Circulating Tumor Cells: From Basic to Translational Research

Abstract

Background

Metastasis is the leading cause of cancer-related deaths. Most studies have focused on the primary tumor or on overt metastatic lesions, leaving a significant knowledge gap concerning blood-borne cancer cell dissemination, a major step in the metastatic cascade. Circulating tumor cells (CTCs) in the blood of patients with solid cancer can now be enumerated and investigated at the molecular level, giving unexpected information on the biology of the metastatic cascade.

Content

Here, we reviewed recent advances in basic and translational/clinical research on CTCs as key elements in the metastatic cascade.

Summary

Findings from translational studies on CTCs have elucidated the complexity of the metastatic process. Fully understanding this process will open new potential avenues for cancer therapeutic and diagnostic strategies to propose precision medicine to all cancer patients.

Introduction

Remarkable advances in the study of circulating tumor cells (CTCs), as representative entities of disseminating tumor subclones and as liquid biopsy analytes, have led to significant progress in cancer diagnostics and therapeutics as well as a better understanding of the biology of the metastatic cascade. Moreover, clinical trials have highlighted their translational potential. A search using the term “circulating tumor cells” on ClinicalTrials.gov yielded 1152 studies among which 482 were completed or terminated. This indicates the interest in exploring the potential of this liquid biopsy biomarker. CTCs have also been included in the TNM staging system of tumors. Indeed, an international consensus panel has suggested to stratify metastatic breast cancer in stage IV indolent and stage IV aggressive diseases according to the CTC counts in blood (1).

Despite low CTC concentrations as a major challenge hindering their widespread clinical application (2), CTCs are now considered as surrogate biomarkers of various solid cancers. The results of several clinical trials support the prognostic value of CTCs and their clinical validity to facilitate risk assessment (tumor staging) and to monitor the response to cancer therapies. So far, only 2 methods for CTC analysis have received clearance for prognosis evaluation in cancer: the CellSearch® system (for metastatic breast, colon, and prostate cancers) and the Parsortix™ Cell Separation System (for metastatic breast cancer). Nevertheless, CTC detection holds potential for the early identification of active minimal residual disease (MRD) during follow-up (3). This has significant implications for treatment strategies to delay or prevent metastatic recurrence.

This review provides an update on CTC clinical relevance, with specific emphasis on prostate, lung, breast, and colorectal cancer (CRC). It also presents recent advances in the understanding of the metastatic cascade biology through the lens of CTCs, and discusses existing knowledge gaps.

CTCs and the Metastatic Cascade

CTC release from the tumor

CTC release from the primary tumor is a crucial step in metastasis formation. However, the mechanisms governing this process and the impact of the different release mechanisms on CTC metastatic competence are not fully understood. Metastasis-competent CTCs might arise from an aggressive clone within the tumor, but other factors (e.g., epigenetic or microenvironmental alterations) also can influence their ability to initiate metastases (4). In tumor areas with limited nutrient and oxygen supply, cancer cells may adapt or migrate to find better microenvironmental conditions. Besides gene mutations, epigenetic changes (e.g., DNA methylation) and transcriptional changes might allow tumor cells to acquire migratory characteristics and enter the blood stream. This adaptation involves cellular processes, such as the epithelial-mesenchymal transition (EMT) that is crucial during embryogenesis and during wound healing (4). During EMT, downregulation of adhesion proteins, such as E-cadherin, allows the detachment of carcinoma cells from the matrix. Moreover, metalloproteinases are secreted to degrade basal membrane components. In physiological conditions, epithelial cells leaving a tissue undergo anoikis, a special form of apoptosis due to the absence of intercellular contacts. Hence, CTCs that have acquired migratory features must resist anoikis (5).

Although EMT and stemness are essential for metastasis-competent CTCs, evidence shows that not all cancer types employ the same mechanisms for metastasis formation. Specifically, the EMT program can vary among different cancer types. For instance, in pancreatic cancer, KRAS mutations are associated to predispose to EMT (6). Moreover, it is generally accepted that cancer cells may present a partial EMT phenotype that promotes metastasis initiation. Indeed, a complete transition to a mesenchymal phenotype would limit necessary protein-to-protein interactions and the epithelial phenotype is associated with higher proliferation rates that are important for the successful colonization of distant organs (7).

