Circulating tumor cells in bladder cancer: Emerging technologies and clinical implications foreseeing precision oncology

Urologic Oncology: Seminars and Original Investigations ] (2018) ∎∎∎–∎∎∎

Review article

Circulating tumor cells in bladder cancer: Emerging technologies and clinical implications foreseeing precision oncology Rita Azevedo, M.Sc.a,b, Janine Soares, B.Sc.a, Andreia Peixoto, M.Sc.a,b,c, Sofia Cotton, M.Sc.a, Luís Lima, Ph.D.a,c,d,e, Lúcio Lara Santos, M.D., Ph.D.a,b,f,g, José Alexandre Ferreira, Ph.D.a,b,c,d,e,h,* a

Experimental Pathology and Therapeutics Group, Research Centre, Portuguese Oncology Institute of Porto (IPO-Porto), R. Dr. António Bernardino de Almeida 62, 4200–162 Porto, Portugal b Institute of Biomedical Sciences Abel Salazar, University of Porto, R. Jorge de Viterbo Ferreira 228, 4050–013 Porto, Portugal c Institute for Research and Innovation in Health (i3S), University of Porto, R. Alfredo Allen, 4200–135 Porto, Portugal d Glycobiology in Cancer, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), R. Júlio Amaral de Carvalho 45, 4200–135 Porto, Portugal e Porto Comprehensive Cancer Centre (P.ccc), R. Dr. António Bernardino de Almeida 62, 4200–162 Porto, Portugal f Health School of University Fernando Pessoa, Praça de 9 de Abril 349, 4249–004 Porto, Portugal g Department of Surgical Oncology, Portuguese Institute of Oncology (IPO-Porto), R. Dr. António Bernardino de Almeida 62, 4200–162 Porto, Portugal h International Iberian Nanotechnology Laboratory (INL), Avda. Mestre José Veiga, 4715 Braga, Portugal Received 22 November 2017; received in revised form 31 January 2018; accepted 12 February 2018

Abstract Context: Circulating tumor cells (CTC) in peripheral blood of cancer patients provide an opportunity for real-time liquid biopsies capable of aiding early intervention, therapeutic decision, response to therapy, and prognostication. Nevertheless, the rare and potentially heterogeneous molecular nature of CTC has delayed the standardization of robust high-throughput capture/enrichment and characterization technologies. Objective: This review aims to systematize emerging solutions for CTC analysis in bladder cancer (BC), their opportunities and limitations, while providing key insights on specific technologic aspects that may ultimately guide molecular studies and clinical implementation. Evidence acquisition: State-of-the-art screening for CTC technologies and clinical applications in BC was conducted in MEDLINE through PubMed. Evidence synthesis: From 200 records identified by the search query, 25 original studies and 1 meta-analysis met the full criteria for selection. A significant myriad of CTC technological platforms, including immunoaffinity, biophysical, and direct CTC detection by molecular methods have been presented. Despite their preliminary nature and irrespective of the applied technology, most studies concluded that CTC counts in peripheral blood correlated with metastasis. Associations with advanced tumor stage and grade and worst prognosis have been suggested. However, the unspecific nature, low sensitivity, and the lack of standardization of current methods still constitutes a major drawback. Moreover, few comprehensive molecular studies have been conducted on these poorly known class of malignant cells. Conclusion: The current rationale supports the importance of moving the CTC field beyond proof of concept studies toward molecularbased solutions capable of improving disease management. The road has been paved for identification of highly specific CTC biomarkers and novel targeted approaches, foreseeing successful clinical applications. r 2018 Elsevier Inc. All rights reserved.

Keywords: Bladder cancer; Circulating tumor cells; Clinical implications; Liquid biopsies; Systematic review; Technologies

1. Introduction Corresponding author. Tel.: þ351225084000 (ext.5111). E-mail address: [email protected] (J.A. Ferreira). *

https://doi.org/10.1016/j.urolonc.2018.02.004 1078-1439/r 2018 Elsevier Inc. All rights reserved.

Advanced bladder cancer (BC) presents significant management hurdles concerning its high recurrence rates,

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rapid progression, poor response to chemotherapy, and lack of novel targeted therapeutics [1]. In addition, significant variations in therapeutic outcomes are observed for tumors of apparently similar histology, mostly due to their high molecular heterogeneity [2]. Prognostication difficulties are often aggravated by the absence of efficient follow-up strategies, especially of noninvasive nature, for real-time monitoring of therapy response, metastatic risk assessment, and early detection of occult micrometastases. Circulating tumor Cells (CTC), derived from primary tumors by passive shedding or dynamic stromal invasion, are regarded responsible for disease dissemination [3]. Once in the bloodstream, CTC capable of overcoming sheer stress and evading the immune system may reach distant organs, whose microenvironment endows its expansion and differentiation or induces temporary quiescence [3] (Fig. 1). Despite being the driving force of metastasis, CTC account for less than 0.004% of all mononucleated blood cells [4]; nevertheless, recent data suggest that CTC counts may be explored to improve disease management. CTC were proven to better predict overall survival than other cancercirculating biomarkers as free DNA [5], holding tremendous potential for aiding early intervention, therapeutic

decision, and therapy response assessment [6]. Nevertheless, it is likely that both CTC and circulating tumor DNA (ctDNA) platforms may have complementary roles in prognosis and metastasis assessment [5]. CTC may also reflect the genetic drift between primary and metastatic tumors [7], paving the way for liquid biopsies in alternative to metastatic biopsies. Moreover, CTC may be selectively recovered from patient’s blood, expanded in vitro, and xenografted into relevant animal models, enabling drug susceptibility evaluation, biomarker discovery, and personalization of targeted therapeutics toward true precision medicine settings [8,9]. However, the scarceness and potentially heterogeneous molecular nature of CTC requires high-throughput capture/enrichment, detection and characterization technologies, ideally at a single cell level, which has delayed the standardization of robust methods for CTC implementation in clinical practice [10]. Nevertheless, in the last decade, numerous platforms have been presented envisaging this end, namely, flow cytometry-based assays and lab-on-a-chip microfluidic devices [10,11] (Fig. 2). This review aims to systematize emerging solutions for CTC analysis in BC, as well as its opportunities and limitations, while providing key insights on specific technologic aspects that may ultimately guide clinical implementation.

