Case report
Case 1. Case 1 involved a 21-year-old Japanese man who presented with a 1-year history of a slowly enlarging, painless mass in the right parotid gland (Table I). Ultrasonography revealed a well-circumscribed, homogeneous, isoechoic mass adjacent to a tiny cyst with posterior acoustic enhancement, measuring 16x12x10 mm (Fig. 1A). Magnetic resonance imaging (MRI) revealed a solid mass in the right parotid gland, showing low signal intensity on T1-weighted imaging (T1WI) (Fig. 1B) and high signal intensity on T2-weighted imaging (T2WI) (Fig. 1C). Fine-needle aspiration cytology (FNAC) demonstrated clusters with epithelial and spindle-shaped cells with interstitial mucin, which was interpreted as intermediate, with features suggestive of pleomorphic adenoma corresponding to category IVA of the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC).
The patient subsequently underwent a right superficial parotidectomy. Histologic examination revealed a lobulated tumor composed of microcystic and reticular growth patterns, with light basophilic secretions within the cystic lumina (Fig. 1D). The tumor cells exhibited mildly atypical, round to oval nuclei with vesicular chromatin and small nucleoli. Few mitotic figures were identified (1-2 mitoses per 10 high-power fields). These histologic findings corresponded to Grade 1 according to the histologic grading system for salivary gland SC proposed by Baněčková et al (3). One intraparotid lymph node was metastasized. Immunoperoxidase staining of formalin-fixed, paraffin-embedded (FFPE) tissue sections with anti-S-100 protein polyclonal antibody (Nichirei; Tokyo, Japan), anti-mammaglobin monoclonal antibody (31A5; Roche Diagnostics, Basel, Switzerland), anti-cytokeratin (CK)7 monoclonal antibody (OV-TL 12/30; Nichirei), anti-cytokeratin antibody (AE1/AE3; Nichirei), anti-p63 antibody (4A4; Nichirei), and anti-DOG1 antibody (DOG1.1; Thermo Fisher Scientific, Waltham, MA, USA) were performed using a Ventana OptiView DAB IHC detection kit (Roche Diagnostics). S-100 protein was weakly and diffusely expressed in the nucleus and cytoplasm of tumor cells (Fig. 1E). Mammaglobin was strongly expressed in the cytoplasm (Fig. 1F), whereas CK7 was expressed in both the membrane and cytoplasm (Fig. 1G). AE1/AE3 staining was positive but staining for p63 and DOG1was negative (data not shown).
Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed according to previously reported method (4) with minor modifications. Total RNA was extracted from formalin-fixed, paraffin-embedded surgical specimens using a NucleoSpin® totalRNA FFPE XS kit (Takara, Tokyo, Japan). A 110-bp fragment corresponding to the ETV6::NTRK3 fusion transcript was amplified using a OneStep RT-PCR kit (QIAGEN, Hilden, Germany) with a reverse primer specific for NTRK3 (5'-CAGTTCTCGCTTCAGCACGATG-3') and forward primer specific for ETV6 (5'-ACCACATCATGGTCTCTGTCTCCC-3'). A synthetic DNA fragment containing the ETV6::NTRK3 fusion junction was used as a positive control. The negative control consisted of RNA from tissue sections without carcinoma and no template. The amplified products were purified and bidirectionally sequenced at AZENTA Life Sciences (Tokyo, Japan). Sanger sequencing identified an ETV6::NTRK3 fusion involving exon 5 of ETV6 and exon 15 of NTRK3 (Fig. 1H). Based on the histologic features, immunohistochemical profile, and molecular findings, the tumor was diagnosed as SC and staged as pT1N1M0 (pathologic Stage III) according to the AJCC/TNM staging system, 8th edition (5). Postoperatively, the patient received radiotherapy to the parotid region with a total dose of 60 Gy. At 7 years of follow-up, the patient remains free of the disease.
