Comparative performance of different methods for circulating tumor cell enrichment in metastatic breast cancer patients

Abstract

The isolation and analysis of circulating tumor cells (CTC) has the potential to provide minimally invasive diagnostic, prognostic and predictive information. Widespread clinical implementation of CTC analysis has been hampered by a lack of comparative investigation between different analytic methodologies in clinically relevant settings. The objective of this study was to evaluate four different CTC isolation techniques-those that rely on surface antigen expression (EpCAM or CD45 using DynaBeads® or EasySep™ systems) or the biophysical properties (RosetteSep™ or ScreenCell®) of CTCs. These were evaluated using cultured cells in order to calculate isolation efficiency at various levels including; inter-assay and inter-operator variability, protocol complexity and turn-around time. All four techniques were adequate at levels above 100 cells/mL which is commonly used for the evaluation of new isolation techniques. Only the RosetteSep™ and ScreenCell® techniques were found to provide adequate sensitivity at a level of 10 cells/mL. These techniques were then applied to the isolation and analysis of circulating tumor cells blood drawn from metastatic breast cancer patients where CTCs were detected in 54% (15/28) of MBC patients using the RosetteSep™ and 75% (6/8) with ScreenCell®. Overall, the ScreenCell® method had better sensitivity.

Fig 1. Summary of methodologies used for CTC enrichment.

Cultured cells were spiked into whole blood to test the recovery efficiency using the different methodologies. (A) The Dynabeads^® and EasySep™ methods rely on the cell surface antigen expression were respectively used first to perform a negative depletion followed by CTC enrichment. (B) The RosetteSep™ and ScreenCell^® Cyto filters were used to evaluate the CTC capture efficiency based on biophysical properties of the tumor cell. (C) The two methodologies, RosetteSep™ and ScreenCell^®, were further used on patient blood samples for CTC enrichment. Four patients had multiple sampling during the course of disease progression. Six patients were isolated with both methodologies in a head-to-head comparison.

Fig 2. Comparison of the various methods used for CTC isolation.

(A) Combined immunomagnetic isolation efficiency using Dynabeads^® and EasySep™ compared to RosetteSep™ at 1000 cells, p = 0.004 between Dynabeads^® and RosetteSep™ (B) The isolation efficiency of RosetteSep™ at 1000, 100, 10 cells and further enrichment with EasySep™ EpCAM at 1000 cells. (C) The data comparison of non-immunomagnetic isolations using RosetteSep™ and ScreenCell^® filters at 100 cells, p = 0.18.

Fig 3. Representative images of controls using spiked MDA-MB-231 cells into whole blood after CTC enrichment and immunocytochemistry.

Top panel: Cytospin preparations from CTC enrichment using the RosetteSep™ method. One hundred MDA-MD-231 cells were spiked into 5 mL of whole blood. Positive cells for cytokeratins are coloured green (white arrow), leukocyte positive for CD45 are coloured red (dashed arrow) and the nuclei stained by DAPI are shown in blue. Images were captured under 200X magnification. Bottom panel: CTC enrichment by ScreenCell^® Cyto filters. One hundred cultured cells were spiked into 3 mL of whole blood. Cells that are CD45^-, pan-CK/CK-7⁺ and with an intact nucleus are considered positive. Filter pores are visually distinct and a CTC caught atop a filter pore is indicated by the red arrow. A leukocyte caught in the filter pore is shown with an opened white arrow. Images were captured under 400X magnification. Scale bar represents 20 μm. RS: RosetteSep™; SC: ScreenCell^®.

Fig 4. Representative images of CTC enrichment and ICCs from four metastatic breast cancer patients.

CTCs were isolated using either the RosetteSep™ (RS) or ScreenCell^® (SC) or with both. Numbers of the left denotes patient ID. CTCs and circulating tumor clusters showing pan-CK/CK-7⁺ (green; white arrow) with an intact nucleus (DAPI⁺, in blue) are CD45^-. Leukocytes are CD45⁺ (dashed arrow) with no CK staining. The arrow head points to un-lysed residual red blood cells from the RosetteSep™ isolation. The red arrow points to a CTCs caught atop a filter pore (8 μm). Images were captured under 400X magnification. Scale bar represents 20 μm. RS: RosetteSep™; SC: ScreenCell^®.

Fig 5. CTC isolations from metastatic breast cancer patients.

(A) Isolation of CTCs by RosetteSep™ and ScreenCell^® methods of all patient samples (p = 0.29). (B) number of CTCs isolated by both the RosetteSep™ and ScreenCell^® methods from 6 metastatic breast cancer patients (p = 0.13).

Fig 6. Patient 38 with circulating tumour clusters isolated by the ScreenCell^® Cyto kit.

ICCs were performed using anti-pan-CK/CK-7/vim and CD45. Cell nuclei are counterstained with DAPI. Circulating tumor clusters (bold arrow) showing pan-CK/CK-7⁺/vim⁺ but CD45^-. CD45⁺ leukocytes are sometimes CK⁺. Cells caught in the filter pores (red arrow). Images were captured under 400X magnification. Scale bar represents 20 μm.

Fig 7. Serial CTC and CT scan monitoring during systemic treatment.

Coloured boxes indicate the duration of therapy. CTC detection may be used to indicate disease progression. CT: computed tomography.