Molecular profiling of single circulating tumor cells with diagnostic intention

Abstract

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA)

“…In the WGA-treated cell, no restriction sites were found in the other three undetected variants. The reason for these missing amplifications as observed in other studies, may be related to the procedural difficulties in the use of the kit, or to the WGA procedure itself (29)(30)(31). Conversely, using our methodological approach, in WGA-untreated CTCs we identified the same 10 variants as in the SK-MEL-28 pellet.…”

“…This technique is based on the DNA cleavage of the TTAA MseI restriction-site motifs (http://www.siliconbiosystems.com/feefor-service) and, in addition to potential drawbacks mentioned above, there is the inconvenient of failure to generate amplicons suitable for subsequent sequencing in case of DNA tracts carrying this motif (19,24,29). For this reason, we planned an experimental design carried out to avoid WGA step in NGS protocol for CTCs.…”

Circulating tumor cell technologies

“…(Carpenter et al, 2014;Fabbri et al, 2013;Fernandez et al, 2014;Manaresi et al, 2003;Peeters et al, 2013;Polzer et al, 2014) Biophysical e Acoustophoresis Separates cells based on acoustophoretic mobility, which is size dependent, by exposing them to acoustic waves.…”

“…DEPArrayä is designed for single-cell recovery and not bulk enrichment of CTCs. Multiple clinical studies have used DEPArrayä to recover single CTCs for subsequent genetic analyses following enrichment using centrifugation or immunoaffinity (i.e., CellSearch) (Carpenter et al, 2014;Fabbri et al, 2013;Fernandez et al, 2014;Peeters et al, 2013;Polzer et al, 2014). The use of DEPArrayä technology for CTC recovery will likely be limited to samples with a relatively high number of CTCs (e.g., metastatic carcinomas) due to a cell-loss of approximately 40% during sample loading (Peeters et al, 2013).…”

Molecular analysis of single circulating tumour cells following long‐term storage of clinical samples

“…It has been observed that the number of bands amplified (0–4) is associated with the integrity of the starting material, with cells with good quality DNA presenting 3 or 4 PCR bands, while cells with degraded DNA present fewer bands (Polzer et al ., 2014). For the mean GII values, the GII value for each individual single cell was determined and used to determine the mean for the entire sample.…”