Mammary analogue secretory carcinoma of salivary glands is a lipid-rich tumour, and adipophilin can be valuable in its identification

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1 Histopathology 2013, 63, DOI: /his Mammary analogue secretory carcinoma of salivary glands is a lipid-rich tumour, and adipophilin can be valuable in its identification Fernanda Viviane Mariano, Harim Tavares dos Santos, 1 Wilson Delgado Aza~nero, 2 Isabela Werneck da Cunha, 3 Claudia Malheiros Coutinho-Camilo, 3 Oslei Paes de Almeida, 1 Luiz Paulo Kowalski 4 & Albina Altemani Department of Pathology, Medical Sciences Faculty, University of Campinas (UNICAMP),Campinas, Brazil, 1 Department of Oral Pathology, Piracicaba Dental School, University of Campinas (UNICAMP), Piracicaba, Brazil, 2 Department of Oral Pathology, Medicine and Surgery, Peruvian University Cayetano Heredia, Lima, Peru, 3 Department of Pathology, AC Camargo Hospital, S~ao Paulo, Brazil, and 4 Department of Head and Neck and Otorhinolaryngology, AC Camargo Hospital, S~ao Paulo, Brazil Date of submission 27 March 2013 Accepted for publication 15 May 2013 Published online Article Accepted 17 May 2013 Mariano F V, dos Santos H T, Aza~nero W D, da Cunha I W, Coutinho-Camilo C M, de Almeida OP, Kowalski L P & Altemani A (2013) Histopathology 63, Mammary analogue secretory carcinoma of salivary glands is a lipid-rich tumour, and adipophilin can be valuable in its identification Aims: Mammary analogue secretory carcinoma (MASC) of salivary glands shows morphological similarities to milk-secreting mammary epithelial. The aim of this study was to analyse the immunohistochemical expression of adipophilin (a component of milk lipid globule membranes) and of proteins related to secretory mechanisms (STAT5a and mammaglobin) in MASC and other salivary tumours. Methods and results: Ten cases of MASC (all with ETV6 translocation) and 83 other salivary carcinomas were studied. In all MASC cases, adipophilin stained numerous large lipid droplets. These droplets were minute in other salivary carcinomas, except for sebaceous carcinoma. Overexpression of STAT5a was Keywords: adipophilin, lipid-rich tumour, mammary analogue secretory carcinoma detected in all MASC cases, but only occasionally in other carcinomas. Mammaglobin expression occurred frequently in MASC (70% of cases), whereas, in other carcinomas, it was uncommon and limited. Only MASC showed cytoplasmic reactivity for p63, particularly in papillary cystic areas. Positivity for S100, vimentin and high molecular weight keratin was observed in 100% of MASC cases. Conclusions: MASC is a lipid-rich tumour containing large lipid droplets covered by adipophilin. This finding can be included among its defining immunohistochemical features, and possibly represents lactation-like secretory differentiation. Strong expression of STAT5a and cytoplasmic p63 in MASC reinforces this hypothesis. Introduction Mammary analogue secretory carcinoma (MASC) of salivary glands is a recently described entity that Address for correspondence: A Altemani, Department of Pathology, Faculty of Medicine, State University of Campinas (UNICAMP), R. Tessalia Vieira de Camargo, 126 Campinas Campinas, SP, Brazil, CEP: aaltemani@uol.com.br shows microscopic, immunohistochemical and molecular features similar to those of mammary secretory carcinoma (SC). 1 Both lesions characteristically harbour an identical balanced translocation, t(12;15) (p13;q25), leading to an ETV6 NTRK3 fusion oncogene, and are considered to be low-grade tumours. 1,2 Morphologically, MASC shows overlap with other salivary tumours, namely acinic cell carcinoma (AciCC), adenocarcinoma/cystadenocarcinoma, not 2013 John Wiley & Sons Ltd.

