Elucidating the nature of acinic cell carcinoma of the breast with high-grade morphology: evidence from case report

Background Acinic cell carcinoma (AciCC) of the breast is a rare subtype of breast cancer. It was considered a low-grade triple-negative breast cancer (TNBC) with the potential to progress or transform into a high-grade lesion because of the molecular similarities with conventional aggressive TNBC in several genetic studies. Microscopically, the coexistence of classical low-grade and high-grade triple-negative components in breast AciCC is not uncommon. However, there is a scarcity of research on the comparative histopathological and genetic aspects of both components. Case presentation A 34-year-old woman with a nontender mass in the upper outer quadrant of the left breast was initially diagnosed with a malignant small round cell tumor (undifferentiated or poorly differentiated carcinoma) based on a preoperative biopsy, which was later identified as breast AciCC with a high-grade solid component. Left breast-conserving surgery with sentinel lymph node biopsy was performed. Microscopically, the breast AciCC consisted of a classical acinic component and a high-grade component. The latter demonstrated a solid sheet-like pattern characterized by large, round, pleomorphic or vesicular nuclei, prominent nucleoli, and frequent mitotic activities. Classical acinic architectures focally merged together to form solid nests and transited into high-grade areas. Remarkably, in the high-grade lesion, conventional immunochemical markers for breast AciCC, such as α1-antitrypsin (AAT), Lysozyme (LYS), Epithelial membrane antigen (EMA), S100 protein (S100), and cytokeratin (CK) were negative, whereas cell cycle protein D1 (cyclin D1) and vimentin showed diffuse expression. Next‑generation sequencing (NGS) revealed that 43.5% of variants were identical in both components. Furthermore, PAK5 mutation; copy number (CN) loss of CDH1, CHEK1, and MLH1; and CN gains of CDK6, HGF, and FOXP1 were identified in the high-grade lesion. The patient was treated with eight cycles of adjuvant chemotherapy (epirubicin combined with cyclophosphamide) and radiotherapy after surgery, and she is currently alive for 43 months with no metastases or recurrences. Conclusions This case demonstrates a comparative analysis of the histopathological and genetic characteristics of classical low-grade and high-grade components of AciCC within the same breast. This information may serve as a morphological and molecular basis for further investigation into the molecular mechanisms underlying high-grade lesions in breast AciCC.

Elucidating the nature of acinic cell carcinoma of the breast with high-grade morphology: evidence from case report Yunjie Ge 1 , Xianping Wei 2 , Jing-Nan Liu 3 , Ping-Li Sun 1* and Hongwen Gao 1

Background
Acinic cell carcinoma (AciCC) of the breast is a rare malignant epithelial tumor that was first reported in 1996 [1] and is classified as a rare and salivary gland-type tumor by the 5th World Health Organization classification of tumors of the breast [2].Although the histological patterns of breast AciCC overlap with those of salivary AciCC, studies have demonstrated that they have different molecular underpinnings [3,4].Some researchers have proposed that breast AciCC should be considered a type of carcinoma developing in microglandular adenosis (MGA) with acinic cell differentiation, rather than as a distinct entity [5].Moreover, there is evidence that the morphological and immunohistochemical transition from typical MGA to atypical MGA and ultimately to AciCC or poorly differentiated components can occur [6,7].Subsequent genetic studies have supported the contention that MGA, atypical MGA, and AciCC may be a part of the same spectrum of lesions and may represent low-grade forms of triple-negative breast cancer (TNBC) with the potential to progress or transform into highgrade TNBC [8].
Additionally, breast AciCC was previously suggested to have a good prognosis.Nonetheless, several cases with recurrence, metastases, or death have been reported successively [9,10].Hence, the true nature and origin of breast AciCC remain yet to be clarified, especially cases with high-grade morphology.Herein, we conducted a comparative investigation of the histopathological and molecular features of low-grade and high-grade components in a single case of breast AciCC.Our aim was to provide a morphological and molecular basis for further exploration of the molecular mechanisms underlying high-grade lesions in breast AciCC.

