Clinicians have long sought rapid and accurate methods to screen for and track the progression of cancer. Current technologies include the use of blood tests to screen for certain biomarkers such as PSA level as an indication of prostate cancer (although there are often problems with the sensitivity and specificity of such tests), along with imaging methods such as computed tomography or magnetic resonance imaging (imaging tests however, often require patients to have tumors above a certain size—usually about 1 cm—to yield positive results).
One approach that has shown promise and is increasingly discussed in clinical settings is the capture and analysis of circulating tumor cells (CTCs). CTCs are cancer cells that break away from either the primary tumor or metastatic sites and circulate in the peripheral blood. Increasingly, CTCs have been viewed as a readily accessible source of tumor cells for molecular analysis (i.e., as a “liquid biopsy”). There is evidence that CTCs may be present even at the earliest stages of cancer, although at very low quantities. In the past 15 years, there have been many different technology platforms and approaches to isolate and analyze CTCs. A number of these technologies function by enriching CTCs based on the presence of surface markers [such as the epithelial cell adhesion molecule (EpCAM)]. In recent years, these technologies have steadily improved by increasing capture efficiency and the purity of the cells captured. This is especially important for downstream applications, such as analysis of cell DNA or RNA, as contaminating blood cells can confound analysis of the tumor cells.
CTCs have the potential to assist medical decision making through both prognostic and diagnostic capacities. However, this potential has been largely unrealized in a clinical setting. Several clinical trials involving CTC identification have noted some prognostic benefit in enumerating CTCs but little or no overall change in patient outcome. Despite these findings, newer studies have suggested potentially powerful clinical applications, particularly in the isolation of genetic information from CTCs that may predict drug response. Such findings, when further validated, will provide additional impetus for the use of CTC analysis in the clinic.
CTCs in the Clinic
To date, the sole U.S. Food and Drug Administration (FDA)-approved instrument for isolation of CTCs is the CellSearch platform. CellSearch is approved only for monitoring CTCs in patients with metastatic breast, prostate, or colorectal cancer. All other applications are still considered investigational. The CellSearch platform functions by isolating and identifying cells that are EpCAM and cytokeratin 8-, 18-, and/or 19-positive.
There have been several clinical studies utilizing this platform to enumerate CTCs to help provide prognostic information. However, these studies were often limited by small sample sizes. Trials in prostate cancer have shown modest prognostic significance of CTC enumeration for predicting outcome; however, this has not translated into significant clinical benefit as measured by overall survival or prognostic-free survival.
With the advent of molecularly targeted agents, however, this will likely change, as CTC analysis may provide actionable clinical information vis-à-vis choice of treatment. In addition, several new platforms are under development to improve the isolation and analysis of CTCs in patients with all types and stages of cancers. For instance, the Vortex chip, being developed by the company Vortex Biosciences, allows for isolation of CTCs by size alone and without the need for capture through external labeling; this could be important, particularly in advanced cancers where the cancer cells often lose expression of specific markers.
How CTCs Can Be Useful in the Clinic
The continued development of molecularly based therapies (i.e., vemurafenib for BRAF V600E/K mutant cancers and afatinib for activating Exon 19 epidermal growth factor receptor deletions) has led to the increased need for genetic analysis of tumor cells to determine a patient’s suitability for such targeted therapies and to determine the overall mutational landscape of the tumor. The rapid drop in sequencing costs, particularly in a clinical setting, means that it may be possible in the near future to complete entire exome, transcriptome, or even whole genome sequencing of tumor tissue to determine the mutations that are present and thus the types of therapies that may be most appropriate, as well as to switch treatments in response to potential mutational changes.
One major limiting factor is being able to obtain sufficient tumor material to enable such studies. In many cases, a patient may have only a single biopsy from one tumor area. Furthermore, serial biopsies are not feasible or practical in many cases, as the tumor may recur in locations that are difficult or dangerous to access, yet such information may be critical to assess for tumor resistance. CTCs may thus provide an important source of tumor material, and their analysis can be an important adjunct test to provide information about tumor mutational status, particularly when repeated tissue biopsies may not be possible.
Prostate Cancer as an Example of CTC Utility
There are many clinically oriented studies underway, and recent reports have shown glimpses of the potential utility of CTC collection and analysis. While the studies described here are not a comprehensive listing, they provide a survey of initial promising data and an impetus for additional work to further characterize the utility of CTC collection.