2 Mammary analogue secretory carcinoma 559 otherwise specified (NOS), and mucoepidermoid carcinoma. 2,3 The former is the main differential diagnosis of MASC, and the separation of MASC from AciCC seems to be relevant 1,4 ; MASC appears to have a higher rate of nodal metastases than AciCC, and a predilection for males. 4 Immunohistochemically, S100 positivity and vimentin positivity are defining features of MASC. 1 In addition, all cases have been found to show diffuse and strong staining for CK7, CK8, and CK18, and most of them for mucins such as MUC1 and MUC4. 1 MASC is also consistently immunoreactive for the transcription factor STAT5a (signal transducer and activator of transcription 5a) and for mammaglobin, proteins involved in cell secretory mechanisms. 1,2 However, expression of these proteins in other salivary tumours has yet to be evaluated. Recently, it has been shown that MASC can exhibit a wider morphological and immunohistochemical spectrum than previously recognized. 1,3 Papillary cystic architecture, focal mucinous differentiation and positivity for high molecular weight keratins (HMWKs) and basal/myoepithelial markers (p63 protein, calponin, and CD10) were features added to those of the original description. 3,5 Absence of S100 expression was observed in a few cases. 3 Up to now, the definitive diagnosis of MASC has relied on molecular studies of the ETV6 NTRK3 fusion. Nevertheless, refinement of the immunohistochemical panel for the diagnosis of MASC has the potential to reduce this need. Because tumour of MASC and breast SC show morphological similarities to milk-secreting mammary epithelial, we hypothesized that MASC could present lactation-like secretory differentiation. Like milk-secreting mammary, MASC and breast SC often show both intracytoplasmic vacuolation and abundant milk-like secretory material. 1,2,6 In line with this idea, we looked for a protein that is expressed in milk-secreting mammary epithelial and could be detected by a monoclonal antibody in paraffin tissues. Adipophilin (a lipid droplet coat protein) fulfils these criteria. It is a component of milk lipid globule membranes, and is highly expressed in milk-secreting mammary. 7 Perilipin, adipophilin, and tail-interacting protein of 47 kda (TIP47) form the PAT family, which is crucial for the formation, maintenance, modification and involution of intracellular lipid droplets. 8 In salivary tumours, this protein has been studied only in lesions with sebaceous differentiation to detect lipid droplets. 9,10 Drawing from this background, the aims of this study were two-fold: (i) to verify whether the pattern and intensity of adipophilin expression could be useful in distinguishing MASC from AciCC and other salivary tumours; and (ii) to analyse the extent and intensity of expression of secretion-related proteins (STAT5a and mammaglobin) in MASC and other salivary tumours. In addition, we investigated whether the recently described morphological and immunohistochemical features of MASC were present in the current series. Materials and methods TUMOURS AND TISSUES This study was approved by the Institutional Ethics Committee. The surgical pathology archives of the Hospital of the University of Campinas (UNICAMP) and AC Camargo Hospital, S~ao Paulo, Brazil were reviewed, and contained 30 tumours that had been diagnosed between 1990 and 2012 as AciCC, and that had available slides and/or blocks. These tumours were reclassified, according to Chiosea et al. 4, as follows: 14 cases of classic AciCC [with discrete unequivocal serous acinar differentiation by either haematoxylin and eosin (H&E) stain or periodic acid Schiff reaction after diastase treatment (PAS-D)]; and 16 cases of zymogen granule-poor carcinomas (without definite serous acinar differentiation, and with some or all