Significance of studying circulating tumor cells

Introduction

Circulating tumor cells (CTC) are tumor cells released into blood. They are considered the pivotal component of the metastatic cascade and are being extensively studied only in the last decade or so. Understanding the biological and clinical impact of CTC is likely to reveal important information of the metastatic process and contribute to better management of cancer. We briefly discuss here the current clinical utility of CTC and their emerging clinical applications.

Current clinical applications

Ashworth first reported tumor cells in a patient’s peripheral blood over one and a half century ago [1]. However, the study of these tumor cells has always been hampered by the rare existence of this cell population amid the excess of hematopoietic cells in blood. Various CTC isolation technologies have been developed only relatively recently, based on various principles such as affinity-based capture technologies including CellSearch™ [2], or non-affinity based technologies such as size based microfiltration [3], density-based gradient centrifugation [4], or electrical property-based dielectrophoresis (DEP) [5]. Most of the clinical data pertaining to clinical utility of CTC was collected utilizing CellSearch™, the only FDA-cleared technology for CTC enumeration for breast, colon, and prostate cancers.

CTC enumeration at baseline as prognostic marker

One well-validated clinical application of CTC is assessment of their prognostic value by CellSearch assay at pretreatment baseline. Patients with higher than 5 CTC per 7.5 mL blood were shown to have shorter progression-free survival and shorter overall survival in a study analyzing baseline CTC level in a cohort of 177 metastatic breast cancer patients in 2004 [2]. Subsequently, similar results were seen for metastatic prostate cancer in 2008 [6] and metastatic colorectal cancer in 2009 [7]. Several follow-up studies have confirmed the prognostic value of CTC, and as a result, CTC has been proposed in the new 2010 edition of the tumor-node-metastasis (TNM) cancer staging system manual as cM0(i+) [8], which is yet to be included in the clinical guidelines. The following paragraphs briefly discuss emerging clinical applications of CTC.

Emerging clinical applications

CTC as surrogate endpoint for clinical trials

One important potential clinical utility of CTC is their use as a surrogate endpoint for clinical trials. If approved to be informative, interrogating CTC with minimally invasive blood draws at follow-up time points to monitor treatment will greatly benefit the cancer management, and several clinical trials are designed to test the feasibility of this notion. For example, CTC enumeration analysis at follow-up visits for the same cohort of patients examined by Cristofanilli et al. above [2] showed CTC to be prognostic of progression-free survival and overall survival [9]. Similarly, another clinical trial in breast cancer also indicated that CTC as a surrogate endpoint is more reproducible and robust than radiographic response [10]. In prostate cancer, a clinical trial conducted in a cohort of 263 metastatic castration resistance prostate (mCRPC) cancer patients—the SWOG S0421 trial also indicated that, patients with rising CTCs at week 3 have significantly worse overall survival as compared with those with less or equal number of CTC at the week 3 follow-up visit [11]. In another study in mCRPC setting, CTC in combination with serum lactate dehydrogenase (LDH) level was shown to be a better surrogate for survival than PSA level [12]. Thus, so far, clinical trials attempting to interrogate CTC as a surrogate endpoint for clinical trials have shown some promising results, prompting further extensive follow-up studies.

CTC as predictive marker to guide treatment

As similarly encouraging evidence supports the value of CTC as prognostic markers in various cancers, an obvious question is: Can we use CTC to guide treatment selection? More specifically, can we use CTC measurement at baseline, or at follow-up time-points, to predict patient’s response to treatment and thus guide therapy? An example is the SWOG S0500 clinical trial [13].

Results from the SWOG S0500 Phase III clinical trial (see Fig. 12.2, Jeffrey B. Smerage, for Trial Schema) were presented at the 2013 ASCO San Antonio Breast Cancer Conference. The trial was designed primarily to determine in a first line chemotherapy setting whether women with metastatic breast cancer and elevated CTCs by CellSearch assay (=5 per 7.5 mL of whole blood) after 3 weeks of first-line chemotherapy derive increased benefit (overall survival and progressionfree survival) from changing to an alternative chemotherapy regimen at the next cycle, instead of waiting for clinical evidence of progressive disease before changing to an alternative chemotherapy regimen. The trial was not designed to compare chemotherapies. The underlying hypothesis was that treatment decisions can be made based on CTC levels, with the belief that a significant number of patients resistant to their first line of therapy would respond to a second-line therapy.

