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.

We used two well-established methods of studying angiogenesis and lymphangiogenesis in tumors. Most blood vessels were properly stained with the anti-CD34 antibody in our study, which is consistent with findings from other studies [30]. Similarly, D2-40 also stained most of the new lymphatic vessels, although this marker has not been studied in MEC tumors before. Several studies have examined the association between angiogenesis and MEC tumor grade and staging with inconsistent results. Etemad et al. (2010) found that angiogenesis may have an important role in the pathogenesis of salivary gland MEC and may be useful in the prediction of its biologic behavior depending on the grade and the method of tumor grading. Similarly, studies have found inconsistent association between lymphangiogenesis and tumor grade and stage in head and neck tumors. We also found that  22% of the tumors had invading cells in the lymphatic system which may explain the ability of MEC to form carcinomatous emboli. This behavior of tumor cells is widespread and other tumors, such as gastric carcinoma, cervical carcinoma, malignant melanoma, oral squamous cell carcinoma, breast carcinoma, and pleomorphic adenoma have also been reported to have tumor emboli [23, 24, 33-39]. Probably the invasion of tumor cells into the lymphatic vessels may reflect a significant route for the spread of tumor cells to regional lymph nodes [33]. Our study adds information to the MEC literature in other ways as well. In our study, we found a preponderance of males with MEC, which is in contrast to the reported sex ratio in the literature [7, 28, 29].

This discrepancy may stem from the fact that we studied a different population and the distribution of risk factors between two sexes may be quite different in our population. Consistent with published literature [43], we found a significant association between the MEC tumor stage and the Brandwein grading system, suggesting that the grading criteria are relevant for tumor staging system as well. Also, we did not find an association between the predominant tumor cell type (squamous, intermediate, or mucous) and tumor grade or stage, in contrast to other studies [36, 43].

This discrepancy may be due to the predominance of low and intermediate grade tumors in our sample; only one case showed a squamous cells predominance and was found to have high tumor stage.

Immunohistochemical findings

Assessment of angiogenesis (CD34 immunostaining)

CD34 was expressed in all 22 cases of MEC (peritumoral and intratumoral). This is in conjunction with a previous  study [17] that found the same result in MEC; however, 6 (27.27%) cases showed stromal positivity and cross reaction with perivascular stromal cells and other stromal elements. Similar cross reactivity findings have been reported by a previous study (Figure 1, 2) [31]. Therefore, careful examination is required in assessing and differentiating positively stained CD34 microvessels from positively stained background and stromal cells. The mean value of MVD in the current study increased with higher grade; however, it did not demonstrate a significant relation with Brandwein grading system. Similarly, no relation was seen with TNM stage while a previous study concluded that MVD expressed by CD34 could be considered as biological markers for invading behavior in salivary gland tumors [32]. On the other hand, Kuo et al. [17] found that there was no significant correlation between peritumoral and intratumoral MVD in MECs and the histological grade, but there was a correlation between intra MVD with its stage.

These conflicting results may be due to different method of assessment and differences in tumor grades, one using Brandwein system and the other using Aucliar system. The grading system by Auclair et al. [7] is based on a scoring system of five histologic features: intracystic component <20% (+2), neural invasion (+2), necrosis (+3), four or more mitoses per 10 high power (+3), anaplasia (+4); and based on the total score a case is categorized as low grade (score 0-4), intermediate (score 5-6) and high grade (score 7-14). On the other hand, Brandwein et al. (20) grading system adds three additional histological features: invasion in the form of small nests or islands (+2), lymphovascular invasion (+3) and bony invasion (+3); and based on the total score a case is categorized as low grade (score 0), intermediate (score 2-3) and high grade (score 4 or more).

 Assessment of lymphangiogenesis (D2-40 immunostaining)

Epidermoid variety of tumor cells were stained by D2-40 marker in all study samples while mucous cells were not stained. The intermediate cells showed a variable staining pattern. This  staining pattern of the intermediate cells may reflect their undifferentiated state with features of both epidermoid and mucous cells. Another implication of this finding may be in differentiating MEC epidermoid cells from cells originating from other tumors. However, studies of D2-40 staining pattern in other tumors are needed before concluding the specificity of D2-40 to MEC epidermoid cells (Figure 3) [16, 29]. The major limitation of our study is its small sample size and its retrospective nature. However, MEC is a relatively rare tumor and we utilized all the samples available to us for this study. Moreover, a prospective study would be prohibitively long. This is the first study of its kind in this population group, which is the strength of this study. Our results show that angiogenesis and lymphangiogenesis are very frequent in MEC and present in all tumor grades. One corollary of the widespread presence of new vessel formation is that MEC tumors may have high metastasis potential irrespective of the tumor grade.