of the morphological features of MASC). For comparison, 67 cases of salivary carcinoma classified as adenocarcinoma/cystadenocarcinoma NOS (A/C NOS), adenoid cystic carcinoma (ACC), clear cell carcinoma (CCC), epithelial myoepithelial carcinoma (EMC), low-grade cribriform cystadenocarcinoma (LGCC), myoepithelial carcinoma (MC), mucoepidermoid carcinoma (MEC), polymorphous low-grade adenocarcinoma (PLGA) and salivary duct carcinoma (SDC) were retrieved from the surgical pathology archives of the Hospital of the UNICAMP. For the analysis of these carcinomas, a pan-tumour tissue microarray (TMA) was built. Three tissue cores of diameter 1 mm were punched (Tissue- Tek Quick-Ray Tissue Microarray System; Sakura, California, USA) as representative of the whole tumour in each case, and placed into receptor blocks of the TMA. Owing to the absence of sebaceous carcinoma in the TMA, two cases of such tumours were also included in the study, and analysed in whole tissue sections. Clinical details and follow-up data were obtained from medical records. IMMUNOHISTOCHEMISTRY Table 1 shows the antibodies that were used in the current series. Briefly, 5-lm sections from the paraffin

3 560 F V Mariano et al. Table 1. Antibodies used in this study Antigen Clone Source Dilution Adipophilin AP125 Fitzgerald Prediluted STAT 5a E289 Epitomics 1:250 Mammaglobin 31A5 Bio SB 1:250 p63 4A4 Dako 1:400 HMWK Cytokeratin Dako 1:100 Vimentine V9 Dako 1:100 S100 Polyclonal Dako 1:100 HMWK, High molecular weight cytokeratin. Manufacturers: Fitzgerald (MA, USA); Epitomics (California, USA); Bio SB (California, USA); Dako (Glostrup, Denmerk). blocks were deparaffinized and hydrated, and endogenous peroxidase activity was quenched by immersing the slides in 3% hydrogen peroxide. Antigen retrieval was achieved by boiling in a steamer, immersed in Tris-EDTA buffer (ph 8.9, 30 min) for mammaglobin and p63, and in citrate buffer (ph 6.0, 30 min) for STAT5a, vimentin, HMWK, and S100. The adipophilin antibody does not require antigen retrieval. Subsequently, the sections were incubated overnight at 14 C with the primary antibody, and then with EnVision polymer HRP and EnVision+ (code K1491; Dako, Glostrup, Denmark) for 1 h at 37 C. Sections were stained for 5 min at 37 C with 3,3 -diaminobenzidine tetrahydrochloride, and counterstained with haematoxylin. Negative controls were run with the omission of primary antibodies. The relative numbers of positive for the different antibodies were considered in relation to all neoplastic observed in each stained section. When the proportion of positive was >10%, the case was judged to be positive. The proportion of positive neoplastic was assessed according to a two-tiered scale: >10% to 50%, and >50% positive. The staining intensity for each antibody was subjectively evaluated as weak/moderate (+) or intense (++). For STAT5a, only nuclear staining was considered to be positive, because, when activated, STAT5a is translocated from the cytoplasm to the nucleus. 11 ETV6 FLUORESCENCE IN-SITU HYBRIDIZATION (FISH) ETV6 FISH with a break-apart ETV6 probe (Abbott- Vysis, Illinois, USA) was performed on sections from the paraffin blocks, according to the manufacturer s recommendations. Briefly, deparaffinized and hydrated sections were incubated, first at room temperature for 20 min, and then at 82 C for 30 min, in 0.2 mmol/l HCl, for 2 min in distilled water, and for 3 min in 2 9 citrate buffer. Thereafter, slides were subjected to protease digestion, immersion in 10% buffered formalin for 10 min, dehydration, and incubation with the probes, first at 75 C for 5 min, and then at 37 C overnight. On the following day, slides were rinsed in post-hybridization solution at 74 C for 3 min, and nuclei were counterstained with DAPI. Slides were analysed by use of a fluorescence