摘要：

TT09. Comparing Sensitivities of Two Somatic Mutation Detection Methodologies for FFPE-Derived DNA K. Rao, S. Shell, S. Bacus Quintiles, Westmont, IL. Introduction: The sensitivity of molecular methods for profiling somatic mutations in fixed solid tumor samples is influenced by varying tumor content, tumor heterogeneity, lymphocyte infiltration, necrosis and degradation of DNA from formalin fixation. Furthermore, detection of somatic mutations at high sensitivity and specificity in limiting materials, whether from small biopsied material or circulating tumor DNA is critical for implementing personalized treatment and monitoring of therapeutic response. In the current study we evaluated the Competitive Allele- Specific TaqMan PCR (castPCR) for sensitive and specific detection of somatic mutations in Formalin-Fixed Paraffin Embedded (FFPE) samples and compared it with Qiagen Scorpions-ARMS assay. Methods: DNA from FFPE cell line pellets and tissue blocks harboring various KRAS or NRAS gene mutations was extracted using QIAamp FFPE tissue DNA extraction kit (Qiagen). The castPCR assays were purchased from Life Technologies and KRAS RGQ PCR Kit was purchased from Qiagen. A cell line control harboring a KRAS G12C mutation at 6% allele frequency and a KRAS wild type control were purchased from Horizon Diagnostics as FFPE curls. DNA extracted from Horizon cell line controls was combined to generate KRAS G12C mutant samples ranging from 6% to 0.03% mutant for limit of detection experiments. Results: The specificity of the KRAS G12C and G12V castPCR assays was assessed using the DNA extracted from 10 FFPE cell line pellets that harbor various KRAS or NRAS mutations. The respective castPCR assays specifically detected KRAS G12C or G12V mutations in these samples. The lower limit of detection was 0.11% mutant for castPCR technology and 0.33% mutant for Scorpions-ARMS technology. DNA dilution studies of cell lines containing KRAS G12C or G12V mutations showed that both castPCR and Scorpions-ARMS assays detect the respective mutations with as little as 600 pg of total input nucleic acids (~ 50 copies of genomic DNA). Subsequently, 15 colon and lung FFPE tissue samples previously characterized for KRAS mutation status by other in-house methodologies were tested using castPCR technology and castPCR showed 100% concordance. Conclusions: Our data show that Life Technologies castPCR and Qiagen Scorpions-ARMS assays are comparable in performance with castPCR demonstrating a slight edge over Qiagen assays for sensitivity. Both technologies can produce viable data with extremely low input of DNA and provide an excellent tool for mutation profiling samples with low DNA yields and/or low tumor burden. Furthermore, the lower limits of detection may be useful for monitoring the mutation status in the circulating tumor DNA.

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