Fig. 1. Illustration of the involvement of circulating tumor cells (CTC) in the metastatic cascade and their potential in basic research and clinical practice. Once in the bloodstream, CTC capable of overcoming sheer stress and evading the immune system may extravasate to distant organs, whose microenvironment endows its expansion and differentiation or induces quiescence. The minimally invasive CTC isolation from patient’s blood provides the opportunity for a “real-time liquid biopsy,” allowing the assessment of genetic and proteomic differences between the primary and metastatic tumors, and the identification of drug-resistant phenotypes as well as CTC subpopulations with actionable molecular characteristics. Moreover, ex vivo model development (cell cultures, animal xenografts) could be a crucial milestone to aid therapeutic decisions and test novel drugs. As such, CTC molecular characterization through basic research can aid early diagnosis, monitoring of disease evolution, prediction of therapy response to targeted approaches, patient stratification, and eligibility for clinical trials, improving prognosis in everyday clinical practice. (Color version of figure is available online.)

R. Azevedo et al. / Urologic Oncology: Seminars and Original Investigations ] (2018) ∎∎∎–∎∎∎

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Fig. 2. Overview of technologies for circulating tumor cells (CTC) capture, enrichment, and characterization. Immunoaffinity-based enrichment technologies either capture CTC by positive or negative selection, typically using antibodies bound to the device surface or to magnetic beads. Positive selection is based on the specific targeting of CTC epithelial biomarkers, whereas negative selection depletes hematopoietic cells by targeting cell-surface antigens not expressed in CTC. Biophysical methods are label-free technologies relying on cell size, shape, density, and electric charge differences between CTC and other blood constituents. Density gradient centrifugation relies in the separation of different cell populations based on their relative densities. Microfiltration consists on size-based cell separation using pores or three-dimensional geometries. Inertial focusing relies on the passive separation of cells by size, through the application of inertial forces that affect positioning within the flow channel in microfluidics devices. Electrophoresis separates cells based on their electrical signatures, using an electric field. Acoustophoresis separates cells based on their acoustophoretic mobility. Direct imaging assays improve the efficiency of imaging or replace enrichment through high-speed fluorescent imaging. Functional assays rely on the enrichment of viable CTC based on the bioactivity of these cells, such as protein secretion or cell adhesion. (Color version of figure is available online.)

2. Evidence acquisition A systematic screening of the state-of-the-art on CTC technologies and its clinical applications in BC was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [12]. 2.1. Search strategy Literature database search for this systematic review was commenced at MEDLINE through PubMed on January 4, 2018, by 2 independent reviewers (RA and JAF). Search strategy included the following query: (“neoplastic cells, circulating”[MeSH Terms] OR (“neoplastic”[All Fields] AND “cells”[All Fields] AND “circulating”[All Fields]) OR “circulating neoplastic cells”[All Fields] OR (“circulating”[All Fields] AND “tumor”[All Fields] AND

“cells”[All Fields]) OR “circulating tumor cells”[All Fields] OR “CTC”[All Fields] OR “Lab-On-A-Chip Devices”[Mesh] OR “Microfluidic Analytical Techniques”[Mesh] OR “Microfluidic”[All fields] OR “Microfluidic System”[All Fields] OR “CellSearch”[All fields] OR “AdnaTest”[All fields] OR “High-throughput imaging”[All fields] OR “circulating tumor cells detection”[All fields] OR “enrichment devices”[All fields]) AND (“urinary bladder neoplasms”[MeSH Terms] OR (“urinary”[All Fields] AND “bladder”[All Fields] AND “neoplasms”[All Fields]) OR “urinary bladder neoplasms”[All Fields] OR (“bladder”[All Fields] AND “cancer”[All Fields]) OR “bladder cancer”[All Fields]). 2.2. Inclusion and exclusion criteria Only original studies and meta-analysis written in English were included, regardless the publishing year.

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A study was considered relevant to this review if it had a patient cohort diagnosed with BC, a cohort in which technological platforms for the capture, enrichment, and analysis of CTC was used, and if it evaluated the association of CTC with tumor detection, tumor staging and grading, metastization, disease recurrence, disease progression, disease-free survival, overall survival, or adverse events (i.e., studies not only based on technology for CTC quantification). Studies with less than 10 participants, preclinical studies, review articles, case reports, editorial commentaries, and meeting abstracts were excluded. Also, if multiple studies with overlapping series and reporting on the same endpoint met the inclusion criteria, the latest study was selected.

3. Evidence synthesis From 215 records identified by the search query, 27 original studies and 2 meta-analyses reporting on the clinical potential of CTC as novel BC biomarkers met full criteria for selection (Fig. 3). In this systematic review, studies were organized based on the core technology for

Fig. 3. Study flow chart. From 200 records identified through Pubmed, 27 original studies and 2 meta-analyses were included in this systematic review. (Color version of figure is available online.)

CTC isolation, namely immunoaffinity by positive or negative selection (n ¼ 17) and biophysical cell properties (n ¼ 1). Also, studies were clustered by CTC detection and characterization technologies (without prior CTC enrichment), including molecular (RNA-based) assays (n ¼ 8) and high-throughput imaging (n ¼ 1). The 2 meta-analyses are addressed in the concluding remarks, as these consolidate current evidence regarding the use of CTC detection assays to diagnose BC (Table). 3.1. Capture and enrichment technologies 3.1.1. Immunoaffinity Immunoaffinity-based enrichment technologies capture CTC by positive or negative selection, typically using antibodies immobilized to inert surfaces. Positive selection frequently targets the cell-surface epithelial cell adhesion molecule (EpCAM), which has been extensively used in proof of concept studies. Conversely, negative selection generally depletes blood samples from hematopoietic cells by targeting cell-surface antigens not expressed in CTC (e.g., leukocyte-specific cell marker CD45). The most reported immunoaffinity methods for CTC isolation and counting are CellSearch (n ¼ 11; Janssen Diagnostics, Raritan, NJ), AdnaTest ProstateCancerSelect kit (n ¼ 2; AdnaGen, AG, Langenhagen, Germany), Isoflux system (n ¼ 1; Fluxion Biosciences, San Francisco, CA), and CELLection Dynabeads (n ¼ 2; Invitrogen, Carlsbad, CA). 3.1.1.1. CellSearch assay. CellSearch is the only FDAapproved method for monitoring CTC in patients with metastatic breast, colorectal, and prostate cancer [42] but not BC. The premise of this semiautomated assay is that CTC are nucleated EpCAMþ/CKþ/CD45- cells, thereby distinguishable from healthy EpCAM-/CK-/CD45þ blood cells [43]. As such, it comprehends a positive immunomagnetic enrichment for CTC using anti-EpCAM functionalized ferromagnetic particles, following the flow cytometry analysis of cells fluorescently labeled with antibodies against CK8, CK18, CK19, and CD45. CellSearch has been the most used platform for CTCbased studies in BC. The Table summarizes the most important findings of all studies. Particularly, 10 out of 11 studies have evaluated CTC in nonmetastatic patients [13,14,16–23], while 7 included metastatic patients [13–16,19,20,23]. Regarding the studies enrolling nonmetastatic patient’s cohorts, 2 were unable to detect CTC in the blood [13,14], whereas the remaining 8 observed CTC in 17% to 30% of patients [16–23]. Such low yields could be associated with the lack of sensitivity of the detection technique; however, the available information is insufficient to corroborate this hypothesis. Conversely, CTC were detected in 44% to 57% of metastatic patients, with the exception of a preliminary study by Rink et al. in which all analyzed patients (5/5) presented with CTC [20]. Irrespective of the metastatic status of patients, average CTC counts

