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Fine needle aspiration cytology of mammary analogue secretory carcinoma of the parotid.

INTRODUCTION

Mammary analogue secretory carcinoma (MASC) is a recently classified entity with unique genetic characteristics of a ETV6-NTRK3 fusion gene, identical to that found in secretory breast carcinoma (1-9). MASC also shares immunohistochemical and histologic features with secretory breast carcinoma (10). Both tumours are strongly positive for S100 protein, vimentin, mammaglobin and cytokeratin markers (10). Histologically both are composed of neoplastic cells arranged in solid, microcystic and papillary patterns associated with intraluminal secretions. These features overlap with those of other salivary gland neoplasms such as pleomorphic adenoma, low-grade mucoepidermoid carcinoma, myoepithelial carcinoma, low-grade cribriform cystadenocarcinoma, oncocytic carcinoma, salivary duct carcinoma, and acinic cell carcinoma.

In this report, we review the cytohistologic features, immunohistochemical and molecular profile of MASC, emphasising those useful in making the diagnosis and distinguishing MASC from the other salivary gland tumours.

CASE REPORT

A 38 year old male presented with a slow growing solitary (11 mm diameter) mass within the superficial aspect of the left parotid gland. There were no other surrounding lesions. The remaining salivary glands were normal. There was no cervical lymphadenopathy and CT imaging showed no abnormality in the pharynx, skull base, larynx, thyroid gland, axilla, mediastinum, lung, liver, or abdomen. Fine needle aspiration (FNA) of the left parotid mass was performed to support the clinical impression of a pleomorphic adenoma. Based on an FNA diagnosis of malignancy a superficial left parotidectomy was performed and subsequently a left preauricular and left neck lymph node excision for diagnostic clarity of tumour type.

MATERIALS AND METHODS

The FNA sample was collected in Surepath preservative (BD Diagnostics TriPath, Burlington, NC) for liquid-based thin-layer preparation. The aspirate sample was spun at 600 ref for 5 minutes. From the sediment, a Surepath slide was prepared and stained (Papanicolaou) using the SurePath Prepstain machine. The remainder of the sediment was used to make a cell block, fixed in 10% formalin, embedded in paraffin, routinely processed and stained with Hematoxylin-Eosin (H & E).

Immunohistochemical studies were carried out on the cell block sections using antibodies to CK AE1/AE3, CK5/6, Cam 5.2, CK7, S100, Melan A, HMB45, EMA, vimentin, CEA and Alpha 1AT. PAS and PASD cytochemistry were also performed on cell block sections.

Immunohistochemical studies were carried out on the biopsy sections using antibodies to CK8/18, CK7, S100, SOX10, CK5/6, GAT A3, GCDFP-15, CD117, p40, SMA, AR, ER, PR, DOG1, Her2. Mucicarmine cytochemistry staining was also performed on histology sections.

RESULTS

Cytological findings

The Surepath and cell block preparations were hypercellular including macrophages, groups of normal salivary gland acinar cells, many dissociated and clustered uniform columnar to polygonal cells with basophilic cytoplasm, round to oval bland nuclei with fine chromatin, and inconspicuous nucleoli. Some cells with plasmacytoid appearances were present. In addition, there were admixed groups of polygonal tumour cells with abundant cytoplasm showing high-grade nuclear feature including enlarged pleomorphic nuclei, abnormal chromatin, and centrally located prominent nucleoli. There were prominent blood vessels in the background, occasional vessels traversing the tumour cell groups (Figures 1a and 1c). Occasional cells demonstrated intracytoplasmic vacuole formation, or intranuclear inclusions (Figure 1b), and focal cytoplasmic haemosiderin pigment mimicking melanin. No matrix material or stromal tissue was present. Based on morphologic features, an initial diagnosis of metastatic malignant melanoma was favoured and immunohistochemical stains were requested on the cell block material.

Immunohistochemical findings

Immunohistochemical staining on the cell block material showed the tumour cells to be positive for vimentin, CKAE1/ AE3, EMA, CAM 5.2 and CK7. They were focally positive for CK5/6 and weakly positive for S100 protein. The tumour cells were negative for Melan A, HMB45, SMA, A1AT, GFAP, CD117 and CEA. The basophilic mucin stained positive with Periodic Acid Schiff and Periodic Acid Schiff Diastase (Figure 1d). Based on the initial immunophenotype, a diagnosis of squamous cell carcinoma was favoured.