Patients may benefit by switching early to a new line of therapy through avoiding the cumulative toxicities of ineffective therapy while spending more time on active therapy, thus improving quality of life and potentially tolerating future therapies better. Contrary to expectation, the patient data from randomized arms did not differ with respect to progression-free or overall survival. Given the very poor survival outcomes for this population, it was concluded that this population likely has a disease that is generally resistant to cytotoxic mechanisms. However, trial data did demonstrate a large, clinically significant, and statistically significant difference in prognosis for patients in whom the CTC remained elevated after one cycle of firstline chemotherapy. This is a population that should be considered for clinical trials of novel agents or novel treatment strategies early in the course of their disease.

In summary, the SWOG-S0500 trial validated the hypothesis that the group of patients with elevated CTC at baseline and 21 days after starting the first chemotherapy has a worse prognosis with regard to progression-free and overall survival, while low baseline CTC levels indicate a very good prognosis. The trial also showed that switching to a different chemotherapy sooner does not improve outcomes. For these patients, a clinical trial to investigate new targeted therapies should be considered, since chemotherapy is not effective in this population of patients.

To address the clinical utility of CTC in another direction, the ongoing METABREAST trial aims at identifying patients without the need for aggressive treatment if they have low CTC at baseline level. In this study, CTC were measured at baseline, and patients receive chemotherapy if they are detected with >5 CTC, otherwise they will receive endocrine therapy [14].

CTC as a marker for early detection of solid tumors

In addition to investigating CTC as a surrogate endpoint and predictive marker, other studies focus on the possibility of using CTC for early detection for solid tumors. As reported in mouse model breast cancer research, tumor cells can “leave home early” [15] and establish metastasis without the necessity of experiencing the steps of transformation at primary sites [16]. Another study in pancreatic cancer transgenic mouse model revealed that CTC can enter blood stream even before tumor formation [17]. These observations encourage the notion that CTC could be used for early detection of cancer, as harbingers of impending malignancy. However, preliminary data from pilot clinical trials has stimulated some disputes. For example, a study probing for CTC in patients with benign colon diseases has detected CTC in 11.3% of the 53 patients analyzed, which could be false-positive results [18]. Another potential problem of using CTC for early detection of cancer is the extremely low CTC count in early stage patients. The cut-off of CTC count in a nonmetastatic breast cancer setting by CellSearch is determined to be 1 per 7.5 mL blood draw, which, although it is prognostic [19], can be easily missed depending on the sampling of the blood and the analysis process. One solution to interrogate such a low level of CTC is to examine larger volume of blood. This can either be achieved by an in vivo CTC capture probe—CellCollector® [20] or by taking advantage of a standard clinical procedure—leukapheresis [21] to harvest CTC from a much larger volume of blood. In general, emerging technologies with ability to interrogate larger volume of blood, or those with higher sensitivity to detect CTC in smaller blood volumes, might shed light on this clinical application of using CTC for early detection of cancer.

In conclusion, although prognostic utility of CTC has been well validated for various cancers, their clinical application as a surrogate endpoint, as a predictive marker to guide therapy, or as an early diagnostic marker is still largely unexplored and will require large scale clinical trials for validation. Although it is still in development awaiting further validation, the future vision of a CTC test is to serve as a “liquid biopsy” that can provide clinicians comprehensive clinical information of the patient in a minimally invasive blood draw.

CTC as a companion diagnostic

As some of the technical hurdles around CTC enumeration and suitability of various CTC capture and analytic platforms for evaluation of biomarkers get resolved, there are ongoing efforts in parallel that address development of CTC assays as companion diagnostic to assess the efficacy, toxicity, and successful targeting of anticancer therapeutics in real time as they are being developed, both in preclinical studies as well as Phase I and II clinical trials. Needless to say, such use of CTC assays must stand the rigor of regulatory hurdles. In a 2012 publication, Punnoose and Lackner review these developments and suggest a path for co-development of anticancer therapeutics with CTC-based diagnostics that could enable clinical validation and quali?cation of CTC-based assays as companion diagnostics [22]. Chapter 15 of this volume also addresses this concept in detail.

New directions for CTC analysis

As CTC research evolves, it is noteworthy that almost all the clinical applications mentioned above have employed CellSearch™ system. Although it is reliable and powerful, CellSearch™ technology is built upon the principle that CTC can be captured via anti-EpCAM antibody. As exhaustive molecular and functional characterization of CTC studies have been carried out, the validity of CTC capture based on EpCAM expression has been questioned. CTC population has been shown to be heterogeneous and the gene expression levels among CTC vary even within the same patient sample [23]. To address this heterogeneity, an increasing number of studies have begun to look beyond CTC enumeration to CTC molecular characterization in order to elucidate the subpopulations within CTC.

