Human salivary gland morphogenesis: myoepithelial cell maturation assessed by immunohistochemical markers

Ianez R F, Buim M E, Coutinho‐Camillo C M, Schultz R, Soares F A & Lourenço S V
(2010) Histopathology 57 , 410–417
Human salivary gland morphogenesis: myoepithelial cell maturation assessed by immunohistochemical markers Aims: Myoepithelial cells are important components of salivary gland structure, aiding the expulsion of saliva from acinar lobules. The aim was to evaluate the expression of smooth muscle actin (SMA), calponin, caldesmon, CD10, CD29, S100 protein, glial fibrillary acidic protein (GFAP) and p63 in myoepithelial cells during salivary gland morphogenesis to understand the maturation process of these cells and their possible use in the diagnosis of salivary gland lesions. Methods and results: Major and minor human salivary glands at various stages of development, derived from fetuses at 8–26 weeks of gestation, were studied immunohistochemically. Fully developed salivary glands were used as controls. The protein p63 was present in all stages of salivary gland morphogenesis from initial bud to terminal bud stage. CD29, S100 and calponin were detected increasingly as salivary gland structure matured and in fully developed salivary gland. Proteins GFAP, CD10 and caldesmon were not observed in myoepithelial cells of salivary glands. Conclusions: The proteins SMA, calponin, CD29, S100 and p63, which are present from the earliest stages of salivary gland maturation, are valuable myoepithelial markers but, although very specific, are not exclusive markers for this cell type.     

Expression of a flt-4 (VEGFR3) splicing variant in primary human prostate tumors. VEGF D and flt-4t(Delta773-1081) overexpression is diagnostic for sentinel lymph node metastasis

Utilizing a cDNA expression library established from human prostate PC-3ML tumor cells, we have cloned a truncated flt-4 gene, termed flt-4t(Delta773-1081). We have then utilized RNase protection and ELISA to measure the relative levels of VEGF B, C, D and flt-1, KDR, flt-4 and flt-4t(Delta773-1081) expression in freshly isolated benign prostatic hyperplasia or BPH tissue (n=21), primary prostate cancers (n=82) and matching sentinel lymph node metastases from stage T2a-T2b/T3 tumors (n=52). Comparisons of the primary tumors with BPH showed that there was a significant upregulation of VEGF-B (P=0.003), VEGF D (P=0.005), flt-1 (P=0.003), KDR (P=0.002), flt-4 (P=0.007), and flt-4t(Delta773-1081) (P=0.001), but not VEGF-C (P=0.543). There was no correlation between VEGF-B and its receptor flt-1 (P=0.545), or VEGF-C and flt-4 (P=0.16) and KDR (P=0.23) receptor expression in tumor specimens. Conversely, there was no significant relationship between VEGF-D and the flt-4t(Delta773-1081) receptor (P=0.516) expression. Statistical analysis further showed that there was no significant correlation between VEGF-B, VEGF-C, VEGF-D, flt-1, KDR, flt-4 and flt-4t(Delta773-1081) with patient age (P>0.10), stage (P>0.10), PSA value (P>0.15) or tumor size (P>0.15). Likewise, there was no significant correlation between VEGF-B, VEGF-C, flt-1, KDR, and flt-4 with Gleason score (P>0.15). In comparison, flt-4t(Delta773-1081) levels clearly increased significantly in Gleason score 7 and Gleason score 8-10 tumors as well as in stage T2a-T2b/T3 tumors. The studies were extended to compare gene expression profiles in T2a-T2b and T3 tumors with (n=26) and without (n=26) matching sentinel lymph node metastases. The data showed that VEGF D and flt-4t(Delta773-1081) expression levels were significantly elevated in primary tumors with sentinel lymph node involvement compared to those lacking lymph node involvement (P>0.0022 and 0.006, respectively). These data suggest that targeting VEGF D and flt-4t(Delta773-1081) receptors may be particularly effective in the prevention of lymph node metastases.     

