Introduction
Mammary analogue secretory carcinoma (MASC) is a new tumour entity described by Skálová et al. in 2010 [
26] that harbours the recurrent translocation t(12;15)(p13;q25) resulting in the
ETV6-
NTRK3 gene fusion, the same cytogenetic abnormality is described earlier in secretory breast carcinoma [
26,
27]. The fusion gene
ETV6-
NTRK3 encodes a chimeric tyrosine kinase, which has potential transforming activity and plays a major role in oncogenesis [
8]. Conceivably, a small molecular tyrosine kinase inhibitor might be a potential treatment for patients of whom the tumour carries this fusion gene [
8]. The resultant fusion protein
ETV6-
NTRK3 has transforming activity, not only in epithelial but also in mesenchymal and blood cell lineages. Earlier, the
ETV6-
NTRK3 translocation has been described in infantile fibrosarcoma [
16], congenital mesoblastic nephroma and acute myelogenous leukaemia [
8,
16].
ETV6 is genetically unstable and fuses not only with
NTRK3 but also with other genes such as
ABL1,
EGFR3,
PAX5,
SYK and
JAK2 in leukaemia, myelodysplastic syndromes and sarcomas [
8,
27].
Since the seminal paper of Skálová et al. [
26], a number of retrospective studies and case reports have been published. They further characterized the tumour in terms of histopathology and immunohistochemistries [
3,
9,
13,
15,
18,
22] as well as cytology [
6,
14,
19]. However, the number of large clinico-pathological studies with long follow-up data describing the full spectrum of salivary gland tumours that may mimic MASC is very limited. Single studies have re-evaluated historical files of acinic cell carcinomas (AciCC) [
13,
18] or other classical mimickers in the light of this newly emerging entity [
10]. Only a single study of a historical retrospective cohort of the whole spectrum of salivary gland tumours has been published so far [
15].
Histomorphologically, MASC is a distinctive entity [
26], and histology in conjunction with an appropriate immunohistochemical profile is sufficient for a diagnosis in most cases. However, several histomorphological features of MASC overlap with those of other salivary gland tumours [
24,
26,
29]. AciCC and adenocarcinomas/cystadenocarcinomas NOS are the most frequent MASC mimics, followed by low-grade mucoepidermoid carcinoma [
3,
24]. The aim of our study is to describe the morphological and clinical features of MASC in seven patients identified retrospectively from a variety of low- and high-grade malignant epithelial salivary gland tumours.
Materials and methods
We reviewed all the primary carcinomas of major and minor salivary glands (183) resected at the Medical University of Gdańsk (Departments of Otolaryngology and Maxillofacial Surgery) between 1992 and 2012 and reclassified them according to the criteria published by WHO 2005 (HM and AS) [
2] based on histomorphology and immunohistochemistry. In cases suspicious for MASC, fluorescence in situ hybridization (FISH) for detection of
ETV6 rearrangement was performed.
The salivary gland tumour material included adenoid cystic carcinoma, (AdCC, n = 61), mucoepidermoid carcinoma (MEC, n = 25), carcinoma ex pleomorphic adenoma (CXPA, n = 24), acinic cell carcinoma (AciCC, n = 17), adenocarcinoma (n = 14), salivary duct carcinoma (SDC, n = 11), polymorphous low-grade adenocarcinoma (PLGA, n = 7), epithelial-myoepithelial carcinoma (n = 6), basal cell carcinoma (n = 4), undifferentiated carcinoma (n = 3), squamous cell carcinoma (n = 3), myoepithelial carcinoma (n = 2), neuroendocrine carcinoma (n = 2), papillary cystadenocarcinoma (n = 2), lymphoepithelial carcinoma (n = 1) and one case of newly recognized entity of cribriform adenocarcinoma of the tongue and other minor salivary glands (CATS). Based on histomorphology and expression of immunohistochemical markers, seven cases of mammary analogue secretory carcinoma (MASC) were retrieved. The original diagnoses in these cases include AciCC (two cases), adenocarcinoma (two cases), cystadenocarcinoma, MEC and SDC (one case each).
Paraffin blocks and recuts were available for histological and immunohistochemical analysis for all the studied cases. Clinical data and follow-up were obtained from the patients or their physicians (DS, CS).
