There has been increased interest in utilizing non-invasive “liquid biopsies” to identify biomarkers for cancer prognosis and monitoring, and to isolate genetic material that can predict response to targeted therapies. Circulating tumor cells (CTCs) have emerged as such a biomarker providing both genetic and phenotypic information about tumor evolution, potentially from both primary and metastatic sites. Currently, available CTC isolation approaches, including immunoaffinity and size-based filtration, have focused on high capture efficiency but with lower purity and often long and manual sample preparation, which limits the use of captured CTCs for downstream analyses. Here, we describe the use of the microfluidic Vortex Chip for size-based isolation of CTCs from 22 patients with advanced prostate cancer and, from an enumeration study on 18 of these patients, find that we can capture CTCs with high purity (from 1.74 to 37.59%) and efficiency (from 1.88 to 93.75 CTCs/7.5 mL) in less than 1 h. Interestingly, more atypical large circulating cells were identified in five age-matched healthy donors (46–77 years old; 1.25–2.50 CTCs/7.5 mL) than in five healthy donors <30 years old (21–27 years old; 0.00 CTC/7.5 mL). Using a threshold calculated from the five age-matched healthy donors (3.37 CTCs/mL), we identified CTCs in 80% of the prostate cancer patients. We also found that a fraction of the cells collected (11.5%) did not express epithelial prostate markers (cytokeratin and/or prostate-specific antigen) and that some instead expressed markers of epithelial–mesenchymal transition, i.e., vimentin and N-cadherin. We also show that the purity and DNA yield of isolated cells is amenable to targeted amplification and next-generation sequencing, without whole genome amplification, identifying unique mutations in 10 of 15 samples and 0 of 4 healthy samples.

Treatment of advanced colorectal cancer (CRC) requires multimodal therapeutic approaches and need for monitoring tumor plasticity. Liquid biopsy biomarkers, including CTCs and ctDNA, hold promise for evaluating treatment response in real-time and guiding therapeutic modifications. From 15 patients with advanced CRC undergoing liver metastasectomy with curative intent, we collected 41 blood samples at different time points before and after surgery for CTC isolation and quantification using label-free Vortex technology. For mutational profiling, KRAS, BRAF, and PIK3CA hotspot mutations were analyzed in CTCs and ctDNA from 23 samples, nine matched liver metastases and three primary tumor samples. Mutational patterns were compared. 80% of patient blood samples were positive for CTCs, using a healthy baseline value as threshold (0.4 CTCs/mL), and 81.4% of captured cells were EpCAM+ CTCs. At least one mutation was detected in 78% of our blood samples. Among 23 matched CTC and ctDNA samples, we found a concordance of 78.2% for KRAS, 73.9% for BRAF and 91.3% for PIK3CA mutations. In several cases, CTCs exhibited a mutation that was not detected in ctDNA, and vice versa. Complementary assessment of both CTCs and ctDNA appears advantageous to assess dynamic tumor profiles.

Genomic characterization of circulating tumor cells (CTCs) may prove useful as a surrogate for conventional tissue biopsies. This is particularly important as studies have shown different mutational profiles between CTCs and ctDNA in some tumor subtypes. However, isolating rare CTCs from whole blood has significant hurdles. Very limited DNA quantities often can’t meet NGS requirements without whole genome amplification (WGA). Moreover, white blood cells (WBC) germline contamination may confound CTC somatic mutation analyses. Thus, a good CTC enrichment platform with an efficient WGA and NGS workflow are needed. Here, Vortex label-free CTC enrichment platform was used to capture CTCs. DNA extraction was optimized, WGA evaluated and targeted NGS tested. We used metastatic colorectal cancer (CRC) as the clinical target, HCT116 as the corresponding cell line, GenomePlex® and REPLI-g as the WGA methods, GeneRead DNAseq Human CRC Panel as the 38 gene panel. The workflow was further validated on metastatic CRC patient samples, assaying both tumor and CTCs. WBCs from the same patients were included to eliminate germline contaminations. The described workflow performed well on samples with sufficient DNA, but showed bias for rare cells with limited DNA input. REPLI-g provided an unbiased amplification on fresh rare cells, enabling an accurate variant calling using the targeted NGS. Somatic variants were detected in patient CTCs and not found in age matched healthy donors. This demonstrates the feasibility of a simple workflow for clinically relevant monitoring of tumor genetics in real time and over the course of a patient’s therapy using CTCs.

