Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer
| Autor: | Shuang Hou, Matthew M. Rochefort, Min Song, Carolyn Hsieh, Xinfang Liao, Zev A. Wainberg, Colin M. Court, James S. Tomlinson, Shonan Sho, Jacob S. Ankeny, Qingyu Li, Thomas G. Graeber, Hsian-Rong Tseng |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Genotype DNA Mutational Analysis Biology medicine.disease_cause Polymerase Chain Reaction Sensitivity and Specificity Pathology and Forensic Medicine Proto-Oncogene Proteins p21(ras) 03 medical and health sciences symbols.namesake 0302 clinical medicine Circulating tumor cell Single-cell analysis Pancreatic cancer Cell Line Tumor Microchip Analytical Procedures medicine Biomarkers Tumor Humans neoplasms Laser capture microdissection Sanger sequencing Reproducibility of Results Regular Article Sequence Analysis DNA medicine.disease Molecular diagnostics Neoplastic Cells Circulating digestive system diseases Pancreatic Neoplasms 030104 developmental biology Molecular Diagnostic Techniques 030220 oncology & carcinogenesis Cancer research symbols Molecular Medicine KRAS Single-Cell Analysis |
| Zdroj: | The Journal of molecular diagnostics : JMD. 18(5) |
| ISSN: | 1943-7811 |
| Popis: | To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs. |
| Databáze: | OpenAIRE |
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