Can Immunohistochemistry Replace Molecular Test in Diagnosing Mammary Analog Secretory Carcinoma?

Abstract

Mammary analog secretory carcinoma (MASC) is a distinct variant of rare, low-grade salivary gland carcinoma with characteristic genetic alteration and the ETV6-NTRK3 gene fusion. MASC is recently described in the new WHO classification of head-and-neck tumors (4^th edition, 2017). The tumor has the similar morphologic, immunohistochemistry (IHC), and molecular features of mammary secretory carcinoma. Immunohistochemically, the tumor cells display strong positivity for CK7, mammaglobin, S100, MUC-4, and STAT5. In resource-constraint centers, IHC plays an important role in diagnosing MASC; however, cases with discrepancies between morphological and immunohistochemical expression, confirmation of ETV6-NTRK3 fusion gene is a must. Herein, we describe this rare entity in a young female with parotid region swelling, which on gross examination was encapsulated and on microscopy had a predominant microcystic pattern comprising polygonal cells, which were immunopositive for S100, mammaglobin, and CK7 while negative for DOG 1. Based on these findings, a final diagnosis of MASC was rendered.

INTRODUCTION

Mammary analog secretory carcinoma (MASC) is a recently described, rare, and distinct variant of low-grade salivary gland malignancy with characteristic immunohistochemical expression and molecular features.[1] MASC has a common translocation, i.e., t (12:15) (p13; q25) and fusion gene ETV6-NTRK3-like secretory carcinoma (SC) of the breast; hence, it bears a synonymous term SC.[2,3] Skalova et al. in 2010 first described the salient molecular features, distinguished it from acinic cell carcinoma (AciCC) and named this new entity as MASC.[1] This novel entity has just been established in the new WHO classification of head-and-neck tumors (4^th edition, 2017).[3] Chiosea et al. in their study highlighted that cases previously misdiagnosed as zymogen poor AciCC were positive for ETV6 translocation in fluorescence in situ hybridization and reclassified them as MASC.[4] According to a major review by Khalele about 279 cases were reported worldwide; in the Indian scenario, few single case reports and one small series of three cases were reported.[5,6] MASC is considered to be a slow-growing, low-grade malignant tumor with few reports of surrounding tissue infiltration and lymph node metastasis.[7,8] It predominantly affects major salivary glands, with the parotid gland being most common, followed by of submandibular gland. However, cases affecting minor salivary glands of lip, soft palate, hard palate, base of tongue, and buccal mucosa are also reported in the literature.[3,9] Immunohistochemically, the tumor cells display strong positivity for CK7, mammaglobin, S100, MUC-4, and STAT5.[3] Herein, we describe this rare entity in a young female with parotid region swelling, which was diagnosed as MASC with detailed immunohistochemical workup.

CASE REPORT

A 39-year-old female presented with a swelling near left parotid region for 3 years. The mass on examination was mobile, nontender, and of size 2.5 cm × 2 cm with no cervical lymphadenopathy. Fine-needle aspiration cytology showed few atypical-looking cells over a mucoid background, and a diagnostic category of suspicious of uncertain malignant potential (Milan category IVb) was rendered. Contrast-enhanced computed tomography revealed a heterogeneously enhancing lesion involving the superficial lobe of the left parotid, and differentials of Warthin’s tumor and pleomorphic adenoma was given. Superficial parotidectomy was performed, and gross examination revealed a capsulated tumor of size 3 cm × 2.5 cm × 2 cm [Figure 1]. No focus of hemorrhage or necrosis was identified.

Figure 1.