Neither expression of VEGF-C/D nor lymph vessel density supports lymphatic invasion as the mechanism responsible for local spread of recurrent salivary pleomorphic adenoma

Recent research suggests that multinodular recurrent pleomorphic adenoma (PA) might result from cell migration through lymphatics. Lymphangiogenesis in malignancies is mediated by vascular endothelial growth factors C and D (VEGF-C/D). We studied the expression of VEGF-C/D in PA by immunohistochemistry as well as lymphatic vessel density (LVD). In 6 non-recurrent, 4 primary-to-recur, and 10 recurrent PAs, VEGF-C/D expression was assessed by immunohistochemistry. Staining was scored in terms of staining intensity (0 = absent to 3 = strong), and the percentage of positive tumor cells (scored as 0 (0–19 %), 1 (20–39 %), 2 (40–50 %), and 3 (60–100 %)) and a sum score were calculated. Intra- and peritumoral LVD was assessed by counting of LV after immunostaining, using the D2-40 antibody. All but one sample were VEGF-C negative. The differences in VEGF-D expression between non-recurrent, primary-to-recur, and recurrent PAs were not significant (p?>?0.05). VEGF-D expression did not correlate with peritumoral LVD (p?>?0.05). Our study revealed a significant difference between intra- and peritumoral LVD values when comparing individual and all sample groups (p?=?0.01). The lack of VEGF-C expression and of significant differences in VEGF-D expression and peritumoral LVD between patients with non-recurrent, primary-to-recur, and recurrent PAs does not support the lymphangiogenic local spread hypothesis.     

The cell with a thousand faces: detection of myoepithelial cells and their contributions in the cytological diagnosis of salivary gland tumors

Myoepithelial cells are an important component of salivary gland tumors and are partly responsible from the diverse histology of them. In this study, we focus on the myoepithelial cell differentiation by using cytological morphology in a various types of salivary gland tumors especially with regard to their contribution to the diagnosis. The relation of myoepithelial cells with stromal matrix and the associated epithelial cells were evaluated. Cytologic slides of one hundred and forty one benign and twenty malignant salivary gland tumors were examined for identification of morphologically different myoepithelial cells such as; spindle-stellate, polygonal-epitheloid, plasmacytoid, basal and clear types. The best examples of myoepithelial cells were detected in pleomorphic adenomas, in some monomorphic adenomas and in the adenoid cystic carcinoma cases. Most of the pleomorphic adenomas were composed more than one type of myoepithelial cells and epitheloid-spindle cell combination was frequent. Basal and clear cell types of myoepithelial cells closely resembled the epithelial cells and their identification was relatively difficult. Identification of myoepithelial cell types was easier when they were associated with stromal matrix material and stood as a secondary layer around tubule-forming epithelial cells. Myoepithelial cell components of various salivary gland tumors may be quite different and identification of myoepithelial cell types may pose difficulties. A confident cytologic identification of myoepithelial cells may be critical part of diagnosing salivary gland tumors.     

Oncocytic lipoadenoma of the parotid gland with sebaceous differentiation. Study of its keratin profile

Oncocytic lipoadenomas of salivary gland are extremely rare tumors with only two previously reported cases. In this paper, we describe an additional example of oncocytic lipoadenoma showing sebaceous differentiation, a hitherto unreported occurrence. The tumor was encapsulated and measured 3 × 2.5 × 2 cm. Microscopically, the tumor comprised a mixture of oncocytes with “light” and “dark” cells intimately associated with mature adipose tissue. The oncocytes were positive for low molecular keratin, epithelial membrane antigen (EMA), and keratin 7, with only a small subgroup of cells expressing high-molecular-weight keratin, keratin 5/6, keratin 19, and p63. Terminally differentiated sebaceous cells were positive for EMA and keratin 14 only. Calponin and actins were negative, indicating a lack of myoepithelial cells in the tumor. The keratin profile and p63 expression of this oncocytic lipoadenoma suggest the presence of a dual cell population somewhat similar to the dual cell population described in some ultrastructural studies of pure salivary gland oncocytomas and may represent partial basal-cell differentiation. The presence and nature of a dual cell population in oncocytic neoplasms of salivary glands merit further investigation to confirm these observations.     