Case 2. Case 2 involved a 79-year-old Japanese man who noticed a mass in the right parotid region 3 weeks prior to presentation (Table I). Ultrasonography revealed an irregularly marginated, heterogeneous, hypoechoic mass measuring 21x16x15 mm (Fig. 2A). MRI revealed a solid tumor located in the anterior part of the right parotid gland, showing low signal intensity on T1WI (Fig. 2B) and a mixture of intermediate and high signal intensity with extension to the masseter muscle on T2WI (Fig. 2C). Fluorodeoxyglucose-positron emission tomography/computed tomography demonstrated increased uptake in the parotid tumor, with no evidence of neck lymph node or distant metastasis (Fig. 2D). FNAC revealed irregular clusters of atypical epithelial cells with nuclear overlapping, vesicular chromatin, and prominent nucleoli, which was interpreted as suspicious for malignancy (Milan category V) (Fig. 2E).
The patient underwent right total parotidectomy with selective neck dissection of levels I, II, and III. Due to tumor invasion, the buccal branch of the facial nerve and a portion of the masseter muscle were resected. Histologic examination demonstrated infiltrative, irregular sheets of atypical epithelial cells with vesicular chromatin. The tumor cells exhibited prominent nucleoli, admixed with microcysts containing light basophilic secretions within the lumina, set in a hyalinized stroma (Fig. 2F). Few mitotic figures were identified (4-5 mitoses per 10 high-power fields). These histologic findings corresponded to Grade 2. No histologic evidence of lymph node metastasis was found; however, the surgical margin was positive. Tumor involvement of the resected masseter muscle was also noted. Immunohistochemically, S-100 protein was diffusely expressed in the nucleus and cytoplasm of tumor cells (Fig. 2G). Mammaglobin was focally expressed in the cytoplasm (Fig. 2H), whereas CK7 was strongly expressed in both the membrane and cytoplasm (Fig. 2I). AE1/AE3 staining was positive but staining p63 and DOG1was negative (data not shown). Sanger sequencing analysis confirmed the presence of an ETV6::NTRK3 gene fusion (Fig. 2J). Based on these findings, the tumor was diagnosed as SC and staged as pT4aN0M0 (pathologic Stage IVA). Postoperatively, the patient received radiotherapy to the parotid gland and neck with a total dose of 60 Gy. As of the 3-year follow-up, the patient remains free of the disease.
Discussion
SC, previously described as MASC and classified as a low-grade malignancy, accounts for approximately 1.5% of all parotid gland carcinomas (6). The mean age at presentation is reportedly 47.5 years, with a male-to-female ratio of approximately 1.4:1(3). Regarding anatomic distribution within the salivary glands, SC most commonly arises in the parotid gland (77.1%), followed by the submandibular gland (6.3%), with the remaining 16.6% of casas occurring in other salivary sites (7). Clinically, parotid gland SC typically presents as a painless, slowly enlarging mass (8).
On ultrasonography, SC typically presents as a predominantly cystic tumor with a solid part of the papillary projection (9). However, in both Case 1 and Case 2 of the present report, no cystic lesions with papillary projections were observed. On MRI of SC, the signal intensities of the solid components varied from low to intermediate on T1WI and from low to high on T2WI (9,10). This variation in signal intensity reflected varying degrees of histological formation of microcysts, a desmoplastic stromal reaction, and cellularity in the tumor. Wang et al (8) described two patterns of MRI of SC: one presenting as a partially cystic, lobulated mass that may mimic a benign tumor, and the other presenting as an irregular mass with a less cystic composition, which was associated with malignant features in their series. In Case 1 of the present report, ultrasonographic and MRI findings of a well-circumscribed, homogeneous tumor were consistent with a benign tumor. In Case 2, by contrast, ultrasonographic findings demonstrated an irregularly marginated, heterogeneous mass, and MRI showed high signal intensity on T2WI with extension into the masseter muscle, suggesting a malignant tumor.