Superficial parotidectomy specimen displaying tumor of maximum size 3 cm (blue double arrow)

On microscopy, the tumor was capsulated well demarcated from the surrounding normal salivary gland tissue [Figure 2a] with small foci of infiltration [Figure 2b]. The tumor displayed a lobular pattern of growth and arranged in predominantly microcystic pattern, also large cysts filled with colloid-like material, in papillary pattern and solid sheets [Figure 2c and d]. The microcystic spaces contained gray-blue mucoid material which was periodic acidSchiff (PAS)-positive and diastase resistant, also Alcian blue with periodic acid Schiff (ALPAS) positive [Figure 2e and f]. The tumor cells are round to polygonal with moderate amount of eosinophilic to bubbly cytoplasm due to intracytoplasmic vacuoles. In ALPAS stain, the luminal secretions were ALPAS positive, whereas the intracytoplasmic secretions were focally PAS-positive. Nuclei are of bland chromatin and inconspicuous nucleoli. Mitosis was 1/10 high-power field.

Figure 2.

(a-f) Light microscopic features of the tumor. Lower magnification displaying well-encapsulated tumor with lobular growth pattern and intervening fibrous septa (a, ×40). Focus of infiltrative border of the tumor (arrow) (b, ×100). Still higher magnification of tumor cell arrangement in macrofollicular pattern with colloid-like secretions (c, ×400), solid, and cribriform pattern (d, ×400), intraluminal secretions were periodic acidSchiff and ALPAS positive (arrow) (e and f, respectively, ×400)

With the above histomorphology, differentials of AciCC (zymogen poor), low-grade mucoepidermoid carcinoma, and MASC were considered. To confirm the diagnosis and proper characterization, extensive immunohistochemical workup was done. The tumor cells were positive for S100 (both nuclear and cytoplasmic) and CK7 (cytoplasmic with membrane accentuation), whereas negative for p63 and CEA; excluding the diagnosis of mucoepidermoid carcinoma (MEC) and AciCC. Again, to support the diagnosis of MASC, a second panel of immunohistochemistry (IHC) was performed with mammaglobin, gross cystic disease fluid protein 15 (GCDFP-15) (EP15), DOG1 and human epidermal growth factor receptor 2 (HER2)/neu. Interestingly, the tumor was positive for mammaglobin (diffuse and strong cytoplasmic), GCDFP (multifocal and cytoplasmic) while negative for HER2/neu and DOG1 stain was nonspecific (both nuclear and cytoplasmic) [Figure 3]. The proliferative index Ki67 was 12% at highest proliferative focus. With these findings, a final diagnosis of MASC was rendered. The patient is on regular follow-up with no clinical or radiological evidence of local recurrence or metastasis.

Figure 3.

Detail immunohistochemical workup. Positive immunostain for CK7 (arrow), S100 (arrow), mammaglobin (arrow), gross cystic disease fluid protein (arrow) (focal), while negative for CEA, p63, human epidermal growth factor receptor 2/neu, and nonspecific nuclear and cytoplasmic staining of DOG1. Proliferative index (Ki67) was approximately 12%

DISCUSSION

Tumors of the salivary gland are rare, and MASC accounts for <0.3% of all salivary gland tumors.[9] According to the WHO 2017 classification of head-and-neck tumors, the mean age of presentation is 46.5 years with no gender predilection.[3] This tumor is usually small, but giant MASC of size 16 cm of the parotid gland is also described in the literature.[10]

Microscopically, the tumor has a lobulated growth pattern with tumor cells having microcystic, papillary cystic, solid, follicular, and tubular patterns of arrangement. The cells usually display small uniform vesicular nuclei with pale eosinophilic or vacuolated bubbly cytoplasm with intraluminal or intracytoplasmic secretions. The luminal secretions are positive for PAS, ALPAS, mucicarmine, and mammaglobin. In our case also, the mucinous luminal secretions and focal intracellular vacuoles were PAS-positive diastase resistant and also highlighted in ALPAS and mammaglobin. In contrast to this, Joshi et al. in their study observed that the large intracellular clear vacuoles are due to fat droplets in most of the cells and not due to mucin. They also described the ultrastructural features of tumor cells and the presence of large intracellular large electron-dense lipid droplets.[10]