Immunophenotypical profiles of salivary gland tumours: a new evidence for their histogenetic origin

The histogenetic origin of salivary gland tumours is not clear. In normal tissues smooth muscle actin (SMA) is expressed in myoepithelial cells, CK14 immunoreactivity is seen in myoepithelial and basal cells and CK10 in keratinized squamous epithelium. In this study, we examine the immunophenotypic properties of salivary gland tumours in order to obtain further insight into their histogenesis. 30 cases of salivary gland tumours (18 pleomorphic adenomas, 8 Warthin's tumours, 2 basal cell adenomas, 2 acinic cell carcinomas) were included in our study. Cytokeratin (CK) 10, CKI4, CKI7, CK18, CK 19, and smooth muscle actin (SMA) immunostains were applied to the sections. Immunoreactivities were detected and the statistical significance was evaluated by chi square test. SMA was not detected in Warthin's tumour (p < 0.0001). CK14 was found in all tumours except acinic cell carcinomas (p < 0.0001). CK10 immunoreactivity was observed in 5 Warthin's tumour. In conclusion, pleomorphic adenomas and basal cells adenomas originate from stem cells. Immunophenotypic profile of Warthin's tumour is suggestive of an embryological remnant origin.     

Expression and regulation of the ΔN and TAp63 isoforms in salivary gland tumorigenesis clinical and experimental findings

The TP63 gene, a TP53 homologue, encodes for two main isoforms by different promoters: one retains (TA) and the other lacks (ΔN) the transactivation domain. p63 plays a critical role in the maintenance of basal and myoepithelial cells in ectodermally derived tissues and is implicated in tumorigenesis of several neoplastic entities. However, the biological and regulatory roles of these isoforms in salivary gland tumorigenesis remain unknown. Our results show a reciprocal expression between TA and ΔN isoforms in both benign and malignant salivary tumors. The most dominantly expressed were the ΔN isoforms, whereas the TA isoforms showed generally low levels of expression, except in a few tumors. High ΔNp63 expression characterized tumors with aggressive behavior, whereas tumors with high TAp63 expression were significantly smaller and less aggressive. In salivary gland cells, high expression of ΔNp63 led to enhanced cell migration and invasion and suppression of cell senescence independent of TAp63 and/or TP53 gene status. We conclude the following: i) overexpression of ΔNp63 contributes to salivary tumorigenesis, ii) ΔNp63 plays a dominant negative effect on the TA isoform in the modulation of cell migration and invasion, and iii) the ΔN isoform plays an oncogenic role and may represent an attractive target for therapeutic intervention in patients with salivary carcinomas.     

Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray

p63, cytokeratin (CK) 5/6 and CK 14 have been employed in diagnostic pathology as markers of basal, squamous and myoepithelial differentiation in several types of human neoplasms; however, there is scant data on the concurrent expression of these markers in large series of human neoplasms. We analyzed the distribution of these three immunohistochemical markers in 51 normal human tissue samples, 350 carcinomas, 25 malignant melanomas (MMs), and 25 glioblastomas using three serial sections of tissue array research program (TARP)-4 multi-tumor tissue microarray. Also, we performed double immunostainings to characterize the differential distribution of p63/CK 5/6 and p63/CK 14 in normal breast, salivary gland and skin. p63, CK 5/6 and CK 14 were expressed in basal cells of the prostate and respiratory epithelia and in breast and bronchial myoepithelial cells. p63 was also expressed in cytotrophoblast cells of human placenta and in scattered cells of lymph node germinal center. CK 5/6 and CK 14 also stained the cytoplasm of basal cells of esophageal stratified squamous epithelium and transitional epithelial cells of the bladder. No mesenchymal, neural, endothelial, smooth muscle or adipose cells were stained by any of the markers. p63, CK 5/6, and CK 14 were respectively expressed in 92.6%, 75.0%, and 52.9% of the squamous cell carcinomas of the lung, 10.2%, 20.0%, and 7.4% of the ductal carcinomas of the breast, 12.9%, 34.4%, and 11.8% of the serous and 25.0%, 0%, and 0% of the endometrioid carcinomas of the ovary. Lung, prostate and colonic adenocarcinomas, as well as MMs and glioblastomas were only rarely decorated by one of the markers. Only matched samples of 16 squamous cell carcinomas and two ductal carcinomas of the breast co-expressed these three markers. In double immunostainings, p63-CK 5/6, as well as p63-CK 14 were co-expressed by basal/myoepithelial cells of the salivary glands and basal cells of the epidermis. Our results demonstrate that p63, CK 5/6 and CK 14 may be used together in immunohistochemical panels to characterize squamous differentiation in poorly differentiated carcinomas or carcinomas of unknown origin.     