Table Prognostic and diagnostic significance of circulating tumor cells Study [Ref]

Technology

Bladder cancer patients

Flaig et al. [16]

Gazzaniga et al. [17]

CellSearch assay UroVysion assay (FISH)

44 patients (14 had metastases) 18 patients set (only for FISH)

CellSearch assay

44 NMIBC patients

% CTC in nonmetastatic patients

% CTC in metastatic patients

Observations (CTC counts correspond to median values)

Metastization

0%

57%

2 CTC/10 ml of blood, range: 0–79

Metastization

0%

55%

0 CTC/7.5 ml of blood, range: 0–27

Metastization

NA

44%

Metastization

17%

50%

0 CTC/7.5 ml of blood, range: 0–87 ¼ 1 metastatic side: 0 CTC ≥2 metastatic sides: 3.5 CTC 1 CTC/7.5 ml of blood, range: 1–177

Overall survival

Recurrence

18% (all T1)

NA

Recurrence Progression-free survival Metastization

20%

NA

Metastization Overall and specific survival Progression-free survival Recurrence Overall and specific survival

30%

23%

NA

NA

tumor staging Gazzaniga et al. [18]

CellSearch assay

102 patients (T1G3 prior to TURBT)

Guzzo et al. [19] Rink et al. [20]

CellSearch assay

43 patients (undergoing RC)

CellSearch assay

55 patients (5 had metastases)

Rink et al. [21]

CellSearch assay IHC

100 patients

21% 100%

PathVysion Kit (FISH) Soave et al. [22]

CellSearch assay

185 patients

Recurrence Overall and specific survival

22%

Abrahamsson et al. [23]

CellSearch assay

88 patients (61 had metastases)

tumor staging Metastization `Progression-free survival

19%

All metastatic patients with detectable CTC died after 337 days of follow-up 5/9 patients with detectable CTC presented same aneusomic chromosomal content of neoplasm 18% of T1 patients had detectable CTC (1 CTC/7.5 ml of blood, range: 1–3) ≥1 CTC associated with shorter time-to-first recurrence and concomitant existence of carcinoma in situ 1 CTC/7.5 ml of blood, range: 1–50 ≥1 CTC associated with shorter time-to-first recurrence and progression-free survival 0 CTC/7.5 ml of blood, range: 0–9 ≥1 CTC was not associated with metastatic potential 2 CTC/7.5 ml of blood, range: 1–372 ≥1 metastatic sides: 34 CTC ≥1 CTC associated with shorter overall, progressionfree and specific survival 1 CTC/7.5 ml of blood, range: 1–100 ≥1 CTC associated with higher disease recurrence and shorter overall and specific survival 14% of patients had HER2-positive CTC HER2-positivity on CTC was consistent with HER2 status on primary tumors and lymph node metastases 1 CTC/7.5 ml of blood, range: 1–163 ≥1 CTC associated with use of adjuvant chemotherapy Subgroup of CTC-positive patients not using adjuvant chemotherapy associated with higher disease recurrence and shorter overall and specific survival 3 CTC/7.5 ml of blood, range: 1–105 CTC associated with increased risk of progression in patients treated with radical cystectomy, metastatic disease and higher stage

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Immunoaffinity—prognostic significance of CTC Naoe et al. CellSearch assay 26 patients (14 had distant [13] metastases) Okegawa et al. CellSearch assay 36 patients (20 had [14] metastases) Gallagher CellSearch assay 33 patients (all had et al. [15] metastases)

Outcomes

5

6

Table Continued Study [Ref]

Fina et al. [24]

Alva et al. [26]

AdnaTest

AdnaTest

Isoflux (CellSearch assay)

Bladder cancer patients

31 patients (all had metastases)

Outcomes Cancer-specific survival Progression-free survival Overall survival

% CTC in nonmetastatic patients

% CTC in metastatic patients

Observations (CTC counts correspond to median values)

NA

55% (before starting MVAC) 65% (after second cycle of MVAC)

CTC-positive MUC1-positive were the most frequent

26 without malignancy

tumor staging

19%

30 40 16 20

7% of patients w/o malignancy 7% of NMIBC 15% of MIBC

tumor staging

NA

NA

54 patients (T1G3)

Disease-free survival

NA

54 patients (T1G3)

Disease-free survival Cancer-specific survival Recurrence Progression-free survival

44% (92% survivinpositive) 44% (92% survivinpositive)

NMIBC MIBC metastatic neoadjuvant patients

11 metastatic 13 healthy controls Gradilone et al. [27] Nicolazzo et al.[28]

CELLection Dynabeads CELLection Dynabeads

Busetto et al. [29]

CELLection Dynabeads

155 patients (T1G3)

Biophysical cell properties—prognostic significance of CTC Fina et al. [30] ScreenCell Cyto 66 patients (47 had metastases) (CellSearch assay)

Metastization

Molecular (RNA-based) assays—diagnostic/prognostic significance of CTC Li et al. [31] RT-PCR (UPII) 60 patients (10 had Metastization metastases) 10 healthy controls

CTC associated with shorter 3-year progression-free and overall survival CTC did not associate with response to MVAC CTC stem cell-specific and EMT-transcripts correlate with clinical tumor stage in MIBC group

Neoadjuvant group: Baseline–13 CTC /7.5ml blood (range: 0–358), Follow-up 5 (range: 0–750). Metastatic group: 29 (range: 0–154). Healthy group: 2 (range: 0–3) Median/high CTC levels associated with higher stage Isoflux method has an increased sensitivity then CellSearch assay ≥1 CTC associated with shorter disease-free survival

NA

≥1 CTC associated with shorter disease-free survival and cancer-specific survival

44%

NA

≥1 CTC associated with shorter time-to-first recurrence and progression-free survival

