSearch System (Menarini-Silicon Biosystems). Enumeration of CTCs is of prognostic significance in metastatic breast cancer (MBC) (5) and early breast cancer (BrCa) when a higher amount of peripheral blood is used for CTC isolation (6).

Beyond enumeration, CTCs can be further characterized by using immunological, molecular, or functional assays (2). Molecular assays based on sensitive and specific reverse transcription quantitative PCR (RT–qPCR) have already revealed a high heterogeneity of gene expression in CTCs, and they offer a promising approach for therapeutic monitoring and discovery of new therapeutic targets on CTCs (7, 8). In patients with MBC, it has already been shown that a signature of 8 genes could discriminate good and poor outcome to first-line aromatase inhibitors (9). Moreover, in MBC, the phenotypic heterogeneity of CTCs during first-line systemic therapy is associated with resistance to systemic therapy (10).

DNA mutations are deeply involved in cancer progression and are related to response of specific therapies. Detection of PIK3CA mutations in primary tissues and/or circulating tumor DNA (ctDNA) in patients who have hormone receptor positive advanced BrCa is critical for the administration of the FDA-approved drug Alpelisib (11). PIK3CA hotspot mutations are present at high frequency in CTCs, and real-time monitoring of PIK3CA in CTCs could have a major clinical impact in patients with BrCa (12). Epigenetic silencing of critical tumor suppressor and metastasis suppressor genes also has been detected in CTCs and is related to worse prognosis (13, 14). Recently we reported that the detection of ESR1 methylation in primary tumors, ctDNA and CTC in patients who are operable and who have MBC is associated with lack of response to endocrine therapy and worse overall survival (15).

In vivo isolation of CTCs was first reported in 2012 (16) and it has been so far successfully tested in lung (17), prostate (18), and colorectal cancer (19). Recently, it was shown that combination of in vivo isolation of CTCs from patients with prostate cancer with downstream highly specific and sensitive RT–qPCR assays is minimally invasive, capable of high throughput, and suitable for the development of molecular diagnostic applications (20). In the present study, we evaluated for the first time the combination of in vivo-CTC isolation with a downstream comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level in breast cancer. We further directly compared our results to those derived by the FDA-cleared CellSearch System and a triple immunofluorescence (IF) assay using the same blood samples.

Methods

Study Outline

An outline of the study is shown in Fig. 1. In this study we enrolled: (a) 42 patients with early BrCa before the administration of adjuvant chemotherapy 13 of whom had a second sample obtained after the completion of therapy, (b) 23 patients with MBC at first diagnosis before the administration of any systemic therapy, 7 of whom had a second sample obtained after 1 treatment cycle. The clinical characteristics of all patients are shown in Table 1 in the online Data Supplement. EpCAM-positive CTCs were isolated in vivo and subjected to molecular analysis, while the same blood samples were used for: (a) CTC enumeration using CellSearch, and (b) triple immunofluorescence (IF)-staining of CTCs. A group of 19 healthy donors (HD), was analyzed in exactly the same way (in vivo isolation) as a control group. The study was carried out in accordance to Declaration of Helsinki. All patients gave written informed consent for participation in this study and publication of results. The present study was approved by the Medical Ethical Committee of 3 different clinical centers: University General Hospital of Heraklion, Crete, Greece (Ethical Allowance: 8756/23-6-2014); Attikon University Hospital, Athens, Greece (Ethical Allowance: ΕΒΔ448/14-10-14); and the University Medical Center Hamburg-Eppendorf, Germany (Ethical Allowance: PV3779).

Molecular Analysis in in Vivo Isolated CTCs

All protocols for the molecular analysis of in vivo isolated CTCs (8, 20–23) are presented in detail in online Supplemental File 1.

CTC Enumeration and Triple Immunofluorescence (IF)

Here, 85 peripheral blood samples at 2 different time points were collected into CellSave tubes in parallel with the in vivo-CTC isolation and further processed for CTC enumeration using the CellSearch. In early BrCa, 47 samples (34 before and 13 after therapy) were collected at the University of Athens (UoA) (22.5 mL of blood) and 8 samples at the University Medical Center Hamburg-Eppendorf (UKE) (7.5 mL of blood), whereas in MBC 30 samples (23 before and 7 after therapy) were collected (7.5 mL of blood). CellSearch analysis was evaluable for 84/85 samples. Triple IF analysis in CTCs was performed using the Ariol system as previously described (online Supplemental File 1) (24). Double staining experiments were also performed in all patients’ samples for creatine kinase (CK)/CD45 to exclude false positive samples (24).