Combined Workflow EGFR in CTCs and cfDNA tissues. Despite the limited size of the patient cohort, results from this non-invasive EGFR mutation analysis are encouraging and this combined workflow represents a valuable means for informing therapy selection and for monitoring treatment of patients with NSCLC.

Background: Lung cancer is the leading cause of cancer-related mortality worldwide. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutation, have shown dramatic clinical benefits. EGFR mutation assays are mainly performed on tumor biopsies, which carry risks and expense and are not always successful. In order to identify the development of secondary EGFR mutations, which cause resistance to 1st and 2nd generation TKI’s and an indication for therapy with a 3rd generation drug, effective and non-invasive monitoring is needed. Liquid biopsy biomarkers, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), allow such monitoring over the course of the therapy. Interestingly, ctDNA or CTC analysis alone had less sensitivity vs. combining both, with a genotyping of 70% and 80% for CTCs and ctDNA respectively, but 100% when combined2. Vortex technology is a platform enabling label-free capture of CTCs from blood samples and genomic assays downstream3. The aim of this study is to demonstrate the sensitivity of a combined CTC and ctDNA assay through Vortex using blood samples spiked with molecularly-characterized lung cancer cell lines and then to apply this technique to matched blood and tumor samples from NSCLC patients. Method: Lung cancer cell lines with different EGFR mutations (A549: wild type, H1975: L858R+ and T790M+, HCC827: 19del+) were used to validate our CTC workflow. Blood samples and matched tumor tissues were collected from NSCLC patients. Plasma was extracted first for ctDNA assay. CTCs were isolated from the plasma-depleted-blood using Vortex technology, immunostained (CK, Vimentin, CD45) and enumerated. DNA from CTCs, plasma and matched tumor tissue was analyzed for EGFR mutations 19del, L858R and T790M using the ctEGFR kit from EntroGen. Results: Mutant DNA could be identified at a quantity as low as 0.5 ng (~83 cells), with a sensitivity ranging from 0.1% to 2% for a total DNA varying from 25ng (~4 CTCs among 4000 WBCs) to 1ng (~4 CTCs among 200 WBCs). We demonstrated the ability of Vortex technology to enrich CTCs from metastatic NSCLC patients. Processing of plasma-depleted-blood showed the same capture efficiency when compared to whole blood. This makes possible the detection of EGFR mutations on CTC samples collected by Vortex technology. &gt; 10 NSCLC patients are being enrolled in this study and results will be presented at AACR. Conclusion: The ctEGFR mutation assay performed well on both Vortex-enriched CTCs and ctDNA, enabling a low cost approach to analyze EGFR mutation from a single blood tube. This non-invasive EGFR mutation analysis will be potentially a useful tool for monitoring treatment and medication guidance of NSCLC patients. 1. Calabuig-Fariñas et al. Transl Lung Cancer Res. 2016. 2. Sundaresan TK et al. Clin Cancer Res. 2016. 3. Kidess-Sigal E et al. Oncotarget 2016. Citation Format: Haiyan E. Liu, Meghah Vuppalapaty, Clementine A. Lemaire, Charles Wilkerson, Steve C. Crouse, Jonathan W. Goldman, Elodie Sollier-Christen. EGFR mutational detection in ctDNA, Vortex-enriched CTCs and comparison to tumor tissue in non-small-cell-lung-cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1715. doi:10.1158/1538-7445.AM2017-1715