microscope (BX61; Olympus, Center Valley, PA, USA), and images were captured using a Q-Color 5 Olympus digital camera. For each case, 50 were counted, and a case was considered to be positive for ETV6 break when at least 20% of showed splitting of the signals. In the zymogen granule-poor AciCC group, 12 cases were tested for the ETV6 translocation; this was technically successful in 10 tumours (all were positive) and failed in two. As a control group, eight classic AciCCs were tested for the ETV6 translocation. Results TUMOUR CLASSIFICATION AND CLINICOPATHOLOGICAL FEATURES The ten cases without definite serous acinar differentiation and with ETV6 translocation were classified as MASC. The other zymogen granule-poor carcinomas (six cases), where FISH failed (two cases) or was not performed, were not evaluated in this study. In the MASC group, the age range was years (mean, 42.8 years); six patients were male, two were female, and gender was not available for two. All tumours but one occurred in major salivary glands. Seven tumours showed papillary cystic architecture, and all showed some microcystic, tubular and solid patterns as well. In all cases, PAS-positive secretion within the microcystic and tubular spaces was observed, and neoplastic showed low-grade nuclei and granular pink vacuolated cytoplasm (Figure 1). Mucinous differentiation was not identified. Fourteen tumours were classified as AciCC because they showed unequivocal serous acinar differentiation; five of these tumours showed no ETV6 translocation, and FISH failed in three cases. In the AciCC group, the age range was years (mean, 49.7 years); six patients were female, five were male, and gender was not available for three. All tumours occurred in major salivary glands. All showed a solid pattern with some

4 Mammary analogue secretory carcinoma 561 A B C D Figure 1. Mammary analogue secretory carcinoma. A, This tumour shows a microcystic growth pattern with abundant bubbly or homogeneous secretion in the cystic spaces (H&E). B, The tumour show eosinophilic or vacuolated cytoplasm (detail in inset). C, Note the absence of cytoplasmic PAS-positive granules. D, ETV6 fluorescence in-situ hybridization showing one fused (yellow) signal and one split (red and green) signal indicative of ETV6 translocation. microcystic or papillary microcystic architecture. TMA cases of salivary carcinomas comprised 17 ACCs, three adenocarcinoma/cystadenocarcinomas NOS, three CCC, one LGCC, six MCs, 13 EMCs, six PLGAs, 14 MECs, and four SDCs. IMMUNOHISTOCHEMICAL FINDINGS Adipophilin expression was not found in normal salivary parenchyma, except for occasional duct with sebaceous differentiation. Among the salivary carcinomas, adipophilin expression was diffuse and strong in all MASC cases and in one of two sebaceous carcinoma cases. Only in MASC and sebaceous carcinoma did adipophilin surround large droplets located in the cytoplasm of neoplastic (Figure 2). In MASC, this protein was not observed in the secretory material. In AciCC, as well as in other types of salivary carcinoma, adipophilin surrounded minute cytoplasmic droplets, which were present in variable proportions of cases and usually in a patchy staining pattern (Table 2; Figure 2). STAT5a expression was often found in epithelial of the intercalated ducts. Few of the acini and striated ducts were STAT5a-positive. Among the salivary carcinomas, all MASC cases showed diffuse STAT5a expression, and the intensity of staining was stronger than that in normal (Figure 3). In other salivary carcinomas, STAT5a was detected in focal areas and/or with an intensity of expression similar to that in normal (Figure 3), except for few cases in which its expression was intense and diffuse. Table 2 shows the extent and intensity of STAT5a staining of other salivary tumours.

