Chromosome analysis suite chas software v 3

Total DNA from an early passage (<6) of the three primary BrC cultures was isolated using the QIAamp^® DNA Micro kit (56304, Qiagen, Gaithersburg, MD, USA) according to the manufacturer's protocol. To verify the quality of DNA, the endogenous β-actin gene was amplified by PCR. The Affymetrix CytoScan HD array (901835, Affymetrix) was used to evaluate copy number and loss of heterozygosity (LOH). This array contains more than 2.6 million copy number markers, of which 750,000 are 'genotype-able' single nucleotide polymorphisms (SNPs) and 1.9 million are non-polymorphic probes. Chromosome Analysis Suite (ChAS) software v3.0 (Affymetrix) was used for data analysis; for gains and losses we considered a minimum length of 100,000 base pairs (bp), and for LOH a minimum length of 3 Mbp. We compared all resulting alterations in the three BrC primary samples against female and male non-cancer control array data. Data from 1038 phenotypically healthy individuals (Affymetrix) and from Database of Genomic Variants were used as reference. Alterations found only in <1% (rare) or never described (new rare) of the reference population were considered.