CTCs may also be released due to physical interventions. For example, needle biopsies of prostate cancer tissue may induce the release of cancer cells into the blood stream, potentially increasing the risk of metastasis (8). However, rigorous clinical trials are required to confirm this mechanism. Moreover, mechanically released cancer cells are not necessarily metastasis-competent CTCs and currently, it is challenging to identify the metastasis-competent CTCs among the cancer cells present in the circulation.

There is increasing evidence that CTC number in blood exhibit circadian fluctuations (9). For instance, Diamantopoulou et al. described variations in CTC counts and metastatic potential in breast cancer patients at different times of the day; notably, CTCs displayed higher counts and metastatic potential at 4:00 AM (10). More studies are needed to fully understand the role of the circadian cycle in CTC release and metastatic potential (11).

CTC survival and extravasation

Various studies have shown that in patients with solid tumors, CTCs can form homotypic or heterotypic clusters (12) and that cell interactions within such clusters can influence CTC metastatic potential (12). The current CTC detection methods predominantly identify single CTCs; however, the presence of CTC clusters has been associated with poorer prognosis (13). Moreover, neutrophils can associate with CTCs (heterotypic clusters) to escort them to distant sites and promote their proliferation (12). However, CTCs are vulnerable to attacks by protective immune cells. In this context, expression of Programmed cell Death-Ligand 1 (PD-L1), which can protect tumor cells from T-cell-mediated attacks (14), has been observed in CTCs from patients with different solid tumor types, including breast (15), lung (16), and urothelial cancer (17). In addition, the association with blood platelets could enhance CTC survival (18). Thus, the interplay between circulating host cells and CTCs contributes to CTC survival in the bloodstream, which is an essential step in successful metastatic progression according to mathematical models (19). Dujon et al. suggested that the most important feature in the metastatic cascade is the capacity of CTCs to overcome the biological and physical pressures (19, 20). Therefore, good viability might be the key (and maybe the only universal) feature to accurately identify metastasis-competent CTCs. Figure 1 summarizes the role of CTCs in the biology of cancer and its implications as a liquid biopsy analyte.

For metastasis formation, the surviving CTCs need to extravasate in distant tissues. Besides the known integrin- and selectin-mediated adhesion to the endothelial cells that line the blood vessels, extravasation is influenced by the blood flow conditions. For instance, Follain et al., showed that hemodynamic forces influence the arrest, adhesion, and extravasation of CTCs implicated in the development of brain metastases (21). Clustering also might affect extravasation because CTC clusters might get more readily trapped in small capillaries from where they can initiate a distant metastasis (22). Moreover, recent work by Fu et al. indicated that bacteria incorporated in CTC clusters promote metastatic progression in a murine breast cancer model (23).

After extravasation, a key question is which CTCs will colonize and proliferate in a distant organ to form overt metastases. Studying CTC expansion in vivo and in vitro might bring some answers. Indeed, CTCs that can grow in culture conditions are most likely to be metastatic-competent CTCs. Several groups developed cell culture and in vivo models to study this important aspect. For example, Cayrefourcq et al. showed that CTC lines from a patient with CRC mirror the clonal evolution during treatment. These CTC lines present different gene expression profiles related to tumor progression and site of origin (primary vs metastatic tumor). The authors also showed that metastasis-competent CTCs are released even during cancer treatment (24). In breast cancer, Koch et al. established an EpCAM⁽⁺⁾ CTC line derived from a patient with metastatic estrogen receptor-positive breast cancer who did not respond to endocrine therapy. The authors detected functionally relevant MAP3K1, MAP3K6, NF1, and PIK3CA mutations in the CTC line, primary tumor, and metastasis (25). Similarly, Zhang et al. described a CTC subpopulation that did not express EpCAM and that could be expanded in vitro. This suggests that some metastasis-competent CTCs might not express EpCAM (26). CTCs from different cancer types have been expanded, however, this topic has been extensively reviewed elsewhere [see ref (27)]. The generation of CTC lines and even the short-term expansion of CTCs are highly challenging and are rarely achieved. Therefore, alternative methods are needed to differentiate metastasis-competent CTCs from other CTCs. One example is the EPIDROP method that evaluates CTC secretion profile as a marker of viability and of metastatic competence (24).

