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.

LightCycler Technology in Molecular Diagnostics

“…High-resolution melting has been used clinically to detect somatic changes in select exons of oncogenes such as EGFR, 53 KRAS, 54 PDGFRA, 55 KIT, 56 BRAF, 57 and TP53. 58 In tumors with mixed populations of tumor and normal cells, the sensitivity of scanning (Ͻ10%) is superior to sequencing (ϳ20%). 59 In genetics, the method has been applied to BRCA1/2, 60,61 cystic fibrosis, 52,62 hereditary hemorrhagic telangiectasia, 37 hemophilia, 63,64 and Charcot-Marie-Tooth disease, 65 and several others.…”

Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling

“…High-resolution melting (HRM or HRMA) is an effective method to screen for sequence variation and has been effectively used to assess genes involving inborn errors of metabolism, cancer susceptibility, and other genes whose dysfunction is associated with disease [Bastien et al, 2008;De Leeneer et al, 2008;Dobrowolski et al, 2007aDobrowolski et al, , 2007bDobrowolski et al, , 2005Erali et al, 2008;Laurie and George, 2009]. HRM profiling identifies the presence of sequence variants by deviation in the shape of a post-PCR melting profile using probe-based and amplicon-based strategies [Dobrowolski et al, 2007a[Dobrowolski et al, , 2007bMontgomery et al, 2007;Zhou et al, 2004].…”

Microsatellite Instability Detection by High-Resolution Melting Analysis

“…Good DNA quality is essential for HRM analysis; therefore, high-yield genomic DNA from peripheral blood leukocytes or saliva is the preferred material for analysis. However, the technique has already been successfully employed to scan for somatic mutations in various genes in archival FFPE tissues prone to DNA fragmentation (33 ), which is particularly appealing for retrospective studies and is within the framework of specimen processing in clinical pathology (34 ). To the best of our knowledge, our study is the first evaluation of 2 different instruments and implemented software modules for HRM assay on tumor DNA extracted from archival tumor material and of MSI detection by HRM.…”