Genetic analysis of microglandular adenosis and acinic cell carcinomas of the breast provides evidence for the existence of a low-grade triple-negative breast neoplasia family

Abstract

Acinic cell carcinoma is an indolent form of invasive breast cancer, whereas microglandular adenosis has been shown to be a neoplastic proliferation. Both entities display a triple-negative phenotype, and may give rise to and display somatic genomic alterations typical of high-grade triple-negative breast cancers. Here we report on a comparison of previously published data on eight carcinoma-associated microglandular adenosis and eight acinic cell carcinomas subjected to targeted massively parallel sequencing targeting all exons of 236 genes recurrently mutated in breast cancer and/or DNA repair-related. Somatic mutations, insertions/ deletions, and copy number alterations were detected using state-of-the-art bioinformatic algorithms. All cases were of triple-negative phenotype. A median of 4.5 (1-13) and 4.0 (1-7) non-synonymous somatic mutations per carcinoma-associated microglandular adenosis and acinic cell carcinoma were identified, respectively. TP53 was the sole highly recurrently mutated gene (75% in microglandular adenosis versus 88% in acinic cell carcinomas), and TP53 mutations were consistently coupled with loss of heterozygosity of the wild-type allele. Additional somatic mutations shared by both groups included those in BRCA1, PIK3CA, and INPP4B. Recurrent (n=2) somatic mutations restricted to microglandular adenosis or acinic cell carcinomas included those affecting PTEN and MED12 or ERBB4, respectively. No significant differences in the repertoire of somatic mutations were detected between microglandular adenosis and acinic cell carcinomas, and between this group of lesions and 77 triple-negative carcinomas from The Cancer Genome Atlas. Microglandular adenosis and acinic cell carcinomas, however, were genetically distinct from estrogen receptor-positive and/or HER2-positive breast cancers from The Cancer Genome Atlas. Our findings support the contention that microglandular adenosis and acinic cell carcinoma are part of the same spectrum of lesions harboring frequent TP53 somatic mutations, and likely represent low-grade forms of triple-negative disease with no/minimal metastatic potential, of which a subset has the potential to progress to high-grade triple-negative breast cancer.

Figure 1. Representative micrographs of histological features of and the expression of lysozyme expression in microglandular adenoses and acinic cell carcinomas

A, B) Carcinoma-associated microglandular adenosis. C, D) Microglandular adenosis-associated invasive carcinoma. E, F) Pure acinic cell carcinoma. G, H) Mixed acinic cell carcinoma, with acinic cell component on the right and non-acinic cell component on the left. Please note lysozyme expression in all lesions. Original magnification 200×.

Figure 2. Non-synonymous somatic mutations detected by targeted capture massively parallel sequencing in microglandular adenoses and acinic cell carcinomas

Heatmap indicating the non-synonymous somatic mutations identified in the pure microglandular adenoses (n=2), carcinoma-associated microglandular adenoses/atypical microglandular adenoses (n=8) and acinic cell carcinomas (n=8) analyzed. Each column represents one sample; mutated genes are reported in rows. Mutation types are color-coded according to the legend. The presence of loss of heterozygosity of the wild-type allele of a mutated gene is represented by a diagonal bar.

Figure 3. Repertoire of copy number alterations identified in microglandular adenoses and acinic cell carcinomas

A) Heatmap depicting the copy number alterations identified in the pure microglandular adenoses (n=2), carcinoma-associated microglandular adenoses/atypical microglandular adenoses (n=8) and acinic cell carcinomas (n=8) analyzed. Samples are represented on the y-axis, copy number alterations are represented along the x-axis according to their respective genomic location. Light red: copy number loss; white: neutral; light blue: copy number gain; dark blue: amplification. B) Frequency plots of recurrent gains and losses in acinic cell carcinomas (top) and carcinoma-associated microglandular adenoses/atypical microglandular adenoses (middle). Significant differences (Fishers’ exact test, p<0.05) are plotted in the bottom panel. On the y-axis the proportion of samples in which gains (green bars) or losses (purple bars) were identified is plotted according to genomic location (x-axis). C) Frequency plots of recurrent amplifications in acinic cell carcinomas (top) and carcinoma-associated microglandular adenoses/atypical microglandular adenoses (middle). Significant differences (Fishers’ exact test, p<0.05) are plotted in the bottom panel. On the y-axis the proportion of samples in which amplifications (green bars) were identified is plotted according to genomic location (x-axis).