Detection of ETV6-NTRK3 gene break by FISH
FISH method
For the FISH study, the LSI ETV6 (TEL) Dual Color, Break Apart Rearrangement Probe (VYSIS/Abott, Abott Park, IL) was used. The specimen, a 4-μm-thick FFPE section, was placed onto a positively charged slide. Tissues were deparaffinized in xylene three times for 5 min and then washed twice in 100 % ethanol once in 95 % ethanol and once in deionized water for 5 min. The slides were then heated in the 1× Target Retrieval Solution (pH 6) (DAKO, Glostrup, Denmark) for 40 min at 95 °C and subsequently cooled for 20 min at room temperature in the same solution. The slides were washed in deionized water for 5 min and covered with the Proteinase K (20 mg/ml) (SERVA, Heidelberg, Germany) for 10 min at room temperature. The slides were then placed into deionized water for 5 min, dehydrated in a series of ethanol solution (70, 85 and 96 % for 2 min each) and air-dried. An appropriate amount of FISH probe was applied onto each specimen, which was then covered with a glass cover slip and sealed with rubber cement. The slides were incubated in the ThermoBriteTM instrument (StatSpin/Iris Sample Processing, Westwood, MA, USA) with co-denaturation parameters at 85 °C for 8 min and hybridization parameters 37 °C for 16 h. The rubber cemented cover slips were then removed, and the slides were placed in a post-hybridization wash solution (2xSSC/0.3 % NP-40) at 72 °C for 2 min. The slides were air-dried in the dark, counterstained with DAPI II (VYSIS/Abbott), cover slipped and immediately examined.
FISH interpretation
Hybridized slides were examined with an Olympus BX51 fluorescence microscope using a ×100 objective and as filter sets triple band pass (DAPI/Spectrum Green/Spectrum Orange), dual band pass (FITC/Texas Red) and single band pass (Spectrum Green or Spectrum Orange) filters. One hundred randomly selected non-overlapping tumour cell nuclei were examined for the presence of yellow (normal) or green and red (chromosomal breakpoint) fluorescent signals. The sample was considered positive if more than 10 % of nuclei showed a breakpoint signal. Molecular genetic analysis (RT-PCR and FISH) was performed in Biopticka Laboratory in Plzen, Czech Republic (AK, PS).
Discussion
According to the 2005 WHO Classification of Head and Neck Tumours [
2], the group of malignant epithelial salivary gland tumours contains many heterogeneous entities. The histomorphological classification of these tumours is complex, and their clinical behaviour is not completely elucidated, partly because they are so rare. Some entities, such as adenocarcinoma/cystadenocarcinoma NOS, might encompass subtypes still to be discovered by molecular analysis. Careful histomorphological examination of cases that did not entirely fulfil the criteria of one given entity, in conjunction with a typical pattern of expression of immunohistochemical markers, enabled Skálová et al. [
26] to define mammary secretory analogue carcinoma (MASC) as a new entity. As a consequence, the recognition and differentiation of MASC from other primary salivary gland tumours are essential in order to clarify its histomorphological features and biological behaviour.
Inspired by the original report and reports from other groups [
9,
13,
15,
18,
26], we reviewed primary salivary gland tumours diagnosed in our department and identified seven tumours which met the criteria for MASC. We collected all available clinico-pathological and follow-up data. All cases diagnosed upon revision as MASCs had been diagnosed histologically as malignancy, as adenocarcinoma/cystadenocarcinoma NOS (in three cases), AciCC (two cases) and MEC and SDC (one case each). Fine needle aspiration biopsy (FNAB) results were available in five of seven cases. Two cases had been diagnosed as benign (cyst and adenoma) and in three as malignant lesion.
On cytological smears, MASCs have been reported as variably cellular and with two different architectural patterns: 1) tissue fragments with isomorphic cells arranged in a sheet-like or papillary configuration and 2) dispersed and dissociated cells. Cells contained abundant vacuolated granular and sometimes vacuolated cytoplasm [
6,
14,
22,
23]. Nuclear atypia was mild to moderate. Mucin was present, sometimes abundant or absent. MASC cytology represents considerable overlap with other tumours such as AciCC, MEC, SDC and oncocytoma [
14] and in the differential diagnosis of low-grade salivary gland neoplasms MASC should be included.
The differential diagnosis of MASC should include AciCC, adenocarcinoma NOS, cystadenocarcinoma and low-grade mucoepidermoid carcinoma. One of our cases (no. 5) was originally diagnosed as salivary duct carcinoma (SDC) due to high-grade transformation prevalent in its morphology [
25]. SDC as MASC mimic has not been reported before. Morphologically, the HG component of MASC in both our cases was composed of anaplastic cells with abundant cytoplasm and large polymorphous nuclei arranged in solid structures with focal comedo-like necrosis. In addition, the tumours showed high mitotic activity and invasion of stroma and of peripheral nerves. The high-grade component did not contain colloid- or protein-like material, but the presence of ETV6 rearrangement was confirmed. Immunohistochemistry may be useful to differentiate MASC with high-grade transformation from SDC, which, in contrast to MASC, typically shows expression of androgen receptor or HER-2/neu but not of S100 protein.