FNAC of SC typically shows moderately cellular smears composed of loosely cohesive sheets, papillary or cribriform tissue fragments, and dispersed tumor cells in a predominantly mucinous background, although hemosiderophages and blood may be present in some cases (11). The tumor cells generally exhibit a low to moderate nuclear-to-cytoplasmic ratio, abundant finely granular or vacuolated cytoplasm, and uniform round to oval nuclei with fine chromatin, distinct single nucleoli, and only mild cytologic atypia. According to the MSRSGC, FNAC specimens of SC are most frequently categorized as malignant (Category VI) (50%), followed by suspicious for malignancy (Category V) (26%), salivary gland neoplasm of uncertain malignant potential (Category IVB) (18%), and atypia of undetermined significance (Category III) (6%) (12). The reported sensitivity of FNAC for diagnosing malignancy in major salivary gland tumors ranges from 38 to 97% (11,13). However, a review by Kala et al (11) reported a sensitivity of FNAC specifically for diagnosing salivary gland SC of only 27.7%, indicating a considerable diagnostic limitation. In many cases, the characteristic cytologic features of SC are recognized retrospectively after a definitive histologic diagnosis, with or without confirmatory molecular analysis. In the present study, FNAC findings in Case 1 were interpreted as intermediate, with pleomorphic adenoma included in the differential diagnosis, whereas in Case 2, FNAC results were suspicious for malignancy but did not allow a specific diagnosis of SC.
Histologically, SC typically exhibits a lobulated growth pattern, with lobules separated by fibrous septa and with composition of variable architectural patterns, including microcystic or solid, tubular, follicular, and papillary-cystic structures with distinctive luminal secretions. The tumor often demonstrates an infiltrative growth pattern with occasional perineural invasion and is associated with abundant fibrosclerotic stroma and prominent, thick, hyalinized septa (14). In 2023, Baněčková et al (3) proposed a 3-tiered grading system for SC featuring 4 histologic parameters: (i) architecture and fibrous septae/fibrosis; (ii) nuclear pleomorphism; (iii) perineural invasion, lymphovascular invasion, and tumor necrosis; and (iv) mitotic activity/Ki-67 index. Each parameter was scored on a scale of 1 to 3 and aggregated to yield a final grade: Grade 1=score 4-6, Grade 2=score 7-9, and Grade 3=score 10-12. Higher-grade tumors were significantly associated with poor prognosis (3). The histologic grade corresponded to Grade 1 with a score of 4 in Case 1 and Grade 2 with a score of 7 in Case 2.
Immunohistochemically, SC reportedly shows CK7 expression in 97-100% of cases (7). In both Case 1 and Case 2, the tumor cells were positive for S-100 protein, mammaglobin, and CK7 but negative for DOG1. This immunophenotypic combination is particularly useful for distinguishing SC from acinic cell carcinoma, which mimics SC and is typically negative for S-100 protein and mammaglobin but strongly positive for DOG1(15).
SC of the salivary glands is typically characterized by a recurrent t(12;15)(p13;q25) chromosomal translocation, resulting in the ETV6::NTRK3 gene fusion, which is considered a defining molecular alteration unique among salivary gland tumors (1). The ETV6::NTRK3 fusion gene encodes a chimeric tyrosine kinase that activates downstream signaling pathways, including the Ras-mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt pathways, thereby promoting tumorigenesis (16). The ETV6::NTRK3 gene fusion has been reported in approximately 94% of salivary gland SC cases (7). Several methods are available for detecting this fusion, including immunohistochemistry using antibodies against pan-tropomyosin receptor kinase (pan-Trk) (17), RT-PCR, fluorescence in situ hybridization (FISH), and DNA- or RNA-based next-generation sequencing (NGS) (18). Nuclear and cytoplasmic staining with anti-pan-Trk antibody showed an excellent sensitivity of 91% and specificity of 100% for the presence of ETV6::NTRK3 gene fusion positive salivary gland SC (17). However, we did not have the opportunity to perform staining with this antibody. RT-PCR is technically feasible, inexpensive and relatively widely accessible, and it can be effectively applied to FFPE tissue samples for the detection of ETV6::NTRK3 fusion transcripts (16). The main limitation of RT-PCR compared with FISH or NGS is the inability to determine novel fusion partners because ETV6 can occasionally fuse with alternative non-NTRK gene partners, including RET (19), MET (20), and MAML3 (21). In the present study, classical ETV6::NTRK3 fusion transcripts with a junction between exon 5 of ETV6 and exon 15 of NTRK3 were identified in both Case 1 and Case 2. This canonical fusion type is reportedly the most frequent in salivary gland SC and associated with less infiltrative histologic features, such as prominent thick fibrous septa, as well as more favorable clinical outcomes compared with other ETV6::NTRK3 exon junction variants (16).