Histologic findings

On gross examination the tumour was ovoid firm, tan-yellow in colour, 11 x 11 x 8 mm in size. H & E stained sections showed parotid gland parenchyma with parotid-associated lymphoid tissue surrounded by fibroadipose tissue. The parotid was subtly infiltrated by a well circumscribed but non-encapsulated neoplasm. The tumour had a solid, largely microcystic, pattern (Figure 2a) associated with evident basophilic mucin. The tumour cells showed ovoid nuclei, discernible nucleoli, occasional intranuclear inclusions, scant mitotic activity, and amphophilic cytoplasm. Similar to the cytology sample there were admixed large neoplastic cells with pleomorphic nuclei and evident nucleoli. The neoplastic nodules were variably surrounded by a conspicuous rim of basal cells and set within a fibroconnective matrix. Fibrosis, chronic inflammation and hemosiderin deposition was identified. No tumour was identified in the lymph node sampling.

Immunohistochemical staining showed the lesional cells were positive for CK 8/18, CK7, S100, GATA3 (diffusely weak), SOX10 (Fig 2c, 2d), CK 5/6, p40 (patchy) and were negative for GCDFP-15, CD117, SMA, AR, ER, PR, Her2 and DOG1. The proliferative index by KI-67 staining was 20%. The rim of basal cells expressed P40 and CK5/6. The basophilic mucin weakly stained with mucicarmine stain (Figure 2b).

Molecular analysis

ETV6 (NTRK 3) analysis was performed at IGENZ, Auckland on formalin-fixed paraffin-embedded tissue sections. Fluorescence in-situ hybridization (FISH) testing revealed gene rearrangement of ETV6 (12p13) in all 100 cells examined (Figure 3).

DISCUSSION

Mammary analogue secretory carcinoma (MASC) is a recently classified tumour first described in 2010 by Skalova et al. which harbours a t(12;15) (p13;q25) translocation, resulting in the ETV6-NTRK3 gene fusion (2, 4, 9). The same cytogenetic abnormality is described in secretory carcinoma of the breast (SCB) (10). The resultant fusion protein ETV6-NTRK3 has transforming activity, not only in epithelial but also in mesenchymal and blood cell lineages (7). Previously, the ETV6-NTRK3 translocation has been described in infantile fibrosarcoma (11), congenital mesoblastic nephroma, and acute myelogenous leukaemia (2, 7, 8). ETV6 is genetically unstable and fuses not only with NTRK3 but also with other genes, including ABL1, EGFR3, PAX5, SYK and JAK2 in leukaemia, myelodysplastic syndromes, and sarcomas (7). Therefore MASC and SCB also share immunohistochemical features including positivity for S100 protein, EMA, and vimentin, while being (ER, PR, Her2) "triple negative" (13).

MASC is more common in males, with a male: female ratio of 1.5: 1 occurring predominantly in the parotid gland followed by the oral cavity (lip, soft palate and buccal mucosa), and submandibular gland (3-8, 12, 13). The lesion typically presents as a painless, slow growing mass. Grossly, all the tumours are circumscribed mimicking salivary gland adenoma (5).

On cytological preparation, MASC have been reported as variably cellular with two different architectural patterns, including sheets of tumour cells arranged in papillary, cystic, tubular, solid growth arrangements (4, 5, 12, 13), and dispersed dissociated cells. These cells contained abundant cytoplasm and round nucleus pushed to periphery giving a plasmacytoid appearances with nuclear atypia and pleomorphism. Tumour cells with intracytoplasmic or intraluminal mucin have been reported to be positive with PAS (diastase resistant) and Mucicarmine (3, 6, 13). Our case contained many abnormal dissociated cells with plasmacytoid appearances, multinucleation, predominant nucleoli, intranuclear inclusions, and pigment, which raises the possibility of metastatic melanoma. In our case melanoma markers were negative, but cells expressed cytokeratins AE1/AE3 and CK5/6 suggesting squamous differentiation. We did not perform more immunohistochemistry and suggested histological assessment for further classification.

The cytopathologic differential diagnosis of MASC includes many low-grade epithelial neoplasms including pleomorphic adenoma, low-grade mucoepidermoid carcinoma, acinic cell carcinoma, myoepithelial carcinoma, low-grade cribriform cystadenocarcinoma, oncocytic carcinoma, and salivary duct carcinoma (2-9).