Oncocytic myoepithelioma and pleomorphic adenoma of the salivary glands

 Twenty oncocytic myoepitheliomas (MEs) and pleomorphic adenomas (PAs) were composed of interlacing fascicles of swollen spindle-shaped or/and epithelioid oncocytic myoepithelial cells showing intense finely granular immunoreactivity with anti-mitochondrial antibody. Focal vacuolation of the cytoplasm of oncocytic myoepithelial cells and their gradual transition into sebaceous metaplasia were observed in 3 cases. Another unusual feature found in 5 cases was the presence of slit-like adenomatoid spaces lined with double-layered oncocytic myoepithelium closely resembling Warthin’s tumour. The nuclei of oncocytic cells were characterized by enlargement, hyperchromasia and polymorphism, which should not be confused with malignancy. Oncocytic change in myoepithelial cells in MEs and PAs can cause pitfalls in the differential diagnosis of salivary gland tumours. We describe some unusual histological features associated with onococytic metaplasia in benign myoepithelial cell-derived salivary gland tumours, hoping to help to avoid the overdiagnosis of malignancy. Received: 24 September 1998 / Accepted: 31 January 1999     

Diagnostic role of DOG1 and p63 immunohistochemistry in salivary gland carcinomas

Background: The differential diagnosis of salivary carcinomas is always difficult and challenging. Salivary neoplasms often shows more than one growth pattern and significant morphologic variability may exist within a single tumor and between different tumors. The aim of this study was to examine the role of DOG1 (discovered on gastrointestinal tumor-1) and p63 immunohistochemistry in the diagnosis and differential diagnosis of salivary carcinomas. Methods: we examined the expression of DOG1 and p63 immunohistochemistry in 33 mucoepidermoid carcinomas (MEC), 9 acinic cell carcinomas (ACC), 10 adenoid cystic carcinomas (AdCC) and 4 myoepithelial carcinomas. Results: All ACC showed strong to moderate positivity for DOG1 (P=0.001) and all were totally negative for p63. All MEC expressed strong to moderate positivity for p63 (P=0.001) while only (9.1%) were weak to moderately positive for DOG1. (80%) AdCC were moderately positive for DOG1 in ductal and myoepithelial components and (100%) showed moderate positivity for p63 in myoepithelial cells only (P=0.001). All myoepithelial carcinomas were DOG1 negative, 2 (50%) were weakly positive for p63 while the other 2 were moderately positive (P=0.5). Conclusion: DOG1 is a sensitive marker in the diagnosis of acinic cell carcinoma, p63 is sensitive in the diagnosis of mucoepidermoid carcinoma, the combined use of both markers is helpful and statistically significant in the differential diagnosis of acinic cell carcinoma versus mucoepidermoid carcinoma, both markers can help in the diagnosis of adenoid cystic carcinoma but they have no role in the diagnosis of myoepithelial carcinoma.     

Immunocytochemical identification of cell types in pleomorphic adenoma, with particular reference to myoepithelial cells

An immunocytochemical study was carried out on normal salivary gland tissue and ten salivary gland pleomorphic adenomas. Antibodies to myosin were used to stain myoepithelial cells. Duct cells were stained using an antibody to total keratin and a subpopulation of basal duct cells with an antibody to 45/46K keratins. Basement membranes were stained with anti-type IV collagen. The results demonstrated that myoepithelial cells are relatively rare in the majority of pleomorphic adenomas and that many of the cells which have been classically described as myoepithelial in routine histological preparations do not clearly show this type of differentiation. However, the tumors presented a spectrum of differentiation patterns from those that were mainly ductal to the rare tumour which was largely myoepithelial. It is further suggested that the 45/46K keratin antibody is capable of identifying a subpopulation of cells which could possibly be important in the histogenesis of this tumour.     

Myoepithelial differentiation markers in salivary gland neoplasia]