With regard to treatment, the National Comprehensive Cancer Network Guidelines (version 1, 2026) recommend complete surgical excision with preservation of the facial nerve for clinically benign lesions and for T1 or T2 salivary gland carcinomas (22). For patients with T3 or T4a tumors, the guidelines recommend total parotidectomy combined with selective neck dissection for clinically N0 disease, and total neck dissection for patients with clinically evident lymph node metastasis (22). Postoperative radiotherapy is generally recommended for patients with incomplete resection, close surgical margins (<5 mm), perineural invasion, tumors classified as T3 or T4a, or regional lymph node metastasis (23). Previous reports have demonstrated that parotidectomy followed by radiotherapy with a total dose exceeding 60 Gy yields excellent local control with minimal treatment-related toxicity in patients with parotid carcinoma (24). In Case 1 of the present study, we performed superficial parotidectomy under a presumed benign diagnosis based on preoperative FNAC. After confirmation of SC, we did not consider reoperation, such as completion parotidectomy or neck dissection, because we were concerned about damage to the preserved facial nerve, and no lymph node swelling was observed in the neck on imaging studies. Postoperative radiotherapy with a total dose of 60 Gy was administered due to close surgical margins. In Case 2, by contrast, a malignant tumor was suspected based on FNAC and imaging findings, and although no cervical lymph node metastasis was observed, total parotidectomy with prophylactic, elective neck dissection of levels I, II, and III was carried out. Radiotherapy was indicated due to positive surgical margins and the presence of perineural and muscle invasion.
More than 75% of patients with salivary gland SC present with early-stage disease (stage I or II), whereas approximately 14 and 8% exhibit regional and distant metastases, respectively, at the first visit (7). The prognosis of patients with SC of the salivary glands is generally favorable. Following complete surgical excision, disease-specific survival rates of approximately 95-98% and disease-free survival rates of 87-89% have been reported (3,25). By contrast, SC with high-grade transformation is associated with a significantly poorer prognosis, with reported postoperative survival typically ranging from 2 to 6 years (26).
Recently, the efficacy and safety of Trk inhibitors have been established for the treatment of NTRK gene fusion-positive tumors, including salivary gland SC. Doebele et al (27) described the results of two phase I trials and one phase II trial of entrectinib involving a total of 54 patients with metastatic or locally advanced NTRK gene fusion-positive tumors, including 7 patients (13%) with salivary gland SC. Although 31 (57%) of the 54 patients showed an overall response rate, 6 (86%) of the 7 patients with salivary gland SC demonstrated an overall response rate to the treatment. A study involving 55 patients with NTRK gene fusion-positive metastatic or locally advanced tumors, including 12 patients (22%) with salivary gland SC, treated with larotrectinib demonstrated an overall response rate of 75%, and no severe side effects necessitating treatment discontinuation were observed (28). If either patient we treated develops unresectable lesions or distant metastasis in the future, these agents would represent promising therapeutic options.
In conclusion, accurate diagnosis of parotid gland SC requires a combined assessment of histologic and immunohistochemical features, with confirmation by detection of the ETV6::NTRK3 gene fusion. Recognition of this entity is important because of its potential diagnostic overlap with other salivary gland neoplasms and its prognostic and therapeutic implications. In our cases, surgical resection followed by radiotherapy resulted in favorable clinical outcomes, with no evidence of recurrence or metastasis observed in either patient during follow-up.