CTC Clinical and Translational Studies

Prostate cancer

In patients with prostate cancer, prostate-specific antigen (PSA) has traditionally been used to monitor the response to treatment and relapse. However, some patients do not show any increase in PSA levels during the follow-up after primary tumor removal (28). Therefore, alternative methods are needed to detect MRD. One potential approach is the detection of CTCs that can provide prognostic information and can be used for the early discovery of tumor recurrence (29). Conversely, in early-stage nonmetastatic prostate cancer, the clinical value of CTC detection has not been established due to the low CTC count in these patients (30); nevertheless, CTCs have shown to strongly associate with localized high grade prostate cancer and PSA levels (31). Thus, CTCs might have also a utility in early stages; however, this should be demonstrating in further multicenter clinical trials. Moreover, higher CTC counts at diagnosis have been associated with high-risk prostate cancer and occult metastases (32). Notably, biochemical recurrence-free survival and therapy-free survival are worse in CTC-positive patients who underwent lymph node dissection (33). This suggests that CTCs can be investigated as a potential liquid biopsy biomarker to assist in selecting patients who could benefit from lymph node dissection upon prostate cancer recurrence.

In localized prostate cancer, Joosse et al. showed that prostate biopsy favors the release of prostate tumor cells into the circulation. In 115 men with elevated serum levels of PSA, CTC count based on the CellSearch® system showed an increase after biopsy and this correlated with worse progression-free survival (PFS) (34). Studies in larger cohorts are ongoing to validate this finding and assess whether mechanically released CTCs are metastasis-competent CTCs. These studies also highlight the importance to identify metastatic-competent CTCs, as not all CTCs released in a biopsy or surgery might be able to initiate metastatic tumors.

In patients with localized high-risk prostate cancer treated with radiotherapy and hormone therapy, variations in CTC patterns have been observed following treatment, particularly a significant association between CTC conversion following treatment and advanced tumor stages (T₃ and N₁). However, in this context, CTC detection was not associated with overall survival (OS) yet (35). Moreover, a study on CTC enumeration using the CellSearch® system during radiotherapy and adjuvant hormone therapy did not find any association between CTC number and known clinical predictors of recurrence in 65 patients with high-risk prostate cancer followed for a median period of 55 months (32). Besides mere enumeration of CTCs, it has been suggested that targeted CTC transcriptome profiling before initiating a new treatment might predict the response to therapy (36).

As prostate cancer progresses from localized to metastatic, CTC number in peripheral blood samples of patients may vary considerably at different stages of treatment. In metastatic prostate cancer, a cutoff of ≥5 CTCs per 7.5 mL of blood has been used in various clinical trials due to its association with shorter OS. The prognostic value of detecting ≥5 CTCs or a change from >5 CTCs to <5 CTCs after treatment has been confirmed in different studies in patients with metastatic castration-resistant prostate cancer (mCRPC) (37).

The clinical significance of CTCs as a biomarker of treatment response and prognosis has been extensively investigated. De Bono et al. (38) demonstrated that CTC enumeration is a superior indicator of treatment response than a 50% reduction in serum PSA levels, particularly in the first 2–5 weeks following treatment initiation. Similarly, Sher et al. (39) found that CTC enumeration outperforms PSA quantification as an early treatment response marker. Moreover, using data from 5 randomized trials including 6081 patients with mCRPC, Heller et al. have shown that absence of CTCs and CTC conversion (from ≥5 CTCs at baseline to ≤4 CTCs at week 13 after baseline measurement) exhibit the highest discriminatory power for predicting OS (40). Consequently, CTCs have been proposed as a surrogate endpoint in clinical trials assessing OS in patients with mCRPC (41).

Mandel et al. (42) examined the prognostic significance of CTC count in patients undergoing radical prostatectomy for oligometastatic prostate cancer. They found that CTC count was a prognostic factor and exhibited a stronger association with OS compared with other biomarkers commonly used in clinical practice.