Results

Specificity of RT–qPCR Assays

The diagnostic specificity of the developed singleplex and multiplex RT–qPCR assays used for gene expression analysis in CTCs, was evaluated by applying the in vivo-CTC isolation step and downstream molecular analysis in the 19 HD control group. CK-8, CK-18, CK-19, ESR1, PR, ERBB2, VEGF, TWIST1, and EGFR transcripts were not detected in any of these samples. CD24, CD44, ALDH1, VIM, and CDH2 transcripts were detected, as expected, due to the presence of contaminating peripheral blood mononuclear cells (PBMC) that were coisolated through EpCAM selection; thus, in all samples, CD24, CD44, ALDH1, VIM, and CDH2 expression was normalized to the expression of B2M (25). For each of these genes, a cutoff value was estimated as Cq_mean ± 2SD, of Cq signals derived from these 19 HD control samples.

Gene Expression in CTCs Isolated in Vivo

Early breast cancer.

Before the initiation of adjuvant chemotherapy, the gene expression profile for the in vivo isolated CTCs from 42 patients and 19 HD samples is shown as a heat map (Fig. 2, A). The expression of at least 1 RNA-based marker was detected in most samples; CK8: 3/42 (7.1%), CK18: 1/42 (2.4%), CK19: 8/42 (19.1%), ESR1: 2/42 (4.8%), PR: 1/42 (2.4%), ERBB2: 2/42 (4.8%), VEGF: 10/42 (23.8%), TWIST1: 1/42 (2.4%). All samples were negative for EGFR, VIM, and CDH2 whereas 2/42 (4.8%) were CD44⁺/CD24⁻ positive and 1/42 (2.4%) was ALDH1^+/high/CD24⁻ positive (online Supplemental Table 2, Fig. 2, A). In total, 23/42 (54.8%) samples were positive for at least one RNA-based marker, while 6/42 (14.3%) and 2/42 (4.8%) samples were positive for 2 and 3 RNA-based markers, respectively.

After the completion of adjuvant chemotherapy, in vivo isolation of CTC was also performed in 13/42 (31.0%) of these patients with early BrCa (Fig. 2, B). In this smaller cohort of patients, before chemotherapy, 1/13 (7.7%) sample was positive for CK8, all samples were negative for CK18 and CK19, 3/13 (23.0%) were positive for VEGF, while no sample was positive for PR, TWIST1, EGFR, CD44^high/CD24^−/low, or ALDH1^high/CD24^−/low. After chemotherapy no sample was positive for CK8, 2/13 (15.4%) were positive for CK18, 3/13 (23.1%) for CK19, 2/13 (15.4%) for VEGF, and 1/13 (7.7%) for PR, EGFR, CD44^high/CD24^−/low, ALDH1^high/CD24^−/low, and TWIST1, while no sample was positive for ESR1 or ERBB2 expression.

Metastatic breast cancer

In MBC, before the first cycle of therapy, 20/23 (87.0%) samples were positive for at least 1 gene target, whereas 5/23 (21.7%) and 1/23 (4.4%) samples were positive for at least 2 and 3 gene targets, respectively (Fig. 2, A). More specifically, samples were positive for: CK8: 2/23 (8.7%), CK18: 1/23 (4.3%), CK19: 6/23 (26.1%), VEGF: 8/23 (34.8%), ALDH1^high/CD24^−/low: 5/23 (21.7%), CD44^high/CD24^−/low: 1/23 (4.3%), VIM: 1/23 (4.3%), and CDH2: 2/23 (8.7%) while no sample was positive for ESR1, PR, ERBB2, EGFR, or TWIST1 (online Supplemental Table 2, Fig. 2, A).

