Clinicopathological characteristics and outcomes of 23 patients with secretory carcinoma of major salivary glands

<jats:title>Abstract</jats:title><jats:p>This retrospective study investigated the clinicopathological characteristics of secretory carcinoma of salivary glands (SCSG) in 23 patients with histopathologically confirmed SCSG between January 2010 and December 2020. In total, 13 males and 10 females (ratio, 1.3:1) aged 10 − 69 years (median, 45 years) were enrolled in this study; the average disease duration was 2.44 years (0.25–20 years). Twenty-one patients (91.3%) had SCSG in the parotid gland, and two (8.7%) in the submandibular gland. All patients had single nodules of diameters 0.8–4.8 cm (average 2.6 cm); five with lymph node metastases, and two with distant metastases. Immunohistochemically, tumors stained positive for S-100, mammaglobin, CK7, GATA3 and pan-Trk, and negative for DOG1, P63, and calponin, with Ki-67 positivity from 1 to 50%. <jats:italic>ETV6</jats:italic> gene rearrangement was confirmed in 15 patients. All patients underwent oncological resection, four had radioactive particles implanted postoperatively, one received chemotherapy, and seven underwent chemoradiotherapy. Six patients had regional recurrences, two distant metastases, and one died before the last follow-up. SCSGs are typically indolent, with a low locoregional recurrence rate and excellent survival. Prognosis is correlated to clinical stage, pathological grade, and surgical procedures.</jats:p>

section_content: The diagnosis of secretory carcinoma of salivary glands (SCSGs), a recently-described rare malignant tumor, was introduced by Skálová et al. in 2010 [1]. These tumors were originally classi ed as mammary analogue secretory carcinomas of salivary glands because their histomorphological and immunohistochemical features are similar to those of secretory carcinoma of the breast [2]. The updated World Health Organization (WHO) Classi cation of Head and Neck Tumors (4th edition, 2017) substituted secretory carcinoma for the previous mammary analogue secretory carcinoma designation [3]. 

These salivary gland tumors are characterized by the presence of a translocation t(12;15) (p13;q25), which leads to fusion of the ETV6 gene on chromosome 12 and the neurotrophic tropomyosin receptor kinase 3 (NTRK3) gene on chromosome 15 [4]. Before introduction of the diagnosis of SCSG, these tumors were mostly diagnosed as acinic cell carcinoma (ACC), mucoepidermoid carcinoma, adenocarcinoma, or not otherwise speci ed [5]. 

Several reports on histopathological features of SCSG have recently been published; however, little is known about the clinical characteristics of this new tumor, including its outcomes [6]. Here, we present a review of 23 patients with SCSG in major salivary glands who were treated in our department at the First A liated Hospital of ZhengZhou University, including detailed discussion of clinicopathological characteristics, outcomes of treatment, and prognosis.

section_content: This was a retrospective study and the protocol of the study was approved by the Medical Ethics Committee of our institution(Ethics Review Number: 2020-KY-230). All patients gave informed consent and signed informed consent forms. We retrospectively enrolled 23 patients with histopathologicallycon rmed SCSG who had been treated between January 2010 and December 2020. The diagnoses of SCSG were blindly reviewed by three independent consultant pathologists, all experts in salivary gland pathology. 

For conventional microscopy, formalin-xed, para n-embedded tissues stained with hematoxylin and eosin were used. Selected immunohistochemical stains were used in all cases, including S-100, mammaglobin, CK7, Gata3, Dog1, P63, calponin, and Ki-67. Fluorescence in situ hybridization was performed to detect the ETV6 gene. 

All patients' clinical data were analyzed for gender, age, size of tumor, symptoms and their duration, means of diagnosis, TNM stage, treatment administered, recurrences, and prognosis.
section_name: Methods

section_name: Results

section_content: The 23 patients with SCSG comprised 13 male and 10 female patients (male:female ratio 1. 3:1). The median age was 45 years (range 10-69 years) and the average duration of disease from initial symptoms to diagnosis was 2. 44 years (range 3 months to 20 years). Twenty-one of the tumors (91. 3%) were located in the parotid gland (four left parotid, 17 right parotid) and two (8. 7%) in the submandibular gland. All 23 patients had single nodules presenting as painless masses of diameter 0. 8-4. 8 cm (average diameter 2. 6 cm). Six patients (26. 1%) had T1 tumors, 14 (60. 9%) T2 tumors, and three (13%) T3 tumors. Five patients had presented with regional lymph node metastases and two had distant metastases at the time of diagnosis (Table 1 ).
section_name: Clinical characteristics

section_content: On color doppler ultrasound, the tumors appeared as well-demarcated, heterogeneous, hypoechoic masses with regular morphology. Most of them were solid, some being a combination of cystic and solid. Punctate blood ow signals were seen within the lesions; these can easily prompt misdiagnosis as pleomorphic adenoma. Typical ultrasonic characteristics are shown in Fig. 2F. 