85%–baseline

84%–baseline

40% after 1st cycle therapy 86% after 2nd cycle therapy

90% after 1st cycle therapy 94% after 2nd cycle therapy

Metastatic cases: Baseline–3 CTC/4 ml blood (range: 1– 9.5). After 1stt cycle therapy 8 (range: 2–23). After 2nd cycle therapy 20 (range: 6.5–30.5). Nonmetastatic cases: Baseline 9.5 (range: 2.5–18.5). After 1st cycle therapy: 0 (range: 0–1). After 2nd cycle therapy: 1.5 (range: 1–3). CTC-positive EPCAM-positive were the most frequent Unclear which method for CTC enrichment performs better

0%

30%

–

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Todenhofer et al. [25]

Technology

RT-PCR (UPII)

Osman et al. [34]

RT-PCR (UPIa, Ib, II, III, EGFR) RT-PCR (CK20)

Retz et al. [35]

Leotsakos et al. [36]

RT-PCR (UPII)

RT-PCR (CK19, CK20, EGFR)

12 patients (3 had metastases) 3 healthy controls 29 NMIBC 14 MIBC 5 locoregional node-positive 8 distant metastases 40 MIBC 22 healthy controls

Metastization

0%

100%

–

Metastization Therapy response

10% NMIBC

Positivity for UPII increased with tumor extension UPII expression disappeared in 2/3 chemotherapy responders

Diagnosis

–

29% MIBC 40% locoregional nodepositive 75% distant metastases –

11 MIBC

Diagnosis

–

–

9 NMIBC 25 healthy controls 169 patients

tumor grade

–

–

39 healthy controls

Diagnosis

Kinjo et al. [37]

RT-PCR (MUC7)

29 NMIBC 9 MIBC

tumor stage and grade

38% NMIBC

78% MIBC

Qi et al. [38]

PCR (FRα)

57 patients 48 healthy controls

Diagnosis

–

–

Overall survival

80%

tumor stage Diagnosis tumor stage and grade Metastization Survival Diagnosis

–

–

–

–

High-throughput imaging - Prognostic significance of CTC Anantharaman EPIC (CK, 25 MIBC et al. [39] CD45, PD-L1)

Meta-analysis—diagnostic/prognostic significance of CTC Msaouel et al. Variable 21 studies [40] Zhang et al. Variable 30 studies [41]

8/22 patients recurred and were UPIa/UPII-positive CTC UPIa/UPIIþ: 75% sensitivity, 50% specificity CTC UPIb/UPIIIþ: 31% sensitivity, 79% specificity Detected preoperatively in 2 patients with advanced tumor stages and postoperatively in 3 patients All healthy controls were negative for CK20 CTC EGFRþ and/or CK20þ, associated with higher tumor grade CTC EGFRþ: low sensitivity but highly specificity CTC CD20þ: high sensitivity but low specificity 0% CTC MUC7-positive grade 1 33% CTC MUC7-positive grade 2 64% CTC MUC7-positive grade 3 CTC FRαþ: 82% sensitivity and 62% specificity

PD-L1þCTCþ: 28% of total CTC High PD-L1þCK- and low apoptotic CTC phenotypes had worse overall survival

35% sensitivity and 89% specificity for BC detection Association with advanced disease 35% sensitivity and 97% specificity for BC detection Association with lymph node and distant metastasis, shorter overall, progression/disease-free and cancerspecific survival

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Yuasa et al. [32] Lu et al. [33]

7

8

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ranged from 0 to 10 CTC/ml of blood, with a detection range of 0 to 372 CTC/ml (Table), suggesting that this platform may lack the necessary sensitivity to address CTC in the blood. Of note, the volume of analyzed blood was variable; which may account for interstudies variability in CTC counts, highlighting the need for protocol standardization. Moreover, the high interstudy variations in detection range suggests heterogeneity in molecular characteristics of CTC, which is not accounted by this technique. Importantly, Abrahamssom et al. [23] demonstrated that the presence of CTC in the blood of patients treated with radical cystectomy (RC) was associated with the presence of metastasis and increased risk of progression after a median follow-up time of 16.5 months. Moreover, Okegawa et al. [14] and Gallagher et al. [15] associated increasing CTC counts with the number of metastatic sites, confirming the utility of this assay for monitoring BC patients in postoperative phase for distant metastasis development. In turn, Guzzo et al. [19] suggested that observable CTC was not a robust predictor of extravesical or nodepositive disease in patients with ≤ T2 BC, excluding CTC as a useful marker for directing therapeutic decisions. This disparity could result from discrepancies and bias in methodologies, study design, and cohorts composition, among other variables. Moreover, Flaig et al. [16] reported that, after a year of follow-up, 100% of metastatic patients with detectable CTC had died compared with 43% of metastatic patients without measurable CTC, suggesting that CTC detection may be a prognostic marker for decreased survival in metastatic BC patients. In agreement with these observations, several studies associated the presence of CTC with shorter time-to-first recurrence, and worse overall, progression-free, and cancer-specific survival [17,18,20–23]. Based on these insights, CTC detection may aid the stratification of patients facing worst prognosis, ultimately guiding therapeutic decisions. Only 2 studies conducted complementary molecular analysis by fluorescence in situ hybridization (FISH) and immunohistochemistry to confirm the molecular origin of CTC, an aspect often disregarded by most studies. Flaig et al. [16] confirmed the presence of DNA copy number variations consistent with neoplasm in 5/9 patients with detectable CTC. In addition, Rink et al. [21] evaluated HER2 gene in captured CTC and confirmed that approximately 14% of patients (3/22) exhibited HER2-positive CTC. However, the presence of CTC and the HER2 status was not associated with any clinicopathological features but all patients presenting with HER2-positive CTC also presented with HER2 in the primary tumors and lymph node metastases. As such, CTC may serve as an indication for multimodal therapy. Again, these results support the importance of complementary molecular analysis for an unequivocal demonstration of CTC origin. In summary, the CellSearch assay is the most explored application addressing CTC in BC. Despite the preliminary nature of the existing studies, all consensually report that