For 7/23 of these patients with MBC, samples after 1 treatment cycle were also available. These 7 samples before therapy were positive for CK8: 2/7 (28.6%), CK18: 1/7 (14.3%), while no sample was positive for CK19. 2/7 (28.6%) samples were ALDH1^high/CD24^−/low, 2/7 (28.6%) were positive for VEGF and 1/7 (14.3%) for CD44^high/CD24^−/low, while all samples were negative for ESR1, PR, ERBB2, TWIST1, and EGFR expression (Fig. 2, B). In the same group after the completion of the first cycle, 2/7 (28.6%) were positive for CK19 and all were negative for both CK8 and CK18. 2/7 (28.6%) samples were positive for VEGF, 2/7 (28.6%) for TWIST1, 1/7 (14.3%) for CD44^high/CD24^−/low, whereas all samples were negative for ALDH1^high/CD24^−/low, VIM, ESR1, PR, ERBB2, CDH2, and EGFR expression. At least 1 gene was expressed in all cases (7/7, 100%) before the first cycle of therapy and in 5/7 (71.4%) cases after the first cycle of therapy.

Detection of PIK3CA Hotspot Mutations by ddCR in CTCs Isolated in Vivo

PIK3CA hotspot mutations (E545K, H1047R) were examined by ddPCR in all 85 genomic DNA (gDNA) samples derived from in vivo isolated CTC and 19 HD, after whole genome amplification (Fig. 2, A). In HD, no mutant signals were observed for the PIK3CA E545K mutation, whereas in 2/19 samples, 2 and 3 positive droplets for the H1047R mutation were detected. Based on this observation, only samples with at least 4 positive droplets were reported as positive. PIK3CA mutations were detected in 11/42 (26.2%) early BrCa samples derived before therapy and in 3/13 (23.1%) after, while in MBC, 11/23 (47.8%) samples were positive before the first cycle of therapy and 2/7 (28.6%) after. Representative 1D plots of ddPCR are shown in Fig. 3, A. More specifically, the PIK3CA E545K mutation was detected in 10/42 (23.8%) early BrCa samples before and in 2/13 (15.4%) samples after therapy, while the PIK3CA H1047R mutation was detected in 1/42 (2.4%) samples before and in 2/13 (15.4%) samples after therapy. In MBC, the PIK3CA E545K mutation was detected in 10/23 (43.5%) samples before the first cycle and in no samples after completion of the first cycle, whereas the PIK3CA H1047R mutation was detected in 5/23 (21.7%) and 2/7 (28.6%) MBC before the first cycle and after completion of the first cycle of therapy.

For 16 patients, paired primary tissue (FFPE) was available with the in vivo isolated CTC samples. In DNA extracted from these primary tumor biopsies, we identified the PIK3CA E545K mutation in 11/16 (68.8%) and the PIK3CA H1047R mutation in 5/16 (31.3%) cases. In gDNA derived from the corresponding in vivo isolated CTCs the presence of PIK3CA E545K and PIK3CA H1047R mutations was confirmed in 10/16 (62.5%) and 9/16 (56.3%) cases, respectively (Table 1). It is important that in patient #2 (metastasis group), both the PIK3CA E545K and PIK3CA H1047R mutations were detected in DNA isolated from in vivo isolated CTCs, but were not detected in the corresponding FFPE, whereas in patient #5 (metastasis group) the PIK3CA H1047R was also detected only in in vivo isolated CTCs but not in the corresponding FFPE.

Detection of ESR1 Methylation in CTCs Isolated In Vivo

After quality control of sodium bisulfite-converted DNAs, samples from 63 patients and 16 HD samples were evaluable for checking ESR1 methylation status (Fig. 2, A). In early BrCa, ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) patients’ samples before and after therapy, respectively. In MBC, ESR1 methylation was detected in 3/15 (20.0%) patients before first-line therapy, while all samples after completion of the first line were negative (Fig. 3, B). DNA was isolated from 10 corresponding primary tumors (FFPEs) that were available for comparison studies, since these were the only common cases that our quality control enabled the analysis for sodium bisulfite-converted DNA in CTCs. ESR1 methylation was detected in all these FFPE samples (10/10, 100%), while in the corresponding CTCs only 1 sample was positive (1/10, 10.0%).

CTC Enumeration and Triple IF Staining

Early BrCa.