Salivary gland tumors frequently present myoepithelial cell differentiation that is not always easily identified on routinely stained sections. Recently novel markers of myoepithelium have been studied, such as calponin (CALP), caldesmon (CALD), and smooth muscle myosin heavy chain. These markers, together with smooth muscle actin may be useful tools for identifying myoepithelial cells. We immunohistochemically studied a series of 23 benign and malignant salivary gland tumors using antibodies to these four markers. The tumors were classified as follows: pleomorphic adenoma (n = 8), basal cell adenoma (n = 3), myoepithelioma with plasmacytoid cells (n = 2), epithelial-myoepithelial cell carcinoma (n = 6) and adenoid cystic carcinoma (n = 4). All tumors were positive for at least one of the four markers. CALP and smooth muscle actin were the markers more frequently expressed. Positivity was mostly located in the myoepithelial cells that constitute the external layer of the glandular or tubular neoplastic structures. In poorly differentiated epithelial myoepithelial carcinomas, composed of solid sheets of neoplastic cells and sometimes of clear cells, immunohistochemical staining for myoepithelial markers evidenced rudimentary glandular structures. CALP and smooth muscle actin were positive in the two cases of myoepithelioma with plasmacytoid cells. In conclusion, the combined staining with four markers helps to disclose myoepithelial cell differentiation and can be a useful tool for the correct histopathological diagnosis of salivary gland tumors. Among the four markers studied, CALP and smooth muscle actin were the most useful to identify myoepithelial cell differentiation.     

Benign myoepithelial tumors of the breast have immunophenotypic characteristics similar to metaplastic matrix-producing and spindle cell carcinomas

We immunohistochemically compared benign myoepithelial tumors (adenomyoepitheliomas [AMEs]) and metaplastic matrix-producing (MMP-CA) and spindle cell (MSC-CA) carcinomas of the breast to identify helpful diagnostic markers. Normal myoepithelial cells (MECs) consistently expressed cytokeratin, alpha-smooth muscle actin (SMA), myosin, S-100, CD10, and maspin. They were variably positive for vimentin and negative for epithelial membrane antigen (EMA), steroid receptors, p53, and HER-2/neu. MECs in AMEs less frequently expressed CD10 (4/8 [50%]) and myosin (6/8 [75%]) but frequently acquired characteristics of luminal cells, such as expression of EMA (5/8 [63%]) and steroid receptors (5/8 [63%]). No abnormal p53 or HER-2/neu expression was seen in AMEs. MMP-CA and MSC-CA were similar to AMEs in cytokeratin, vimentin, S-100, maspin, and HER-2/neu expression. MMP-CAs expressed less alpha-SMA (2/8 [25%]) and myosin (2/7 [29%]) and lacked estrogen receptor (0/9 [0%]). MSC-CAs were consistently CD10+ (4/4 [100%]) yet failed to express myosin (0/3 [0%]). p53 overexpression was seen frequently in MMP-CAs (4/8 [50%]) and MSC-CAs (1/3 [33%]). Benign myoepithelial mammary tumors differ immunophenotypically from normal MECs; a panel of immunohistochemical markers may be required to establish their myoepithelial origin. A similarly altered myoepithelial phenotype also is characteristic of metaplastic mammary carcinomas. The abnormal expression of oncogenes or antioncogenes, such as p53, may be more useful for distinguishing between those entities than the expression of the classic myoepithelial markers.     

Immunoelectron Microscopic Study of Podoplanin Localization in Mouse Salivary Gland Myoepithelium

We have recently reported that salivary gland cells express the lymphatic endothelial cell marker podoplanin. The present study was aimed to immunohistochemically investigate the expression of the myoepithelial cell marker α-smooth muscle actin (SMA) on podoplanin-positive cells in mouse parotid and sublingual glands, and to elucidate podoplanin localization in salivary gland myoepithelial cells by immunoelectron microscopic study. The distribution of myoepithelial cells expressing podoplanin and α-SMA was examined by immunofluorescent staining, and the localization of reaction products of anti-podoplanin antibody was investigated by pre-embedded immunoelectron microscopic method. In immunohistochemistry, the surfaces of both the mucous acini terminal portion and ducts were covered by a number of extensive myoepithelial cellular processes expressing podoplanin, and the immunostaining level with anti-podoplanin antibody to myoepithelial cells completely coincided with the immunostaining level with anti-α-SMA antibody. These findings suggest that podoplanin is a salivary gland myoepithelial cell antigen, and that the detection level directly reflects the myoepithelial cell distribution. In immunoelectron microscopic study, a number of reaction products with anti-podoplanin antibody were found at the Golgi apparatus binding to the endoplasmic reticulum in the cytoplasm of myoepithelial cells between sublingual gland acinar cells, and were also found at the myoepithelial cell membrane. These findings suggest that salivary gland myoepithelial cells constantly produce podoplanin and glycosylate at the Golgi apparatus, and transport them to the cell membrane. Podoplanin may be involved in maintaining the homeostasis of myoepithelial cells through its characteristic as a mucin-type transmembrane glycoprotein.