The Sensitivity and Specificity of Novel Primers for Detection of BRAFV600E Mutation.

OBJECTIVE: This study aimed to evaluate the sensitivity and specificity of a PCR-based novel technique for the detection of BRAF mutation in early stages of the cancer. METHODS: Different lengths of primer sets, ranging from 8 bp to 20 bp, were designed and used in this study. These primers were developed by applying on  cancer cell lines. After that, the sensitivity and specificity of the methodology  was evaluated by making serial dilutions. RESULTS: The quantitative allele specific discrimination PCR (QUASAqPCR) primer with 14 bp length was sensitive enough to detect significantly 1:1,000 ratio of BRAFV600E to wild-type background  (P = 0.011), when using 150 nanograms of DNA from cell lines in the reactions. CONCLUSION: High sensitivity and specificity levels of QUASA-qPCR method can improve diagnostic accuracy for BRAF mutation testing in patients at early stages of cancers and help stratify the appropriate choice of treatment.

Circulating tumor DNA in patients with colorectal adenomas: assessment of detectability and genetic heterogeneity.

Improving early detection of colorectal cancer (CRC) is a key public health priority as adenomas and stage I cancer can be treated with minimally invasive procedures. Population screening strategies based on detection of occult blood in the feces have contributed to enhance detection rates of localized disease, but new approaches based on genetic analyses able to increase specificity and sensitivity could provide additional advantages compared to current screening methodologies. Recently, circulating cell-free DNA (cfDNA) has received much attention as a cancer biomarker for its ability to monitor the progression of advanced disease, predict tumor recurrence and reflect the complex genetic heterogeneity of cancers. Here, we tested whether analysis of cfDNA is a viable tool to enhance detection of colon adenomas. To address this, we assessed a cohort of patients with adenomas and healthy controls using droplet digital PCR (ddPCR) and mutation-specific assays targeted to trunk mutations. Additionally, we performed multiregional, targeted next-generation sequencing (NGS) of adenomas and unmasked extensive heterogeneity, affecting known drivers such as APC, KRAS and mismatch repair (MMR) genes. However, tumor-related mutations were undetectable in patients' plasma. Finally, we employed a preclinical mouse model of Apc-driven intestinal adenomas and confirmed the inability to identify tumor-related alterations via cfDNA, despite the enhanced disease burden displayed by this experimental cancer model. Therefore, we conclude that benign colon lesions display extensive genetic heterogeneity, that they are not prone to release DNA into the circulation and are unlikely to be reliably detected with liquid biopsies, at least with the current technologies.