Emerging Uses for Circulating Tumor Cell Technology

Detection of circulating tumor cells (CTCs), which are released from primary or metastatic lesions into the bloodstream and are the “seeds” for distant metastatic lesions, has been of interest in cancer research and treatment because these cells can potentially provide information on cancer detection, prognosis, and likelihood of treatment response with minimally invasive methods (eg, a blood draw).¹ Development of validated technologies to isolate and detect CTCs has been slow because of their relatively low counts in the bloodstream. However, refinement of assays have improved the ability to isolate and enumerate CTCs using their biological or physical properties and/or microfluidic- or nanotechnology-based techniques.¹

Use of these techniques has shown CTCs to be useful biomarkers for detection of early-stage and metastatic cancer, prediction of overall survival and disease-free survival, and assessment of treatment response.^1,2 As liquid biopsy further solidifies its role as a clinically important tool for a variety of prognostic applications, an understanding of the mechanisms of action of CTCs and the optimal detection methods is needed to improve widespread adoption.² 

CTCs vs Circulating Tumor DNA

Circulating tumor DNA (ctDNA) is fragmented DNA that originates from necrotic or apoptotic tumor cells or is actively secreted from intact cells. Like CTCs, ctDNA offers a source of potential biomarkers that can be obtained through standard minimally invasive liquid biopsy collection (eg, blood samples) and has been shown to have prognostic capability in certain tumor types.³

Unlike ctDNA, which consists of a mere fraction of cellular DNA, CTCs are intact, viable tumor cells with metastatic potential and thus offer more clinically relevant information.⁴

Additional advantages of CTCs, once isolated, include having the ability to analyze tumor cells via several methods (eg, DNA, RNA, protein), as well as to perform functional assays and single-cell analyses.³ Analyses of CTCs can also provide information on the biology and potential weaknesses of aggressive cancer clones that promote metastases, which can improve understanding about the mechanisms of metastatic spread and development of therapies that target these mechanisms.¹

Approaches for CTC Assays

Technologies for isolation and enrichment of CTCs tend to target their biological or physical properties.² Technologies that use biological properties concentrate on antibody/antigen interactions; they tend to focus on antigens that are uniquely expressed on CTCs combined with CD45-based negative selection to eliminate the contaminant hematopoietic cells.¹ Epithelial cell adhesion molecule (EpCAM) is expressed on the surface of undifferentiated stem cells and is the most commonly used marker for antibody-dependent detection of CTCs in the bloodstream.^2,5

Although EpCAM is considered a “universal” marker of epithelial cancers, it is downregulated during epithelial-mesenchymal transition, and it is minimally or not expressed on the CTCs of some types of cancer (eg, neurogenic cancers).² Therefore, important subgroups of EpCAM-negative cells are likely to be missed when using EpCAM alone to isolate CTCs, so adding additional markers of epithelial and mesenchymal cells and antibody-independent methods of detection are needed to improve CTC yield.²

Antigen-independent methods use physical properties (eg, size, electrical charge, density, or elasticity) to isolate CTCs; some of these methods include filter-based devices, density gradient centrifugation, capture surfaces, and microfluidic systems.¹ After isolation and enrichment based on differences between CTCs and blood cells, fluorescence microscopy, fluorescence spectrophotometry, flow cytometry, surface-enhanced Raman scattering, or electrical impedance are used for detection of isolated CTCs.² Once identified, CTCs can be extracted from the sample analyzed using genomics, transcriptomics, proteomics, and cell culture.²

What follows are details on several systems available for CTC detection. 

CellSearch System

The CellSearch System was the first method of detection and enumeration of CTCs to be cleared by the FDA, in 2004, to predict outcomes in patients with metastatic breast cancer; its clearance was expanded in 2008 for patients with metastatic prostate cancer.⁶ The system isolates CTCs in 7.5 mL of blood using ferrofluid nanoparticle beads that target EpCAM; this is followed by staining the CTCs with anti-cytokeratin (CK) antibodies—which also target EpCAM, an anti-CD45 antibody—as a negative stain to identify contaminating leukocytes and with DAPI to highlight the nuclear DNA of CTCs and leukocytes.^1,7 The samples are then placed in a magnetic cartridge and analyzed for the presence of tumor cells that are stained positive for CK and DAPI.⁷