Case presentation
A 34-year-old woman discovered a painless mass through self-palpation in the left breast for 5 days.Physical examination revealed a hard mass in the upper outer quadrant of the left breast, measuring 30 mm in diameter.She was previously healthy and denied a family history of cancer.The nipple and the skin overlying the mass were normal.There were no palpable axillary and supraclavicular lymph nodes.Ultrasonography revealed an irregular hypoechoic mass in the upper outer quadrant of the left breast (BIRADS: 4B), and enhanced magnetic resonance imaging revealed an ill-defined, roundish signal shadow (BIRADS: 6) (Fig. 1A).Based on preoperative core needle biopsy, the diagnosis was a malignant small round cell tumor (undifferentiated or poorly differentiated carcinoma) (Fig. 1B, C).We performed modified radical mastectomy and axillary lymph node excision on the patient.Intraoperative sentinel lymph nodes and surgical margins were negative.

Clinicopathological examination
Grossly, the tumor was a grayish-white, well-defined nodule with soft texture and measured 27 mm in its greatest axis (Fig. 1D).Microscopically, the tumor grew infiltratively and comprised a classical breast AciCC component and a solid sheet-like architecture with necrosis (Fig. 2A).SRY-box transcription factor 10 (SOX10) staining revealed the distribution of two components (Fig. 2B).The classical AciCC component was composed of irregular acinar or glandular architectures lined by one to several layers of neoplastic epithelial cells (Fig. 2C).Neoplastic cells in the classical AciCC component were round or cuboidal in shape with eosinophilic granular cytoplasm and unclear cytoplasmic borders (Fig. 2D).There were no myoepithelial cells or basal lamina, and neoplastic glands were focally surrounded by a capillary network (Fig. 2D).Nuclei were round, oval-to-irregular with granular chromatin.Eosinophilic secretions were observed in the lumen (Fig. 2D), and approximately three mitotic counts were detected per 10 high-power fields.Between the two components, some acinic architectures merged together into small solid or cribriform nests (Fig. 2E, F).We also detected the transition from neoplastic glands or confluent nests to the large solid region (Fig. 2G, H, and I) and the faint outline of the remaining glands in the high-grade lesion (Fig. 2I).Furthermore, a large number of stromal tumor-infiltrating lymphocytes were observed in acinic and transition areas (Fig. 2E, F,  and G).In the high-grade region, the tumor exhibited a diffuse solid sheet-like pattern with marked atypia (Fig. 3A).Neoplastic cells had weak eosinophilic cytoplasm and round, ovoid, or polygonal nuclei (Fig. 3A).These round or ovoid nuclei contained coarse granular chromatin and a distinct perinuclear halo, and pleomorphic nuclei were deeply stained with little cytoplasm (Fig. 3A).In the high-grade region, there were up to 20 mitotic counts per single high-power field, and the Ki-67 labeling index was 95%.Interestingly, neoplastic cells in the focal highgrade region gradually transited into the lesion with larger vesicular pleomorphic nuclei, more prominent red nucleoli, frequent mitotic activities, and abundant cytoplasm (Fig. 3B, C, D, E, and F).

Molecular findings
Next-generation sequencing (NGS) targeting all the exons of 769 cancer-related genes was performed separately on the classical and high-grade components of this case.The results (Table 2) revealed 23 gene mutations or copy number variants in the two components, of which 10 (43.5%) variants were identical in both components, including the mutations of TP53, LMO1, MDC1, MSH3, KMT2D, and CCND3 and the copy number gains of CCND1, FGFR2, MYC, and IDH1.Remarkably, ) were expressed merely in the acinic component, but their expression was absent in the high-grade lesion.Cyclin D1 (I, EnVision, 400×) showed diffuse expression, and the Ki-67 labeling index (J, EnVision, 200×) was 95% in the high-grade lesion.Vimentin (K, EnVision, 200×) showed diffusely positive expression, and p53 (L, EnVision, 400×) staining was approximately 80% positive in the high-grade area each mutant allele frequency (MAF) or the copy number (CN) of these shared variants was higher in the highgrade lesion.Moreover, KMT2C (c.161 + 1G > A) was identified in the classical component, whereas KMT2C (c.250 + 1G > A) was identified in the high-grade component.Both the classical and high-grade components were microsatellite-stable with tumor mutation burden of 8.78 and 5.85 mutations/Mb, respectively.No gene fusions or rearrangements were detected in both components.
Furthermore, both components had their unique genetic variants.ALOX12B, KDM5A, PIK3CD, and POLE mutations were identified in the classical component.PAK5 mutation; CN loss of CDH1, CHEK1, and MLH1; and CN gains of CDK6, HGF, and FOXP1 were identified in the high-grade component.All identical and different gene mutations or copy number variants detected in different components of the tumor are listed in Table 2.
The patient was finally diagnosed with breast AciCC with a high-grade solid component.After surgery, she received eight cycles of adjuvant chemotherapy (epirubicin combined with cyclophosphamide) and radiotherapy.She was followed up closely with periodic rechecks.Currently, she is alive for 43 months with no metastases or recurrences.