Two randomized trials (COU-AA-301 that includes patients with disease progression after docetaxel treatment; and ELM-PC4 that includes patients with chemotherapy naive mCRPC) that enrolled 1195 patients quantified CTC number and serum PSA level in the treatment and control arms at baseline and after 13 weeks to determine the predictive accuracy of baseline and post-baseline CTC enumeration. Absence of CTC and CTC conversion (from ≥5 CTCs at baseline to ≤4 at 13 weeks) had the highest discriminatory power for OS prediction (41). Lorente et al. investigated the clinical significance of any increase in CTC count as an indicator of disease progression in 511 patients with mCRPC and data on CTC number before treatment initiation. They found that increased CTC count during the first 12 weeks of treatment was independently associated with worse OS in patients treated with abiraterone or chemotherapy (43).

CTCs have a role in identifying patients for hormone therapy. A recent study in patients with metastatic hormone-sensitive prostate cancer (mHSPC) from the SWOG S1216 trial (androgen deprivation plus orteronel or bicalutamide) showed that patients without CTCs were significantly more likely to attain complete biochemical response and had a significantly lower risk of disease progression and death after adjusting for clinical covariates (disease burden, bone metastases, age, race, alkaline phosphatase, hemoglobin, PSA, treatment arm) compared with patients with ≥5 CTCs. This study also confirmed that CTC count at treatment start is highly prognostic of the PSA response, PFS, and OS in both treatment arms. Moreover, baseline CTC count strongly correlated with progression. This suggests that baseline CTC count is a valuable prognostic marker in patients with mHSPC at therapy initiation to discriminate between patients with favorable and unfavorable response and OS (44). Similarly, variants in the androgen receptor (AR) have been associated to therapy resistance. In particular, as first shown by Antonarakis et al., the androgen receptor splice variant 7 (AR-V7) is frequently present in CTCs of mCRCP patients and it predicts therapy resistance against enzalutamide and abiraterone—both agents that block the AR pathways (45). In a more recent multicenter prospective blinded study, Armstrong et al., assessed the association between AR-V7 status in CTCs and clinical outcomes in 118 patients with low-risk mCRPC. They found that the presence of CTCs^AR-V7(+) before treatment was independently associated with worse PFS and OS and also with lower probability of confirmed PSA responses during treatment with abiraterone or enzalutamide (46). Sharp et al. analyzed 227 peripheral blood samples from 181 patients with mCRPC and heterogeneous treatment regimens. They found that patients with CTCs^AR-V7(+) had higher CTC counts and AR-V7 protein expression in tumor tissue biopsies compared with patients with CTCs^AR-V7(−) samples. Interestingly, OS was shorter in patients with CTCs^AR-V7(+) than without CTCs; conversely, OS was not worse in patients with CTCs^AR-V7(+) than with CTCs^AR-V7(−) (47). These findings indicate that the CTC AR-V7 status has a prognostic relevance.

Despite the wealth of data on the prognostic values of CTCs detected by the CellSearch^® system, which uses epithelial markers for CTCs enrichment, the presence of CTCs with mesenchymal features has also been reported by devices using epithelial independent enrichment methods. For example, Xu et al. showed the existence of CTCs enriched by the label-independent Parsortix™ system that express both mesenchymal vimentin and epithelial cytokeratins (48). Moreover, the same group has shown that the presence of CTCs expressing both cytokeratin and vimentin is more significantly associated with the presence of metastatic tumors than CTCs expressing only cytokeratin (49). Recently, Yang et al. categorized CTCs into epithelial, mesenchymal, and epithelial/mesenchymal subgroups and found a positive correlation between the mesenchymal CTC count and the number of bone metastases (50). These mesenchymal CTCs better predicted mCRPC-free survival and cancer-specific survival compared with the other subgroups. Patients with ≥5 epithelial CTCs per blood sample and patients with ≥2 mesenchymal CTCs per blood sample exhibited significantly worse outcomes in terms of mCRPC-free survival and cancer-specific survival after undergoing cytoreductive radical prostatectomy.