With computed tomography (CT), the lesions appeared oval or lobulated, with clear boundaries, regular edges, and uneven density. Some had low-density cystic areas and foci of calci cation(Fig. 2A-C ). The average HU was 42 (41. 69 ± 16. 5) with plain scanning and 76 (76. 1 ± 24. 7) with enhanced scanning (Fig. 2D-E ). The degree of enhancement varied, mostly being uneven and marked (> 40 HU). However, a few tumors showed mild enhancement (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) or no obvious enhancement (< 10 HU). Most of the recurrences showed marked enhancement. Slightly enlarged lymph nodes were found in the ipsilateral submaxillary region and carotid sheath, potentially resulting in the misdiagnosis of pleomorphic adenoma. 

We found that SGSCs consistently had the following characteristics on magnetic resonance imaging (MRI): (1) a tendency to be roundish in shape, with clear boundaries; and (2) most T1 or T2 images showed long signals or mixed long and short signals. Fat suppression images showed high signals. On diffusion weighted imaging, there was little high b value diffusion, with high signals and uneven enhancement ranging from slight to signi cant. As with the CT ndings, slightly enlarged lymph nodes were found in the ipsilateral submaxillary region and carotid sheath, as shown in Fig. 3A-C. 

Moreover, two patients in our study group had undergone magnetic resonance hydrography (MRH), which showed that in both cases the lesions were located in the posterior lower pole of the parotid gland, more than 3 mm away from the main duct, which appeared normal. Regional parotid gland resection was therefore performed to preserve the secretory function of the parotid duct and part of that gland, as shown in Fig. 3D-F.
section_name: Imaging ndings

section_content: Grossly, the tumors were isolated solid nodules with light-tan or grayish cut surfaces (Fig. 4A ). Their texture was hard, they had clear boundaries, and their average diameter was 2. 6 cm (0. 8-4. 8 cm). A few of them harbored cysts containing yellowish-white uid (Fig. 4B ). 

The tumors exhibited a lobulated growth pattern with brous septa and were composed of microcystic/solid, tubular, follicular and papillary cystic structures with distinctive luminal secretions (Figs. 4C-F ). At high magni cation, the tumor cells were round or oval with little atypia. They had eosinophilic granular or vacuolated cytoplasm with small, uniform nuclei (Fig. 4C, green box). 

In our study group, immunohistochemical staining showed strong positivity for S-100 (Fig. 5A ), mammaglobin (Fig. 5B ), CK7 (Fig. 5C ), and Gata3 (Fig. 5D ), whereas staining was negative for calponin (Fig. 5E ), P63 (Fig. 5F ), and Dog1 (Fig. 5G ). Ki-67 positivity (Fig. 5H ) ranged from 1-50%, being ≤ 20% in 19 patients and ≤ 10% in 14 patients. Fluorescence in situ hybridization data were available for 15 patients (Fig. 6 ). The tumors of 15 patients that had not been de nitely diagnosed by immunohistochemistry were diagnosed on the basis of ETV6 gene rearrangement, this being found in all 15 patients.
section_name: Pathological characteristics

section_content: SCSGs are generally low-grade salivary gland carcinomas, and are characterized by morphological resemblance to mammary analogue secretory carcinoma and ETV6-NTRK3 gene fusion [2, 7]. In 2017, SCSGs were added to the WHO classi cation of head and neck tumors [3]. 

The age of onset of SCSGs reportedly varies widely. These tumors usually develop in adults, but can occur in children and adolescents; there is no signi cant sex difference [8]. In the present group, the median age was 45 years (range: 10-69 years) (male:female ratio 1. 3:1). The parotid gland is the most common site of SCSGs; small salivary glands such as the buccal, upper lip, and palatal glands can also be involved [9, 10, 11]. The tumors of 21 of the 23 patients in the present study were located in the parotid gland, 17 being in the right parotid and four in the left parotid gland, differing slightly from that previously reported [12]. SCSGs most commonly present as painless, slow-growing, well-circumscribed masses, and can be misdiagnosed as pleomorphic adenoma or acinar cell carcinoma [13]. Consistent with previous reports, most patients presented after nding a painless mass, with only a few recurrences being sensitive to touch. In the present study, no speci c pre-operative ultrasonography, CT or MRI ndings were identi ed. The tumors presented as well-demarcated masses of uneven density, sometimes with cystic changes, that could easily be misdiagnosed as pleomorphic adenomas. There were only three patients with T3 tumors in our cohort, the others being T1 or T2 tumors. However, T4 tumors have been reported by others [12]. 