The other MASC case was previously diagnosed as mucoepidermoid carcinoma (MEC) with intermediate differentiation, mostly due to focal but unequivocal PAS-positive mucinous differentiation. This feature and variable expression of myoepithelial markers (HMWCK, p63 and CD10) make MEC an important differential diagnosis from MASC [
10,
17]. However, the basal/myoepithelial markers (calponin, p63 and CD10) are usually diffusely and strongly expressed in MEC, while weak and focal in MASC [
17]. Additionally, lack of squamoid areas with intercellular bridges and/or basal-like intermediate cells supports a diagnosis of MASC. Moreover, MEC often (in 38 to 81 % of cases) harbours a t(11;19) translocation resulting in
CRTC1-
MAML2 fusion transcript [
12,
20]. This differs from MASC, which tends to have the t(12;15)(p13;q25) translocation resulting in the
ETV6-
NTRK3 fusion transcript.
The most common mimic of MASC is zymogen granule-poor AciCC [
13,
18]. We also found two cases (out of 17, 12 %) formerly diagnosed as AciCC: one with papillary cystic (case 3) and the other with macrocystic (case 4) growth pattern. AciCC is characterized by a wide variety of architectural patterns, some of which (microcystic, follicular and papillary cystic) need to be differentiated from MASC. Strong and diffuse S100 protein expression and positive mammaglobin staining should favour a diagnosis of MASC [
4,
21].
Adenocarcinoma/cystadenocarcinoma not otherwise specified (ANOS) is a poorly defined entity of otherwise unclassifiable salivary gland carcinoma. Its diagnosis should be made by exclusion of other salivary gland carcinoma types. The differentiation from MASC requires evidence of the
ETV6-
NTRK3 translocation through which many cases diagnosed as ANOS were reclassified as MASC [
3,
5,
9,
13,
15,
24,
29]. Our series contained 14 cases of ANOS, three of which (21 %) were reclassified as MASC.
We performed both FISH and RT-PCR for molecular genetic analysis. In our study, on FISH, all but one case (6/7) was positive for ETV6 gene rearrangement. In the cystic tumour with delicate cellular lining (case 1), the neoplastic material was very limited and, thus, insufficient for analysis. By RT-PCR, only three out of seven cases were positive for the t(12,15) (
ETV6-
NTRK3) fusion transcript. Petersson et al. proposed as possible explanation for negative RT-PCR results a different fusion partner for the
ETV6 gene [
22]. In haematopoietic malignant disorders,
ETV6 fusions other than with
NTRK3 have been described with
ABL1,
RUNX1 or
FLT3 [
9]. Moreover, the
ETV6-
NTRK3 fusion is not found in 100 % of secretory carcinomas of the breast [
22].
The majority of MASC arose in the parotid gland, followed by the oral cavity (lip, soft palate and buccal mucosa) and submandibular gland [
5,
26]. Of our cases, all but one (85 %) developed in the parotid gland. The remaining tumour arose in a small salivary gland of the hard palate. The male predominance in our series (2.5:1) is more prominent than in other reports which found MASC to be only slightly more common in males (53 %) [
3]. Age varied widely in our cases from 17 to 73 years (median 51.4) corresponding to earlier data (range from 14 to 78 years) [
3]. The size of MASC ranged from 0.2 to 5.5 cm [
3,
6,
9,
13‐
15,
18,
19,
22,
26,
29]. In our series, the smallest tumour (2.0 cm) was located in the hard palate (case 7), whereas others ranged from 2.2 to 4 cm. This is consistent with data from the literature in that MASC in the oral cavity tends to be smaller (mean 0,9 cm) than in the parotid gland (mean 2, 2 cm) [
3].
The limited number of cases of MASC with full clinical correlation and follow-up data precludes assessment of its prognosis and response to treatment. Although MASC is currently treated as a low-grade carcinoma with overall favourable prognosis, it has potential for regional lymph node metastasis. In cases with positive surgical margins [
9,
26], the tumour often recurs locally, and therefore, adjuvant radiotherapy is recommended. Two of our patients with MASC with high-grade transformation (cases 5 and 6) died of neoplastic disease, one with distant metastasis 20 and 79 months after primary surgery. MASC has a capacity for an aggressive course, and the
ETV6-
NTRK3 translocation might provide a potential therapeutic target [
9].
In conclusion, MASC is a morphologically and molecularly well-defined salivary gland neoplasm. MASC may share microscopic features with AciCC, adenocarcinoma/cystadenocarcinoma NOS and low-grade MEC. In rare cases, MASC with high-grade transformation may morphologically mimic high-grade salivary gland malignancies, such as salivary duct carcinoma.