CTC presence is associated with worst prognosis. Moreover, increased CTC counts appear to be associated with recurrence, metastization, and shorter survival. In addition, the CellSearch assay may hold potential for early identification of nonmetastatic NMIBC patients facing higher risk of disease progression and dissemination, which could allow early interventions such as RC or systemic treatment or both. However, there are sensitivity and protocol standardization issues that need to be addressed before clinical implementation. Moreover, some studies have presented complementary analysis by FISH and immunohistochemistry, highlighting molecular similarities between the primary tumor, lymph node metastasis, and CTC. These observations reinforce CTC potential in the context of liquid biopsies and may provide key insights on metastasis formation dynamics, contributing to the development of novel strategies for disease management. These preliminary findings now require validation in clinical studies. The low number of CTC counts displayed by CellSearch may derive from the fact that it explores solely the expression of EpCAM, which may be significantly reduced or absent in certain CTC subpopulations, especially those undergoing epithelial-to-mesenchymal transition (EMT) [44], a crucial milestone for metastasis. 3.1.1.2. AdnaTest. The AdnaTest is a nonautomated immunoaffinity-based enrichment technology based on the positive immunomagnetic selection of CTC from peripheral blood. Unlike CellSearch, it uses a cocktail of antibodies specific to the type of cancer under study (e.g., antiEpCAM, anti-mucin 1 (MUC1), anti-human epidermal growth factor receptor 1 (EGFR1), and anti-human epidermal growth factor receptor 2 (HER2/ErbB2)). Captured cells are submitted to molecular profiling for various cancer-associated tumor markers by reverse-transcription polymerase-chain reaction (RT-PCR). CTC-positivity is defined when at least one of the cancer-associated tumor markers is above a previously defined threshold. Fina et al. [24] analyzed blood samples of 31 metastatic BC patients receiving first-line MVAC chemotherapy, using the AdnaTest for EpCAM and HER2 as well as multiplexPCR assays for EPCAM, MUC1, and HER2. CTC-positivity was evaluated a day before chemotherapy (baseline – at T0) and after the second cycle of chemotherapy (at T2). About 55% (17/31) of patients presented with CTC at T0 and 65% (17/26) were CTC-positive at T2. The most frequently detected cancer-associated tumor marker in CTC-positive samples was MUC1 both at T0 and T2 endpoints. Patients with persistent CTC-positive status (CTC-positive at T0 and T2) demonstrated shorter 3-year progression-free and overall survival, while no associations were found between CTC detection and response to MVAC. In addition, Todenhfer et al. [25] used AdnaTest for EPCAM, HER2, and EGFR (AdnaTest EMT2/SC) as well as multiplex-PCR assays for HER2, MUC1, and EPCAM (BreastCancerDetect system) in 4 patient groups: Twenty-six heathy controls, 30 NMIBC,

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40 MIBC, and 16 metastatic patients. CTC were present in 7% of controls, 7% of NMIBC, 15% of MIBC, and 19% of metastatic patients. Stem cell-specific and EMT-associated transcripts were detected in increasing percentages in control, NMIBC, MIBC and metastatic groups. The number of transcripts correlated with tumor stage in MIBC patients and was increased in the metastatic group. Despite promising, these are preliminary prospective studies, which now require validation in larger cohorts. Moreover, the test is based on a predefined biomarker panel, neglecting the molecular heterogeneity of CTC and increasing falsepositive results. Importantly, unlike automated systems as CellSearch, AdnaTest is unable to provide CTC quantification, and CTC-positivity is only based on the assumption that at least one of the cancer-associated tumor markers is above a previously defined threshold. 3.1.1.3. Isoflux system. The Isoflux is an automated EpCAM-based immunoaffinity microfluidic system for CTC enrichment, consisting of 3 fluidic reservoirs connected by a microfluidic system [45]. It uses a continuous low velocity sample flow to maximize the contact between the CTC coated with anti-EpCAM magnetic beads and a high magnetic field that enables cell sorting [45]. Similarly to Cellsearch, it relies on the assumption that CTC are nucleated EpCAMþ/CKþ/CD45- cells. So far, only 1 study has evaluated CTC prognostic significance in BC using Isoflux [26]. It comprehended a cohort of 20 BC patients elected for cisplatin-based neoadjuvant chemotherapy, which collected blood before and after chemotherapy. For comparison purposes, 11 metastatic BC patients and 13 healthy controls were also analyzed. Median CTC counts were 13/7.5 ml of blood at baseline (range: 0–358), 5 in the neoadjuvant group at follow-up (range: 0–750), 29 in the metastatic group (range: 0–154), and 2 in the healthy control group (range: 0–3). The authors also compared Isoflux and CellSearch at baseline and follow-up, in 5 patients of the neoadjuvant group. Results demonstrated that 44% (4/9) of samples had ≥10 CTC using Isoflux, while no CTC were detected using CellSearch. After 4 months of follow-up, all patients with median/high CTC levels, at both baseline and follow-up, progressed to higher stage disease. Moreover, Alva et al. [26] explored the possibility of downstream molecular analysis of CTC using Isoflux. Accordingly, a sequencing assay with sufficient sensitivity to detect somatic variants in CTC of 50% (4/8) of patients with medium/high CTC levels was presented. In summary, these preliminary studies suggest that Isoflux may present increased sensitivity for CTC detection in BC patients compared to CellSearch. Moreover, increased CTC counts appear to be associated with higher disease stage, demonstrating prognostic potential in BC. 3.1.1.4. CELLection Dynabeads. Contrasting with CellSearch and Isoflux, CELLection Dynabeads is a nonautomated

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EpCAM-based immunoaffinity technique for CTC enrichment. CTC are selectively captured by anti-EpCAM functionalized Dynabeads and lysed for posterior RT-PCR analysis to confirm CD45 negative and CK8 positive expressions. Three studies evaluated the prognostic significance of CTC in BC using CELLection Dynabeads: Gradilone et al. [27] and Nicolazzo et al. [28] screened a series of 54 NMIBC T1G3 patients for CTC expressing survivin, an inhibitor of apoptosis frequently overexpressed in bladder tumors but not detected in normal bladder epithelium [46]. Half of the primary tumors were survivin-positive. CTC were detected in 44% of cases (24/54), 92% of which were also survivin-positive. The presence of CTC was associated with shorter disease-free survival, independent of survivin status. Furthermore, Busetto et al. [29] evaluated 155 NMIBC T1G3 patients using both CELLection Dynabeads and CellSearch. Of note, 44% (24/54) of patients were rendered CTC-positive through CELLection Dynabeads analysis, while only 20% of patients (20/101) presented CTC according to CellSearch. CTC-positivity was correlated with time-to-first recurrence, with 83% and 75% of CTC-positive patients relapsing in the CELLection Dynabeads and CellSearch assay groups, respectively. The same correlation was observed for progression-free survival, where 65% and 29% of CTC-positive patients progressed in the CELLection Dynabeads and CellSearch assay groups, respectively. The authors highlight the difficulty in comparing CELLection Dynabeads and CellSearch, as both assays had different starting groups; however, data suggest that CellSearch is likely to be more reliable and efficient for prognostication, besides allowing CTC quantification. Even though the 2 studies involving CELLection Dynabeads hold potential for improving risk stratification in NMIBC patients, large-scale clinical trials are required for validation. In summary, all immunoaffinity methods for analyzing bladder CTC explore EpCAM expression, mostly relying on the assumption that the molecular profile of CTC (EpCAMþ/CKþ/CD45-) differs from normal blood cells. Although the FDA-approved CellSearch has been the most used approach, comparative studies suggest that other automated (Isoflux) or nonautomated (AdnaTest and CELLection Dynabeads) platforms may constitute valuable alternatives. Despite the need for upfront investments, standardized methods envisaging clinical implementation should be envisaged. Irrespective of the technological background, most mentioned studies consensually associated the presence of CTC with worst prognosis, with some demonstrating the utility of CTC counts in predicting the risk of metastasis in superficial bladder tumor patients. Clinical trials are now warranted to validate these preliminary findings and ultimately provide a useful tool for managing NMIBC patients facing higher risk of progression. Nevertheless, most reports fail to provide more indepth molecular studies, thereby concealing the full