Pleomorphic adenoma and MASC contain bland cells and vacuolated cells but we did not observe typical chondromyxoid stroma and fibrillary extracellular matrix admixed with spindle myoepithelial cells, as seen in pleomorphic adenoma. Additionally, the tumour cells were negative for GFAP making pleomorphic adenoma unlikely. Low-grade mucoepidermoid carcinoma and MASC share the features of hypocellular samples, vacuolated cytoplasm, macrophages, and proteinaceous material in the background. MASC does not have the intimate mixture of epidermoid cells, intermediate cells and mucinous cells that characterise mucoepidermoid carcinoma. Also, a lack of squamoid areas with intercellular bridges and/or basal like intermediate cells support a diagnosis of MASC. Mucoepidermoid carcinoma often harbours at (11;19) translocation resulting in CRTC1-MAML2 fusion transcript (3).

Acinic cell carcinoma may be distinguished by DOG1 positivity. Myoepithelial carcinoma will show positivity for smooth muscle actin. Oncocytic carcinoma, salivary duct carcinoma, and mucoepidermoid carcinoma all are SOX10 negative. Metastatic clear renal cell carcinoma could be considered, given the low-grade appearance with clear cells and histiocyte-like cells observed in MASC. Additionally, CD10 and PAX8 immunostains on cell block material would help to resolve the differential diagnosis.

GCDFP-15 or BRST-2 has variable staining strength. In the study by Skalow et al. eight out of eleven samples were positive, while others have found positive GCDFP-15 staining restricted to a minority of cases (14). Our case was negative for GCDFP-15. The proliferative index of our case is in keeping with the majority of studies which demonstrate a low proliferation index by Ki67 staining (5-28%) (14). Most studies have detected negative staining for calponin, CK15, smooth muscle actin, ER, PR, HER2, and CK5/6 (14). Our case was positive for CK5/6 and a weak expression of GATA3. p63 is generally negative in tumour cells; however, Chiosa et al. found that 28% of MASC cases had positive staining for peripherally located entrapped non-tumour basal cells, which could represent intraductal extension or possibly a ductal epithelial origin for this tumour (14, 15).

CONCLUSIONS

We have presented the typical cytologic features of a relatively recently recognised salivary gland tumour which displays cytological, histologic, immunophenotypic, and genetic features similar to secretory breast carcinoma. MASC and secretory breast carcinoma share a t (12;15) (p13; q25) translocation and both are positive for S100 protein and GATA3, but with a "triple negative" (ER/PR/Her2) phenotype. FNA is a common investigation in the workup of salivary gland tumours and it is important to recognise the cytoarchitectural features of this tumour to avoid confusion from other primary or secondary tumours at these sites. Failure to appreciate these features initially led us to an erroneous diagnosis of metastatic squamous cell carcinoma and the true diagnosis was revealed only on subsequent definitive surgery.

ACKNOWLEDGEMENT

The authors acknowledge Louise Goossens for her excellent photographic assistance.

AUTHOR INFORMATION

Sharda Lallu, BSc CT(FIAC), Cytotechnologist

Sarla Naran, BSc CT(FIAC), Cytotechnologist

Peter Bethwaite, MBChB PhD FFSc(RCPA) FRCPA, Cytopathologist

Department of Anatomic Pathology, Wellington Southern Community Laboratories

Author for correspondence: Dr Peter Bethwaite

Email: Peter.Bethwaite@wellingtonscl.co.nz

REFERENCES

(1.) Skalova A, Vanecek T, Sima R, Lako J, Weinreb I, Perez-Ordonez B, et al. Mammary analogue secretory carcinoma of salivary glands containing the ETV6-NTRK3 fusion gene: a hitherto undescribed salivary gland tumour entity. Am J Surg Pathol 2010; 34: 599-608.

(2.) Bishop JA, Yonescu R, Batista DA, Westra WH, Ali SZ. Cytopathologic features of mammary analogue secretory carcinoma. Cancer Cytopathol 2013; 121: 228-233.

(3.) Levine P, Fried K, Krevitt LD, Wang B, Wenig BM. Aspiration biopsy of mammary analogue secretory carcinoma of accessory parotid gland: another diagnostic dilemma in matrix-containing tumors of the salivary glands. Diagn Cytopathol 2014; 42: 49-53.

(4.) Takeda M, Kasai T, Morita K, Takeuchi M, Nishikawa T, Yamashita A, et al. Cytological features of mammary analogue secretory carcinoma--review of literature. Diagn Cytopathol 2015; 43: 131-137.

