title: High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation calling.
abstract: Identifying mutations in the TP53 gene is important for cancer prognosis, predicting response to therapy, and determining genetic risk. We have developed a high-throughput scanning assay with automatic calling to detect TP53 mutations in DNA from fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues. The coding region of the TP53 gene (exons 2-11) was PCR-amplified from breast cancer samples and scanned by high-resolution fluorescent melting curve analyses using a 384-well format in the LightCycler 480 instrument. Mutations were confirmed by direct sequencing. Sensitivity and specificity of scanning and automatic mutation calling was determined for FF tissue (whole genome amplified [WGA] and non-WGA) and FFPE tissue. Thresholds for automatic mutation calling were established for each preparation type. Overall, we confirmed 27 TP53 mutations in 68 primary breast cancers analyzed by high-resolution melting curve scanning and direct sequencing. Using scanning and automatic calling, there was high specificity (>95%) across all DNA preparation methods. Sensitivities ranged from 100% in non-WGA DNA from fresh tissue to 86% in WGA DNA and DNA from formalin-fixed, paraffin-embedded tissue. Scanning could detect mutated DNA at a dilution of 1:200 in a background of wild-type DNA. Mutation scanning by high-resolution fluorescent melting curve analyses can be done in a high-throughput and automated fashion. The TP53 scanning assay can be performed from a variety of specimen types with high sensitivity/specificity and could be used for clinical and research purposes.
authors.affiliation: Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84112-5550, USA.
authors.forename: Roy
authors.initials: R
authors.lastname: Bastien
authors.forename: Tracey B
authors.initials: TB
authors.lastname: Lewis
authors.forename: Jason E
authors.initials: JE
authors.lastname: Hawkes
authors.forename: John F
authors.initials: JF
authors.lastname: Quackenbush
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Robbins
authors.forename: Juan
authors.initials: J
authors.lastname: Palazzo
authors.forename: Charles M
authors.initials: CM
authors.lastname: Perou
authors.forename: Philip S
authors.initials: PS
authors.lastname: Bernard
mesh_terms: D001331:Automation; D001943:Breast Neoplasms / Q000235:genetics*; D005453:Fluorescence; D006801:Humans; D009154:Mutation; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D016159:Tumor Suppressor Protein p53