Mutation Profiling of Key Cancer Genes in Primary Breast Cancers and Their Distant Metastases

Abstract

Although the repertoire of somatic genetic alterations of primary breast cancers has been extensively catalogued, the genetic differences between primary and metastatic tumors have been less studied. In this study, we compared somatic mutations and gene copy number alterations of primary breast cancers and their matched metastases from patients with estrogen receptor (ER)-negative disease. DNA samples obtained from formalin-fixed paraffin-embedded ER-negative/HER2-positive (n = 9) and ER-, progesterone receptor (PR-), HER2-negative (n = 8) primary breast cancers and from paired brain or skin metastases and normal tissue were subjected to a hybridization capture-based massively parallel sequencing assay, targeting 341 key cancer genes. A large subset of nonsynonymous somatic mutations (45%) and gene copy number alterations (55%) was shared between the primary tumors and paired metastases. However, mutations restricted to either a given primary tumor or its metastasis, the acquisition of loss of heterozygosity of the wild-type allele, and clonal shifts of genes affected by somatic mutations, such as TP53 and RB1, were observed in the progression from primary tumors to metastases. No metastasis location-specific alterations were identified, but synchronous metastases showed higher concordance with the paired primary tumor than metachronous metastases. Novel potentially targetable alterations were found in the metastases relative to their matched primary tumors. These data indicate that repertoires of somatic genetic alterations in ER-negative metastatic breast cancers may differ from those of their primary tumors, even by the presence of driver and targetable somatic genetic alterations.Significance: Somatic genetic alterations in ER-negative breast cancer metastases may be distinct from those of their primary tumors, suggesting that for treatment-decision making, genetic analyses of DNA obtained from the metastatic lesion rather than from the primary tumor should be considered. Cancer Res; 78(12); 3112-21. ©2018 AACR.

Figure 1:

Somatic genetic alterations identified in 17 ER-negative/HER2-positive and TN breast tumors and matched brain or skin metastases by massively parallel sequencing of 341 cancer-related genes. a) Heatmap of most frequent somatic mutations and gene copy number alterations identified in this study according to subtype. Mutation types and gene copy number alterations are color-coded according to the legend. Please note that copy number alterations could not be assessed in samples from cases 7 and 19. b) Frequency of PIK3CA and TP53 mutations according to clinical subtype of the primary tumor. c) Percentage of primary only, metastasis only and shared mutations per clinical subtype and metastasis location. d) Sanger sequencing validation of TP53 mutation p.Cys238Phe (Exon 7, Fw primers) in patient #5 (triple-negative primary tumor with brain metastasis). ER, estrogen receptor; TNBC, triple-negative breast cancer.

Figure 2:

Somatic mutations and copy number alterations shared between ER-negative/HER2 positive and TN breast tumors and matched brain or skin metastases. a) Number of variants identified in the primary tumor only, in the metastasis only and shared between paired tumors and metastases; shown are total variants and (non)synonymous variants. b) Gains, amplifications, losses and homozygous deletions in all primary tumor samples and metastases. c) Number of amplifications and homozygous deletions in the primary tumor only, in the metastasis only and shared between paired tumors and metastases. d) Copy number profiles of the primary tumor and metastasis of patient #9 (HER2-positive primary tumor with brain metastasis). CDK12 and ERBB2 are coamplified in the primary tumor as well as the metastasis.

Figure 3:

Cancer cell fractions of the somatic mutations identified using ABSOLUTE for 15 ER-negative/HER2-positive and TN breast tumors and matched brain or skin metastases. Color-coding according to the legend. Loss of heterozygosity (LOH) is depicted by a diagonal bar, clonal mutations by an orange box. Please note that copy number alterations and cancer cell fractions could not be assessed in samples from cases 7 and 19.