Here, we offer a proof-of-concept that ROS1 rearrangement can be detected in CTCs from ROS1-rearranged NSCLC patients using filter-adapted-fluorescent in-situ hybridization (FA-FISH), a FISH assay optimized for CTC analysis on filters [ 18].
We did not perform CTC analysis in the current study because no standardized method for CTC screening had been established when samples were taken 6 to 10 years ago.
To evaluate the extent of cell loss that occurs during loading of a DEPArray cartridge and sample injection into the main microchamber, counting experiments were performed using CellSearch CTC control samples (CellSearch CTC control kit, Veridex LLC), processed with the CellSearch CTC kit as an internal quality control for routine CellSearch CTC analysis in our lab.
The same IHC protocol and antibodies used for ISET CTC analysis was adopted to determine tumour EpCAM and CK expression in tumour samples.
CTC analysis has the unique potential to offer a minimally invasive 'liquid biopsy' sample, easily obtainable at multiple time points during disease history, thus providing valuable information on the very early assessment of treatment efficacy and helping towards establishing individualised treatment approaches that will improve efficacy with less cost and side effects for BC patients.
SJ performed the CellSearch® CTC analysis.
CTC enumeration and analysis on the microfluidic CTC-chips were run at two reference labs that manufacture their own CTC-chip platforms: OncoCEE microchannel (Biocept Inc, San Diego, CA) and On-Q-ity's CTC-Chip (On-Q-ity, Boston MA and previously CELLective Dx, Menlo Park, CA).
As we did not apply subclone analysis on primary tumor samples, we cannot exclude the existence of CTC-associated mutations that are detected in a minority of primary tumor cells, as it has been suggested by recent sequencing studies of primary tumors revealing molecular heterogeneity of different subclones within individual tumors (Gerlinger et al, 2012).
The Phoenix is capable of performing the same chemical analysis on three more samples.
Several new methods have been developed to count the number of CTCs in blood, and to perform limited marker analysis on isolated CTCs using immunohistochemistry and FISH.
Methods for the analysis of CTCs in clinical samples should be designed to detect both epithelial and mesenchymal-like subpopulations.
