Q: Liquid biopsy is “all the rage” in the cancer diagnostic space these days. You have worked with Circulating Tumor Cells (CTCs) for quite some time. Beyond R and D, is there a practical clinical usefulness for CTC at this time, what is it and how does one go about using it well?
A: Yes, I believe there are several important practical clinical applications for CTCs beyond R&D, including the early diagnosis of lethal cancers, such as lung cancer. If discovered at an early stage in the disease and treated appropriately with video-assisted transthoracic resection or stereotactic body radiation therapy (SBRT), the early diagnosis of lung cancer would result in a much improved survival rate. If there is a sensitive and specific CTC test for lung cancer, that can be performed as an adjunct to an indeterminate nodule on spiral CT scan or as a primary screening test in high risk individuals and that can be validated and replicated in laboratories worldwide, it would have a major impact on the devastating morbidity and mortality that lung cancer currently wreaks.
Unfortunately, while CTCs are a subject of intense interest to the scientific community, and a current avid pursuit of many researchers, with thousands of publications appearing in the last few years, there is still a state of confusion regarding their reliability as a biomarker for the presence of early cancers, as well as to what constitutes the best platform for the isolation and enumeration of CTCs. Numerous techniques to detect CTCs have been described ranging from different EpCAM capture antibody methods via magnetic beads, with subsequent positive staining for Cytokeratin’s (CK8,CK18, CK19) and negative staining for CD45, to filter methods that rely on isolation of CTCs by size and morphology. Other methods include negative selection of CTCs by depletion of leukocytes from the blood stream. All the aforementioned methods have their pros and cons, with lack of sensitivity being the most prominent disadvantage, as early in dissemination CTCs adopt an EMT phenotype and most cells will escape detection via EPCAM. At MD Anderson Cancer Center, our lab has devised a sensitive and specific CTC test for lung cancer, that relies on a gradient enrichment step, followed by a multi-probe DNA FISH test that has been designed to detect genetic aberrations common in lung cancers irrespective of histologic subtype, including small cell and non-small cell lung cancers. The objective of CTC detection is for an unambiguous test that is specific for rare CTC detection, without inadvertent detection of accompanying background lymphocytes and mononuclear cells. Using a method known as FICTION, in which we perform dual staining for combinations of cytokeratin, SNAIL1, ALDH1 and CD45 followed by FISH for multiple different DNA probes, we have shown that genetically abnormal cells undergo a constant phenotypic evolution or lineage plasticity throughout different time points after tumor resection. In addition there is also genetic heterogeneity amongst the CTCs. Therefore to overcome this phenotypic variance, we have focused on an antigen independent or label-free assay, in which only aneuploid cells with gains of extra genetic material can be scored as CTCs. We have defined a threshold of CTCs, above which a sample can be called positive, and in this way we have been able to detect needle biopsy confirmed lung cancer < 1 cm on spiral CTC scan We also demonstrate that CTCs disappear over time in patients undergoing removal of their tumors either by surgery or by SBRT.
Other important applications involve detecting CTCs that carry ligands which can be targeted by specific antibodies that are conjugated to deliver a lethal dose of the conjugate. For example, in small cell lung cancer (SCLC) an antibody drug- conjugate targeted to a novel protein called delta-like protein 3 (DLL3) (Rovalpituzumab tesirine (Rova-T),Stemcentrx) may bind selectively to the CTCs of SCLC and deliver a drug that kills these cells, while not affecting the normal blood cells. Although not yet approved as a companion diagnostic for CTCs, it would seem that the next logical step for exploiting the presence CTCs would be making drugs of this class that would be effective in fighting both small cell and non-small cell lung cancer. This would take CTCs into the realm of application.
In summation, I would say that CTCs may imminently be used in practical application both in the early detection of lethal cancers as well as the creation of drugs targeting CTCs themselves. There is still work to be done, and we should bear in mind that in order to be acceptable to laboratory professionals, as well as useful to the general public, tests for CTCs for lung cancer should be fairly easy to perform and replicate, and should be validated in a CLIA approved laboratory. We are on the cusp of an exciting new phase in oncology and precision medicine.