Microglandular adenosis associated with triple-negative breast cancer is a neoplastic lesion of triple-negative phenotype harbouring TP53 somatic mutations

Abstract

Microglandular adenosis (MGA) is a rare proliferative lesion of the breast composed of small glands lacking myoepithelial cells and lined by S100-positive, oestrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative epithelial cells. There is evidence to suggest that MGA may constitute a non-obligate precursor of triple-negative breast cancer (TNBC). We sought to define the genomic landscape of pure MGA and of MGA, atypical MGA (AMGA) and associated TNBCs, and to determine whether synchronous MGA, AMGA, and TNBCs would be clonally related. Two pure MGAs and eight cases of MGA and/or AMGA associated with in situ or invasive TNBC were collected, microdissected, and subjected to massively parallel sequencing targeting all coding regions of 236 genes recurrently mutated in breast cancer or related to DNA repair. Pure MGAs lacked clonal non-synonymous somatic mutations and displayed limited copy number alterations (CNAs); conversely, all MGAs (n = 7) and AMGAs (n = 3) associated with TNBC harboured at least one somatic non-synonymous mutation (range 3-14 and 1-10, respectively). In all cases where TNBCs were analyzed, identical TP53 mutations and similar patterns of gene CNAs were found in the MGA and/or AMGA and in the associated TNBC. In the MGA/AMGA associated with TNBC lacking TP53 mutations, somatic mutations affecting PI3K pathway-related genes (eg PTEN, PIK3CA, and INPP4B) and tyrosine kinase receptor signalling-related genes (eg ERBB3 and FGFR2) were identified. At diagnosis, MGAs associated with TNBC were found to display subclonal populations, and clonal shifts in the progression from MGA to AMGA and/or to TNBC were observed. Our results demonstrate the heterogeneity of MGAs, and that MGAs associated with TNBC, but not necessarily pure MGAs, are genetically advanced, clonal, and neoplastic lesions harbouring recurrent mutations in TP53 and/or other cancer genes, supporting the notion that a subset of MGAs and AMGAs may constitute non-obligate precursors of TNBCs.

Keywords: TP53; breast cancer precursor; mutations; targeted capture massively parallel sequencing; triple-negative breast cancer.

Figure 1. Microglandular adenosis and associated synchronous breast cancers are S100-positive/triple-negative breast lesions

Representative micrographs of a MGA (A, case 8) lacking ER (B), PR (C) and HER2 (D) expression, and expressing S100 protein (E). Its associated invasive breast cancer (F) displayed a similar immunohistochemical profile, lacking ER (G), PR (H) and HER2 (I) expression, and expressing S100 protein (J). Original magnification 200×.

Figure 2. Non-synonymous somatic mutations and copy number alterations detected by targeted capture massively parallel sequencing in microglandular adenosis and atypical microglandular adenosis

Heatmap indicating the non-synonymous somatic mutations, and selected gene amplifications and homozygous deletions identified in the MGAs and AMGAs analyzed. Each column represents one sample; altered genes are reported in rows. The types of somatic genetic alterations are color-coded according to the legend. No homozygous deletions in the targeted genes were detected in this cohort. The presence of loss of heterozygosity of the wild-type allele of a mutated gene is represented by a diagonal bar. The presence of each gene in three cancer gene datasets, Kandoth et al.[33], Cancer Gene Census[34] and Lawrence et al.[35], is depicted beside the heatmap (grey). AMGA, atypical MGA; indel, small insertion/deletion; MGA, microglandular adenosis; SNV, single nucleotide variant.

Figure 3. Repertoire of copy number alterations identified in microglandular adenosis and atypical microglandular adenosis

Repertoire of copy number alterations as defined by targeted capture massively parallel sequencing. Samples are represented on the y-axis, chromosomes are represented along the x-axis. Light red: copy number loss; white: neutral; light blue: copy number gain; dark blue: amplification. AMGA, atypical MGA; MGA, microglandular adenosis; TNBC, triple-negative breast cancer.

Figure 4. Genomic profiling of the microglandular adenosis and atypical microglandular adenosis components of case 13

Representative micrographs of MGA and AMGA components of case 13, their respective genome plots and repertoire of non-synonymous somatic mutations. In the genome plots, smoothed Log₂ ratios (y-axis) were plotted according to their genomic positions (x-axis). In the middle, a chart representing the non-synonymous mutations identified in each component, color-coded based on their cancer cell fractions as defined by ABSOLUTE[38]. On the right, clonal evolution is illustrated in the phylogenetic tree, where the length of the branches is representative of the number of synonymous and non-synonymous mutations that distinguishes a given clone from its ancestral clone. The coloured branches represent each of the subclones identified, and selected somatic mutations that define a given subclone are illustrated along the branches. AMGA, atypical MGA; MGA, microglandular adenosis.

Figure 5. Genomic profiles of microglandular adenosis and atypical microglandular adenosis and associated invasive triple-negative breast cancers

Each panel shows representative micrographs of each component of cases 7, 8, 14 and 27, its respective genome plot and repertoire of non-synonymous somatic mutations. In the genome plots, smoothed Log₂ ratios (y-axis) were plotted according to their genomic positions (x-axis). In the middle, a chart representing the non-synonymous mutations identified in each component, color-coded based on their cancer cell fractions as defined by ABSOLUTE[38]. On the right, clonal evolution is illustrated in the phylogenetic tree, where the length of the branches is representative of the number of synonymous and non-synonymous mutations that distinguishes a given clone from its ancestral clone. The coloured branches represent each of the subclones identified, and selected somatic mutations that define a given subclone are illustrated along the branches. AMGA, atypical MGA; IDC, invasive ductal carcinoma, no special type; MBC, metaplastic breast cancer; MGA, microglandular adenosis.

Figure 6. Phenotypic and genomic profiling of microglandular adenosis and associated in situ and invasive components of case 5

Representative micrographs of MGA (A), DCIS (D) and IDC (G) components of case 5, their respective genome plots and repertoire of non-synonymous somatic mutations. Loss of p53 protein expression was seen in MGA (B) and IDC (H) consistent with the presence of frameshift TP53 mutations in these samples; whereas the DCIS, which harboured a TP53 missense mutation, displayed nuclear p53 expression (E). Lack of CK5/6 expression was seen in both MGA (C) and IDC (I) components; in contrast strong CK5/6 immunoreactivity (F) was detected in the DCIS. In the genome plots, smoothed Log₂ ratios (y-axis) were plotted according to their genomic positions (x-axis). In the middle, a chart representing the non-synonymous mutations identified in each component, color-coded based on their cancer cell fractions as defined by ABSOLUTE[38]. On the right, clonal evolution is illustrated in the phylogenetic tree, where the length of the branches is representative of the number of synonymous and non-synonymous mutations that distinguishes a given clone from its ancestral clone. The coloured branches represent each of the subclones identified, and selected somatic mutations that define a given subclone are illustrated along the branches. DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma, no special type; MGA, microglandular adenosis.