Table 3.

Characterization of molecular biomarkers of CTCs and their clinical significances in diverse aggressive and metastatic cancers

Results and clinical significance
Cancer type	CTC detection method	Number of CTCs detected by patients/samples	Biomarkers and their diagnostic, prognostic, and predictive potentials	Ref.
Breast cancer	AdnaTest Breast Cancer  Select for detection of  EpCAM, MUC1, HER2  and β-actin transcripts	CTCs were detected by  AdnaTest in 19% of 502  blood samples from  patients with breast  cancer. Blood samples  from 502 primary patients  with breast cancer versus  30 healthy donor  samples	29% samples positive for CTCs were  characterized by RT-PCR by the  expression of at least one EMT marker  (twist, Akt2, and PI3Kα) whereas 97% of  30 healthy donor samples investigated  were negative. Moreover, 14% samples  positive for CTCs expressed ALDH1  stem cell–like marker whereas 95% of 30  healthy donor samples were negative.	(38)
Breast cancer	AdnaTest Breast Cancer  Select for detection of  EpCAM, MUC1, and  HER2 transcripts	226 blood samples from 39  patients with metastatic  breast cancer obtained  during their follow-up of  chemotherapy,  hormonal, or  trastuzumab antibody-  based treatment	CTCs were detected by AdnaTest in 31%  of 226 blood samples. The samples  positive for CTCs were characterized by  RT-PCR by the expression of at least one  EMT marker (twist, Akt2, and PI3Kα) and  ALDH1 in 62% and 69% cases as  compared with 7% and 14% in blood  samples negative for CTCs, respectively.  The EMT and ALDH1 expression levels in  blood samples from patients unresponsive  to therapy were of 62% and 44% relative  to only 10% and 5% in responders.	(39)
Breast cancer	CellSearch EpCaM-based  immunocapture method	Blood samples from 16  women with metastatic  breast cancer with  disease progression	Data from analyses performing by  CellSearch system and immunostaining  have revealed that over 75% of CTCs  detected in blood samples from patients  with metastatic cancers breast cancer  coexpressed epithelial markers (EpCAM  and E-cadherin) and mesenchymal  proteins (vimentin and N-cadherin).	(34)
Breast cancer	Microfluidic capture of  CTCs with epithelial- and  tumor-specific  antibodies directed  against EpCAM, EGFR,  and HER2	Blood samples from 41  patients with metastatic  breast cancer at various  stages of treatment.	CTCs were detected in 41% of blood  samples from patients with breast  cancer. CTCs expressed epithelial  (cytokeratins, EpCAM, and cadherin 1)  and/or mesenchymal markers  (fibronectin, cadherin 2, and serpin  peptidase inhibitor, clade E). The  enrichment of mesenchymal CTCs was  associated with disease progression  and expression of TGF-β signaling  pathway components.	(37)
Prostate cancer	CellSearch EpCaM-based  immunocapture method	Blood samples from 41 men  with metastatic  castration-resistant  prostate cancer with  disease progression	Data from analyses performing by Cell  Search System and immunostaining  have indicated that over 80% of CTCs  detected in blood samples from  metastatic CRPC patients coexpressed  epithelial markers (EpCAM, cytokeratin,  and E-cadherin), mesenchymal proteins  (vimentin, N-cadherin, and O-cadherin),  and CD133 stem cell–like marker.	(34)
Ovarian cancer	The monocyte blood  fraction containing CTCs  was enriched by 2-layer  density gradient and RNA  extracted from the  enriched cell fraction	Blood samples from 216  patients with epithelial  ovarian cancer obtained  before primary treatment  and 6 months after  adjuvant platinium-  based chemotherapy  and 39 cases of healthy  women	CTCs were detected in 24.5% of the  baseline and 20.4% of the follow-up  samples. The detection of CTCs  correlated with the presence of ascites,  suboptimal debulking and elevated CA-  125, and human epididymis protein 4  (HE-4) levels. Moreover, the CTC  detection was more frequent in  platinium resistant patients than in the  group sensitive to platinium treatment  and indicative of poor outcome of  patients.	(187)