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biomarker potential of CTC. Although CTC are rare, we note the relatively low number of cell counts in EpCAMbased (semi)automated methods CellSearch (median o2 CTC/sample) and Isoflux (median o29 CTC/sample). Targeting an epithelial marker such as EpCAM, generally underexpressed or absent in aggressive cancer cells undergoing EMT, may account for these low yields. It may also significantly limit the spectra of captured CTC and hamper the access to yet unknown subpopulations holding clinical value. Moreover, EpCAMþ/CKþ/CD45- cells have been observed in the bloodstream of patients with benign lesions and healthy controls [17,47], contributing to false positives. Similarly, EpCAMþ/CKþ/CD45þ cells have been recently reported in some solid tumors [48]. Such observations demand the introduction of a more clinically relevant biomarker panel. Furthermore, most studies fail to present complementary molecular analysis unequivocally demonstrating the malignant nature and origin of isolated cells, as well as a relevant more in-depth biomarker signature of metastasis, urging more in-depth studies. 3.1.2. Biophysical cell properties Biophysical CTC enrichment methods are label-free technologies exploring differences on the physical properties of CTC, such as cell size, shape, density, and electric charge. These are designed to avoid bias and potentially low yields presented by immunoaffinity approaches targeting a single or multiple CTC surface antigens [49]. Most biophysical technologies assume that the CTC can be sorted from blood cells due to their significantly larger and more rigid shape. The most common CTC enrichment strategy is size-based microfiltration, which has been improved by the introduction of nano- to micron-sized filter pores [49]. However, the recent introduction of lab-on-a-chip microfluidics devices have significantly improved CTC yields compared to conventional membrane microfiltration and EpCAM-based immunoaffinity assays [49,50]. Moreover, these solutions have provided improved in situ platforms for molecular analysis by FISH [51], or immunofluorescence [52], as well as for extraction of biomolecules [52] for downstream genomic, transcriptomic, and proteomic studies [53]. These platforms also provide the oportunity for CTC retriavel and ex vivo expansion [9,54]. There were only 2 reports exploring nontargeted approaches for CTC isolation in BC. The first study uses the commercially available ScreenCell microfiltration size exclusion technology for CTC isolation from whole blood, with minor contaminations by white blood cells. This technique is compatible with downstream cytological (ScreenCell Cyto), functional (ScreenCell CC), and molecular (ScreenCell MB) studies based on the kits provided by the vendor [49]. Accordingly, Fina et al. [30] explored the prognostic significance of ScreenCell Cyto and provided a comparative analysis with immunoaffinity AdnaTest assay combined with multiplex-PCR analysis of EPCAM, MUC1, and HER2. This study used a 66-patient cohort (47

metastatic) and evaluated the outcomes at baseline and during treatment. According to ScreenCell Cyto, 80% of cases were CTC-positive regardless of metastatic status. At baseline, median CTC counts of metastatic cases were 3/ 4 ml of blood (range: 1–9.5) and 9.5/4 ml of blood (range: 2.5–18.5) in nonmetastatic cases. After the first cycle of therapy, CTC numbers significantly increased in metastatic cases (median CTC count of 8/sample, range: 2–23), while decreasing in nonmetastatic cases (median 0/sample, range: 0–1). After the second therapy cycle, the same trend was observed (metastatic cases: 20 CTC/4 ml of blood, range: 6.5–30.5; nonmetastatic cases: 1.5 CTC/4 ml of blood, range: 1–3). Thus, CTC counts decreased in nonmetastatic patients during treatment, while increasing in metastatic cases, suggesting distinct susceptibilities to chemotherapy and probable distinct molecular profiles. Moreover, more numerous CTC aggregates were observed in metastatic patients before treatment. Regarding AdnaTest, the global contribution to CTC positivity was derived from EPCAM, MUC1 and the combination of both markers. The comparison between ScreenCell and AdnaTest resulted in a concordance in detecting CTC inferior to 30%, possibly due to differences in what both tests considered the CTC. Ultimately, more samples were rendered positive by ScreenCell Cyto than AdnaTest (84% vs. 55%, at baseline). Such fluctuations in CTC detection could be from treatmentinduced modifications in CTC phenotypes, affecting the performance of the test. Following these observations, our group has recently used a size-based microfluidics device to isolate CTC from metastasized MIBC patients [55]. Downstream immunofluorescence and RT-PCR analysis demonstrated that, in mimicry of the primary tumor, most CTC expressed the cell-surface sialyl-Tn glycan, responsible for modulating cell motility and tolerogenic immune responses [56], and presented a basal-like phenotype associated with poor response to treatment [57]. Altogether, these preliminary reports demonstrate the potential of nontargeted approaches for accessing the multiplicity of CTC subpopulations and their clinical relevance, ultimately driving biomarker discovery to guide targeted therapeutics. 3.2. Detection and characterization technologies (without prior CTC enrichment) 3.2.1. Molecular (RNA-based) assays Some studies have explored the indirect detection of CTC using rapid, well implemented, sensitive, and low-cost molecular assays (e.g., RT-PCR) for detection of BC CTCrelated genes. Contrasting with enrichment technologies, these methods do not require CTC isolation; however, the possibility of detecting tumor-derived circulating RNAs constitutes a major limitation. Accordingly, several PCRbased studies have focused on uroplakins (UPs), i.e., urothelium-specific proteins that may indicate tumor cells dissemination. Li et al. [31] extracted total RNA from the blood of 60 BC patients (50 nonmetastatic and 10

