The Landscape of Somatic Genetic Alterations in Metaplastic Breast Carcinomas

Abstract

Purpose: Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic type of breast cancer, predominantly of triple-negative phenotype, and characterized by the presence of malignant cells showing squamous and/or mesenchymal differentiation. We sought to define the repertoire of somatic genetic alterations and the mutational signatures of MBCs.Experimental Design: Whole-exome sequencing was performed in 35 MBCs, with 16, 10, and 9 classified as harboring chondroid, spindle, and squamous metaplasia as the predominant metaplastic component. The genomic landscape of MBCs was compared with that of triple-negative invasive ductal carcinomas of no special type (IDC-NST) from The Cancer Genome Atlas. Wnt and PI3K/AKT/mTOR pathway activity was assessed using a qPCR assay.Results: MBCs harbored complex genomes with frequent TP53 (69%) mutations. In contrast to triple-negative IDC-NSTs, MBCs more frequently harbored mutations in PIK3CA (29%), PIK3R1 (11%), ARID1A (11%), FAT1 (11%), and PTEN (11%). PIK3CA mutations were not found in MBCs with chondroid metaplasia. Compared with triple-negative IDC-NSTs, MBCs significantly more frequently harbored mutations in PI3K/AKT/mTOR pathway-related (57% vs. 22%) and canonical Wnt pathway-related (51% vs. 28%) genes. MBCs with somatic mutations in PI3K/AKT/mTOR or Wnt pathway-related genes displayed increased activity of the respective pathway.Conclusions: MBCs are genetically complex and heterogeneous, and are driven by a repertoire of somatic mutations distinct from that of triple-negative IDC-NSTs. Our study highlights the genetic basis and the importance of PI3K/AKT/mTOR and Wnt pathway dysregulation in MBCs and provides a rationale for the metaplastic phenotype and the reported responses to PI3K/AKT/mTOR inhibitors in these tumors. Clin Cancer Res; 23(14); 3859-70. ©2017 AACR.

Figure 1. Repertoire of non-synonymous somatic mutations of metaplastic breast cancers (MBCs) and triple-negative invasive carcinomas of no special type (IDC-NSTs)

Clinico-pathologic and immunohistochemical features, and non-synonymous somatic mutations identified in 35 MBCs subjected to whole-exome sequencing (left), and in 69 triple-negative IDC-NSTs from TCGA breast cancer study (right) (9). The effects of the mutations are color-coded according to the legend, with hotspots (32) colored in red. Likely passenger mutations and mutations of indeterminate pathogenicity are marked using a hatched pattern. The presence of multiple non-synonymous mutations in the same gene is represented by an asterisk. For MBCs, the presence of loss of heterozygosity of the wild-type allele of a mutated gene is represented by a diagonal bar, and mutations found to be clonal by ABSOLUTE (24) are indicated by a black box. Genes recurrently mutated in MBCs and displaying at least one likely pathogenic mutation are presented. Gene names highlighted in purple were significantly more frequently altered in MBCs as compared to triple-negative IDC-NSTs. Percentages to the right of the mutation heatmaps indicate the percentage of cases affected by non-synonymous somatic mutations in a given gene. Bar charts (top) indicate the number of non-synonymous and synonymous somatic single nucleotide variants (SNVs) and the number of somatic insertions and deletions (indels) for each sample. The dominant mutational signatures (35, 36) were assigned using deconstructSigs (37). Large-scale transitions (LST)-^high and LST-^low status was determined in accordance with Popova et al. (39). TCGA, The Cancer Genome Atlas.