Epithelial–mesenchymal transition (EMT) in CTC

One important process involved in tumor metastasis that calls EpCAM-based CTC capture in question is the Epithelial–Mesenchymal Transition (EMT). EMT phenomenon has been described as the process whereby tumor cells gradually transition from epithelial phenotype into mesenchymal phenotype during metastatic progression, ostensibly via downregulation of expression of epithelial markers (EpCAM, E-cadherin, cytokeratin, etc.) and upregulation of mesenchymal gene expression (e.g., vimentin), to achieve a more invasive phenotype [24]. EMT process has been extensively investigated in primary tumors but to a much lesser extent in CTCs. EpCAM-based technologies may tend to capture and enrich “epithelial” CTC, thus potentially missing the CTCs with mesenchymal phenotype that may be metastasis-initiating. A study by Lin et al. demonstrated that by employing a sizebased isolation strategy without relying on EpCAM expression, CTC can be detected at higher sensitivity [25]. In addition, a study by Harouaka et al. has demonstrated that a mesenchymal phenotype CTC can be detected using size-based isolation technologies [26]. A 2013 study by Zhang et al. isolated viable breast cancer CTC using four target markers—HER2+/EGFR+/HPSE+/Notch1+, cultured the CTC and derived a population that metastasized to brain in a mouse model [27]; this specific population was EpCAM-negative, and would have been missed if EpCAM was employed as the sole target molecule for CTC capture. In contrast, it is likely that antigen-agnostic CTC capture methods (such as those based on cell size) or capture methods that exploit other target antigens (including epithelial and/or mesenchymal antigens) will likely provide more insights into this phenomenon. For example, various studies employing cell size-based CTC capture have reported mesenchymal-like CTCs expressing the mesenchymal marker Vimentin [26, 28]. Another study investigated EMT status of CTC captured from breast cancer patient samples using EpCAM, HER2 and EGFR as capture target antigens, and discovered that mesenchymal cells were highly enriched in the CTC population. The proportion of mesenchymal CTC increased during chemotherapy treatmentss [29]. A converse interesting notion barely examined in the context of cancer but worth studying in CTC is the concept of Mesenchymal–Epithelial Transition (MET), wherein mesenchymal CTC may revert back to an epithelial phenotype once at the secondary site, expressing cell attachment protein such as such as E-cadherin, thereby regaining ability to form proliferative epithelial growths in distant organ sites. In contrast, cells without this capability to revert back to epithelial status seem to be unable to initiate metastasis effectively [30]. This hypothesis could be the explanation for the observation that many EpCAM-based CTC capture technologies seem to be capturing CTC in an intermediate status that is neither epithelial nor mesenchymal but rather a transitional status, also referred to as epithelial-mesenchymal plasticity (EMP) [31]. Cells that have the EMP capability seem to be able to switch between epithelial and mesenchymal status and might be population of the utmost importance in circulation [14]. One of the many studies that supports this hypothesis is a clinical study looking at EMT status on CTC captured from metastatic breast cancer and mCRPC patients, where 75% CTCs were found to co-express Cytokeratin (epithelial), Vimentin (mesenchymal), and N-cadherin (mesenchymal), along with a stem cell marker CD133 expressed at a high frequency [32]. Although the association between CTC EMT status and clinical outcome is still unclear, such studies will be critical not only to choose an appropriate CTC capture technology (EpCAM versus non-EpCAM-based capture) but also to elucidate the biological nature of CTC and the clinical relevance of mesenchymal CTC subpopulations.

Cancer stem cell subpopulation in CTC

Another potential phenomenon that is worth studying in CTC besides EMT is the existence of cancer stem cell subpopulation. It has been well demonstrated that the CD44+/CD24-/low population can form tumor with much higher efficiency as compared with the other subpopulations in breast cancer [33]. It has been previously shown that disseminated tumor cells in bone marrow possess such putative stemlike phenotype (CD44+/CD24-/low) at a proportion that is significantly higher than that in the primary tumors [34]. It will be of interest to look for this subpopulation in breast cancer CTCs. A study in a pilot cohort of 30 breast cancer patients analyzed for CTC subpopulations found 35.2% of the CTCs to be CD44+/CD24-/low, while another cohort was shown to contain 17.7% CTCs that were ALDH1+/CD24-/ low [35]. A different group of researchers attempting to detect metastasis-initiating cells (MICs) using a xenograft assay demonstrated a subpopulation of CTC from luminal breast cancer patients that could initiate metastasis in mice, where they manifest a EpCAM+, CD44+, CD47+, MET+ phenotype [36]. Thus, preliminary data has shown that there is a subpopulation of CTC, which possesses “stem-like” phenotype and can be responsible for metastasis initiation. Further studies interrogating these features in larger cohort of clinical trials and their correlation with patient clinical outcome can be informative and reveal more information about the “real culprit” CTC subpopulation that is responsible for metastasis, and the one that potentially could prove to be the valuable therapeutic target.

