Abstract 750: Detection of mutations in single circulating tumor cells using MALDI-TOF mass spectrometry
| Autor: | Melinda Duplessis, Eric Kaldjian, Ryan T. Birse, Jackie L. Stilwell, Arturo Ramirez, Brennan Enright, Darryl Irwin |
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| Rok vydání: | 2017 |
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| Zdroj: | Cancer Research. 77:750-750 |
| ISSN: | 1538-7445 0008-5472 |
| Popis: | Background: As cancer therapeutics are increasingly selected based upon molecular genetic information, having reliable, accurate, rapid and inexpensive methods for mutational analysis are extremely desirable. Circulating tumor cells (CTCs) allow non-invasive “liquid biopsy” access to intact cells for molecular analysis. Here we demonstrate the successful detection of mutations in model CTCs individually isolated from blood (AccuCyte® - CyteFinder® system, RareCyte) using MALDI-TOF Mass Spectrometry (MassARRAY, Agena Bioscience). Methods: Breast (MDA-MD-231) and lung (NCI-H1975) cancer cells with a set of known mutations were spiked into blood and processed by AccuCyte onto microscope slides and stained on an automated immunostainer. Slides were imaged using the CyteFinder digital fluorescence scanning microscope and mCTCs were identified by positive nuclear, EpCAM, and cytokeratin staining, and negative CD45 staining. mCTCs and white blood cell (WBC) negative controls were picked from the slides and put into PCR tubes using the CytePicker® module. DNA from individual or small pools of cells (3-5) was amplified using the PicoPLEX® (Rubicon) whole genome amplification (WGA) kit; alternatively cells were lysed and directly entered into the ensuing iPLEX® Pro workflow. Specific regions surrounding 5 different mutations in each of the mCTC lines were amplified from the WGA product or the lysed cells and the products were detected and scored for the mutations using a single PCR reaction iPLEX® Pro panel that includes a combination of 10 common lung and breast cancer mutations using the MassARRAY® platform. Results: Five point mutations in four different genes (CDKN2A, EGFR, PIK3CA, and TP3) were measured in the NCI-H1975 lines and four point mutations in four genes (BRAF, KRAS, NF2, and TP53) and a deletion in one gene (CDKN2A) were measured in the MDA-MB-231 cells by iPLEX® Pro chemistry on the MassARRAY® system. All mutations were accurately detected in the WGA single and pooled cell samples and most were also detected in cells that did not undergo WGA before PCR with with iPLEX Pro panel. Allelic frequency observed was consistent with known zygosity of the mutation. Conclusions: MassARRAY successfully detected mutations in single model CTCs that were individually picked from a blood sample processed by the AccuCyte - CyteFinder system. Integrating CTC isolation with MassARRAY may be a practical way to identify and monitor known cancer mutations non-invasively. Citation Format: Jackie L. Stilwell, Ryan T. Birse, Arturo Ramirez, Melinda Duplessis, Brennan Enright, Darryl Irwin, Eric Kaldjian. Detection of mutations in single circulating tumor cells using MALDI-TOF mass spectrometry [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 750. doi:10.1158/1538-7445.AM2017-750 |
| Databáze: | OpenAIRE |
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