Antibody
Classical component  Genetic studies have shown that MGA and atypical MGA may be a part of the same spectrum of lesions harboring frequent TP53 somatic mutations and MGA or atypical MGA with associated carcinoma might being the nonobligate precursor lesion of breast AciCC [8].In the present case, no MGA or atypical MGA components were observed though comprehensive immunohistochemical and histological evaluation.This is perhaps because there existed other precursor lesions for breast AciCC besides MGA or atypical MGA.It is necessary to accumulate additional cases for further investigation.Moreover, carcinomas developing in MGA often have a metaplastic carcinoma component [16], whereas no metaplastic carcinoma components were identified in our case despite detailed pathological sampling and observation.
In the present case, the transitional histology of the classical breast AciCC component and high-grade solid component was observed in the same mass of left breast, and based on the distribution of both components, they were presumed to be the same tumor with different stages of differentiation.Nevertheless, the morphological and immunohistochemical features varied significantly between the two components.This leads us to the concept "high-grade transformation" or "dedifferentiation, " which has been previously used in parotid AciCC that is characterized by the abrupt transformation or progression of low-grade carcinoma into high-grade carcinoma [17,18].In salivary AciCC with high-grade transformation, the high-grade component was composed of solid cribriform patterns of neoplastic cells with large vesicular pleomorphic nuclei, prominent nucleoli, frequent mitoses, and a higher Ki-67 labeling index [19].Moreover, the high-grade component of salivary AciCC was characterized by strong nuclear staining for cyclin D1, whereas classical diagnostic immunophenotypic markers, such as S100, AAT, and LYS, were absent [18], similar to that in the present case.
Furthermore, CN loss of CDH1 and CN gain of CDK6 were detected in the high-grade lesion.The CDH1 gene located on 16q22.1 encodes a cell adhesion protein, E-cadherin, that plays a vital role in gland formation, cell differentiation, polarity, and maintaining the integrity of epithelial cells [20].Studies have shown that the abnormality of CDH1 gene and the loss of expression of membrane E-cadherin are common in the lobular carcinoma of the breast [21,22].However, recent studies showed that 21% of invasive ductal carcinomas also exhibited the CN loss of CDH1 and 27% of high-grade invasive ductal carcinomas exhibited reduced or loss of E-cadherin membranous expression [23].The loss or reduced expression of E-cadherin may result in cellular dedifferentiation and facilitate cancer invasion and metastasis in breast cancer [24].Furthermore, cyclin-dependent kinases 4 and 6 (CDK4/6) play a significant role in regulating cellcycle progression from the gap phase to the DNA synthesis phase [25].Therefore, in the high-grade lesion of the present case, the CN loss of CDH1 and the CN gain of CDK6 may partially explain the poor morphological differentiation of the high-grade lesion or the dedifferentiation from classical to high-grade components.

Conclusions
Overall, we have described in detail the histopathological and genetic features of a breast AciCC with classical lowgrade and high-grade components.The growth pattern, loss of immunophenotypic markers, and complex genetic variants were identified in the high-grade lesion, which may provide a morphological and molecular basis for further investigating the possible molecular mechanisms underlying high-grade lesions in breast AciCC.

Fig. 2
Fig. 2 Histopathological features of the tumor.Microscopically, the tumor consisted of a classical acinic component and a solid high-grade component (A, H&E, 20×).SOX10 (B, EnVision, 20×) staining highlighted the distribution of the two components.The acinar architectures arranged irregularly were composed of one to several layers of neoplastic epithelial cells (C, H&E, 200×).There were no myoepithelial cells or basal lamina, and neoplastic glands were focally surrounded by a capillary network (D, H&E, 400×).Between both components, acinic architectures merged together into small solid or cribriform nests, and the number of stromal tumor-infiltrating lymphocytes was high (E, H&E, 100×; F, H&E, 200×).The transition from neoplastic glands or confluent nests to the large solid region (G, H&E, 100×; H, H&E, 200×; I, H&E, 400×) and the faint outline of the remaining glands in the high-grade lesion (I, H&E, 400×) were observed

Table 2
Identical and different gene mutations or copy number variants detected in different components of the tumor