CTC in clusters

While studies in subpopulations in CTC can be riveting, another interesting observation is the CTC clusters, also known as Circulating Tumor Microemboli (CTM). Their existence was first reported in Small Cell Lung Cancer (SCLC) patients using a size-based CTC isolation strategy. In this study, presence of CTM was shown to correlate with worse clinical outcome as an independent prognostic marker [37]. In addition, recent studies have revealed that CTC travel with other blood components as heterogeneous clusters including immune cells [38], macrophages [39], and platelets [40]. In addition, mouse model studies have shown tumor cells traveling with stromal cells, potentially cancer associated fibroblasts as its own “soil” to establish distant metastasis [41], although these are to date not shown to exist in peripheral blood in human cancer patients. Study of CTC companion cells in circulation could reveal important information on metastasis initiation and expand the definition of “liquid biopsy” to include other cell types beyond CTC.

Fate of CTC in circulation

Another important question to be answered is the fate of CTC in circulation. It is reasonable to assume that there can be three potential fates for tumor cells in circulation. The first fate is that a given CTC will be “permanently non-productive” such that it will undergo either anoikis or apoptosis or necrosis, be eliminated by immune surveillance or simply remain unable to home to metastatic niche or unable to initiate the intravasation process. The second fate is that a CTC can be “temporarily non-productive,” either successfully invading into secondary site and staying “dormant” (either stay in G0 phase of cell cycle or maintain an equilibrium of proliferative and apoptotic rates), or staying locked in mesenchymal status [30] and failing to colonize and form metastasis. The third fate is that the CTC is “productive,” not only capable of invading into a secondary site but also forming metastasis by rapid proliferation. It is possible that CTC subpopulations are not committed to one certain fate. Thus, cells from “temporarily non-productive” fate can transform into “productive” fate and form metastasis after long-term dormancy under certain environmental cues or additional genetic mutations.

To interrogate the fate of CTCs, one study looked at expression of apoptotic marker (M-30) and proliferative marker (Ki-67) in breast cancer CTC, and the data supported the hypothesis that there were proliferative as well as apoptotic subpopulations of CTC in circulation. Apoptotic CTC were seen more in early stage breast cancer patients [42]. Another study looking at M-30 and Bcl-2 expression in CTC indicated that, surprisingly, apoptotic CTC with M-30 expression is associated with worse prognosis in patients with elevated CTC level, whereas patients with Bcl-2 CTC had better clinical outcomes in contrast to the notion that Bcl-2 will lead to anti-apoptotic effect on CTC and worse outcomes [43]. While the data on apoptotic CTC looks confounding, other groups have also examined proliferative subpopulations in CTC. Thus, a study investigating Ki-67+ CTC concluded that proliferative CTC, independent of disease stage or treatment, is a rare population in circulation, and a fraction of non-proliferative CTC seem to be more chemoresistant [44].

Since data on apoptotic CTC remains elusive, and proliferative CTC seems to be a rare population in circulating, the key distinctive characteristics between the first “permanently non-productive” fate and the other two fates could lie in the homing to secondary site and initiation of extravasation process. Mechanisms of establishing micrometastasis at secondary sites can be a combination effect of physical trapping and chemical homing. Whereas physical trapping at secondary organ can be correlated to organ vasculature and tumor cell clustering (CTM lodging), chemical homing can be correlated with chemokines, micro-RNAs, and other tumor microenvironment signaling [45]. In 1889, Stephen Paget first brought up the notion that tumor cells form metastasis at secondary organs as “seeds” on congenial “soils” [46]. Later on, recent research has indicated that, certain “tumor tropism” signatures can be established to predict the potential secondary sites [47, 48]. Studying these signatures on CTC might reveal important traits that could shed light on the homing mechanisms of CTC to secondary organs.

It has been indicated that the half-life for CTC was 1–2 h. However, CTC can be detected in dormancy patients 8–22 years post mastectomy [49]. It is highly likely that CTC can be shed from micrometastasis and circulates in the blood, even seed back to the primary/metastatic lesions [50]. This indicates that, secondary organs, especially bones [51], can possibly serve as reservoirs for CTC, and thus will be critical to monitor for patients with metastatic dormancy.