Initial studies conducted using CellSearch technology identified associations between presence of CTCs and poor clinical outcomes in metastatic breast, colorectal, and castration-resistant prostate cancer; additionally, higher concentration of CTCs in the blood sample appear to portend shorter survival in all these cancers.⁸ Associations between CTCs detected using the CellSearch System and clinical outcomes have also been identified in small cell and non–small cell lung, bladder, pancreatic, head and neck, ovarian, neuroendocrine, and hepatocellular cancers.⁸

CTC counts obtained using the CellSearch System could also be used to monitor treatment response in patients with metastatic disease.⁴ In a pooled analysis of data from patients who underwent CTC isolation and quantification using the CellSearch System before and after starting treatment for metastatic breast cancer, high CTC counts after start of treatment (adjusted for baseline CTC count) were associated with shorter progression-free survival and overall survival.⁹ Further, addition of baseline CTC count, CTC change at 3 to 5 weeks, and CTC change at 6 to 8 weeks to clinicopathologic models improved survival prediction, whereas addition of other blood-based prognostic tests (ie, carcinoembryonic antigen and cancer antigen 15-3 concentrations) did not improve the prognostic ability of this model.⁹

CTC detection using the CellSearch System may also have clinical applicability in predicting recurrence and survival in patients with nonmetastatic disease.¹⁰ In patients with high-risk, node-negative, HER2-negative operable breast cancer enrolled in the phase 3 E5103 trial (NCT00433511), the risk of recurrence was 13.1-fold higher among patients who had at least 1 CTC per 7.5 mL of blood.¹⁰ Additionally, analysis of patients with early-stage, high-risk breast cancer in the phase 3 SUCCESS-A trial (NCT02181101) showed that after controlling for CTC status at baseline, the presence at least 1 CTC per 7.5 mL of blood in a CellSearch System assay 2 years after chemotherapy was an independent predictor of decreased overall survival and disease-free survival.¹¹

Parsortix PC1

The Parsortix PC1 device detects CTCs based on their physical attributes without the use of antibodies or other cell surface affinity agents; this allows the device to isolate CTCs regardless of their immunophenotype, including CTCs undergoing epithelial-mesenchymal transition and other rare cell types.¹² The Parsortix device isolates the desired cells based on size and resistance to compression, with cells captured in a specialized separation cassette. The system’s design allows staining of cells while they are still inside the cassette (the captured cells can also be harvested into a buffered solution for evaluation).¹²

The Parsortix system was given a De Novo class II classification by the FDA for harvesting and subsequent analysis of cancer cells from the blood of patients with metastatic breast cancer.¹³ This classification offers a marketing pathway for novel medical devices that have shown reasonable evidence of safety and effectiveness for their intended use but do not have legally marketed predicate devices and was based on findings from the HOMING study (NCT03427450).¹⁴ Study findings showed that the Parsortix PC1 system was able to isolate CTCs from patients with metastatic breast cancer that could subsequently be evaluated using cytology, quantitative real-time polymerase chain reaction, RNA sequencing, and fluorescence in situ hybridization.¹³

Early research suggests that cell size–based methods such as the Parsortix system could also be an effective option for CTC isolation in cancers that do not have cell surface markers targeted by other commonly used systems.¹⁵ One study that compared 4 approaches for isolation of CTCs in clear cell renal cell carcinoma (RCC) lines and patient samples found that the Parsortix cell size–based system had numerically higher recovery rates than EpCAM and CD45-based approaches.¹⁵ EpCAM expression was identified in only 29% of clear cell RCC, which led the authors to conclude that a label-independent platform such as the Parsortix system may be more suitable for this and other cancer types with low or absent EpCAM expression.¹⁵

TriNetra

The TriNetra system uses label-free, nonmechanical methods to isolate and enrich viable apoptosis-resistant circulating tumor–associated cells and circulating ensembles of tumor-associated cells for analysis with immunohistochemistry.¹⁶ The system has been assessed in the case-control TRUEBLOOD trial (CTRI/2019/03/017918) and the subsequent blinded, prospective RESOLUTE study (CTRI/2019/01/017219). In an analysis of the complementary studies, blood samples from enrolled patients were enriched and then detected using the TriNetra system, which isolated breast adenocarcinoma–associated CTCs, which were then analyzed via profiling for GCDFP15, GATA3, EpCAM, pan-CK, and the absence of CD45 using multiplexed fluorescence immunocytochemistry.¹⁷

