Circulating tumor cell analysis: technical and statistical considerations for application to the clinic

Abstract

Solid cancers are a leading cause of death worldwide, primarily due to the failure of effective clinical detection and treatment of metastatic disease in distant sites. There is growing evidence that the presence of circulating tumor cells (CTCs) in the blood of cancer patients may be an important indicator of the potential for metastatic disease and poor prognosis. Technological advances have now facilitated the enumeration and characterization of CTCs using methods such as PCR, flow cytometry, image-based immunologic approaches, immunomagnetic techniques, and microchip technology. However, the rare nature of these cells requires that very sensitive and robust detection/enumeration methods be developed and validated in order to implement CTC analysis for widespread use in the clinic. This review will focus on the important technical and statistical considerations that must be taken into account when designing and implementing CTC assays, as well as the subsequent interpretation of these results for the purposes of clinical decision making.

Figure 1

Specific molecular features of CTCs can be analyzed via secondary phenotyping using the CellSearch system (Veridex) (a)–(c). Breast cancer CTCs in 7.5 mL of blood were processed on the CellTracks AutoPrep system using the CellSearch CTC kit and additional characterization antibodies against either (a) EGFR (Veridex), (b) CD44 (4 μg/mL; BD BioSciences), or (c) M30 (3.7 μg/mL; Alexis Biochemicals). Samples were then analyzed by the CellTracks Analyzer II. CTCs were identified and enumerated via positive staining for CK and DAPI (respective red and blue staining in left panels), negative staining for CD45 (not shown), and size and morphological characteristics. The additional FITC channel (green staining in right panels) was exploited for identification of molecular characteristics in individual CTCs. Expression of markers such as (a) EGFR and (b) the cancer stem cell marker CD44 may provide information regarding disease aggressiveness and/or indicate patient suitability for specific targeted therapies. Apoptosis markers such as (c) M30 (caspase-cleaved CK18) and the corresponding morphological characteristic of apoptotic membrane blebbing could provide information in patients on active treatment regarding efficacy of therapy and antitumor effects. The sample shown in (c) was exposed to palitaxel chemotherapy prior to CTC analysis.