Figure 2. Repertoire of somatic non-synonymous mutations of metaplastic breast cancers (MBCs) of different histologic subtypes

Non-synonymous somatic mutations identified in 16, ten and nine chondroid, spindle and squamous MBCs, respectively, by whole-exome sequencing are color-coded by their effect according to the legend, with hotspots (32) colored in red. Likely passenger mutations and mutations of indeterminate pathogenicity are marked using a hatched pattern. The presence of multiple non-synonymous mutations in the same gene is represented by an asterisk. The presence of loss of heterozygosity of the wild-type allele of a mutated gene is represented by a diagonal bar, and mutations found to be clonal by ABSOLUTE (24) are indicated by a black box. Genes recurrently mutated in MBCs and displaying at least one likely pathogenic mutation are presented. Genes highlighted in purple were significantly differentially altered in the given subtype of MBC. Percentages to the right of the mutation heatmaps indicate the percentage of cases affected by non-synonymous somatic mutations in a given gene. Bar charts (top) indicate the number of non-synonymous and synonymous somatic single nucleotide variants (SNVs), and the number of somatic insertions and deletions (indels) for each sample. The dominant mutational signatures (35, 36) were assigned using deconstructSigs (37). Large-scale transitions (LST)-^high and -^low status was determined in accordance with Popova et al (39).

Figure 3. Repertoire of non-synonymous somatic mutations affecting genes associated with the PI3K/AKT/mTOR and Wnt pathways in metaplastic breast cancers (MBCs) and triple-negative invasive carcinomas of no special type (IDC-NSTs)

(a) Non-synonymous somatic mutations identified in 35 MBCs subjected to whole-exome sequencing and 69 triple-negative IDC-NSTs from TCGA (9) are color-coded by their effect according to the legend, with hotspots (32) colored in red. Likely passenger mutations and mutations of indeterminate pathogenicity are marked using a hatched pattern. Genes associated with the KEGG PI3K/AKT/mTOR and Gene Ontology Wnt pathways (including FAT1, see Supplementary Table S6) and mutated in at least one MBC or triple-negative IDC-NST are included and ordered in decreasing order of mutational frequency in MBCs. The presence of multiple non-synonymous mutations in the same gene is represented by an asterisk. For MBCs, the presence of loss of heterozygosity of the wild-type allele of a mutated gene is represented by a diagonal bar, and mutations found to be clonal by ABSOLUTE (24) are indicated by a black box. Percentages to the right indicate the percentage of cases affected by non-synonymous somatic mutations in a given gene. Gene names highlighted in purple were significantly more frequently altered in MBCs. (b) Overall survival of patients with MBCs or triple-negative IDC-NSTs from TCGA that harbored and did not harbor somatic non-synonymous or likely pathogenic mutations in the KEGG PI3K/AKT/mTOR and Gene Ontology Wnt pathways (including FAT1, see Supplementary Table S6) using the Kaplan–Meier method. TCGA, The Cancer Genome Atlas.

Figure 4. Activation of Wnt/β-catenin and PI3K/AKT/mTOR pathways in metaplastic breast cancers (MBCs)

Wnt signal transduction pathway activity scores (a) between MBCs with somatic mutations in genes related to the Wnt signaling pathway (Gene Ontology GO:0060070, including FAT1) and wild-type MBCs and (b) between MBCs with likely pathogenic mutations in genes related to the Wnt signaling pathway (Gene Ontology GO:0060070, including FAT1) and wild-type MBCs (Supplementary Table S6). PI3K/AKT pathway activation scores (c) between MBCs with somatic mutations in genes related to the PI3K/AKT/mTOR (KEGG pathways hsa04150, hsa05151 and hsa04012) and wild-type MBCs and (d) between MBCs with likely pathogenic mutations in genes related to the PI3K/AKT/mTOR (KEGG pathways hsa04150, hsa05151 and hsa04012) and wild-type MBCs, and mTOR pathway activation scores (e) between MBCs with somatic mutations in genes related to the PI3K/AKT/mTOR (KEGG pathways hsa04150, hsa05151 and hsa04012) and wild-type MBCs and (f) between MBCs with likely pathogenic mutations in genes related to the PI3K/AKT/mTOR (KEGG pathways hsa04150, hsa05151 and hsa04012) and wild-type MBCs (Supplementary Table S6). Significance was assessed using Mann-Whitney U-test.