(5.) Oza N, Sanghvi K, Shet T, Patil A, Menon S, Ramadwar M, et al. Mammary analogue secretory carcinoma of parotid. Is preoperative cytological diagnosis possible? Diagn Cytopathol 2016; 44: 519-525.

(6.) Bajaj J, Gimenez C, Slim F, Aziz M, Das K. Fine--needle aspiration cytology of mammary analogue secretory carcinoma masquerading as low-grade mucoepidermoid carcinoma: case report with a review of the literature. Acta Cytol 2014; 58: 501-510.

(7.) Majewska H, Skalova A, Stodulski D, Klimkova A, Steiner P, Stankiewicz C, et al. Mammary analogue secretory carcinoma of salivary glands: a new entity associated with ETV6 gene rearrangement. Virchows Arch 2015; 466 : 245-254.

(8.) Pinto A, Nose V, Rojas C, Fan YS, Gomez-Fernandez C. Searching for mammary analogue secretory carcinoma of salivary gland among its mimics: Mod Pathol 2014: 27: 30-37.

(9.) Salat H, Mumtaz R, Ikram M, Din NU. Mammary analogue secretory carcinoma of the parotid gland: A third world country perspective-a case series. Case Rep Otolaryngol 2015; 2015: 697254.

(10.) Skalova A. Mammary analogue secretory carcinoma of salivary gland origin: an update and expanded morphologic and immunhistochemical spectrum of recently described entity. Head Neck Pathol 2013; 7 Suppl 1: S30-S36.

(11.) Bourgeois JM, Knezevich SR, Mathers A, Sorensen PH. Molecular detection of the ETV6-NTRK3 gene fusion differentiates congenital fibrosarcoma from other childhood spindle cell tumors. Am J Surg Pathol 2000; 24: 937-946.

(12.) Stevens TM, Kovalovsky AO, Velosa C, Shi Q, Dai Q, Owen RP, et al. Mammary analogue secretory carcinoma, low-grade salivary duct carcinoma, and mimickers: a comparative study. Mod Pathol 2015; 28: 1084-1100.

(13.) Jackson BS, Pratt TL, Van Rooyen A. Mammary analogue secretory carcinoma: a rare salivary gland tumour. S Afr Med J 2017; 107: 304-306.

(14.) Sethi R, Kozin E, Remenschneider A, Meier J, VanderLaan P, Faquin W, et al. Mammary analogue secretory carcinoma: update on a new diagnosis of salivary gland malignancy. Laryngoscope 2014; 124: 188-195.

(15.) Chiosea SI, Griffith C, Assaad A, Seethala RR. Clinicopathological characterization of mammary analogue secretory carcinoma of salivary glands. Histopathology 2012; 61: 387-394.

Sharda Lallu, Sarla Naran and Peter Bethwaite

Department of Anatomic Pathology, Wellington Southern Community Laboratories

Caption: Figure 1a. Surepath preparations showing groups of cells with low-grade and high-grade nuclear features and blood vessels traversing the tumour cell groups (Papanicolaou stain x 400).

Caption: Figure 1b. Surepath preparations showing rare cells with intranuclear inclusions (Papanicolaou stain x 400).

Caption: Figure 1c. Cell block preparations showing groups of cells with low-grade and high-grade nuclearfeatures (Haematoxylin-eosin stain x 400).

Caption: Figure 1d. Histochemical stain on cell block section, cells showing positive staining for PASD (PASD stain x 400).

Caption: Figure 2a. Section of parotidectomy specimen showing the tumour with microcystic pattern, admixed low-grade, high-grade nuclearfeatures (Haematoxylin-eosin stain x 200).

Caption: Figure 2b. Histochemical stains on sections of parotidectomy specimen, secretions within cystic space showing positive staining for mucicarmine (Mucicarmine stain x 200).

Caption: Figure 2c. Immunohistochemical stain on section of parotidectomy specimen, tumour cells, showing nuclear positive staining for GATA3 (GATA3 x 200).

Caption: Figure 2d. Immunohistochemical stain on section of parotidectomy specimen, tumour cells showing nuclear positive stains for SOX10 (SOX10 x 200).

Caption: Figure 3. ETV6 FISH analysis for the 12q13 cytogenetic location showing positive translocation.
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Title Annotation:CASE STUDY
Author:Lallu, Sharda; Naran, Sarla; Bethwaite, Peter
Publication:New Zealand Journal of Medical Laboratory Science
Article Type:Report
Date:Apr 1, 2018
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