Before the initiation of adjuvant chemotherapy, 42 patient samples were analyzed in the CellSearch; 5/34 (14.7%) were found to have at least one CTC/22.5 mL of PB (collected in UoA), while 2/8 (25%) were found to have at least one CTC/7.5 mL of PB (collected in UKE) (Fig. 2, A). Triple IF staining was also performed in 34/42 of these samples; 8/34 (23.5%) samples were positive for CKs (8, 18, 19), 5/34 (14.7%) were found to have the phenotype ER⁻/HER2⁻/CK⁺, 3/34 (8.8%) the phenotype ER⁺/HER2⁻/CK⁺, 2/34 (5.9%) the phenotype ER⁻/HER2⁺/CK⁺, while none of the CTCs was positive for the ER⁺/HER2⁺/CK⁺ phenotype (online Supplemental Table 3, Fig. 2, A). For 13/42 of these patients with early BrCa, samples were available after the completion of adjuvant chemotherapy (online Supplemental Table 4). CellSearch analysis in this group has shown that 2 patients (patient #4 and patient #7) were CTC positive both before and after chemotherapy, while 3 patients (patient #5, patient #12, and patient #3) were positive only after chemotherapy (online Supplemental Table 4). Triple IF staining in this group revealed that 4/13 (30.7%) had CTCs before and 3/13 (23.1%) had CTCs after therapy. Only one patient (patient #7) was positive both before and after treatment (online Supplemental Table 4). More specifically 2/13 (15.4%) were found to have the phenotype ER⁻⁻/HER2⁻/CK⁺, 1/13 (7.7%) the phenotype ER⁺/HER2⁻/CK⁺, while there were no CTCs bearing the ER⁺/HER2⁺/CK⁺ or the ER⁻/HER2⁺/CK⁺ phenotypes (Fig. 2, B, online Supplemental Table 3). Results for CTC in vivo molecular analysis, IF staining, CellSearch analysis and clinical outcome in patients with early BrCa before and after the completion of therapy are shown in online Supplemental Table 5. Ηowever, since the follow-up period was short, it was not possible to make any correlations with the clinical outcome.

MBC

CTC enumeration was performed in 22 MBC samples and 9/22 (40.9%) had more than 5CTC/7.5 mL of PB (Fig. 2, A). Triple IF staining was performed in 12 of these samples; 3/12 (25.0%) were positive for CKs (8, 18, 19), 1/12 (8.3%) was ER⁻/HER2⁻/CK⁺, 2/12 (16.7%) were ER⁺/HER2⁻/CK⁺ and ER⁻/HER2⁺/CK⁺ while there was no CTC positive for ER⁺/HER2⁺/CK⁺ (Fig. 2, B, online Supplemental Table 3). For 7 of these patients with MBC, samples before second cycle of therapy were available (Supplemental Table 5). Two of these 7 samples were also positive according to the CellSearch before the first cycle of therapy, but all these samples were negative for CTCs after completion of the first cycle (Fig. 2, B, online Supplemental Table 5). Triple IF staining revealed that 2 and 1 samples were positive before and after first cycle of therapy, respectively (Fig. 2, B, online Supplemental Tables 3 and 5). Our results on CTC in vivo molecular analysis, IF staining, CellSearch analysis, and clinical outcome in patients with MBC before the first and second line of therapy are shown in online Supplemental Table 5. Six out of seven (85.7%) patients with MBC relapsed and 5/7 (73%) died. In the group of patients with MBC that relapsed 5/6 patients (patients #1, #2, #3, #6, and #7) were found positive based on in vivo molecular analysis, while only one out of these 5 patients had CTC counts according to CellSearch analysis (patient #1: 4 CTCs/7.5 mL of PB). Moreover, IF showed viable CTCs only in one case after chemotherapy (patient #3).

Comparison between Molecular Analysis, CellSearch, and IF Staining

Our molecular analysis results were directly (same blood samples) compared with CTC enumeration in the CellSearch and triple IF (online Supplemental Table 2). Our results demonstrated a higher sensitivity for the combination of in vivo-CTC isolation with downstream comprehensive molecular analysis based on gene expression, ESR1 methylation, and PIK3CA hotspot mutation analysis. The concordance between these 3 different CTC detection systems is shown in Fig. 4, A. The concordance between: (a) CellCollector^TM/molecular analysis and CellSearch was 34/84 (40.5%), (b) CellCollector/molecular analysis and triple IF was 24/66 (36.4%), and (c) CellSearch and triple IF was 44/65 (69.2%) (Fig. 5). However, since these 3 CTC detection systems are based on different isolation systems and different markers for CTC detection, we also evaluated this comparison by using only the common epithelial markers CK8, CK18, and CK19. In this case, a better agreement was found, between in vivo-CTC isolation/comprehensive molecular analysis and CellSearch (46/84, 54.8%), while the agreement between in vivo-CTC isolation/comprehensive molecular analysis and triple IF was 34/66 (40.5%) (Fig. 4, B).