Phenotypic, genotypic features of CTC for clinical applications

As we are looking into more and more in-depth biological characterization of CTC population, let us take a step back and ask the question: how can the phenotypic and genotypic studies of CTC feed back into clinical applications to benefit cancer management? An intuitive thinking will be assessing the therapeutic targets on CTC to monitor dynamic changes during treatment. One study looking at Androgen Receptor (AR) signaling in CTC from Castration-Resistant-Prostate-Cancer (CRPC) indicated that, “AR-on” signature was predominant in CTC population pretreatment. Post-treatment, a more heterogeneous population of CTC is observed. The increase of percentage of “AR-on” CTC population is correlated with worse outcome despite administration of abiraterone acetate therapy [52]. Another study looking at ALK-rearrangement status on CTC in non-small-cell lung cancer (NSCLC) patients indicated that ALK-rearranged CTCs were detected ALK-negative patients and ALK-rearranged CTCs harbored a unique pattern whereas primary tumor harbored more heterogeneous patterns [53]. This information can be useful in guiding therapies since ALK-rearrangement positive NSCLC patients do not benefit from EGFR Tyrosine-Kinase-inhibitor (TKI) and need to be treated with ALK inhibitor [54]. Another study conducted whole-exome sequencing for CTC in metastatic prostate cancer and compared it with that in the primary tumor and lymph node metastasis. This proof-of-principle study provides support for the notion that CTC can be used as a minimally invasive window to give us a peek at the mutational landscape of prostate cancer [55].

Functional characterizations of CTC and CTC culture

In addition to molecular characterization of CTC, another interesting direction is the functional characterization of CTC. Technologies that allow viable CTC capture enable such assays [56–59]. One study demonstrated development of oligoclonal CTC culture from six metastatic luminal breast cancer patients, indicating that CTC culture can be potentially used for Next-Gen sequencing and, more importantly, for drug sensitivity screening [60]. Another study tested drug sensitivity on chip without establishing CTC culture in prostate cancer, where CTCs from docetaxel resistant CRPC patients do not respond to on-chip docetaxel and paclitaxel treatment [56]. A third study showed that xenograft assays of patient-derived CTC into immunocompromised mice can provide valuable prognosis information [36]. However, attempts to culture CTC are still at very early stage and have low efficiency. The CTC culture method reported by Yu et al. managed to establish 6 CTC culture from 36 patient samples attempted, while the xenograft assay only managed to establish CTC xenograft mice from patients pre-screened with more than 1000 CTCs per 7.5 mL blood. If CTC culture can be developed into a high-efficiency method and validated in larger cohorts to faithfully reflect patient treatment response without introducing culture-induced artifacts, it could be a powerful tool to guide therapy and greatly benefit cancer management.

Conclusions

The field of CTC analysis has grown exponentially in the past decade. As more and more researches have looked into the biological aspects of CTC, the field is going in divergence into two separate while equally important directions: studies focusing on clinical utility of CTC and studies focusing on biological nature of CTC. For clinical applications, the CTC assay needs to be standardized, reliable and robust. Thus, the clinical utility of CTC is still largely bound to be based on CTC enumeration since the CellSearch™ system is still the only FDA-cleared technology and it is so far only cleared for CTC enumeration, although a fourth channel on the device can be used for molecular characterization of CTC [61]. By utilizing CellSearch system, clinical trials, as described above, have looked extensively into clinical utility of CTC count beyond prognosis, and have begun to address various clinical applications including using CTC to monitor treatment efficacy, using CTC as surrogate endpoint for clinical trials and using CTC for early detection of solid tumor. Meanwhile, other studies are also investigating the biological features of CTC by attempting to answer questions about subpopulations of CTC (EMT, stem cell, clusters etc.), the fate of CTC, and how molecular and functional assays of CTC could benefit patients from the clinical perspective. These questions, if answered, can then feed back into the clinical applications, and expand or improve the clinical utility of CTC. Although it is a rapidly advancing field, there is still a lot to learn about CTCs. More ongoing research will likely be expanding the definition of “liquid biopsy,” and we believe the ultimate goal will be developing a “universal test” that allows us to not only look at CTC counts but also phenotypic, genotypic, and functional features of CTC, as well as possibly other circulating blood-based biomarkers including associated cells, cell-free DNA and microRNA, etc., all studied in one or serial simple and minimally invasive blood draw(s).

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