Clinicopathological and Molecular Characteristics of Mammary Adenoid Cystic Carcinoma With Adipocytic Differentiation With Emphasis on the Identification of a Novel BRAF Mutation

Case Report

Clinical characteristics. A 52-year-old Korean woman was referred to our Institution because of a non-palpable mass in the left lower breast. The mass was detected during screening ultrasonography at a local clinic. Neither the patient nor her family had any history of breast-related health problems. Initial mammographic series revealed a 1.3 cm-sized indistinct, irregular-shaped, isodense mass in the left lower breast. Magnetic resonance imaging revealed a breast mass with an irregular margin measuring 2.5 cm in the greatest dimension. After confirmation of the pathological diagnosis by core needle biopsy, surgical excision with sentinel node biopsy was carried out. Two Board-certified pathologists specialized in breast pathology made a final diagnosis of primary adenoid cystic carcinoma of the breast with clear surgical resection margins. The sentinel lymph nodes exhibited no evidence of metastasis. The patient received postoperative adjuvant chemotherapy and radiation therapy. During the 4-year follow-up period, the patient remained recurrence-free. The surgically-resected breast lesion was subjected to pathological examination and immunohistochemical staining.

Pathological characteristics. Grossly, the resected breast tissue measured 6.8×5.5×3.1 cm. The cut section showed a well-circumscribed, firm mass surrounded by ill-defined, soft, yellow bulging nodules and measuring 2.5 cm in the greatest dimension. Histologically, the central portion of the tumor showed complex glandular architectures, composed predominantly of solid or cribriform growth patterns and accompanied by desmoplastic stroma, whereas the peripheral portion of the tumor consisted of solid nests of tumor cells embedded in adipose tissue (Figure 1A). In particular, the central portion displayed cylindromatous neoplastic glands, the lumina of which were filled with basophilic basement membrane-like material characteristic of adenoid cystic carcinoma. The peripherally located yellow nodules showed scattered, small tumor cell nests surrounded by a periductal cuff of cellular myxoid stroma and bound by adjacent adipose tissue. Slender spindle-shaped cells forming the periductal cuff went beyond the boundary and merged imperceptibly with surrounding adipocytes (Figure 1B). These two morphological components appeared to be intermingled.

Immunohistochemistry. The formalin-fixed, paraffin-embedded tissue sections were immunostained using a Bond-maX automated immunostainer (Leica Biosystems, Melbourne, Australia) and a Bond Polymer Refined Detection Kit (Leica Biosystems). The primary antibodies targeted estrogen receptor (1:200; Novocastra, Newcastle upon Tyne, UK), progesterone receptor (1:100; Novocastra), HER2 (1:200; Ventana Medical Systems, Tucson, AZ, USA), cytokeratin 5/6 (1:100; Dako, Carpinteria, CA, USA), epidermal growth factor receptor (EGFR; 1:100; Novocastra), smooth muscle actin (SMA; 1:1,000; Dako), and p63 (1:200; Biocare Medical, Concord, CA, USA). Briefly, antigen retrieval was conducted at 97°C for 20 min in ER1 or ER2 buffer. After blocking endogenous peroxidase activity with 3% hydrogen peroxide for 10 min, primary antibodies were reacted for 15 min at room temperature. To verify antibody specificity, anti-mouse immunoglobulin was used instead of the primary antibody as a negative control. Estrogen and progesterone receptor expression status was assessed using the Allred scoring method (6). HER2 expression was evaluated using American Society of Clinical Oncology/College of American Pathologists guideline recommendations (7). For cytokeratin 5/6 immunostaining, staining with a moderate-to-strong intensity in the cell membrane was interpreted as positive expression. For SMA and p63 immunostaining, staining with a moderate-to-strong intensity in the cytoplasm and nuclei, respectively, was

Immunohistochemical staining results. We observed a basal-like, triple-negative phenotype. The tumor cells were negative for estrogen receptor, progesterone receptor, and HER2. The tumor cells were positive for cytokeratin 5/6 (Figure 1C), SMA (Figure 1D), EGFR, and p63. The peritumoral spindle-shaped cells were positive for cytokeratin 5/6, EGFR, and p63, and the slender spindle-shaped cells within adipose tissue were positive for cytokeratin 5/6, EGFR, and SMA.

Next-generation sequencing. Next-generation sequencing was conducted on an Ion Torrent Personal Genome Machine using Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies, Grand Island, NY, USA), as previously described (8, 9). The panel contains 2,855 cancer-related mutations in 50 commonly mutated oncogenes and tumor suppressor (please see https://www.thermofisher.com/order/catalog/product/4475346 for complete list). To construct the sequencing library, 10 ng DNA from a formalin-fixed, paraffin-embedded tumor section was subjected to single-tube, multiplex polymerase chain reaction amplification using the Ion AmpliSeq Cancer Primer Pool (Life Technologies) and Ion AmpliSeq Kit reagents (Life Technologies). An Ion 316 Ion Personal Genome Machine chip (Life Technologies) was used. The sample was then treated with α-fluoro-β-ureidopropionic acid reagent to partially digest the primers and phosphorylate the amplicons, which were then ligated to Ion Adapters and purified. Barcoded adapters from the Ion Xpress Barcode Adapters 1-96 Kit (Life Technologies) were substituted for the nonbarcoded adapter mix supplied in the Ion AmpliSeq Library Kit (Life Technologies). The ligated DNA was subjected to nick translation and amplification to complete the linkage between the adapters and amplicons and to generate sufficient material for downstream template preparation. Two rounds of Agencourt AMPure XP Reagent (Beckman Coulter Diagnostics, Brea, CA, USA) binding at 0.6 and 1.2 bead/sample volume ratios were used to remove the input DNA and primers from the amplicons. Final library molecules of 125-300 bp were obtained. The library was then transferred to the Ion OneTouch System (Life Technologies) for automated template preparation. Sequencing was carried out on the Ion Personal Genome Machine sequencer (Life Technologies), according to the manufacturer's recommendations. Ion Torrent software version 5.0 was used for automated data analysis, as previously described (9).

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Figure 1.

Histopathological features of primary mammary adenoid cystic carcinoma with extensive adipocytic differentiation. A: The tumor tissue exhibited two morphologically distinct components: classic solid and cribriform neoplastic glands in a desmoplastic background (central portion; lower half) and atypical tumor cell nests within adipose tissue (peripheral portion; upper half). B: At the periphery, individual tumor cell nests possessed a periductal cuff of cellular myxoid stroma and adipose tissue (right lower corner). Some nests exhibited spindle-shaped stromal cells only in the remaining myxoid stroma (upper half). A few spindle-shaped cells scattered and merged with adipocytes in the remaining area can be seen. C and D: The tumor nests within the nests, peritumoral spindle-shaped cells, and scattered slender spindle-shaped cells embedded in adipose tissue were positive for cytokeratin 5/6 (C) and smooth muscle actin (D). Staining method: A and B, hematoxylin and eosin; C and D, polymer method. Original magnification: A, 40×; B, 100×; C, 100×; D, 200×.

Molecular characteristics. Nucleotide sequence alignment revealed a homozygous missense mutation (NM_004333.4: c.1742A>G; p.N581S) in exon 15 of the BRAF gene (allelic frequency, 45.7%; depth of coverage, 644) (Figure 2).

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Figure 2.

Nucleotide sequence alignment of exon 15 of the v-Raf murine sarcoma viral oncogene homolog B (BRAF) gene, visualized by the Integrative Genomics Viewer. Next-generation sequencing revealed a pathogenic missense mutation in the BRAF gene (NM_004333.4:c.1742A>G; p.N581S) of the patient in this study.