Atsuko Ashida1, Kaori Sakaizawa1, Asuka Mikoshiba1, Hisashi Uhara1, Ryuhei Okuyama2. 1. Department of Dermatology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan. 2. Department of Dermatology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan. rokuyama@shinshu-u.ac.jp.
Abstract
BACKGROUND: BRAF V600E is a common mutation in melanoma, and BRAF inhibitors are effective in treating of BRAF mutation-positive melanoma. DNA carrying this mutation is released from melanoma cells into the circulation. As such, circulating tumor-derived DNA (ctDNA) in peripheral blood represents a novel biomarker for evaluating tumor features in cancer patients. However, ctDNA is present in the peripheral blood at very low levels, which makes the detection of specific mutations in this DNA a challenge. Competitive allele-specific TaqMan PCR (castPCR), a straightforward commercially available assay, is a sensitive technique for quantitating a small amount of DNA. METHODS: The level of BRAF V600E ctDNA was quantified by castPCR in 26 consecutive plasma samples from six melanoma patients. RESULTS: The castPCR assay was performed using a mixture of BRAF V600E DNA and BRAF wild DNA and found to be able to detect BRAF V600E at a fractional abundance of ≥0.5 % in 2- to 10-ng samples of genomic DNA. Cell-free DNA was then extracted from peripheral blood samples collected from six patients with melanoma harboring the BRAF V600E mutation. BRAF V600E ctDNA was detected in three patients, at a fractional abundance of between 1.28 and 58.0 % of total BRAF cell-free DNA. The abundance of BRAF V600E ctDNA correlated with tumor burden, as determined by computed tomography imaging. In two cases, an increase in the level of BRAF V600E ctDNA preceded exacerbation of clinical symptoms. CONCLUSION: The castPCR assay can detect and quantitate small amounts of BRAF V600E ctDNA in samples containing large amounts of BRAF wild cell-free DNA. Thus, we suggest that the castPCR assay is suitable for monitoring ctDNA in the plasma of melanoma patients.
BACKGROUND:BRAFV600E is a common mutation in melanoma, and BRAF inhibitors are effective in treating of BRAF mutation-positive melanoma. DNA carrying this mutation is released from melanoma cells into the circulation. As such, circulating tumor-derived DNA (ctDNA) in peripheral blood represents a novel biomarker for evaluating tumor features in cancerpatients. However, ctDNA is present in the peripheral blood at very low levels, which makes the detection of specific mutations in this DNA a challenge. Competitive allele-specific TaqMan PCR (castPCR), a straightforward commercially available assay, is a sensitive technique for quantitating a small amount of DNA. METHODS: The level of BRAFV600E ctDNA was quantified by castPCR in 26 consecutive plasma samples from six melanomapatients. RESULTS: The castPCR assay was performed using a mixture of BRAFV600E DNA and BRAF wild DNA and found to be able to detect BRAFV600E at a fractional abundance of ≥0.5 % in 2- to 10-ng samples of genomic DNA. Cell-free DNA was then extracted from peripheral blood samples collected from six patients with melanoma harboring the BRAFV600E mutation. BRAFV600E ctDNA was detected in three patients, at a fractional abundance of between 1.28 and 58.0 % of total BRAF cell-free DNA. The abundance of BRAFV600E ctDNA correlated with tumor burden, as determined by computed tomography imaging. In two cases, an increase in the level of BRAFV600E ctDNA preceded exacerbation of clinical symptoms. CONCLUSION: The castPCR assay can detect and quantitate small amounts of BRAFV600E ctDNA in samples containing large amounts of BRAF wild cell-free DNA. Thus, we suggest that the castPCR assay is suitable for monitoring ctDNA in the plasma of melanomapatients.
Authors: Frank Diehl; Kerstin Schmidt; Michael A Choti; Katharine Romans; Steven Goodman; Meng Li; Katherine Thornton; Nishant Agrawal; Lori Sokoll; Steve A Szabo; Kenneth W Kinzler; Bert Vogelstein; Luis A Diaz Journal: Nat Med Date: 2007-07-31 Impact factor: 53.440
Authors: Geoffrey R Oxnard; Cloud P Paweletz; Yanan Kuang; Stacy L Mach; Allison O'Connell; Melissa M Messineo; Jason J Luke; Mohit Butaney; Paul Kirschmeier; David M Jackman; Pasi A Jänne Journal: Clin Cancer Res Date: 2014-01-15 Impact factor: 12.531
Authors: Carrie C Lubitz; Tiannan Zhan; Viswanath Gunda; Salma Amin; Benjamin J Gigliotti; Abbey L Fingeret; Tammy M Holm; Heather Wachtel; Peter M Sadow; Lori J Wirth; Ryan J Sullivan; David J Panka; Sareh Parangi Journal: Thyroid Date: 2018-02-27 Impact factor: 6.568
Authors: Christina M Wood-Bouwens; Derrick Haslem; Bryce Moulton; Alison F Almeda; Hojoon Lee; Gregory M Heestand; Lincoln D Nadauld; Hanlee P Ji Journal: J Mol Diagn Date: 2019-12-16 Impact factor: 5.568
Authors: Parisa Momtaz; James J Harding; Charlotte Ariyan; Daniel G Coit; Taha Merghoub; Billel Gasmi; Daoqi You; Agnes Viale; Katherine S Panageas; Aliaksandra Samoila; Michael A Postow; Jedd D Wolchok; Paul B Chapman Journal: Oncotarget Date: 2017-09-16