Before their recognition, SCSGs were frequently classi ed as ACCs because of the nearly identical histological growth patterns of these tumor types [14]. There are no signi cant clinical differences between SCSGs and ACCs. However, these tumors may show considerable morphological differences histopathologically. Unlike ACCs, SCSGs have no secretory zymogen cytoplasmic granules that show true positive periodic acid-Schiff reactions [15]. The nuclei of SCSGs have prominent pseudo-inclusion bodies and SCSGs do not have the other cell types, such as serous, intermediate, and clear cells, that are seen in ACCs [16]. As to immunophenotypes, SCSGs can express various markers of breast secretory carcinomas, including mammaglobin, S-100 protein, STAT5A, MUC1, MUC4, GCDFP-15, as well as CK7, CK8, CK18, CK19, and epithelial cell membrane proteins [17]. However, they characteristically do not express DOG-1, estrogen, androgen, progesterone receptors, or HER2 [18]. In contrast with SCSGs, ACCs do not express mammaglobin, or express it weakly locally, whereas they express DOG-1 diffusely [18]. Khalele et al. showed that SCSG can be diagnosed when a salivary gland tumor expresses mammaglobin and S-100 protein strongly, and does not express DOG-1 [19]. It has also been reported that if mammaglobin and S-100 are strongly positive, SCSG can be diagnosed without resorting to molecular biological methods [20]. In particular, mammaglobin has become a very important tumor marker for diagnosing SCSGs. 

Similarly to secretory carcinomas of the breast, SCSGs have been shown to have the translocation t(12; 15) (p13; q25), which results in fusion of the ETV6 gene on chromosome 12 with the NTRK3 gene on chromosome 15 [4]. ETV6-NTRK3 gene fusion has not been found in other types of salivary gland tumor. However, it has been found in congenital brosarcoma, mesodermal renal tumor, and acute myeloid leukemia [21, 22]. Recent ndings have expanded the molecular pro le of SCSG to include multiple novel ETV6 fusion partners, including ETV6-RET and EGFR-SEPT14 [23]. Rupture of ETV6 and its fusion with NTRK3 may lead to production of functional tyrosine kinases or affect the signaling pathways of some transcriptional activators, leading to strong expression of mammaglobin and S100, but not of DOG-1 [24]. 

It is therefore considered that detection of ETV6 gene rearrangement is the most speci c method, and therefore the gold standard, for diagnosing SCSGs. Additionally, gene therapy may offer new hope to patients with this tumor in the future [25]. In our study, the tumors of 15 patients that had not been de nitely diagnosed by immunohistochemistry were diagnosed on the basis of ETV6 gene rearrangement, this being found in all 15 cases. Chiosea reviewed 81 cases of ACC for ETV6 uorescence by in situ hybridization and found that all classical ACCs had intact ETV6, whereas 10 of 17 zymogen granule-poor ACCs showed ETV6 translocation; these were accordingly reclassi ed as mammary analog secretory carcinomas [26]. Other differential diagnoses include adenoid cystic carcinoma, mucoepidermoid carcinoma, and salivary ductal carcinoma, all of which can identi ed by immunohistochemistry and ETV6 gene detection. Before immunohistochemistry and ETV6 gene detection had resulted in the correct diagnoses of SCSG being made, four of the present 23 patients' tumors were misdiagnosed as acinar cell carcinomas, three as mucoepidermoid carcinomas, two as adenoid cystic carcinomas, one as low-grade ductal carcinoma, and one as squamous cell carcinoma. Thus, the rate of misdiagnosis with routine pathology only was 47. 8%. 

Because SCSGs behave similarly to ACCs, the treatment of SCSGs is mainly surgical. However, there is currently no consensus on optimal treatment of these tumors. Some researchers believe that SCSGs are indolent tumors that rarely produce distant metastases and that surgery alone is su cient [27]. There have also been reports that surgery combined with postoperative radiotherapy is more effective [28]. In the present study, 11 patients were treated with surgery alone, one of whom had a recurrence postoperatively. Radioactive I 125 implantation was performed under MRI navigation in four patients, none of whom developed recurrences. In addition, one patient received postoperative chemotherapy and later had a recurrence. Four of seven patients who received postoperative chemoradiotherapy had recurrences. The overall recurrence rate was 26. 1%. 

Interestingly, six of the patients with recurrences had undergone their rst surgeries in local primary hospitals. Their recurrences were considered to have resulted from incomplete resection by the rst surgery. There are currently few reports of different surgical procedures for SCSGs. Some studies advocate simultaneous total resection of the affected lobe of the parotid gland and cervical lymph node dissection [29]. Chiosea et al. reported a higher incidence of lymph node metastases from SCSGs than from ACCs (33% vs. 8%); despite this, cervical dissection is still rarely performed [30]. Five of the present 23 patients developed cervical lymphatic metastases and two developed distant metastases (both to the lung) after having local recurrences. None of the patients who had undergone excision of the tumor and super cial lobe of the parotid gland had recurrences or distant metastases. All six patients with local recurrences had only undergone tumor enucleation or partial parotid gland resection, which suggests that preoperative determination of the size and extent of the tumor is vital to the success of surgery. 

