CTCs include those with stem-cell or stem cell–like properties or any degree of differentiation. They may originate from the primary tumor or metastatic sites and are estimated to represent at most one cell in 1 billion of the circulating cells in the blood.49
Presently, there are a range of assays and devices in various stages of development and clinical testing. These approaches generally include an enrichment step followed by different detection and characterization methods.49,50
Enrichment is achieved using the following methods: first, morphologic or physical methods; second, positive selection with antibodies to cell surface markers on CTCs; or third, negative selection with antibodies to the common leukocyte antigen CD45 to deplete mononuclear cells or those that eliminate RBCs and mononuclear cells. Detection and characterization methods use tumor- or tissue-specific markers or various cytometric methods.49,50
Importantly, there is no standardized definition of a CTC and no single feature that distinguishes CTCs from normal, nonmalignant blood elements. Consequently, different assays do not measure the same malignant cell population, and as such, each reports a different biomarker, not all of which are useful in a given context.
Our interest in CTCs in prostate cancer is based on promising early results showing that the presence of mRNA for PSA in the mononuclear cell fraction by real-time polymerase chain reaction could provide information distinct from PSA51
and serve as a non–PSA-based outcome measure.52
Presently, CellSearch (Veridex) is the only CTC assay cleared by the FDA for use in the clinic.7
With this assay, a CTC is defined as a cell that is morphologically intact; has a nucleus surrounded by cytoplasm when stained with 4′,6-diamidino-2-phenylindole; and expresses cytokeratin 8, 18, or 19 but does not express the mononuclear cell determinant CD-45.53,54
Assay results are reported as the number of cells meeting the analytically valid definition per 7.5 mL of blood, which represents only a proportion of cytokeratin-positive events.55
The cells isolated are confirmed to have molecular features of prostate cancer.54
CTC counts trend toward higher values in relation to PSA level and extent of disease in bone, but the association is modest, suggesting that each parameter provides independent information.56
Clearance for use was obtained first for breast followed by colorectal and later prostate cancers based on trials of similar design that enrolled patients who were about to start a new line of chemotherapy57–59
and that showed baseline CTC number was prognostic for survival in univariate and multivariate analyses before treatment and a range of times post-treatment including, 2 to 5, 6 to 8, 9 to 11, and 13 to 20 weeks. For prostate cancer, a cutoff of four or fewer cells per 7.5 mL of blood was considered favorable and five or more cells per 7.5 mL of blood unfavorable. At all time points evaluated, the post-therapy CTC number was more predictive than a decline of 50% or more in PSA.58
In separate analyses of CTC number as a continuous variable,60
it is noteworthy that a low count alone did not assure a long survival.57–59,61,62
In a multivariate analysis, only CTC count and lactate dehydrogenase were prognostic; PSA was no longer predictive.63
The FDA clearance states that “presence of CTC in the peripheral blood, as detected by the CellSearch Circulating Tumor Cell Kit, is associated with decreased progression free survival and decreased overall survival in patients treated for metastatic breast, colorectal or prostate cancer … Serial testing for CTC should be used in conjunction with other clinical methods for monitoring [these cancers].”7(p3)
It has not been shown to be a surrogate for survival, nor can it replace other methods of assessing disease. It should be noted that many investigators are not yet using CTC enumeration testing in day-to-day practice; they are waiting until more data and more standardized methods of measuring CTCs become available.
One way to use CTC counts is to obtain a baseline sample before starting a new therapy. In our experience, the proportion of patients with unfavorable counts has been approximately 25% in the prechemotherapy population, 50% in patients receiving first-line chemotherapy, and 70% in those receiving second-line chemotherapy (Danila et al, unpublished data). If the counts are unfavorable at baseline, the approach of our group is to serially monitor them after each cycle of therapy. Conversions from unfavorable to favorable tend to occur early, and in one study, they were maximal at 8 weeks.63
Patients in whom CTC counts do not decline tend to do poorly. Trials are ongoing in breast cancer to address whether an early change in therapy for patients who do not show a CTC conversion will affect outcome. If the count is favorable after two baseline determinations, we repeat the assessment at the time of progression.
At this point, it is too early to rely solely on CTC enumeration results to guide patient management. Going forward, use of the CTC assay in specific contexts will require further qualification. These efforts are ongoing through a formal collaboration with the Center for Devices and Radiologic Health branch of the FDA. CTC enumeration was studied in the phase I/II trial of abiraterone, with favorable conversion rates.10,64
The trial subsequently showed a significant survival benefit, and analyses are ongoing to define a biomarker panel including CTCs that is prognostic for survival after treatment and which explains a sufficient portion of the survival benefit observed. Once determined, the panel will be prospectively studied in a phase III trial of similar design, with the CTC end point embedded, as it is in trials of MDV3100 (AFFIRM; NCT00974311), TAK-700 (NCT01193257), and ipilimumab (NCT01057810). There is also an ongoing effort to qualify CTCs as a surrogate outcome measure.
It is hoped that in the future, CTCs will provide a noninvasive, real-time liquid biopsy for just-in-time disease characterization to guide treatment selection. Several new technologies are in development that could detect cells in more patients and more cells overall with greater purity. Techniques include fluorescence-activated cell sorting,67
although further analytic validation will be required before they can be considered for clearance or qualification.49,50,73
The specific assay needed will depend on the context of use and the biologic determinant being measured.