Tumor tissue biopsies are invasive, costly, and collect a limited cell population not completely reflective of patient cancer cell diversity. Circulating tumor cells (CTCs) can be isolated from a simple blood draw and may be representative of the diverse biology from multiple tumor sites. The VTX-1 Liquid Biopsy System was designed to automate the isolation of clinically relevant CTC populations, making the CTCs available for easy analysis. We present here the transition from a cutting-edge microfluidic innovation in the lab to a commercial, automated system for isolating CTCs directly from whole blood. As the technology evolved into a commercial system, flexible polydimethylsiloxane microfluidic chips were replaced by rigid poly(methyl methacrylate) chips for a 2.2-fold increase in cell recovery. Automating the fluidic processing with the VTX-1 further improved cancer cell recovery by nearly 1.4-fold, with a 2.8-fold decrease in contaminating white blood cells and overall improved reproducibility. Two isolation protocols were optimized that favor either the cancer cell recovery (up to 71.6% recovery) or sample purity (≤100 white blood cells/mL). The VTX-1's performance was further tested with three different spiked breast or lung cancer cell lines, with 69.0% to 79.5% cell recovery. Finally, several cancer research applications are presented using the commercial VTX-1 system.
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:The integrin binding-defective mutant of IGF1 (R36E/R37E) is functionally defective and does not induce ternary complex formation (integrin-IGF1-IGF1R). Results: R36E/R37E suppressed signaling induced by WT IGF1, the binding of WT IGF1 to cells, ternary complex formation, cell viability, and tumorigenesis. Conclusion: R36E/R37E is a dominant-negative antagonist of IGF signaling. Significance: R36E/R37E has potential as a therapeutic agent.
This study investigated the delayed circulating leptin response to maximal and prolonged treadmill exercise. Six healthy untrained males performed three sessions after an overnight fast: control, maximal exercise, and prolonged exercise at 50% of maximal oxygen consumption. Blood samples were collected prior to exercise, at the end of exercise, and at 60, 120, 180, and 240 min following exercise and control sessions. Blood samples were analyzed for serum leptin, insulin, glucose, free fatty acids, and glycerol. Hemoglobin and hematocrit were measured to correct for plasma volume changes. Resting energy expenditure (REE) and body fat (BF) were also assessed. Immediately at the end of maximal and prolonged exercise, and during the 4 hours of recovery, serum leptin levels did not change significantly compared to their respective baseline values. At 240 min of recovery serum leptin decreased 7% and 9% (p>0.05) from the baseline in the maximal and prolonged sessions, respectively. In the control experiment serum leptin decreased 27% from the baseline at 240 min of the recovery (p < 0.05). No significant differences were found in leptin values between the control and exercise sessions. Control serum leptin was positively correlated (p < 0.05) to BF (r = 0.88) and glucose (r=0.96), and negatively correlated to REE (r= -0.81). In conclusion, maximal or prolonged exercise do not appear to have an influence on circulating serum leptin in the delayed (4 hr) post exercise recovery period.
Background Lung cancer is the leading cause of cancer-related mortality worldwide and 85% cases are NSCLC. Epidermal growth factor receptor (EGFR) mutations occur in 10-30% of NSCLC patients1. EGFR tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutation, have shown dramatic clinical benefits. EGFR assays are mainly performed on tumor biopsies, which carry risks and expense and are not always successful1. 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 biopsies containing circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), allow such monitoring over the course of the therapy2. The Vortex Biosciences' VTX-1 Liquid Biopsy System enables the label-free capture of CTCs from blood samples, with high CTC recovery, purity and compatibility with downstream genomic assays on CTCs and plasma3,4. Our combined CTC and ctDNA EGFR assay was applied to blood samples from NSCLC patients. Method ctEGFR kit (EntroGen) was selected and validated for the detection of EGFR major mutations in a single PCR reaction, for both ctDNA and CTCs. Several blood collection tubes (BCT: EDTA, CellSave, LBGard and Streck) were tested using spiked cells, considering CTC recovery, DNA yield, and EGFR profiling. 20 blood samples were studied from 15 NSCLC patients. Plasma was extracted for ctDNA assay. CTCs were isolated from the plasma-depleted-blood using the VTX-1, immunostained and enumerated5. EGFR mutations were then detected in CTC+ctDNA, and compared to the tissue results. Results The VTX-1 provided a similar CTC recovery from plasma-depleted-blood and whole blood, enabling CTC+ctDNA EGFR profiling from the same tube of blood. Among the BCTs tested, LBGard obtained the best CTC capture and EGFR mutation detection performance. 10/15 patients showed the same mutations between tissue and CTC+ctDNA. For one patient with two blood draws without mutation in tissue, no mutation was detected in the first draw, while a T790M mutation was identified 6 months later. For another patient, an Exon19 deletion was detected in the ctDNA+CTC but not in the tissue, and a repeat draw confirmed the result. 4/15 patients had mutations in tissue that were missed in CTC+ctDNA. Conclusion Performing EGFR mutation analysis on the combination of ctDNA and VTX-1-collected CTCs may offer an improved sensitivity of detection over analysis of only the ctDNA, and 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. [4] Haiyan E L et al. npj Genomic Medicine (2017) 2:34. [5] Che J. et al., Oncotarget (2016) Citation Format: Elodie Sollier-Christen, Haiyan E. Liu, Meghah Vuppalapaty, Michael Chiu, James Che, Charles Wilkerson, Nasim Barzanian, Steve Crouse, James Carroll, Melissa Matsumoto, Edward B. Garon, Jonathan W. Goldman. EGFR mutational detection in vortex-enriched CTCs, ctDNA, 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 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1598.
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