5 562 F V Mariano et al. A B C D Figure 2. Immunohistochemical expression of adipophilin. A, Absence of expression in salivary parenchyma. B, Adipophilin surrounds large cytoplasmic droplets in mature sebocytes of sebaceous carcinoma. C, Numerous large droplets covered by adipophilin in mammary analogue secretory carcinoma tumour (detail in inset). D, Minute cytoplasmic droplets in several tumour of acinic cell carcinoma. Mammaglobin expression was not detected in normal salivary glands. In the MASC group, mammaglobin expression was seen in tumour in seven of 10 cases (70%); it was strong in all cases, and in four of seven (57.1%) showed a focal pattern (Figure 3). Mammaglobin was negative in other salivary carcinomas, except for one EMC and three PLGAs (Table 2; Figure 3). However, in these tumours, the proportion of positive was small (<20%). Immunoreactivity for S100 protein, vimentin, HMWK and p63 was examined in MASC and AciCC but not in other salivary carcinomas (Table 3). In MASC, S100, vimentin and HMWK expression was strong and diffuse in all cases (Figure 4), except for two cases with focal expression of vimentin and HMWK (one case each). Cytoplasmic but not nuclear expression of p63 was observed in tumour of MASC (Figure 3). This cytoplasmic expression was strong, and usually seen in areas with papillary cystic architecture. In AciCC, S100 expression and vimentin expression comparable with that of MASC (diffuse and strong) were found in three and one cases, respectively (Figure 4). Expression of HMWK and p63 was not found in ACiCC. Discussion In the current study, we showed that, like milksecreting mammary, all cases of MASC showed diffuse and strong staining for adipophilin. As adipophilin staining mirrors the lipid droplet accumulation in normal and neoplastic, 12 our findings also indicate that MASC is a lipid-rich tumour. Furthermore, the adipophilin staining pattern was useful in separating MASC from other salivary carcinomas. In

6 Mammary analogue secretory carcinoma 563 Table 2. Immunoreactivity for adipophilin, STAT 5a and mammaglobin in salivary carcinomas Adipophilin STAT 5a Mammaglobin Histological subtypes cases/(%) Pattern of staining Qt. pos. Qt. pos. Qt. pos. Int.Stain Int.Stain + ++ cases/(%) cases/(%) MASC 10/10 (100) LD 10 10/10 (100) /10 (70) AciCC 12/14 (85.7) MD /8 (37.5) 3 3 0/8 (0) ACC 4/15 (26.7) MD 4 5/17 (29.4) /17 (0) EMC 5/12 (41.7) MD 4 1 8/13 (61.5) /13 (7.7)* 1 1* MC 5/6 (83.3) MD 2 3 5/6 (83.3) /6 (0) PLGA 1/6 (16.7) MD 1 0/6 (0) 3/6 (50)* 3 3* MEC 4/10 (40) MD 4 0/14 (0) 0/14 (0) SDC 3/4 (75) MD 3 2/14 (50) 2 2 0/4 (0) SEC 1/2 (50) LD 1 CCC 0/3 (0) A/C NOS 1/3 (33.3) MD 1 LGCC 0/1 (0) MASC, Mammary analogue secretory carcinoma; AciCC, Acinic cell carcinoma; ACC, Adenoid cystic carcinoma; EMC, Epithelial-myoepithelial carcinoma; MC, Myoepithelial carcinoma; PLGA, Polymorphous low grade adenocarcinoma; MEC, Mucoepidermoid carcinoma; SDC, Salivary duct carcinoma; SEC, Sebaceous carcinoma; CCC, Clear cell carcinoma; A/C NOS, Adenocarcinoma/Cystadenocarcinoma not otherwise specified; LGCC, Low-grade cribriform cystadenocarcinoma; LD, large droplets: MD, minute droplets; Int. Stain: Intensity of staining: + (weak/moderate), ++ (intense); Qt. pos. : quantity of positive : + (<50% of positive ), ++ (>50% of positive ). *In these cases, the quantity of positive was <20% of all tumor.

