title: Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer.
abstract: 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.
authors.affiliation: Department of Surgery, University of California Los Angeles, Los Angeles, California; Department of Surgery, Greater Los Angeles Veterans Affairs Administration, Los Angeles, California; Department of Molecular, Cellular, and Integrative Physiology, University of California Los Angeles, Los Angeles, California.
authors.forename: Colin M
authors.initials: CM
authors.lastname: Court
authors.affiliation: Department of Surgery, University of California Los Angeles, Los Angeles, California; Department of Surgery, Greater Los Angeles Veterans Affairs Administration, Los Angeles, California.
authors.forename: Jacob S
authors.initials: JS
authors.lastname: Ankeny
authors.affiliation: Department of Surgery, University of California Los Angeles, Los Angeles, California; Department of Surgery, Greater Los Angeles Veterans Affairs Administration, Los Angeles, California.
authors.forename: Shonan
authors.initials: S
authors.lastname: Sho
authors.affiliation: Department of Surgery, University of California Los Angeles, Los Angeles, California; Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California.
authors.forename: Shuang
authors.initials: S
authors.lastname: Hou
authors.affiliation: Department of Surgery, University of California Los Angeles, Los Angeles, California.
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Surgery, University of California Los Angeles, Los Angeles, California.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Hsieh
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California.
authors.forename: Min
authors.initials: M
authors.lastname: Song
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California.
authors.forename: Xinfang
authors.initials: X
authors.lastname: Liao
authors.affiliation: Department of Surgery, University of California Los Angeles, Los Angeles, California; Department of Surgery, Greater Los Angeles Veterans Affairs Administration, Los Angeles, California.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Rochefort
authors.affiliation: Center for Pancreatic Disease, University of California Los Angeles, Los Angeles, California.
authors.forename: Zev A
authors.initials: ZA
authors.lastname: Wainberg
authors.affiliation: Department of Molecular, Cellular, and Integrative Physiology, University of California Los Angeles, Los Angeles, California; Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Graeber
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California.
authors.forename: Hsian-Rong
authors.initials: HR
authors.lastname: Tseng
authors.affiliation: Department of Surgery, University of California Los Angeles, Los Angeles, California; Department of Surgery, Greater Los Angeles Veterans Affairs Administration, Los Angeles, California; Center for Pancreatic Disease, University of California Los Angeles, Los Angeles, California. Electronic address: jtomlinson@mednet.ucla.edu.
authors.forename: James S
authors.initials: JS
authors.lastname: Tomlinson
mesh_terms: D014408:Biomarkers, Tumor; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D005838:Genotype; D006801:Humans; D046208:Microchip Analytical Procedures / Q000379:methods*; D025202:Molecular Diagnostic Techniques; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; C117307:KRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)