Pyromark platform

For in situ mutation detection, the tumors K229, K255, and K386 were identified as the best candidates, as NGS data indicated genetic heterogeneity. Before applying in situ mutation detection, pyro-sequencing was performed of the tumors K229 and K386 to confirm NGS data. Pyro-sequencing was performed using the Qiagen Pyromark platform with the appropriate kits and according to the manufacturer’s recommendations. Pyro-sequencing assays were designed for the DPYD (R886H) (Tumor K229) and FANCD2 (E912Q) (Tumor K386) mutations using the Pyromark Assay Design software. Detection and quantitative mutation analysis were performed using the Qiagen Pyromark software.

Frozen glycerol stocks served as template material for PCR reactions. The PyroMark platform from QIAGEN was used for pyrosequencing and reactions were performed as per manufacturer’s instructions. The region surrounding the point mutations was amplified and biotinylated with the use of a biotinylated reverse primer. To detect the ratio of SNPs in the population, single stranded primers were designed to terminate before the codon containing the mutation.

Genomic DNA was extracted from frozen tissue samples using the QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. DNA was quantified in a spectrophotometer. The presence of mutations in KRAS exon 2, BRAF exon 15 and in the catalytic subunit of PI3K, PIK3CA, were evaluated by pyrosequencing (Pyromark platform^®; Qiagen) following amplification by Polymerase Chain Reaction (PCR). MSI status was determined following PCR amplification from tumoral DNA analyses of six microsatellite markers, NR21, NR24, NR27, BAT25, BAT26 and CAT25. The tumor was considered MSI-High when three or more of these markers showed instability, while MSI-Low was defined as taking place when one or two of the six markers showed instability [50 (link)]. Sequencing analyses were performed using the Applied Biosystems^® 3130 Genetic Analyzer (Thermo Fisher Scientific, Illkirch, France,). In sporadic (non-familial) CRCs, MMR defect results from the silencing of MLH1 transcription by cytosine methylation. Histological sections of tumors were first processed for immunodetection of MLH1. In tumors with a lack of MLH1 protein, MLH1 promoter methylation status was then measured by real-time PCR following sodium bisulfite conversion of tumor DNA, as previously described [51 (link)].