Lung cancer

In early-stage non-small cell lung cancer (NSCLC), the TRACERx study investigated CTC enumeration in pulmonary vein blood samples (collected at surgery) using the CellSearch® system. These CTCs represented subclones responsible for tumor relapse and remained an independent predictor of relapse in the multivariate analysis adjusted for tumor stage (51). Genomic profiling of single CTCs isolated from pulmonary vein blood samples collected during surgery revealed that their mutation profile was more similar to that of metastasis detected 10 months later than to the primary tumor mutation profile (51). Additionally, in patients with NSCLC and CTC detection before radiotherapy, the risk of nodal and distant tumor recurrence was increased (52). Higher pretreatment CTC counts and CTC persistence after treatment were significantly associated with increased risk of recurrence outside the targeted treatment site (52).

In metastatic NSCLC, CTC counts ≥2 and ≥5 have been associated with the most unfavorable clinical outcome (53). Furthermore, increased CTC count and tumor metabolic parameters (i.e., 18F-fluorodeoxyglucose positron emission tomography/computed tomography) during treatment have been identified as a prognostic factor for PFS and OS (54).

The prognostic potential of CTC enumeration in metastatic NSCLC was assessed through a pooled analysis of patients from 9 European NSCLC CTC centers. The results confirmed that CTC counts ≥2 and ≥5 per 7.5 mL of blood are associated with reduced PFS and OS, respectively (53). Moreover, in patients with advanced-stage NSCLC after one cycle of chemotherapy, CTC-based surveillance revealed that prognosis was worse in patients with >5 CTCs than with <5 CTCs. CTC number could be modulated by therapeutic intervention, and lower CTC counts were correlated with better clinical outcomes (55). Besides CTC number, CTC molecular phenotypes have strong prognostic value. For instance, PD-L1 expression in CTCs of patients with advanced NSCLC has been associated with poor prognosis (16). The differential expression of PD-L1 and Ki67 on CTCs can offer additional predictive information in patients with advanced NSCLC who received pembrolizumab. Changes in the PD-L1^low subpopulation during early treatment have been associated with disease control (decreased number) or resistance to the immunotherapy (increased number) (56).

In small cell lung cancer (SCLC), CTCs can be detected in approximately 70%–95% of patients (57). The CTC threshold for prognosis in this cancer type varies widely across studies, from >2 to >50 CTCs (57). Higher CTC number after the initial cycle of chemotherapy is associated with poorer PFS and OS (58). In SCLC, CTC-based surveillance demonstrated that the baseline CTC number and CTC number change after one cycle of chemotherapy are independent prognostic factors (59).

Breast cancer

In patients with breast cancer, CTC count at diagnosis is strongly associated with relapse, indicating that the release and survival of malignant cells in the circulation are crucial processes for the development of overt metastases (60).

Bidard et al. analyzed data from 17 different centers, involving 1944 patients with metastatic breast cancer, and demonstrated that ≥5 CTCs per 7.5 mL of blood is associated with shorter OS and PFS, thus establishing the clinical validity of CTC enumeration (61).

CTC characterization based on gene expression, DNA methylation, and DNA mutation analysis, in combination with CTC count and phenotypic analysis, can identify MRD up to 4 years before clinically detectable metastatic disease (62). Interestingly, the detection of prostate-specific membrane antigen (PSMA)-positive CTCs in patients with nonmetastatic triple-negative breast cancer before and after neoadjuvant chemotherapy has shown clinical significance in identifying patients at high risk for relapse. Moreover, the presence of full-length AR-positive CTCs and of CTCs^AR-V7(+) is associated with therapeutic failure, suggesting that AR inhibition may not be effective in primary breast cancer (63).

Magbanua et al. developed a novel latent mixture model to stratify groups with similar CTC trajectory patterns during chemotherapy, revealing that the repeated analysis of CTCs can stratify patients with poor prognosis into distinct prognostic subgroups that may benefit from more effective treatment. This prognostic classification approach can be used to refine CTC-based risk stratification strategies and guide future prospective clinical trials in patients with metastatic breast cancer (64).