Figure 4. Comparison of somatic mutations detected by targeted capture massively parallel sequencing in carcinoma-associated microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas cases with those in triple-negative breast cancers from the The Cancer Genome Atlas

(A) Lollipop plots illustrating the prevalence and type of TP53 mutations in acinic cell carcinomas (top, n=8), carcinoma-associated microglandular adenoses/atypical microglandular adenoses (middle, n=8) and triple-negative breast cancers from the The Cancer Genome Atlas (bottom, n=77). The structure of p53 protein is shown with a transactivation domain (green), a DNA binding domain (black) and a tetramerization domain (blue). Each ‘lollipop’ represents a mutation that occurs at the amino acid location labeled along the x-axis. The height of each lollipop indicates the frequency of the mutation and the color of the lollipop represents the type of mutation. Mutation types are color-coded according to the legend. (B) Bar plots depicting the prevalence and type of mutations affecting other genes, detected in acinic cell carcinomas, carcinoma-associated microglandular adenoses/atypical microglandular adenoses and triple-negative breast cancers from The Cancer Genome Atlas. Each row represents a mutated gene; horizontal bars represent the prevalence of mutations affecting each gene in triple-negative breast cancers from The Cancer Genome Atlas (left) and carcinoma-associated microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas (right). Sample histologic types are color-coded according to the legend. Mutation types are texture-coded according to the legend. Due to the high number of genes mutated at low frequency in triple-negative breast cancers, only genes affected by mutations in carcinoma-associated microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas carcinomas, and genes recurrently mutated (n≥2) in triple-negative breast cancers are plotted. TCGA, The Cancer Genome Atlas.

Figure 5. Comparison of copy number alterations identified in carcinoma-associated microglandular adenosis/atypical microglandular adenosis and acinic cell carcinoma cases with those identified in triple-negative breast cancers from The Cancer Genome Atlas

A) Frequency plots of recurrent gains and losses in carcinoma-associated microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas (top, n-16) and triple-negative breast cancers from The Cancer Genome Atlas (middle, n=77). Significant differences (Fishers’ exact test p<0.05) are plotted in the bottom panel. On the y-axis the proportion of samples in which gains (green bars) or losses (purple bars) were identified is plotted according to genomic location (x-axis). B) Frequency plots of recurrent amplifications in carcinoma-associated microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas (top) and triple-negative breast cancers from The Cancer Genome Atlas (middle). Significant differences (Fishers’ exact test p<0.05) are plotted in the bottom panel. On the y-axis the proportion of samples in which amplifications (green bars) were identified is plotted according to genomic location (x-axis). TCGA, The Cancer Genome Atlas.

Figure 6. Comparison of the frequencies of mutations affecting selected genes in carcinoma-associated microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas, as a group, and in ER-positive/HER2-negative, HER2-positive and triple-negative breast cancers from The Cancer Genome Atlas

ER, estrogen receptor.

Figure 7. Hypothetical evolution model of triple-negative breast neoplasms

Microglandular adenosis, atypical microglandular adenosis and acinic cell carcinoma likely constitute a low-grade triple-negative breast neoplasia family (middle pathway), characterized by recurrent TP53 mutations, 5q losses and 8q gains, and high levels of genetic instability, hallmark features of conventional high-grade triple-negative breast cancers (top pathway). Despite the lack of a myoepithelial cell layer, microglandular adenosis and atypical microglandular adenosis are not considered invasive lesions; therefore the progression from ductal carcinoma in situ to microglandular adenosis/atypical microglandular adenosis remains hypothetical as indicated by the dashed lines. Nevertheless, evidence favoring clonal relatedness between ductal carcinoma in situ and microglandular adenosis/atypical microglandular adenosis has been documented (7, 9). A second group of low-grade triple-negative neoplasms of the breast is underpinned by specific/pathognomonic genetic alterations, display low to intermediate levels of genetic instability and can be broadly categorized as salivary gland-like tumors of the breast, encompassing, most likely among others, secretory and adenoid cystic carcinomas. These special histologic types of triple-negative breast cancer are underpinned by ETV6-NTRK3 and MYB-NFIB fusion genes, respectively. *In their salivary gland counterparts, distinct genetic alterations but with likely similar functional effect have been described, such as MYBL1-NFIB fusion gene in adenoid cystic carcinomas (54) and ETV6 rearrangements with an unknown partner (ETV6-X) in mammary-analogue secretory carcinomas (55), which theoretically also occur in the breast lesions. ACC, acinic cell carcinoma; AMGA, atypical microglandular adenosis; MGA, microglandular adenosis; TN, triple-negative; TNBC, triple-negative breast cancer. Solid lines, associations between lesions supported by molecular evidence; dashed line, hypothetical evolutionary associations.