Discussion

We present for the first time a comprehensive molecular analysis of in vivo isolated CTCs in BrCa at the gene expression, DNA mutation, and DNA methylation level, and directly compared our results to those derived by CellSearch and triple IF using identical blood samples. Molecular characterization of CTCs is highly important, since new insights into the biology of cancer cell dissemination and identification of novel gene targets and signaling pathways relevant to therapeutic interventions can be achieved (26, 27). One major limitation of the analysis of CTCs is their heterogeneity, clearly shown by the fact that different isolation and detection procedures detect different cell populations (18, 20, 28). CTC heterogeneity is the main reason for important discrepancies found when different methodologies for CTC isolation and detection are compared (18, 28–30).

A major advantage of our approach is that the whole procedure for in vivo-CTC enrichment is easily applicable in many hospitals, since it does not require any specific instrumentation. Moreover, since captured CTCs are immediately lysed in Trizol-LS where nucleic acids (RNA/DNA) are stable even at room temperature, the samples can be safely transported to central labs for downstream molecular analysis. The proposed combination is less prone to preanalytical errors that usually result from the instability of CTCs in peripheral blood during transportation of samples to central laboratories. The tumor nature of these in vivo captured EpCAM-positive cells has been demonstrated by genomic analyses in many reports including ours (18, 20).

As revealed by our results, in CTCs isolated in vivo using the CellCollector, a high heterogeneity in gene expression was detected in individual patients. Moreover, many samples found negative for the presence of CTCs by the CellSearch and IF based on CK-expression, were found positive for the expression of a variety of these genes in in vivo isolated CTCs. In early BrCa, the clinical relevance of CTCs as enumerated in the CellSearch was only shown when 22.5 mL of peripheral blood was used, since based on the Poisson distribution, in these cases the number of events is low, and a relatively higher amount of sample should be used (6). However, when using Abs for CTC analysis at the protein level such as in CellSearch, detection of multiple protein markers on CTCs is very limited, since the number of antibodies that can be used in parallel in this type of experiment is limited (31). CTC analysis using IF is more time consuming, but it can provide information at the single cell level (32).

Serial monitoring of PIK3CA mutations status provides insights about biology of detected resistance alterations and tumor genomic evolution (33). Our molecular analysis for PIK3CA hotspot mutations, confirms that detection of DNA mutations is feasible in in vivo isolated CTC and could provide important information. We found PIK3CA mutations at a higher percentage in the MBC group (47.8%) than in the early BrCa group (26.2%). Similar to our results, PIK3CA mutations that are mainly localized on exon 9 and 20 hotspots are detected in 25%–40% of BrCa (34), while there is a net gain of PIK3CA mutations in tumor biopsies obtained from MBC sites in patients with MBC in comparison to the primary tumor (35).

We have shown for the first time that detection of ESR1 methylation is also feasible in gDNA extracted from in vivo isolated CTC, and the detected frequency of ESR1 methylation was consistent with our previous findings for both patients with early and MBC (15). ESR1 methylation, represents a novel liquid biopsy-based biomarker, since its detection in CTCs has been associated with lack of response to everolimus/exemestane regimen (15). ESR1 methylation has been shown to be an adverse prognostic factor in patients with BrCa (36), while its detection is predictive of clinical response in patients treated with the antiestrogen tamoxifen (37).

Our results are consistent with previous studies, where by using in vivo isolation of CTCs in prostate cancer, about 2-fold more CTC-positive samples were detected compared to using the FDA-cleared CellSearch System (17, 18, 20). This could possibly be explained by the higher blood volume used for CTC isolation in the in vivo approach (18, 20), compared to the in vitro isolation protocols (28). This is likely very important, especially in early BrCa where the frequency of CTCs is low. Moreover, the in vivo isolation of CTCs is more effective, since obtaining an adequate volume of blood from patients with cancer is not always feasible. The discrepancies found between in vivo isolation/molecular analysis, CellSearch, and IF staining are to be expected since these 3 CTC detection systems are based on different isolation systems and different markers for CTC detection. We already have shown this clearly in our previous studies on prostate cancer (18, 20).