The system was found to be 100% specific and 92.07% sensitive for distinguishing patients with breast cancer from healthy controls in the case-control study, and findings from the prospective study showed a specificity of 93.1% and a sensitivity of 94.64% for distinguishing breast cancer (n = 112) from benign breast conditions (n = 29).¹⁶ In the case-control study, the system was also able to detect stage 0 (sensitivity, 70%) and stage I (sensitivity, 89.36%) breast cancer. TriNetra was granted a breakthrough device designation by the FDA for detection of early-stage breast cancer.¹⁸

In addition, the TriNetra-Prostate system, which immunostains isolated prostate adenocarcinoma–associated CTCs for pan-CK, prostate-specific membrane antigen, α-methyl-acyl coenzyme A racemase, EpCAM, and CD45 negativity, received breakthrough device designation in February 2022.^19,20 Like the breast cancer system, the TriNetra-Prostate system was validated by case-control and prospective studies.

The case-control study mixed VCaP prostate cancer cells into healthy donor blood and found a sensitivity of 100% (95% CI, 89.1%-100.0%) and a specificity of 100% (95% CI, 97.7%-100.0%) for distinguishing prostate cancer from noncancer samples; the prospective clinical study found a sensitivity of 91.2% (95% CI, 81.8%-96.7%) and a specificity of 100% (95% CI, 97.4%-100.0%) for discerning prostate cancer (n = 68) from benign prostate conditions (n = 142).²⁰ The test was also able to detect localized disease with a sensitivity of 75% (95% CI, 50.9%-91.3%), and the high specificity may make it a more effective and less invasive option for detecting prostate cancer and reduce the need for prostate biopsies.²⁰ This in turn may decrease the likelihood of overdiagnosis and overtreatment compared with traditional prostate-specific antigen–based approaches.²⁰

Lastly, the TriNetra-Glio blood test enriches for circulating glial cells (CGCs) in the peripheral blood and uses immunostaining to identify GFAP-positive, S100-/Nestin-positive, and CD45-negative cells; it was given FDA breakthrough device designation in 2023 for patients who require a brain biopsy but are unable to undergo the procedure or had a previously unsuccessful biopsy.^21,22

This analysis also leveraged findings from 2 case-controlled studies conducted by the device sponsor. The test had 98% sensitivity and 98% specificity for detecting glial malignancies and distinguishing them from benign central nervous system conditions, and it was also able to distinguish glial malignancies from healthy brain tissue and brain metastases.²³ In the prospective clinical study portion, the TriNetra-Glio system confirmed glial malignancies in all patients found to have CGCs (n = 56) and confirmed a benign central nervous system condition in all of those who did not have CGCs (n = 12).²³ The device developers also noted that brain biopsies are unable to be performed in up to 40% of advanced cases, so having a technique that is less invasive and requires fewer resources than a brain biopsy could be a helpful tool in the diagnosis of glial malignancies.²²

Challenges and Future Directions of CTCs

CTCs have promise in the diagnosis of cancer, prognostication of outcomes, and assessment of treatment response.1 Further work to improve sensitivity, specificity, and CTC yield and to address unanswered questions about the biology and clinical relevance of CTCs will be important to increase the utility of CTC assays in clinical practice.¹

Areas of future research may include the degree to which CTCs captured using current assays reflect the aggressive clones that contribute to metastases, the relative contribution of interactions between CTCs and noncancerous cells in the circulation to metastatic potential, and the ability to use CTCs to identify early relapses and targeted treatments that are most likely to yield a response.¹

References

1. Ring A, Nguyen-Sträuli BD, Wicki A, Aceto N. Biology, vulnerabilities and clinical applications of circulating tumour cells. Nat Rev Cancer. 2023;23(2):95-111. doi:10.1038/s41568-022-00536-4

2. Lin D, Shen L, Luo M, et al. Circulating tumor cells: biology and clinical significance. Signal Transduct Target Ther. 2021;6(1):404. doi:10.1038/s41392-021-00817-8

3. Lowes LE, Bratman SV, Dittamore R, et al. Circulating Tumor Cells (CTC) and Cell-Free DNA (cfDNA) Workshop 2016: Scientific Opportunities and Logistics for Cancer Clinical Trial Incorporation. Int J Mol Sci. 2016;17(9):1505. doi:10.3390/ijms17091505

4. Pantel K, Alix-Panabières C. Crucial roles of circulating tumor cells in the metastatic cascade and tumor immune escape: biology and clinical translation. J Immunother Cancer. 2022;10(12):e005615. doi:10.1136/jitc-2022-005615

