Immunohistochemical Expression of CD34 as Biological Marker of Angiogenesis and Expression of D2-40 as Marker of Lymphangiogenesis in Mucoepidermoid Carcinoma of Salivary Glands

Salivary gland tumors (SGTs) constitute an important area in the field of oral and maxillofacial pathology. Its incidence around the world ranges from about 1.0 to 6.5 cases per 100,000 people per year, and it represents 2-4% of head and neck neoplasms [1]. Mucoepidermoid carcinoma (MEC) is the most common malignant salivary gland tumors, with uniform distribution between the ages of 20 and 70 years [1, 2]. It is the most common malignant salivary gland tumors in children [3, 4]. Histopathologically, MEC is composed of a mixture of mucus-producing, intermediate and squamous (epidermoid) cells [5, 6, 7, 8]. Due to their high metabolic needs, malignant tumors have to induce formation of new blood and lymphatic vessels [10, 11, 12, 13, 14, 15].  Angiogenesis is the process of forming new blood vessels [9]. Immunohistochemical staining for CD34 is a sensitive and well-studied marker of vascular epithelium and  a useful tool to determine microvessel density within tumors. CD34 staining has been established to be useful in predicting tumor relapse or metastasis [16]. Microvessel density evaluated by CD34 immunostaining had been studied as prognostic marker relevant in MEC [17]. Lymphangiogenesis, the formation of new lymphatic vessels, has also been implicated in metastasis. Recent evidence suggests an active role of malignant tumors in the induction of intratumoral and peritumoral lymphangiogenesis [18]. D2-40 is a novel new selective immunomarker specific for lymphatic endothelium; it does not stain vascular endothelium [19]. While tumors induce both angiogenesis and lymphangiogenesis, their metastatic potential may be related to the extent of these two processes within tumors. While angiogenesis within MEC tumors has been studied with CD34, lymphangiogenesis with D2-14 has not been examined. Only after examining both processes in a tumor, the true metastatic potential of a tumor can be predicted.  Thus, we hypothesized that the differences in the malignant and metastatic behavior of MECs can be explained by the differences in the extent of angiogenesis and lymphangiogenesis. To test this hypothesis, we examined the association of MEC tumor grade and stage with its CD34 and D2-40  staining.

Twenty two formalin-fixed paraffin-embedded tissue blocks of salivary gland MEC were collected from the Department of Oral Diagnosis, College of Dentistry, University of Baghdad from the period of 1972 to 2010. Four micrometer thick sections were cut from each paraffin tissue block and stained with hematoxylin and eosin for diagnostic confirmation and histological grading. Tumors were classified into low, intermediate and high grade MEC according to Brandwein grading system [20]. TNM staging was applied to 18 cases only in which the required clinical data relevant to tumor stage were properly mentioned in the case sheet. Another 4µm thick section was cut from each tissue block and mounted on positively charged slides (Esco, USA) to be stained with CD34 monoclonal antibodies (USBiological-C2386-10). Negative and positive tissue controls were included into each immunohistochemical run.

Immunohistochemical staining procedure

Slides were baked in hot air oven at 65°C overnight. Sections were sequentially dewaxed through a series of xylene, graded alcohol, and water immersion steps. For CD34, endogenous peroxidase activity was blocked with 0.03% hydrogen peroxide followed by blocking the nonspecific antibody binding with normal goat serum (USBiological-I7506A). Primary CD34  antibodies at a dilution of 1:40 were applied to all slides. The slides were then incubated for 1 hour at 37°C and kept at 4°C in a humid chamber overnight. Next day, after washing the sections with phosphate buffer solution (PBS), biotinylatedantimouse IgG was applied to slides followed by incubation and rinsing with a stream of PBS. Conjugated antibodies were visualized with diaminobenzidine (DAB) chromogen stain. Sections were counterstained with Mayer’s hematoxylin for 1–2 minutes, dehydrated and mounted.

Assessment of immunohistochemical results

 Microvessel Density Determination (MVD)

To determine the microvessel density, the stained normal and cancer tissue sections were initially screened microscopically at low power (X10) to identify the areas of highest vascularization (“hotspots”). Five intratumoral and peritumoral high power (X40) fields were then chosen randomly, and the number of microvessels in each high power field was counted for each sample. MVD for each sample was taken as the mean of the five values obtained. Both peritumoral and intratumoral MVD were counted separately, and total MVD was obtained [21, 22].

Lymphatic Vessel Density Determination (LVD)

All MEC slides were scanned at low power (X10) to select six fields with the highest number of stained lymphatic vessels that were identified as “hotspots” (the area of greatest number of highlighted lymphatic vessels). In three intratumoral and three peritumoral (within an area of 1mm from the invasion front), the LVD was countered as the number of stained vessels per optical field  and the number of D2-40 positive vessels was calculated in each hotspot at a higher magnification (X40) and the average of them was obtained as total LVD. In addition, the whole tumor area was scanned to determine lymphatic vessel invasion LVI (the presence of tumor cells within a lymph vessel) in each case. Intratumoral or peritumoral LVI was considered evident if at least one tumor cell cluster was clearly visible inside a D2-40 positive vessel. [23, 24]. D2-40 expression was also evaluated in tumor cells. Only the positivity of staining was assessed. Cytoplasmic and/or membranous immunoreactivity were considered a positive. The positivity was evaluated as follows; – <10%, + 10-25%, ++ 26-50%, +++51-100% [25]. The data was analyzed with SPSS (Statistical Package for the Social Sciences) statistical software (Version 17). Chi square and ANOVA tests were applied to compare variables as needed. Pearson’s correlation coefficient was applied to plot a correlation matrix among the different immunohistochemical markers expression values. P values of less than 0.05 were considered significant, and less than 0.01 were considered highly significant.

The sample comprised 14 males and 8 females. The age range of the patients with MEC was between 19 and 65 years (mean=45.9±10.53). Submandibular gland was most affected (7 cases) followed by palate (6 cases), parotid gland (5 cases) and the buccal mucosa (4 cases). According to Brandwein grading system, 7 cases were found as low grade, 8 were intermediate and 7 cases were high grade. TNM staging system of MEC (only 18 cases) showed 7 cases being stage I, 3 cases stage II, 4 cases stage III and 4 cases stage IV. The stage of MEC had a highly statistically significant relation with Brandwein grading system (P=0.039). There was no significant relationship between the predominant cell type and grade or stage of the tumor (Table 3).

Data showed that the stage of MEC had a significant relationship with Brandwein grading system (P=0.039). The mean of microvessel density count measured in all cases by CD34 immunomarker was 10.74±5.97 (Figure 1, 2); however, no significant relationship was found with tumor grade or stage; P=0.58, P=0.48, respectively (Table 1).

In this study, we showed that there is no association between angiogenesis or lymphangiogenesis and tumor grade or stage in MEC tumors.