The repertoire of somatic genetic alterations of acinic cell carcinomas of the breast: an exploratory, hypothesis-generating study

Abstract

Acinic cell carcinoma (ACC) of the breast is a rare form of triple-negative (that is, oestrogen receptor-negative, progesterone receptor-negative, HER2-negative) salivary gland-type tumour displaying serous acinar differentiation. Despite its triple-negative phenotype, breast ACCs are reported to have an indolent clinical behaviour. Here, we sought to define whether ACCs have a mutational repertoire distinct from that of other triple-negative breast cancers (TNBCs). DNA was extracted from microdissected formalin-fixed, paraffin-embedded sections of tumour and normal tissue from two pure and six mixed breast ACCs. Each tumour component of the mixed cases was microdissected separately. Tumour and normal samples were subjected to targeted capture massively parallel sequencing targeting all exons of 254 genes, including genes most frequently mutated in breast cancer and related to DNA repair. Selected somatic mutations were validated by targeted amplicon resequencing and Sanger sequencing. Akin to other forms of TNBC, the most frequently mutated gene found in breast ACCs was TP53 (one pure and six mixed cases). Additional somatic mutations affecting breast cancer-related genes found in ACCs included PIK3CA, MTOR, CTNNB1, BRCA1, ERBB4, ERBB3, INPP4B, and FGFR2. Copy number alteration analysis revealed complex patterns of gains and losses similar to those of common forms of TNBCs. Of the mixed cases analysed, identical somatic mutations were found in the acinic and the high-grade non-acinic components in two out of four cases analysed, providing evidence of their clonal relatedness. In conclusion, breast ACCs display the hallmark somatic genetic alterations found in high-grade forms of TNBC, including complex patterns of gene copy number alterations and recurrent TP53 mutations. Furthermore, we provide circumstantial genetic evidence to suggest that ACCs may constitute the substrate for the development of more aggressive forms of triple-negative disease.

Keywords: TP53; breast cancer; immunohistochemistry; massively parallel sequencing; triple-negative.

Figure 1. Pure acinic cell carcinoma of the breast

Representative micrographs of a pure acinic cell carcinoma (Case 17, A, B) displaying lysozyme (C) and intermediate Ki67 expression (D). Note that this case, which harbored a TP53 somatic frameshift mutation and LOH of the wild-type allele, lacked p53 expression (E). Original magnification x40, A, B, C, D and E.

Figure 2. Mixed acinic cell carcinoma of the breast

Representative micrographs of the acinic (A) and high-grade triple-negative invasive ductal carcinoma of no special type (B) components of a mixed acinic cell carcinoma (Case 9), where both components expressed lysozyme (C, D), Ki67 (E, F) and p53 (G, H). Note the substantially higher Ki67 labeling index in the high-grade triple-negative invasive ductal carcinoma of no special type component (F) than in the acinic cell carcinoma component (E). Original magnification x40, A, B, C, D and E; x100, F, G and H.

Figure 3. Non-synonymous somatic mutations detected by targeted capture massively parallel sequencing in acinic cell carcinomas of the breast

Heatmap indicating the non-synonymous somatic mutations considered not to be neutral by either MutationTaster or CHASM. Cases are represented in columns; genes are depicted in rows. Mutation types are color-coded according to the legend. The presence of loss of heterozygosity of the wild-type allele in association with the somatic mutation is depicted by a diagonal bar. On the right, the membership of each gene in three cancer gene datasets, Kandoth et al. [41], Cancer Gene Census [42] and Lawrence et al. [43], is reported. ACC, acinic cell carcinoma; CC, clear cell focus; G1=grade 1; G2=grade 2.

Figure 4. Mixed acinic cell carcinomas of the breast displaying morphologic and genetic heterogeneity

Each panel depicts representative micrographs of each component, its respective genome plot and repertoire of non-synonymous somatic mutations. In the genome plots, smoothed Log₂ ratios were plotted on the y-axis according to their genomic positions indicated on the x-axis. Gains and losses are plotted in green and red, respectively. On the right, diagrams depicting the mutations identified in each component of each case. In these diagrams, each mutation is color-coded on the basis of its mutant allele fraction, as explained in the color key. A, case 14; B, case 16. Scale bars: 500μm. ACC: acinic cell carcinoma; CC: clear cell ACC component; G1, grade 1; G2, grade 2; IDC-NST: invasive ductal carcinoma of no special type; MC: metaplastic carcinoma.

Figure 5. Genomic profiling of mixed acinic cell carcinomas of the breast

Each panel depicts representative micrographs of each component, its respective genome plot and repertoire of non-synonymous somatic mutations. In the genome plots, smoothed Log₂ ratios were plotted on the y-axis according to their genomic positions indicated on the x-axis. Gains and losses are plotted in green and red, respectively. On the right, diagrams depicting the mutations identified in each component of each case. In these diagrams, each mutation is color-coded on the basis of its mutant allele fraction, as explained in the color key. A, case 9; B, case 15. Scale bars: 500μm. ACC: acinic cell carcinoma; IDC-NST: invasive ductal carcinoma of no special type.

Figure 6. Cancer cell fractions and clonal composition of histologically distinct components of clonally-related mixed acinic cell carcinomas

(A) Representative cancer cell fraction (i.e. estimated percentage of cancer cells harboring a given validated somatic mutation) as defined by ABSOLUTE [44] through the integration of tumor cellularity, ploidy, gene copy number and mutant allele fractions in each histologically-distinct component of mixed acinic cell carcinomas where the acinic and non-acinic components were found to be clonally-related. (B) Putative evolution of clones in each lesion, where each circle represents a (sub)clone with a specific subset of genetic alterations. Each black arrow represents the acquisition of somatic genetic alterations, listed above or below it, which define the emergence of the respective clone. Red solid arrows depict the divergence of a cell population from one lesion to another. Decomposition of genetically distinct clones and clonal evolution in lesions from case 9 and case 16 were performed using the results from ABSOLUTE [44] and SubcloneSeeker [45]. The percentages indicate the prevalence of each clone in each morphologically distinct component of each case. ACC, acinic cell carcinoma; CC, clear cell acinic cell carcinoma; IDC, invasive ductal carcinoma of no special type; MC, metaplastic carcinoma.