Genomic alterations in matching primary tumors and metastasis in breast cancer.

Abstract

1549 Background: We examined DNA sequence alterations in matching normal tissues, primary breast cancers and metastatic lesions to study the genomic evolution of breast cancer. We also assessed technical reproducibility. Methods: DNA was extracted from formalin fixed paraffin embedded tissues obtained during autopsies from 3 patients including 12 specimens. We performed paired-end whole exome sequencing with SOliD V4 Genome Analyzer using the Agilent Sure Select All Exon Kit including technical replicates. Nucleotide variants (NV) were detected by Varscan V2.2 and functional impact was predicted by ANNOVAR. Results: After 4 QC steps (duplicate and strand bias removal, base quality and minor allele frequency filters) that removed 98.81% of variants per sample (range 96.50%-99.99%). We observed on average 2648 new NVs per cancer sample (range 10-5541) at a mean coverage of 72 (range 19-283) and mean library size of 48270660 base (range 7764034 base - 60752085 base). Concordance for high quality NVs reached 95% (range 92%-98%) in replicates. Mutation rate in the primary cancer differed from patient to patient (range 10.41per Mb to 18.29 per Mb) with more mutations seen in patients with longer disease history and extensive prior therapy. We also observed more differences in mutations between primary tumors and metastasis than expected by technical nose alone. On average, 234(83.53%) mutations were shared between a primary tumor and a metastasis (range 4 – 947; 62.5%-97.63%) and the number of site-specific mutations ranged from 0 to 251. An index for assessing mutational heterogeneity was greater in primary cancers compared to their metastasis. Mapping of variants with predicted functional impact yielded different targetable anomalies for different tumor sites. Conclusions: Mutation rates differed among patients according to their disease history. Difference of mutation between primary tumor and metastatic lesion were larger than technical noise and the heterogeneity of primary cancers was larger than that of metastasis. Metastasis often differed from the primary tumor in the spectrum of potentially targetable anomalies.