7 564 F V Mariano et al. A B C D E F G H I Figure 3. Immunohistochemical expression of STAT5a, mammaglobin and p63 in mammary analogue secretory carcinoma (MASC) and in other salivary tumours. A, B, C, STAT5a: nuclear expression in of the intercalated ducts (arrows) of the salivary parenchyma (A), strong and diffuse expression in MASC (B), and weak expression in epithelial myoepithelial carcinoma (C). D, E, F, Mammaglobin: absence of expression in salivary parenchyma (D), strong expression in MASC (E), and weak and limited expression in polymorphous low-grade adenocarcinoma (F). G, H, I, p63: strong cytoplasmic expression in tumour of MASC (G,H); note nuclear expression in myoepithelial of ducts (arrows) of the adjacent salivary parenchyma (inset in G); absence of expression in acinic cell carcinoma (I). all MASC cases, this antibody stained the rims of numerous large cytoplasmic lipid droplets, whereas in the other salivary carcinomas these droplets were minute. Only sebaceous carcinoma showed a staining pattern similar to that of MASC. Considering the fact that sebaceous carcinoma has distinct histological characteristics (holocrine secretion and sebocytes) that do not overlap with MASC, adipophilin could be helpful in supporting the diagnosis of the latter. Expression analysis of PAT proteins has shown that lipid droplet formation is a very frequent phenomenon in carcinoma, suggesting that up-regulation of lipogenic pathways is a common mechanism in different tumours. 12,13 Our finding of minute lipid droplets in different types of salivary carcinoma but not in normal salivary is in agreement with such an idea. However, apart from reflecting up-regulation of lipogenesis in cancer, PAT protein expression may also reflect cellular differentiation in some tumours. 12,13 For instance, in adipogenic, hepatocellular and sebaceous tumours, perilipin expression has been proposed to be linked to cellular differentiation rather than metabolic phenotype. 12 In salivary glands, it is likely that adipophilin expression in both MASC and sebaceous lesions is associated with cellular differentiation. Regarding MASC, the lipid droplet accumulation could result from lactation-like secretory differentiation. In fact, the formation of lipid droplets in the mammary alveolar epithelium has been observed as an early sign of lactogenesis, and

8 Mammary analogue secretory carcinoma 565 Table 3. Immunoreactivity for S100 protein, vimentin, HMWK and p63 protein in MASC and AciCC Histological subtypes S100 Protein Vimentin HMWK P63 protein cases/(%) Int. of stain Qt. pos. Qt. pos. Qt. pos cases/(%) + ++ cases/(%) + ++ cases/(%) Pattern of staining MASC 10/10 (100) /10 (100) /10 (100) 1 9 9/10 (90) cytoplasm AciCC 13/14 (92.8) 9 4* 6 7* 9/14 (64.3) 8 1 0/14 (0) 0/14 (0) HMWK, High molecular weight cytokeratin; MASC, Mammary analogue secretory carcinoma; AciCC, Acinic cell carcinoma; Int. Stain, Intensity of staining, + (weak/moderate), ++ (intense); Qt. pos., quantity of positive, + (<50% of positive ), ++ (>50% of positive ). *In these cases, S100 reactivity was strong and diffuse only in 3 of them. A C E B D F Figure 4. Immunohistochemical expression of S100, vimentin and high molecular weight keratin (HMWK) in mammary analogue secretory carcinoma (MASC) and in acinic cell carcinoma. A, B, S100: strong expression in MASC, similar to that of nerves localized in the adjacent parenchyma (arrows) (A), and weak expression in acinic cell carcinoma as compared with that of the adjacent nerve (arrow) (B). C, D, Vimentin: diffuse expression in MASC (C); a smaller number of are positive in acinic cell carcinoma (D). E, F, HMWK: diffuse expression in MASC tumour (E), and absence of expression in acinic cell carcinoma (F); note positive duct in the adjacent salivary parenchyma (arrow). their dramatic increase in size (large lipid droplets) is considered to be the most obvious histological change marking the beginning of lactation. 6,14 Milk fat droplets are surrounded by a complex membrane, of which adipophilin and MUC1 are major components. 15,16 Interestingly, expression of MUC1 and MUC4 (which is also abundant in the lactating mammary gland 17 ) was reported in the original description of MASC. 1 The strong and diffuse nuclear expression of STAT5a detected in all MASC cases in the current series, as well as in that reported by Skalova et al., 1 is in line with our hypothesis that this tumour shows lactation-like secretory differentiation. In mice, STAT5a has been demonstrated to be mandatory for lactogenesis. 14 This protein is responsible for the transcription of a variety of regulatory and differentiation proteins, and can be activated by many cytokines