It has recently been shown that MRH of salivary gland ducts can clearly establish the three-dimensional relationship between the tumor and parotid duct and detect dilation, stenosis, displacement, and destruction of main ducts and branch ducts, potentially guiding preoperative planning [31]. If the tumor has invaded the main duct, retaining the gland is pointless and super cial or total parotid gland resection should be performed where possible. If the tumor is su ciently distant from the main duct, regional parotid gland resection with preservation of part of the gland can be considered. Thus, detailed MRHs can accurately guide the planning of parotid gland preservation surgery, potentially enabling retention of some functional gland while completely resecting the tumor. In the present study, two patients had undergone preoperative magnetic resonance hydrography and regional resection of the parotid gland without any other postoperative treatment. Thus far, they remain well, their average follow-up time being 26 months. 

The prognosis of SCSG is related not only to the rst operative procedure, but also to age, clinical stage, and Ki-67 proliferation index. Although six of 23 patients in this study had local recurrences (mean age 53 years) and one patient died, the overall prognosis was good, which is consistent with reported results [32]. Additionally, the prognosis is age-dependent. Children and young patients have better prognoses, adult patients having more aggressive tumors. Older patients have the most aggressive tumors and highest recurrence rate [33]. This may be related to differences in pathological features at different ages and the characteristically poor immune function of older patients. Furthermore, previous studies have shown that high clinical stage and high grade are the main adverse prognostic factors, lymph node involvement and extra-parenchymal glandular invasion being associated with a greater risk of local recurrence and metastasis [34]. In the present study, ve patients developed lymph node metastases, all of whom underwent therapeutic neck dissection and postoperative radiotherapy and/or chemotherapy. However, they all had postoperative recurrences, with pulmonary metastases in two of them, one of whom died. Additionally, the tumor of the patient who died had a Ki-67 proliferation index of 50%+ and had recurred three times. This is consistent with reports that patients with a Ki-67 proliferation index of more than 10% have poor prognoses, suggesting that the Ki-67 proliferation index affects the prognosis of secretory carcinomas [35].
section_name: Discussion

section_content: In summary, SCSGs are a rare type of low-grade malignant salivary gland tumor. They commonly occur in the parotid gland, rarely invade surrounding tissues, and have a good prognosis. Histomorphological and immunohistochemical characteristics are the key to distinguishing SCSGs from other salivary gland tumors, detection of ETV6 translocation being the gold standard for their diagnosis. Surgical resection is the main treatment and the thoroughness of the rst operation is the major determinant of prognosis. 

Considering the low rate of cervical lymphatic metastasis, functional excision of SCSGs with facial nerve preservation is generally performed, depending on preoperative MRH ndings concerning salivary gland duct status. When considered indicated, postoperative I 125 implantation or local radiotherapy can be performed to reduce the recurrence rate. If distant metastases occur, neck dissection and postoperative treatment are necessary. Because SCSG is a relatively newly-described tumor type, few clinical ndings have been reported. There were only a small number of patients in our study cohort. Thus, de nitively ascertaining the optimal treatment and outcomes of SCSG still requires large studies and long-term follow-up.
section_name: Conclusions

section_content: SCSG secretory carcinoma of salivary gland SCSGs secretory carcinoma of salivary glands Figures

On
section_name: List Of Abbreviations

section_name: Acknowledgment

section_content: Not applicable.
section_name: Acknowledgment

section_name: References

section_name: Funding

section_content: This was supported by Natural Science Foundation of China ( 81901039 ).
section_name: Funding

section_name: Availability of data and materials

section_content: The datasets analysed during this study are included in this published article.
section_name: Availability of data and materials

section_name: Declarations

section_content: NTRK3 neurotrophic tropomyosin receptor kinase 3 ACC acinic cell carcinoma CT computed tomography MRI magnetic resonance imaging MRH magnetic resonance hydrography

section_content: This was a retrospective study and the protocol of the study was approved by the Medical Ethics Committee of our institution(Ethics Review Number: 2020-KY-230). All patients gave informed consent and signed informed consent forms.
section_name: Ethics approval and consent to participate

section_content: All participants provided informed written consent.
section_name: Consent for publication

section_content: The authors declare that have no competing interests. Authors' contributions JJ. S contributed to the manuscript preparation and performed signi cantly the data analyses and wrote the manuscript; SR. L, K. F, N. G and R. L helped perform the analysis with constructive discussions; W. H and ZJ. G contributed to the conception of the study and manuscript preparation.
section_name: Competing interests