One of the first applications in prostate cancer was assaying for the presence of the ERG (TMPRSS2-ERG) fusion and for androgen receptor and PTEN status . In prostate cancer, the ERG fusion is present in about 50% of cases and is an early sentinel event. More recent studies have utilized CTC isolation in combination with next-generation exome (DNA-based) or transcriptome (RNA-based) sequencing. As an example, Emmanuel Antonarakis and his colleagues at Johns Hopkins University utilized CTC isolation followed by quantitative polymerase chain reaction of the androgen receptor to identify the androgen receptor splice variant 7 (AR-V7) mRNA among CTCs. They utilized this information to determine whether it could help predict resistance to the novel prostate cancer drugs enzalutamide and abiraterone in patients with known metastatic castration-resistant prostate cancer . The AR-V7 variant lacks the binding domain, and so it was hypothesized that the presence of this splice variant would signify reduced responsiveness to the drugs. Indeed, Antonarakis found that those patients with AR-V7-positive CTCs had reduced response to either enzalutamide or abiraterone and, thus, decreased overall survival—though these findings will need to be validated in a larger prospective trial.
How CTCs Can Help Clinicians with Decision Making
The development of molecularly guided therapies as discussed previously has prompted the need for companion diagnostics to ascertain the presence or absence of that specific genetic variant. This knowledge can be particularly critical, as several drugs have unwanted off-target effects in the absence of targeted mutation (e.g., vemurafenib). Given the cost of these newer drugs, it is critical to have assays that can reliably confirm the presence of the actionable target.
Particularly critical is the ability to follow treatment response through CTCs. An increase in CTCs over the course of treatment may signify a treatment failure; furthermore, these CTCs can be analyzed to see what genetic changes may have occurred in response to the initial treatment. A decline in CTC count followed by a rise while on a particular treatment may indicate early resistance and prompt consideration of alternate treatment strategies; this may occur even before changes are noted on imaging studies.
The ability to capture intact tumor cells is a significant benefit of CTC isolation. While many CTCs may be undergoing apoptosis or necrosis, some have not and maintain relatively intact DNA and RNA. Such cells are of particular interest as they may contain more recent information about tumor status. Analysis of the CTC genome (DNA) or transcriptome (RNA) could yield information about genetic changes in the tumor, such as the AR-V7 splice variant in prostate cancer, which may predict resistance to the next-generation of the anti-androgen drugs enzalutamide and abiraterone.
What’s Next for CTCs in the Clinic?
There are several competing platforms under development to enable CTC isolation and analysis. Space limitations prevent a comprehensive summary, although there have been several reviews that nicely summarize the state of the field .
To be clinically applicable, any platform that seeks to be translated must undergo rigorous evaluation and validation with clinical samples in multiple rigorous clinical trials to gain FDA approval. Furthermore, these trials should also test the ability of CTCs to predict drug sensitivity or resistance through genetic analysis. This process will require several years, and it is not yet clear which platforms or technologies can overcome this significant barrier. Several companies already have efforts in this direction, and I expect to see fully functional and approved platforms in a few years.
Finally, and most importantly, these tests have to be transparent to the end user, the clinician. Medicine is in a constant state of flux, and these test results must be incorporated seamlessly into the clinician’s knowledge base; otherwise, they will not be used. Given the number and types of platforms in preclinical testing, I am confident that there will be at least one that can meet these criteria.
CTCs do hold the potential to significantly assist with clinical decision making. However, for CTC analysis to achieve clinical uptake, there need to be standardized platforms and protocols for isolating and analyzing CTCs as well as more focused and relevant prognostic information, which will require several additional controlled clinical trials to determine the true benefit of CTC isolation and analysis. Such studies—several of which are currently underway, with many more planned in the near future—will likely yield actionable information so that clinicians can utilize CTC analysis as an additional piece of information with which to guide treatment determinations and help predict outcomes.
- G. Attard, J. F. Swennenhuis, D. Olmos, A. H. Reid, E. Vickers, R. A’Hern, R. Levink, F. Coumans, J. Moreira, R. Riisnaes, N. B. Oommen, G. Hawche, C. Jameson, E. Thompson, R. Sipkema, C. P. Carden, C. Parker, D. Dearnaley, S. B. Kaye, C. S. Cooper, A. Molina, M. E. Cox, L. W. Terstappen, and J. S. de Bono, “Characterization of ERG, AR and PTEN gene status incirculating tumor cells from patients with castration-resistant prostate cancer,” Cancer Res., vol. 69, no. 7, pp. 2912–2918, 2009.
- E. S. Antonarakis, C. Lu, H. Wang, B. Luber, M. Nakazawa, J. C. Roeser, Y. Chen, T. A. Mohammad, Y. Chen, H. L. Fedor, T. L. Lotan, Q. Zheng, A. M. De Marzo, J. T. Isaacs, W. B. Isaacs, R. Nadal, C. J. Paller, S. R. Denmeade, M. A. Carducci, M. A. Eisenberger, and J. Luo, “AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer,” N. Eng. J. Med., vol. 371, no. 11, pp. 1028–1038, 2014.
- D. A. Haber and V. E. Velculescu, “Blood-based analyses of cancer: circulating tumor cells and circulating tumor DNA,” Cancer Discov., vol. 4, no. 6, pp. 650–661, 2014.