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metastatic) as well as from 10 healthy controls, and screened it for human uroplakin II gene (UPII). UPII mRNA was detected in 20% of untreated metastatic patients, in 12.5% of metastatic patients that underwent chemotherapy, and in none of the nonmetastatic patients or normal controls, denoting its cancer-specific nature. Despite the suggested association with metastasis, we highlight the significantly low percentage of positive cases in comparison to previously discussed reports using CTC-isolation technologies. We also note the heterogeneous nature of sampling and the lack of complementary molecular analysis, which compromises the possibility of more in-depth conclusions. Similarly, Yuasa et al. [32] investigated UPII gene expression in the peripheral blood of 12 BC patients (3 metastatic), and 3 healthy controls by nested RT-PCR, detecting UPII in all metastatic patients but not in healthy controls or nonmetastatic patients. The sensitivity of the assay allowed the detection of 1 CTC/5 ml of blood. Using the same strategy, Lu et al. [33] detected UPII mRNApositive cells in 10% (3/29) of NMIBC patients, 29% (4/14) of MIBC patients, 40% (2/5) of locoregional node-positive patients, and 75% (6/8) of distant metastases patients. UPII expression increased with tumor progression but decreased in 2/3 of patients that responded to chemotherapy. Altogether, these results suggest that overexpression of blood UPII may be associated with advance stage disease; however, its association with metastasis and prognosis remains to be fully disclosed. Moreover, additional studies are required to determine the origin of detected UPII (circulating RNA vs CTC). Following these observations, Osman et al. [34] evaluated the expression of UPIa, UPIb, UPII, UPIII, and EGFR in 40 advanced BC patients and 22 healthy controls by nested RT-PCR. The sensitivity and specificity of CTC detection for each individual marker and in UPIa/UPII and UPIb/UPIII combinations was also determined. Eight out of 22 patients with refractory disease were positive for UPIa/UPII at presentation; moreover, the combination of both UPs provided the best sensitivity (75%) for CTC detection, with a specificity of 50%. In turn, the combination of UPIb/UPIII was the most specific (79%) but had a modest sensitivity (31%). Detection of EGFR-positive cells alone and in combination with UPs was inferior to UPIa/UPII combination to uncover CTC. These authors suggest that the combination of UPs may improve advanced BC detection, reinforcing the importance of multiplex solutions. Other studies explored CK, EGFR, or MUC7 expression presumably from CTC in blood samples. Retz et al. [35] evaluated CK20 in 11 patients undergoing RC, 9 NMIBC patients undergoing TUR, and 25 healthy controls. CK20 was detected preoperatively in 2 patients with advanced tumor stages (2/5) and postoperatively in 3 patients with low tumor stages (3/20). All healthy controls were negative for CK20, suggesting that this method could aid BC detection. In a larger study, Leotsakos et al. [36] evaluated the expression of CK19, CK20, and EGFR in blood samples

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from 169 patients and 39 healthy controls. Urine samples were also screened for survivin (BIRC5), human telomerase reverse transcriptase (hTERT), and CK20. EGFR and/or CK20 detection in blood samples correlated with tumor grade. The presence of EGFR in the blood was highly specific (95%) but modestly sensitive (23%) for diagnosing BC, whereas CK20 detection had high sensitivity in both blood and urine (54% and 33%, respectively). Moreover, all urine markers were highly specific for detecting BC (495%), and their positivity was associated with shorter progression-free survival, and hTERT expression correlated with tumors T≥3. In addition, in a series of 38 patients (29 NMIBC and 9 MIBC), Kinjo et al. [37] demonstrated the presence of MUC7 in 38% of NMIBC patients and 78% of MIBC. Moreover, MUC7 expression increased with tumor grade. Namely, all grade 1 patients were MUC7-, 33% of grade 2 were MUC7þ, and 64% of grade 3 were MUC7þ. However, this was a small and heterogeneous cohort, warranting careful future validation. Finally, the folate receptor α (FRα) was validated as a tumor marker to detect CTC through tumor-specific ligand PCR (LT-PCR). Moreover, the quantitation of CTC through FRα ligand-PCR was demonstrated to be a promising method for noninvasive diagnosis of BC with 82% sensitivity and 62% specificity [38]. In summary, transcript analysis, particularly for different UPs, may provide important tools for noninvasive BC detection and assessment of tumor progression with reasonable sensitivity and specificity. However, the preliminary nature of these findings has delayed its introduction in clinical practice. Moreover, the association of these biomarkers with CTC and the risk of metastasis development remains to be fully disclosed. 3.2.2. High-throughput imaging High-throughput imaging tools also provide rapid and low-cost alternatives for CTC detection. Anantharaman et al. [39] evaluated the expression of programmed death ligand-1 (PD-L1) on CTC isolated from MIBC and metastatic patients, and explored the prognostic value of PDL1þCTC. Twenty-five blood samples from MIBC patients (21 with metastases) were analyzed using the EPIC platform. Samples were processed in microscope slides, screened by automated immunofluorescence for CK, CD45, and PD-L1 and analyzed through a digital phatology algorithm considering protein expression and morphology to differentiate CTC from surrounding blood cells. CTC were detected in 20/25 (80 %) of patients, including CKþCTC (13/25, 52 %), CK-CTC (14/25, 56 %), CKþCTC Clusters (6/25, 24 %), apoptotic CTC (13/25, 52 %), and PD-L1þCTC (7/25, 28%). Exploratory analysis demonstrated that patients with high PD-L1þCD45- CTC burden and low apoptotic CTC levels had worse overall survival. Moreover, in metastatic patients, PD-L1 expression was demonstrated in both CKþ and CK-CTC, whose genomic analysis supported their tumor origin. As such, this

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methodology highlighted the heterogeneous molecular nature of CTC subpopulations, with possible clinical implications. It is possible that cell-surface PD-L1 expression may be part of an array of molecular mechanisms enabling CTC to escape immune surveillance. In addition, these observations suggest that CTC monitoring may allow sorting patients for Tecentriq (atezolizumab) therapy, a recently FDA-approved immunotherapy based on PD-L1 for BC (2016), among other immunotherapies [58].