Pancreatic cancer	CellSearch system	Blood samples from 26  patients with pancreatic  cancer	CTCs expressing cytokeratins were  detected in 42% of blood samples and  patients with pancreatic cancer with  CTCs exhibited significantly shorter  overall survival.	(188)
Pancreatic cancer	CellSearch system or  isolation by size of  epithelial tumor cells  (ISET)	Blood samples from 54  patients with pancreatic  cancer	CTCs were detected by CellSearch  EpCaM-based immunocapture  method and ISET in 40% versus 93% of  patients with pancreatic cancer,  respectively. The immunostaining  analyses have also indicated that CTCs  expressed variable levels of EpCaM,  pan-cytokeratin, E-cadherin, vimentin,  and cytokeratin 7.	(186)
Colorectal cancer	Pre- and postoperative  EpCAM based-flow  cytometry analyses	Blood samples from 76  patients with colorectal  cancer undergoing  surgical resection	CTCs were detected in 71% patients  preoperatively, and all metastatic  patients showed high levels of CTCs.  Colorectal tumor surgical resection was  associated with a significant decrease  in CTCs. A high postoperative level of  CTCs was also related to cancer  relapse. The progression-free survival  rate of patients with colorectal  cancer without CTCs was increased  from 16% to 86%, with a reduction in  the risk of disease relapse greater than  90%.	(185)
Lung cancer	Centrifugal force-based  separation technique  termed dean flow  fractionation (DFF)	Blood samples from 20  patients with metastatic  lung cancer at different  stages of treatment  versus 20 blood samples  from healthy individuals	5 to 88 CTCs were detected by DFF in all  blood samples analyzed from patients  with metastatic lung cancer whereas  healthy subjects had a negligible  number of CTCs. Moreover, the data  from immunostaining have revealed  that CTCs detected in blood samples  from patients with advanced lung  cancer coexpressed cytokeratin and  CD133 stem cell–like marker.	(40)
Non–small cell lung  cancer (NSCLC)	CellSearch system or  mutation detection in  circulating tumor DNA	Blood samples from 41  patients with relapsed or  refractory NSCLC  enrolled in a single-arm  phase II multicenter trial  with erlotinib and  pertuzumab	CTCs were detected using CellSearch  system in 78% of patients with NSCLC  and a greater baseline of CTC counts  was associated with a response to  treatment by response evaluation  criteria in solid tumor (RECIST). A  decrease of CTC counts upon  treatment of patients was also related  with FDG-PET and RECIST response  and longer progression-free survival.  Data from RT-PCR analyses have also  indicated that the detection of mutation  in EGFR in ctDNA and DNA isolated  from CTCs was associated with a most  substantial decrease in CTC counts  over the course of treatment of NSCLC  patients with erlotinib and pertuzumab.	(184)
Epithelial  malignancies	Multimarker quad-  μ-nuclear magnetic  resonance (μ-NMR)	Biopsies of 58 patients with  epithelial malignancies  including breast (n = 4),  gastrointestinal (n = 17),  genitourinary (n = 4),  gynecologic (n = 7),  pancreatic (n = 10), lung  (n = 9), and poorly  differentiated  adenocarcinoma (n = 7)  and 6 patients with  benign diagnosis	Data from quad μ-NMR expression  profiles of the biopsies obtained by the  fine needle aspiration of 58 patients  with different epithelial malignancies  have revealed that the combined  analyses of EpCAM, HER2, EGFR, and  MUC1 markers may be used to achieve  the diagnosis of patients with cancer in  99.2% of samples.	(189)
Melanoma	RNA isolation from whole  blood samples	Blood sample from 230  melanoma patients,  including 154 patients at  early stages I–II and 76   patients at late stages  III–IV versus 152 cases of  healthy individuals	Data from quantitative RT-PCR analyses  have indicated that 92% of melanoma  patients expressed MLANA, TGF-β2,  PAX3d, MCAM, and ABCG5 multidrug  transporter. Importantly, the  expression of MLANA and ABCG5  could predict disease recurrence  whereas MLANA expression was  associated with a poor outcome of  patients with melanoma after  treatment.	(41)