Acinic cell carcinoma (AcCC) and MASC show similar histologic features but PAS-positive basophilic zymogen granules of AcCC are absent in MASC. Retrospective analysis of the literature revealed many cases of MASC were previously diagnosed as zymogen poor AcCC.[1] Such cases can be distinguished by S100 and mammaglobin immunostain, as AcCCs are S100 and mammaglobin negative, while in MASC, the expression is strong and diffuse. MASC is typically negative for high molecular cytokeratin and basal/myoepithelial markers such as p63, CK5/6, CK14, calponin, and Smooth Muscle Actin (SMA). Due to the presence of cystic foci and cells looking like intermediate cells, one differential was low-grade MEC, but negative immunostain for p63 and positive IHC expression of S100 in our case ruled out MEC.

Breast and salivary glands are derived from the same embryonic ectoderm with similar ductuloacinar histology, attributing multiple similar neoplasms such as, pleomorphic adenoma, adenoid cystic carcinoma, epithelial-myoepithelial carcinoma, and MEC in both the sites.[11] The common characteristic oncogenic fusion gene ETV6-NTK3 in SC of breast and salivary glands is also identified in other tumors such as infantile fibrosarcoma, congenital mesoblastic nephroma, and acute myeloid leukemia.[1,3] This fusion encodes a chimeric oncoprotein tyrosine kinase that activates the Ras-MAP kinase and phosphatidylinositol–3–kinase–Akt pathways.[12] This chimeric tyrosine kinase activates cell proliferation and increases the survival of tumor cells playing a fundamental role in its oncogenesis.[13] Literature review of case series and reports of MASC cases depicts the absence of ETV6-NTK3 fusion gene rearrangement does not rule out the diagnosis of MASC.[1,14] This signifies for diagnosing MASC, molecular study of ETV6 rearrangement is not absolutely required in classic morphologic and immunophenotypic cases; however, in unusual morphologic and immunophenotypic cases, molecular testing for ETV6 rearrangement studies should be done.[15] Furthermore, positive immunohistochemical stain for S100, mammaglobin, and STAT5 is useful in diagnosis even in negative test for fusion gene.[8,12]

MASC is usually an indolent salivary gland malignancy and usually does not infiltrate the surrounding tissue. However, cases with local recurrence, lymph node, and distant metastasis are also described in the literature.[3,8] High clinical stage and high-grade transformation with infiltrative growth pattern, brisk mitotic activity, significant nuclear atypia, and/or necrosis are the main adverse prognostic factors.[3] Skalova et al. first time described the high-grade transformation, which on IHC revealed strong membrane staining for EGFR and β-catenin, cytoplasmic/nuclear staining for S100 protein, and nuclear staining for cyclin-D1.[16] However, our case did not reveal any high-grade morphology, and the tumor was limited within the parotid gland. The patient is doing well without any adjuvant therapy on her follow-up after 3 years.

Due to a limited number of cases being reported with follow-up, the standard treatment protocol is yet to establish. Lymph node dissection is recommended only for T3/T4 stage and patients with clinical nodal disease. The value of postoperative radiotherapy (RT) is unclear due to the paucity of treatment-specific survival data. Postoperative RT is reserved for close margins (<0.5 mm), T3/T4 stage, incomplete excision, and perineural invasion.[17] Chemotherapy is restricted to cases with distant metastasis. To prognosticate biologic behavior of this rare molecularly defined distinct entity, further larger studies with long-term follow-up are needed. Molecular-targeted therapy inhibiting the ETV6-NTRK3 fusion tyrosine kinase may be useful in selected cases along with surgery.

In resource-constraint centers, IHC plays an important role in diagnosing MASC; however, cases with discrepancies between morphological and immunohistochemical expression, molecular confirmation of ETV6-NTRK3 fusion gene is mandatory. Further, larger studies can unfold the biological behavior and the role of molecular-targeted therapy of the novel entity.

Declaration

Herein, we declare that we have ethical approval from our IEC.

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Conflicts of interest

There are no conflicts of interest.