The clinical utility of CTC enumeration (with a cutoff of ≥5 CTCs/7.5 mL of blood) for early changes in first-line chemotherapy regimen (after 21 days) was not demonstrated in an early clinical trial (65). However, in the recent STIC-CTC trial (NCT01710605), a cutoff of ≥5 CTCs per 7.5 mL of blood predicted therapy outcomes (chemotherapy vs endocrine therapy) in patients with receptor-positive, HER2-negative metastatic breast cancer (66). However, the standard of care for this group of patients has changed since this publication, limiting its clinical utility. Nonetheless, this study was the proof-of-principle that CTCs can be used as an independent biomarker of breast cancer progression and prediction.

Colorectal cancer

The clinical utility of CTC detection in CRC has not been demonstrated yet; however, a cutoff of ≥3 CTCs per 7.5 mL of blood has been validated as a reliable predictor of PFS and OS (67).

Van Dalum et al. prospectively monitored CTC changes using the CellSearch® platform in patients with nonmetastatic CRC for a median of 5.1 years after the initial diagnosis. They detected CTCs in 24% of patients before surgery, and found that CTC presence was significantly associated with unfavorable outcomes, compared with patients without detectable CTCs (68). CTC presence after surgery and before adjuvant therapy initiation did not influence the clinical outcomes. Conversely, CTC detection 2–3 years after surgery predicted an unfavorable prognosis (68). Therefore, CTC detection may suggest CRC recurrence and long-term persistence of MRD (68).

Wang et al. showed that patients with early-stage CRC and detectable CTCs after surgery had a significantly higher risk of recurrence and thus reduced recurrence-free survival rate. In early-stage CRC, patients with a preoperative CTC count ≥4 exhibited a significantly higher recurrence risk compared with those with <4 CTCs. They used postoperative CTC count, TNM staging and carbohydrate antigen 72–4 to identify patients at high risk who could benefit from adjuvant chemotherapy (69). They also found that, irrespective of the clinical risk status and preoperative CTC levels, if postoperative CTC number remained ≥4 for more than 3 consecutive time points during 2–6 months, the recurrence rate was 100% (70).

A recent meta-analysis demonstrated that CTC presence in patients with CRC (with and without metastasis) has a strong predictive value for OS and PFS and significantly increases the risk of cancer-related death and recurrence (71).

To determine the optimal first-line treatment for patients with metastatic CRC, valuable guidance can be obtained from clinicopathologic characteristics and prognostic and predictive factors. Two large cohorts (NCT01640405 and NCT01640444) were designed to assess associations between baseline CTC count, molecular tumor profile, and clinicopathologic features of chemotherapy-naive patients with metastatic CRC. Overall, 41% of the 1202 included patients had a baseline CTC count ≥3 that was associated with low performance status (Eastern Cooperative Oncology Group scale), stage IV at diagnosis, at least 3 metastatic sites, and elevated levels of carcinoembryonic antigen (72).

In patients with metastatic CRC, the chemotherapy regimen FOLFOXIRI plus bevacizumab is more effective than FOLFOX/FOLFIRI plus bevacizumab as first-line therapy. However, it is not widely used due to concerns about toxicity and the lack of predictive biomarkers. Analysis of the role of CTC counts as a biomarker for patient selection showed that in patients with metastatic CRC and baseline CTC count ≥3, first-line FOLFOXIRI-bevacizumab significantly improved PFS compared with FOLFOX-bevacizumab. Thus, the CTC count may help to select patients for intensive first-line therapy. Changes in CTC trajectory patterns in patients with metastatic CRC during treatment confirmed the clinical value of CTC positivity, even with a cutoff of ≥1 CTC per 7.5 mL of blood. It also demonstrated that CTC trajectories over time are a better prognostic indicator than CTC enumeration at baseline (73).

Conclusion

The presence of metastasis-competent CTCs is a key factor in the metastatic process. Various micro- and macro-environmental stimuli can support viable CTCs in initiating a new tumor at a distant site. Therefore, the identification and characterization of metastasis-initiator CTCs are of utmost importance. It is generally accepted that only a small fraction of CTCs can form metastases. CTC survival in the bloodstream is an important factor in the metastatic cascade and explains the strong prognostic significance of CTC counts in many tumor types, particularly breast cancer, as demonstrated by large clinical studies. CTC counts can be used for risk stratification to improve the current tumor staging algorithms. Personalized treatments may be proposed to patients at higher risk of relapse.