9 566 F V Mariano et al. and hormones. 18 In the human breast, STAT5a expression has been detected in the terminal duct lobular unit and large ducts. 19 This expression is lost in the usual ductal carcinoma, but not in the SC subtype, leading to the hypothesis that STAT5a is necessary for maintenance of the secretory state in the human breast. 19 In our series, we showed that, in normal salivary glands, STAT5a was expressed in duct (intercalated and striated) and acini. Among the salivary carcinomas, only in MASC was overexpression of STAT5a detected in all cases. This pattern of expression was only seen occasionally in other salivary carcinomas; however, several salivary carcinomas showed STAT5a expression similar to that of normal parenchyma, suggesting that, in these lesions, the protein could be involved in functions other than secretory differentiation. On the other hand, as MASC and breast SC show overexpression of STAT5a and the ETV6 NTRK3 fusion oncogene, it is tempting to speculate that there could be a relationship between these two events and lactation-like secretory differentiation. Nevertheless, it should be mentioned that, to the best of our knowledge, adipophilin expression has not so far been investigated in SC of breast. Mammaglobin is a secretory protein that is overexpressed in breast carcinomas (usual and SC types) and in some non-breast lesions, such as endometrioid and sweat gland carcinomas and salivary tumours. 1,20,21 In relation to salivary tumours, mammaglobin has been studied only in small series containing both benign and malignant lesions. 20,21 In the original description of MASC, mammaglobin expression was observed in 100% of cases. 1 In our series of salivary carcinomas, mammaglobin expression was mainly found in MASC cases (70% of cases), reinforcing the importance of this protein for the diagnosis of this entity. PLGA and EMC showed mammaglobin expression as well, but in these it was only focal (<20% of ). However, although mammaglobin is believed to be involved in cell secretory mechanisms, its expression does not seem to be related to lactation-like secretory differentiation. Indeed, mammaglobin has been detected in other types of cell secretion, e.g. admixed with cytoplasmic mucin of normal endocervical and within glandular secretions of endometrioid carcinomas. 21 Regarding the recently recognized morphological and immunohistochemical features of MASC, 3,5 in the current series we confirmed the presence of papillary cystic architecture (seven of 10 cases) and positivity for HMWK keratin (10 of 10 cases). However, we did not observe focal mucinous differentiation or absence of S100 expression. All cases showed intense and diffuse S100 positivity. Nevertheless, our findings do not support the hypothesis that S100 intensity might be important in distinguishing MASC from AciCC. 4 In our series, three of 14 cases (21.4%) of classic AciCC showed intense S100 positivity, similar to that of MASC. S100 expression has been reported as a defining immunophenotypic feature of MASC, even though it can be observed in other salivary tumours. Interestingly, in MASC, it is likely that S100 expression is related to lactation-like secretory differentiation. This protein belongs to a family that has a broad range of functions, including secretion, and has been localized at high levels in exocrine of lactating bovine mammary glands Finally, in the current series, in MASC tumour we looked for nuclear expression of p63 as an indicator of basal/myoepithelial differentiation. Surprisingly, only cytoplasmic reactivity for p63 was detected. It was strong, and occurred mainly in tumour with papillary cystic architecture. This finding is in agreement with observations in breast SC and showing lactational change. 25 Although the mechanisms of cytoplasmic p63 expression in MASC and SC still need to be clarified, it reinforces our hypothesis that the secretory differentiation of these tumours is lactation-like. In conclusion, MASC is a lipid-rich tumour containing large lipid droplets covered by adipophilin. This finding can be included among the defining immunohistochemical features of MASC, and possibly represents lactation-like secretory differentiation. The strong expression of STAT5a and cytoplasmic p63 in MASC reinforces this hypothesis. Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgements This work was supported by the following FAPESP grants: Processo FAPESP 2011/ ; Processo FAPESP 2011/ ; Processo FAPESP 2012/ ; and Processo FAPESP 2012/ References 1. Skalova A, Vanecek T, Sima R et al. Mammary analogue secretory carcinoma of salivary glands, containing the ETV6 NTRK3 fusion gene: a hitherto undescribed salivary gland tumor entity. Am. J. Surg. Pathol. 2010; 34;

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