In conclusion, we found no association between histological grade or stage of MEC tumors and angiogenesis or lymphangiogenesis. Additional immunohistochemical characteristics of the tumors must be sought that are predictive of tumor grade and/or stage.

1. Neville B.W., Damm D.D., Allen C.M., and Bouquot J.E.: Oral and maxillofacial pathology; Salivary gland pathology, Third Edition, Saunders (2009).
2. Elizabeth J. Rosen, MD, Salivary Gland Neoplasms, Grand Rounds Presentation, UTMB, Dept. of Otolaryngology, (2002); 26.
3. Carvalho AL, Nishimoto IN, Califano JA, et al. Trends in incidence and prognosis for head and neck cancer in the United States: a site-specific analysis of the SEER database. Int J Cancer 2005; 114:806–816.
4. Leon Barenes: Surgical Pathology of the Head and Neck 2009; 1,3rd.
5. Saw D, Lau WH, Ho JH, et al. Malignant lymphoepithelial lesion of the salivary gland. Hum Pathol 1986; 17:914–923.
6. Laane CJ, Murr AH, Mhatre AN, et al. Role of Epstein-Barr virus and cytomegalovirus in the etiology of benign parotid tumors. Head Neck 2002; 24:443–450.
7. Auclair PL, Goode RK. Ellis GL. Mucoepidermoid carcinoma of intraoral salivary glands: evaluation and application of grading criteria in 143 cases. Cancer 1992; 69:2021-2030.
8. Goode RK, Auclair PL, Ellis GL. Mucoepidermoid carcinoma of the major salivary glands: clinical and histopathologic analysis of 234 cases with evaluation of grading criteria. Cancer 1998; 82:1217–1224.
9. Igin RK, Camrmeliet PF. Vascular endothelial growth factor (VEGF). Scientific American 2001; 29-35.
10. Nicholsen B, Theodoresco D. Angiogenesis and prostate cancer tumor growth. J Cell Biochem 2004; 91:125-150.
11. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor path in tumor growth and angiogenesis. J Clin Oncol 2005; 10:1011-1027.
12. Shang ZJ, Li JR. Expression of endothelial nitric oxide synthase and vascular endothelial growth factor in oral squamous cell carcinoma: its correlation with angiogenesis and disease progression. J Oral Pathol Med 2005; 34:134.
13. Miyazaki H, Patel V, Wang H. Ensley JF, Gutkind JS, Yeudall WA. Growth factor sensitive molecular targets and metastatic head and neck-squamous cell carcinoma using microarray analysis. Oral Oncol. 2006; 42:240-256.
14. Folkman J. Tumor angiogenesis: Therapeutic implications. N Engl J Med 1971; 285:1182–1186.
15. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis–correlation in invasive breast carcinoma. N Engl J Med 1991; 324:1–8.
16. Folkman J. Fighting cancer by attacking its blood supply. Am 1996; 275:150-4.
17. Chou KC, Chang LC, Su HC, Lee SH, Lee HS, Lee JY, et al. Immunohistochemical Study of Tumor Angiogenesis in Mucoepidermoid Carcinoma. J Med Sci 2005; 25:285-290.
18. Oliver G, Detmar M. Review. The formation of lymphatic vessels and its importance in the setting of malignancy. The Journal of Experimental Medicine 2002; 16:773-783.
19. Miller RT. Immunohistochemistry utility of immunostains for D2-40 in diagnostic pathology. The Focus 2005.
20. Brandwein MS, Ivanov K, Wallace Dl, et al. Mucoepidermoid carcinoma: a clinicopathologic study of 80 patients with special reference to histological grading. Am J Surg Pathol 2001; 25:835-845.
21. Vermeulen PB, Gasparini G, Fox SB, Toi M, Martin L, McCulloch P, et al. Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation. Eur J Cancer 1996; 32:2474-2484.
22. Ranieri G, Labriola A, Achille G, Florio G, Zito A, Grammatica L, Paradiso A. Microvessel density, mast cell density and thymidine phosphorylase expression in oral squamous cell carcinoma. Int J Oncol. 2002; 21:1317-1323.
23. Soares AB, Ponchio L, Juliano PB, de Aravjo VC, Altemani A, Lymphatic vascular density and lymphangiogenesis during tumor progression of carcinoma ex pleomorphic adenoma. J Clin Pathol 2007; 60:995-1000.
