Assessment of angiogenesis by CD105 antigen in epithelial salivary gland neoplasms with diverse metastatic behavior

Malignant salivary gland tumors constitute a major challenge in head and neck oncology because of its frequency, varied histological typing, difficult surgical approach, and poor response to other therapies. It is further complicated by the absence of clear parameters to preview biological behavior, in particular to predict development of metastasis. In this sense, there is few information regarding pathways implicated in tumor dissemination of salivary gland cancer.

Tumor growth is limited by the balance between neoplastic demand for oxygen and nutrients and diffusion from preexisting surrounding vascularization, and therefore formation of new blood vessels is an essential step in progression of cancer [1]. In general, this process is named angiogenesis, requires release of activating mediators as well as suppression of inhibitory mechanisms, and may evolve through several mechanisms, such as sprouting, intussusception, recruitment, cooption, and mimicry [2]. Both angiogenesis and neoplastic invasion and metastasis are associated with increasing vascular surface, remodeling of extracellular matrix, and secretion of growth factors [3].

In situ quantification of microvessel density by immunohistochemistry is a usual form to assess angiogenesis for different types of neoplasia. However, inconsistencies and contradictions have been pointed out, in particular because of the use of endothelial markers which are unable to distinguish true angiogenic endothelial activity [4–6]. CD105, also known as endoglin, is a receptor for TGFβ signaling, and play an important role in angiogenesis and fibrogenesis [7, 8]. It is essential for endothelial cell proliferation, thus promoting the activation phase of angiogenesis [9]. More important, CD105 expression is a prominent feature of newly formed tumor vessels but minimally expressed in quiescent preexisting ones [7, 10]. It is also a prognostic marker for squamous cell carcinoma of the head and neck [11, 12].

Finally, there is few information translating the relationship between angiogenesis and dissemination of salivary gland tumors to clinical-driven parameters. In this sense, this work was conducted to investigate whether lesions with diverse clinical course also differ in their neovascular content.

Metastasis is an important cause of death for patients with cancer and there is evidence pointing out for a role of angiogenesis in the dissemination of malignant tumors [15]. In an effort to investigate possible association between formation of new blood vessels and development of metastasis in salivary gland tumors, the present work investigated the most frequent of these lesions (PA) as a conceptual standard for non-metastasizing disease, as well as three of the most important malignant neoplasms for which metastasis is possible, but less (PLGA) or more (ACC, MEC) frequent [16, 17]. These lesions also diverge regarding to differentiation and myoepithelial content [18]. Another considerably common salivary gland cancer, adenocarcinoma NOS, was not included because of the prominent and characteristic morphologic and behavioral heterogeneities of this group of lesions. Work covering prognosis and new insights into biology of this rare lesion has been recently released [19]. Other aggressive tumors were not included because of its rarity and, in spite of one of the largest number of primary samples in the literature, it was not possible to obtain a representative sample of metastatic implants because many were only detected by imaging exams in organs such as lungs, bones, liver, or brain. In addition, interpretation of the present data is limited by the acknowledgement that an overall five years follow-up time is too short to fully assess metastatic behavior for ACC, since late distant manifestations is a prominent finding in this disease [13, 17, 18].

CD105 expression has been demonstrated almost exclusively in venous and arterial vessels surrounding and permeating tumors [10]. It also has been detected in non-neoplastic tissues with increased angiogenic activity, such as embryonic development and wound healing [2, 4]. Greater accuracy (mainly specificity) of CD105 in demonstrating new blood vessels compared with other molecules such as CD34 and factor VIII is clear in the literature [20–22]. As expected, in the present work CD105-positive vessels were absent in normal salivary gland. Even almost half of the tumors did not present such finding, corroborating the specificity of CD105 in demonstrating newly formed blood vessels, but it also evidences that neoplastic development does not always require prominent angiogenesis. Costa et al. [23] observed discrepancy between proliferation of salivary gland tumor cells and closeness to vessels stained by CD34, suggesting that possibly tumors such as ACC maintain metabolic activity via an oxygen-independent process. Anyway, there was a noticeable increase in the mean IMD from benign to malignant lesions, therefore indicating that vascularization contributes to or reflects aggressiveness in salivary gland tumors. Increased demand for blood supply because of accelerated growth or acquired ability of tumor cells to produce angiogenic factors could explain this finding [2].

In the present work, mean IMD assessed by CD105 was not significantly different between the entire samples of malignant tumors that did or not metastasized, and even all of the PLGA and more than half of the ACC that metastasized did not present CD105 positive vessels, suggesting that angiogenesis is neither an absolute determinant nor required for acquisition of metastatic phenotype in these salivary gland tumors. However, only metastasizing ACC and all of metastasizing MEC were positive for CD105, suggesting that angiogenesis would be a priming step for at least some of these lesions to disseminate, specially for ACC. Zhang and Peng [24] observed that ACC cell lineages with higher invasive and metastatic potential were also more efficient than other with low metastatic behavior in promoting migration and tube formation of endothelial cells in vitro.

Another interesting finding of the present work was the continuous increase of IMD from ACC to PLGA and then to MEC. It is not apparently correlated to the aggressiveness of these lesions, but their myoepithelial content may help to explain such difference. There is evidence that myoepithelial cells inhibit angiogenesis [25, 26]. It could greatly impair angiogenesis in PA and ACC. This effect would be only partial in PLGA, where myoepithelial content is intermediate between ACC and MEC. The latter does not present myoepithelial differentiation, and therefore would not present their inhibitory effect. Similarly to the present results, Costa et al. [23] observed less frequent staining of vessels by CD105 in ACC than MEC. In addition, these authors observed almost identical mean IMD in non-metastasizing and metastasizing ACC, in the same way of the present work, but did not evaluate PLGA or metastasizing MEC.

The results of the present work contribute to the knowledge on the biology of salivary gland tumors, since increased angiogenesis was observed in malignant tumors. Further, it was also higher in MEC than in PLGA and particularly than in ACC and PA, supporting a likely role of myoepithelial cells as regulators of the formation of new blood vessels. Diagnostic implications are also present, since those ACC with CD105 positive vessels seems to be at increased risk for metastasis.