5. Ng VY, Ang SN, Chan JX, Choo AB. Characterization of epithelial cell adhesion molecule as a surface marker on undifferentiated human embryonic stem cells. Stem Cells. 2010;28(1):29-35. doi:10.1002/stem.221

6. FDA clears CellSearch circulating tumor cell test. News release. Veridex LLC. February 27, 2008. Accessed July 26, 2023. bit.ly/3QePuFT

7. How does the CELLSEARCH System work? CellSearch. Accessed June 12, 2023. bit.ly/3OxFaYu

8. Andree KC, van Dalum G, Terstappen LW. Challenges in circulating tumor cell detection by the CellSearch system. Mol Oncol. 2016;10(3):395-407. doi:10.1016/j.molonc.2015.12.002

9. Bidard FC, Peeters DJ, Fehm T, et al. Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data. Lancet Oncol. 2014;15(4):406-414. doi:10.1016/S1470-2045(14)70069-5

10. Sparano J, O’Neill A, Alpaugh K, et al. Association of circulating tumor cells with late recurrence of estrogen receptor-positive breast cancer: a secondary analysis of a randomized clinical trial. JAMA Oncol. 2018;4(12):1700-1706. doi:10.1001/jamaoncol.2018.2574

11. Trapp E, Janni W, Schindlbeck C, et al; SUCCESS Study Group. Presence of circulating tumor cells in high-risk early breast cancer during follow-up and prognosis. J Natl Cancer Inst. 2019;111(4):380-387. doi:10.1093/jnci/djy152

12. Miller MC, Robinson PS, Wagner C, O'Shannessy DJ. The Parsortix Cell Separation System-a versatile liquid biopsy platform. Cytometry A. 2018;93(12):1234-1239. doi:10.1002/cyto.a.23571

13. Cohen EN, Jayachandran G, Moore RG, et al. A multi-center clinical study to harvest and characterize circulating tumor cells from patients with metastatic breast cancer using the Parsortix PC1 system. Cancers (Basel). 2022;14(21):5238. doi:10.3390/cancers14215238

14. Evaluation of automatic class III (De Novo) designation summaries. FDA. Updated June 11, 2023. Accessed June 12, 2023. bit.ly/477YId1

15. Maertens Y, Humberg V, Erlmeier F, et al. Comparison of isolation platforms for detection of circulating renal cell carcinoma cells. Oncotarget. 2017;8(50):87710-87717. doi:10.18632/oncotarget.21197

16. Akolkar D, Patil D, Crook T, et al. Circulating ensembles of tumor-associated cells: a redoubtable new systemic hallmark of cancer. Int J Cancer. 2020;146(12):3485-3494. doi:10.1002/ijc.32815

17. Crook T, Leonard R, Mokbel K, et al. Accurate screening for early-stage breast cancer by detection and profiling of circulating tumor cells. Cancers (Basel). 2022;14(14):3341. doi:10.3390/cancers14143341

18. FDA grants breakthrough designation for early-stage breast cancer detection blood test developed by Datar Cancer Genetics. News release. Datar Cancer Genetics Inc. November 19, 2021. Accessed June 12, 2023. bit.ly/44IjfDw 

19. Datar’s TriNetra-Prostate blood test gets FDA breakthrough designation. Inside Precision Medicine. February 14, 2022. Accessed June 12, 2023. bit.ly/3Ybjmop

20. Limaye S, Chowdhury S, Rohatgi N, et al. Accurate prostate cancer detection based on enrichment and characterization of prostate cancer specific circulating tumor cells. Cancer Med. 2023;12(8):9116-9127. doi:10.1002/cam4.5649

21. Gaya A, Crook T, Plowman N, et al. Evaluation of circulating tumor cell clusters for pan-cancer noninvasive diagnostic triaging. Cancer Cytopathol. 2021;129(3):226-238. doi:10.1002/cncy.22366

22. US FDA grants breakthrough designation for blood test to help diagnose inaccessible brain tumors. News release. Datar Cancer Genetics. January 2, 2023. Accessed June 12, 2023. bit.ly/3YciDmU

23. Anichini G, Fulmali P, O'Neill K, Datta V, Crook T, Syned N. PATH-24. accurate identification of glial malignancies from peripheral blood. Neuro Oncol. 2022;24(suppl 7):vii155. doi:10.1093/neuonc/noac209.597