Despite the limitations of the current methods to identify metastasis-competent CTCs, CTCs represent the only biomarker in blood that fully reflects the tumor biology. Additionally, CTCs represent solid tumors in their “liquid phase” during the metastatic cascade, suggesting their potential as targets for therapy to prevent cancer cell dissemination.

Technologies that increase CTC capture rates are currently being developed and validated. This may allow the reliable detection of very few CTCs (and also of other biomarkers, such as circulating tumor DNA) to be used in clinical settings, for instance for the early detection of primary or metastatic cancer (74). The capture of sufficient CTCs will also allow downstream analyses (e.g., DNA sequencing, epigenome profiling). In this context, tumor-draining blood vessels are a source of higher amounts of CTCs compared with peripheral blood (75).

Moreover, the clinical utility of any biomarker needs to be demonstrated in interventional clinical trials. The first promising results on CTCs as a tool to help therapy decision-making have been obtained in breast cancer (66). Clinical trials are now needed to test this CTC role in patients with other tumor types. Initiatives, such as the European Liquid Biopsy Society (ELBS, www.elbs.eu), promote enhanced collaboration and integration among basic, translational, and clinical research disciplines. These collaborations are crucial for the widespread CTC implementation in the clinic.

Nonstandard Abbreviations

CTC, circulating tumor cell; CRC, colorectal cancer; MRD, minimal residual disease; EMT, epithelial-mesenchymal transition; PD-L1, Programmed cell Death-Ligand 1; PSA, prostate-specific antigen; PFS, progression-free survival; OS, overall survival; mCRPC, metastatic castration-resistant prostate cancer; mHSPC, metastatic hormone-sensitive prostate cancer; AR-V7, Androgen Receptor Splice Variant 7; NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; PSMA, prostate-specific membrane antigen.

Author Contributions

The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (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. Nobody who qualifies for authorship has been omitted from the list.

Luis Enrique Cortes-Hernandez (conceptualization—equal, data curation—equal, investigation—equal, writing—original draft—equal, writing—review and editing—equal), Zahra Eslami-S (conceptualization—equal, data curation—equal, investigation—equal, writing—original draft—equal, writing—review and editing—equal), Klaus Pantel (conceptualization—equal, data curation—equal, investigation—equal, project administration—equal, supervision—equal, writing—original draft—equal, writing—review and editing—equal), and Catherine Alix-Panabières (conceptualization—equal, data curation—equal, investigation—equal, supervision—equal, writing—original draft—equal, writing—review and editing—equal).

Authors’ Disclosures or Potential Conflicts of Interest

Upon manuscript submission, all authors completed the author disclosure form.

Research Funding

C. Alix-Panabières and K. Pantel received funding from the European IMI research project CANCER-ID (115749-CANCER-ID), Horizon Europe programme 2020 Research and Innovation program under the Marie Skłodowska-Curie grant agreement no. 765492 and ERA-NET EU/TRANSCAN 2 JTC 2016 PROLIPSY (Early detection of PROstate cancer via LIquid bioPSY) and PANCAID (PANcreatic Cancer Initial Detection via liquid biopsy) funded by European Union. C. Alix-Panabières is also supported by la Fondation ARC pour la Recherche sur le cancer. K. Pantel also received funding from Deutsche Krebshilfe (no. 70112504), Deutsche Forschungsgemeinschaft (DFG) SPP2084 µBone and ERC Advanced Investigator Grant INJURMET (no. 834974). L.E. Cortés-Hernández has received funding from ERC Advanced Investigator Grant INJURMET (no. 834974).

Disclosures

C. Alix-Panabières is one of the patent holders (US Patent Number 16,093,934) for detecting and/or characterizing circulating tumor cells; is a scientific advisor for, and received an honorarium from, Menarini; Guest Editor, Clinical Chemistry, ADLM. K. Pantel is scientific advisor for, and received an honorarium from, Menarini; Associate Editor, Clinical Chemistry, ADLM.

Acknowledgments

We thank Dr. Elisabetta Andermarcher for assistance with her comments and proofreading that greatly improved the manuscript. The figure was created with BioRender.com.

References

Author notes