The DNA was extracted from paraffin-embedded tissue blocks by the Cobas DNA Sample Preparation Kit (Roche) according to manufacturer’s protocol. Mutation analysis was performed by multiparallel sequencing (NGS). Indexed Illumina NGS library was constructed from 100 ng tumor DNA by KAPA Library Preparation Hyper Plus Kit (Kapa Biosystems). Hybrid selection was performed with a custom SeqCap EZ Choice Library (Roche NimbleGen). The library was designed using genome build hg19 NCBI. Build 37.1/GRCh37 input genomic regions are listed as follows: AKT, BRAF (exons 11,15), BRCA1, BRCA2, EGFR (exons 18, 19, 20, 21), ESR, GNAS (exon 8), KRAS, KIT, MLH1, MSH2, MSH6, NRAS, PDGFRA (exons 8, 10, 12, 14, 18), PIK3CA, PMS2, PTEN, RET and TP53.
Paired-end cluster generation and sequencing were performed according to standard protocols from Illumina, using v2 kits. Sequencing data analysis and variant classification were performed by NextGENe software (Softgenetics) with minimum 5% variant allele frequency filtering.
Paired-end cluster generation and sequencing were performed according to standard protocols from Illumina, using v2 kits. Sequencing data analysis and variant classification were performed by NextGENe software (Softgenetics) with minimum 5% variant allele frequency filtering.