4. Concluding remarks A significant myriad technological platforms, including immunoaffinity, biophysical and direct molecular approaches, have arisen to address the complex biological nature and tremendous clinical potential of CTC in the context of disease dissemination and liquid biopsies (Fig. 4). Despite their preliminary nature, most studies, irrespective of the applied technology, concluded that CTC analysis provides a noninvasive strategy for BC detection. Moreover, associations between CTC detection and advanced tumor stage, grade, metastasis, and worst prognosis have been presented. In addition, a meta-analysis based on 21 studies using different techniques for CTC capture/detection (immunoaffinity, biophysical methods, molecular (RNA-based) assays, and high-throughput imaging) [40], has concluded that the overall sensitivity of CTC detection assays was 35.1% (95% CI: 32.4%–38.0%) and specificity was 89.4% (95% CI: 87.2%–91.3%) for BC diagnosis. Moreover, CTC-positive patients were significantly more likely to have advanced (stage III-IV) disease compared with CTC-negative patients (OR, 5.05; 95% CI: 2.49–10.26). A more recent meta-analysis, based on 30

studies [41], demonstrated similar sensitivity (35%, 95% CI: 28.0%–43.0%) and specificity (97%, 95% CI: 92.0%– 99.0%). Moreover, CTC-positivity was significantly associated with higher tumor stage (stage III-IV), grade (III), locoregional or distant metastasis and decreased overall, progression/disease-free, and cancer-specific survival. Zhang et al. also addressed CTC as diagnosis or prognosis markers of upper tract BC, a very uncommon disease subtype (5%–10%) with identical behavior to BC; nevertheless the potential of CTC in this context remains unclear [41]. In conclusion, even though the low sensitivity of CTC detection assays limits early-stage screening, CTC evaluation can confirm tumor diagnosis and identify patients with advanced bladder cancer. In addition, several follow-up studies demonstrate that CTC detection may determine response to chemotherapy and suggest the existence of chemoresistant subpopulations, which warrants a comprehensive interpretation. We also highlight a report demonstrating PD-L1þCTC subpopulations that may be electable for anti-PD-L1 immunotherapy, recently approved for BC. Nevertheless, the preliminary nature of these findings, stemming from a limited number of patients of heterogeneous clinical nature, now demands validation by large multicentre clinical studies. Translation into BC clinical practice will also require the establishment of robust and reproducible technologies, which remains to be accomplished due to the lack of clinically relevant biomarkers and scarce information on CTC biology. Most studies use immunoaffinity methods targeting cell-surface EpCAM, which significantly decrease CTC representation and ultimately hamper their full clinical potential. On the contrary, nontargeted approaches focusing on CTC biophysical properties provide a broader overview of existing subpopulations with minor blood cell

Fig. 4. Overview of the circulating tumor cells technology in bladder cancer and the potential of these cells in the development of precision medicine settings. (Color version of figure is available online.)

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contaminants. These may constitute good starting points for addressing CTC’s molecular nature, which has been a critical matter for the development of more reliable targeted therapeutics. Moreover, it may pave the way for designing more sensitive and specific affinity-based solutions targeting clinically relevant CTC subpopulations. Nevertheless, it becomes pressing to explore the utility of CTCs beyond enumeration, including the analysis of molecular information or biomarkers to aid in clinical management. Nevertheless, while most of presented technologies are compatible with downstream morphological and molecular analysis, few have confirmed the malignant nature of isolated CTC and undergone more comprehensive studies. Moreover, no studies have attempted to isolate viable CTC for ex vivo model development (cell cultures, animal xenografts), which would be a crucial milestone to aid therapeutic decisions and test novel drugs. In fact, the biomarker potential of CTC has been mainly restricted to cell counts on peripheral blood, failing to fulfill its enormous clinical utility. A comparative analysis involving the primary tumor, CTC, and the metastasis through the course of disease is now required for defining molecular signatures for CTC phenotypes and metastasis dynamics. These aspects will certainly provide key insights on the selective pressure played by the microenvironment and the

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clonal evolution experienced by CTC at metastasis sites, ultimately helping define CTC clinical utility for accessing the molecular nature of the metastasis in noninvasive settings. Nevertheless, we believe the field will strongly benefit from advances in integrative panomics (genomics, transcriptomics, and proteomics), especially at a single cell level (Fig. 5). The integration of CTC with molecular information arising from circulating DNA, RNA, and tumor exosomes, responsible for formation of premetastatic niches, will also be critical for understanding the mechanisms underlying disease dissemination. Particularly, recent publications support circulating tumor DNA (ctDNA) analysis as a cost-effective tool for diagnosing BC progression and metastasis by mutation assays [59–61]. Even though no studies exist comparing CTC and ctDNA, we believe these biomarkers may reflect biologically different aspects of the disease. Although ctDNA may be shed from tumor sites at initial stages of the disease, its temporal evolution may ultimately reflect tumor progression, dissemination, and response to treatment. Therefore, multiple mutation analysis of ctDNA via next-generation sequencing (NGS) platforms will likely become a gold standard tool for early diagnosis, while providing real-time molecular information on treatment response and relapse. This will ultimately generate key information for unveiling treatment

Fig. 5. SWOT analysis of circulating tumor cells in bladder cancer. (Color version of figure is available online.)

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resistant mechanisms. On the contrary, CTC enumeration alone has been proven to have prognostic value, and may directly indicate disseminated disease, even in patients with radiological occult micrometastasis. However, CTC’s potential goes beyond genotyping, as these cells may also be object of phenotypical, trancriptomic, proteomic, and metabolomic characterization. Moreover, CTC may allow generating primary cell line cultures, and patient-derived xenograft models, paving the way for tailored treatment schemes and therapeutic development. Accordingly, these 2 liquid biopsy biomarkers provide complementary information, and therefore, should be addressed in parallel in the context of tumor metastasis and disease evolution. In summary, the current rationale supports the importance of moving CTC analysis field beyond proof of concept studies toward solutions capable of improving advanced-stage disease management. Key starting points should focus on exploring nontargeted approaches backed by comprehensive molecular studies, envisaging clinically relevant CTC biomarkers. This will allow focusing CTC analysis on clinically relevant subpopulations and more sensitive and specific analytical solutions. Ultimately, it may provide novel therapeutic targets to address the metastasis. The comprehensive inclusion of CTC with emerging biomarkers, namely ctDNA, will most likely contribute to tailor clinical practice toward molecular-based precision medicine.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgments The authors wish to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the human resources grants: PhD grant SFRH/BD/105355/2014 (Rita Azevedo), SFRH/BD/111242/2015 (Andreia Peixoto), and Postdoctoral grants SFRH/BPD/101827/2014 (Luis Lima) and SFRH/BPD/111048/2015 (José Alexandre Ferreira). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Program (POPH) from National Strategic Reference Framework (NSRF). The authors also acknowledge the Portuguese Oncology Institute of Porto Research Centre (CI-IPOP-29–2014; CI-IPOP-58–2015) and PhD Programs in Biomedicine and Pathology and Molecular Pathology of ICBAS-University of Porto for support in the acquisition of reagents and scientific equipment.

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