24. Kyzas PA, Stefanou D, Batistatou A, Agnantis NJ, Nakanishi Y, Hirohashi S, Charalabopoulos K. Dysadherin expression in head and neck squamous cell carcinoma association with lymphangiogenesis and prognostic significance. Am J Surg Pathol 2006; 30:185-193.
25. Browning L, Bailey D, Parker A. D2-40 is a sensitive and specific marker in differentiating primary adrenal cortical tumors from both metastatic clear cell renal cell carcinoma and phaeochromocytoma. J Clin Pathol. 2008; 61:293-296.
26. Naka T, Boltze C, Kuester D, Schulz TO, Samii A, Herold C, et al. Expression of Matrix Metalloproteinase (MMP)-1, MMP-2, MMP-9, Cathepsin B, and Urokinase Plasminogen Activator in Non–Skull Base Chordoma. Am J Clin Pathol 2004; 122:926-930.
27. Stewart FW, Foote FW, Becker WF. Mucoepidermoid tumors of salivary glands. Ann Surg 1945; 122:820–844.
28. Ellis GL, Auclair PL. Tumors of the salivary glands. In Atlas of Tumor Pathology, 4th series, fascicle 9. Washington, DC, Armed Forces Institute of Pathology, 2008.
29. Triantafillidou K, Dimitrakopoulos J, Lordanidis E, Koufogiannis D. Mucoepidermoid carcinoma of minor salivary glands: a clinical study of 16 cases and review of the literature. Oral Dis 2006; 12:364-70.
30. Hannen EJ, Riediger D. The quantification of angiogenesis in relation to metastasis in oral cancer: a review. Int J Oral Maxillofac Surg 2004; 33:2-7.
31. Hasan J, Byers R, Jayson GC. Intra-tumouralmicrovessel density in human solid tumours. Br J Cancer 2002; 86:1566-1577.
32. Huang ZQ, Chen WL, Li HG, Li JS, Xu ZY, Lin ZY. Extracellular Matrix Metalloproteinase Inducer Expression in Salivary Gland Tumors: A Correlation With Microvessel Density. J Craniofac Surg 2010; 21:1855-1860.
33. Siriwardena BSMS, Kudo Y, Ogawa I, Udagama MNGPK, Tilakaratne WM, Takata T. VEGF-C is associated with lymphatic status and invasion in oral cancer. J Clin Pathol 2008; 61:103-108.
34. Braun M, Flucke U, Deblad M, Walgenbach-Bivenagel G, Walgenbach KJ, Holler T, et al. Detection of lymphovascular invasion in early breast cancer by D2-40 (Podoplanin) a clinically useful predictor for axillary lymph node metastasis. Breast Cancer Res Treat 2008; 112:503-11.
35. Arigami T, Natsugoe S, Uenosono Y, Arima H, Makati Y, Ehi K, et al. Lymphatic invasion using D2-40 monoclonal antibody and its relationship to lymph node micrometastasis in PN gastric cancer. British J Cancer 2005; 93:688-693.
36. Arnaout – Alkarain A, Kahn HJ, Narod SA, Sun PA, Marks AN. Significance of lymph vessel invasion identified by the endothelial lymphatic marker D2-40 in node negative breast cancer. Mod Pathol 2007; 20:183-91.
37. Gombos Z, Xu X, Chu CS, Zhang PJ, Acs G. Peritumoral lymphatic vessel density and vascular endothelial growth factor C expression in early- stage squamous cell carcinoma of uterine cervix. Clin Cancer Res 2005; 11:8364-8371.
38. Niakosari F, Kahn HJ, Marks A, From L. Detection of lymphatic invasion in primary melanoma with monoclonal antibody D2-40: A new selective immunohistochemical marker of lymphatic endothelium. Arch Dermatol 2005; 141:440-444.
39. Ito M, Mariya T, Ishida T, Usami S, Kasajima A, Sasano H, Ohuchi N. Significance of pathological evaluation for lymphatic vessel invasion in invasive breast cancer. Breast Cancer 2007; 14:381-387.
40. Ordonez NG. D2-40 and podoplanin are highly specific and sensitive immunohistochemical markers of epithelioid malignant mesothelioma. Human Pathology 2005; 36:372-380.
41. Roy S. D2-40, a novel monoclonal antibody against the M2A antigen as a marker to distinguish hemangioblastomas from renal cell carcinomas. Acta Neuropathol 2005; 109:497-502.
42. Zeboon ZA. Expression of D2-40 immunohistocemical marker in testicular genu cell tumors. A thesis for the degree of followship of Iraqi council for medical specialities in pathology, 2008.
43. Seethala RR. An Update on Grading of Salivary Gland Carcinomas. Head Neck Pathol. 2009; 3:69–77.