Another interesting finding of our study is that the status of molecular markers changes during the progression of the disease, since in both early and MBC BrCa, patients who were CTC negative turned out to be CTC positive, while those who were CTC positive turned out to be CTC negative. The detection of CK-19 mRNA-positive CTCs after adjuvant chemotherapy is an independent risk factor indicating the presence of chemotherapy-resistant residual disease (38) while relative changes in PIK3CA ctDNA level after treatment strongly predict long-term clinical outcomes on palbociclib (39). Our findings verify that CTCs are highly heterogeneous and that different isolation and detection systems do not give identical but complementary results, as already previously suggested (40).

In conclusion, a comprehensive molecular analysis of in vivo isolated CTC by using specific and sensitive molecular assays at the RNA, DNA, and DNA methylation level is feasible. This approach is minimally invasive, not prone to preanalytical errors and transportation and sample storage considerations, capable of high throughput, and thus suitable for the development of molecular diagnostic applications in patients with BrCa. Future clinical studies will estimate the clinical significance of the combination of in vivo-CTC isolation with downstream RNA and DNA analysis.

Supplemental Material

Supplemental material is available at Clinical Chemistry online.

Author Contributions

All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.

E. Politaki, provision of study material or patients; T. Gorges, administrative support, provision of study material or patients; S. Joosse, statistical analysis; V. Mueller, provision of study material or patients; N. Poulakaki, provision of study material or patients; A. Psyrri, provision of study material or patients; D. Mavroudis, Provision of study material or patients; K. Pantel, financial support, administrative support, provision of study material or patients; E. Lianidou, administrative support.

Authors’ Disclosures or Potential Conflicts of Interest

Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership

None declared.

Consultant or Advisory Role

K. Pantel, Menarini SAB.

Stock Ownership

None declared.

Honoraria

K. Pantel, Menarini, Illumina, Agena.

Research Funding

This research has been cofinanced by the European Union (European Regional Development Fund—ERDF) and Greek national funds through the Operational Program “Competitiveness and Entrepreneurship” of the National Strategic Reference Framework (NSRF)—Research Funding Program: “Liquid biopsy: In vivo capturing and molecular characterization of circulating tumor cells as a novel tool for improving tertiary prevention in BrCa”.

Expert Testimony

None declared.

Patents

None declared.

Role of Sponsor

The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation of manuscript, or final approval of manuscript.

References

Nonstandard Abbreviations:

 
• CTC

  circulating tumor cells

 
• RT–qPCR

  reverse transcription quantitative PCR

 
• ctDNA

  circulating tumor DNA

 
• BrCa

  breast cancer

 
• MBC

  metastatic breast cancer

 
• SB

  sodium bisulfite

 
• UoA

  University of Athens

 
• UKE

  University Medical Center Hamburg-Eppendorf

 
• HD

  healthy donors

 
• FFPEs

  formalin-fixed paraffin-embedded tissue sections

 
• DAPI

  4′,6-diamidino-2-phenylindole

 
• IF

  immunofluorescence

 
• PBMC

  peripheral blood mononuclear cells

 
• ddPCR

  droplet digital PCR

Human Genes

Human Genes
 
• CK8

  cytokeratin 8

 
• CK18

  cytokeratin 18

 
• CK19

  cytokeratin 19

 
• ERBB2

  Human Epidermal Growth Factor Receptor 2

 
• TWIST1

  BHLH Transcription Factor 1

 
• VEGF

  Vascular Endothelial Growth Factor A

 
• ESR1

  estrogen receptor 1

 
• PR

  progesterone receptor

 
• EGFR

  Epidermal Growth Factor Receptor

 
• CD44

  antigen (homing function and Indian blood group system)

 
• CD24

  antigen (small cell lung carcinoma cluster 4 antigen)

 
• ALDH1

  aldehyde dehydrogenase 1 family member A1

 
• VIM

  vimentin

 
• CDH2

  cadherin

 
• B2M

  beta-2-microglobulin

 
• PIK3CA

  phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha