Therascreen braf pyro kit

DNA was extracted from three 10 μm-slides from FFPE tumor tissue samples using the QIAamp DNA FFPE Tissue Kit (QIAGEN, Milan, Italy), following the manufacturer’s protocol. In order to minimize contamination by normal cells, the tumor areas dissected for DNA extraction contained at least 70% of tumor cells. KRAS, NRAS and BRAF mutational analyses were carried out by pyrosequencing on chemo-naïve primary or metastatic samples, as previously described [26 (link)]. RAS and BRAF mutational analysis were performed using the therascreen KRAS Pyro Kit, therascreen RAS Extension Pyro Kit and therascreen BRAF Pyro Kit (Qiagen, Milan, Italy). Mutation status was determined by pyrosequencing on the Qiagen PyroMark Q24.

All primary tumors were analyzed for KRAS exon 2 mutations. For metastases, molecular analyses of KRAS, NRAS and BRAF were carried out by using the Qiagen therascreen kits (therascreen BRAF Pyro Kit, therascreen RAS Extension Pyro Kit, therascreen KRAS Pyro Kit, therascreen NRAS Pyro Kit) as previously described [49 (link)]. PIK3CA was analyzed by applying high resolution melting analysis (HRM), mutations were confirmed by Sanger sequencing as described beforehand [50 (link)]. Mismatch repair deficiency/microsatellite instability (MSI) was evaluated by means of immunohistochemistry following the locally established protocol with the ready to use antibodies MLH1 (Clon ES05), MSH2 (Clon FE11), MSH6 (Clon Epi 49) and PMS2 (Clon EP51) (Dako, Agilent technologies, Glostrup, Denmark).

DNA extraction was performed with the QiAmp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) and pyrosequencing analysis was performed as previously described with the therascreen BRAF Pyro Kit (Qiagen) and the therascreen NRAS Pyro Kit (Qiagen) [47 (link)–49 (link)]. The platform of the LCEP, (Nice, France) has been accredited for molecular testing by pyrosequencing of exons 11 and 15 of the BRAF gene and for codon 12, 13 and 61 of the NRAS gene. The laboratory holds full COFRAC (“Comité Français d'Accréditation”) accreditation for these assays, according to the ISO 15189 norm (http://www.cofrac.fr). The analytical sensitivity of the pyrosequencing method was considered to be 5% according to the manufacturer (Qiagen). Pyrogram profile analyses and interpretation of the results were done blindly with PyromarkQ24 (Qiagen) by five independent assessors (EL, MI, VL, VH OB).

Pyrosequencing was performed with the therascreen® BRAF Pyro Kit (Qiagen) detecting certain mutations in codon 600 of the BRAF gene according to manufacturer’s instructions. 1 μl of each isolated DNA was analyzed per run. Pyrosequencing was performed on the PyroMark Q24 platform (Qiagen) using the PyroMark Gold Q24 reagents. Pyrograms were generated with the PyroMark Q24 software (v. 2.0.6.) and data were analyzed manually or with a plug-in tool provided by Qiagen. Sequences surrounding the site of interest served as normalization and reference peaks for quantification and quality control. Dispensation order was as follows: 5’- GCT ACT GTA GCT AGT ACG AAC TCA-3’. Two different “sequence to analyze” were used: 5’- YAY TGT AGC TAG ACS AAA AYC ACC -3’ or 5’- CHC TGT AGC TAG ACS AAA ATY ACC -3’ for manual analysis. Samples with 5% mutated alleles or more were scored as mutation positive.

Pyrosequencing of BRAF codon 600 was carried out with the therascreen® BRAF Pyro Kit (Qiagen, cat.no 971470) on the PyroMark Q24 platform (Qiagen) according to manufacturer’s instructions. Pyrograms were generated and analyzed with the PyroMark Q24 software (version 2.0.6.). The light signal (in relative light/fluorescent units, RLU) generated by the pyrosequencing reactions is proportional to the amount of DNA template and the number of nucleotides incorporated into a target DNA strand. Data are displayed in the form of a pyrogram, a series of peaks on a graph. The height ratio between the peaks allows for the estimation of allele frequencies (in % units). The mutant allele frequency is the proportion of a particular variant allele among all allelic copies of a sample. Samples with a mutation frequency greater than the limit of detection (LOD) plus 3 % units were scored as mutation positive. Evaluation of potential low level mutations (mutation frequency ≥ LOD and ≤ LOD + 3 % units) was performed taking into account histological tumor cell content and VE1 immunohistochemical findings.