Rui P L Neves1, Katharina Raba2, Oliver Schmidt3, Ellen Honisch4, Franziska Meier-Stiegen4, Bianca Behrens1, Birte Möhlendick1, Tanja Fehm4, Hans Neubauer4, Christoph A Klein5, Bernhard Polzer6, Christoph Sproll7, Johannes C Fischer2, Dieter Niederacher4, Nikolas H Stoecklein8. 1. Department of General, Visceral and Pediatric Surgery. 2. Institute for Transplantation Diagnostics and Cell Therapeutics. 3. Chair of Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany; 4. Department of Obstetrics and Gynecology, and. 5. Chair of Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany; Fraunhofer Institute for Toxicology and Experimental Medicine, Regensburg, Germany. 6. Fraunhofer Institute for Toxicology and Experimental Medicine, Regensburg, Germany. 7. Department of Maxillo- and Facial Plastic Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany; 8. Department of General, Visceral and Pediatric Surgery, nikolas.stoecklein@uni-duesseldorf.de.
Abstract
BACKGROUND: Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level. METHODS: Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene. RESULTS: One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene. CONCLUSIONS: This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.
BACKGROUND: Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level. METHODS: Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene. RESULTS: One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene. CONCLUSIONS: This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.
Authors: C F Pixberg; K Raba; F Müller; B Behrens; E Honisch; D Niederacher; H Neubauer; T Fehm; W Goering; W A Schulz; P Flohr; G Boysen; M Lambros; J S De Bono; W T Knoefel; C Sproll; N H Stoecklein; R P L Neves Journal: Oncogene Date: 2017-01-09 Impact factor: 9.867
Authors: Lukasz A Adamczyk; Hannah Williams; Aleksandra Frankow; Hayley Patricia Ellis; Harry R Haynes; Claire Perks; Jeff M P Holly; Kathreena M Kurian Journal: Front Neurol Date: 2015-08-10 Impact factor: 4.003