Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 389
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size


 
ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 3  |  Page : 403-407
Proliferative activity in oral pyogenic granuloma: A comparative immunohistochemical study


1 Department of Oral and Maxillofacial Pathology, Dental School of Shiraz University of Medical Science, Shiraz, Iran
2 Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
3 Department of Pathology, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran

Click here for correspondence address and email

Date of Web Publication22-Oct-2010
 

   Abstract 

Context: Pyogenic granuloma (PG) is one of the most common reactive vascular lesions in the oral mucosa, which has been divided into the lobular capillary hemangioma (LCH) and the non lobular type (non-LCH) as two distinct entities, on the basis of some investigations. Aims: This study aims to compare the proliferative and angiogenic activity of two histological types of PG to determine whether they have two distinct types of biological behavior. Settings and Design: In this retrospective cross-sectional study, immunostaining was performed on 10 cases of each type of PG. Materials and Methods: About 4μm sections were cut from formalin-fixed paraffin-embedded blocks and each specimen was stained with both anti-CD31 and anti-Ki-67 antibodies simultaneously. Labeling index (LI) was determined for both types by counting Ki-67 and CD31 positive cells separately and simultaneously in 1000 stromal and luminal cells. Micro vessel count (MVC), the mean number of micro vessels in five areas at Χ200 magnification, was also determined for both groups. Statistical Analysis: The results were statistically compared using the Mann-Whitney U-test. Results: Ki-67 LI in LCH (5.4 ± 2.4) was higher than non-LCH (3.9 ± 3.9). The percentage of CD31 positive cells in LCH (28.5 ± 22) was lower than non-LCH (37.1 ± 20.8) and simultaneously immunostaining for both markers in LCH type (2.4 ± 2.1) was higher than non-LCH (1.2 ± 1). The MVC was approximately 77.35 ± 34.6 and 82.6 ± 42.7 in the lobular areas of LCH and central areas of non-LCH PG, respectively. These differences were not statistically significant. Conclusions: These results demonstrate a higher proliferation activity in endothelial cells of LCH PG than in non-LCH.

Keywords: CD31, immunohistochemistry, Ki-67, lobular capillary hemangioma, pyogenic granuloma

How to cite this article:
Rezvani G, Azarpira N, Bita G, Zeynab R. Proliferative activity in oral pyogenic granuloma: A comparative immunohistochemical study. Indian J Pathol Microbiol 2010;53:403-7

How to cite this URL:
Rezvani G, Azarpira N, Bita G, Zeynab R. Proliferative activity in oral pyogenic granuloma: A comparative immunohistochemical study. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Mar 25];53:403-7. Available from: http://www.ijpmonline.org/text.asp?2010/53/3/403/68242



   Introduction Top


Pyogenic granuloma is a common tumor-like lesion of the oral cavity. It is considered to be non-neoplastic and shows a highly vascular proliferation, sometimes organized in lobular aggregates. [1] Due to some types of behavior such as rapid growth, multiple occurrence and frequent recurrence of pyogenic granuloma (PG), some investigators regard it as a benign neoplasm but it is mostly considered to be a reactive tumor-like lesion as a response to various stimuli such as traumatic injury, hormonal factors or certain kinds of drugs. [2]

The term lobular capillary hemangioma was first introduced in 1980 by Mills et al, [3] as an underlying lesion of PG, but nowadays this term is mostly used synonymously with PG. [4] On the other hand, in some publications two histological types of PG have been described. The first type consists of highly vascular proliferation which resembles granulation tissue, whereas the second type is characterized by proliferation of blood vessels with lobular aggregates which is called lobular capillary hemangioma (LCH). [1],[4],[5]

Although there are many studies about clinicopathologic and immunohistochemical features of PG, the two histological types of PG have rarely been compared in the literature. [2],[3],[4],[5],[6],[7],[8],[9] Apostosis et al, [5] used CD34, a-SMA, MSA to compare LCH and non-LCH PG and suggested that the two histological types of PG represent distinct entities.

Meanwhile, based on some studies, the proliferative activities of juvenile hemangioma, a benign vascular tumor of infancy and lobular capillary hemangioma were relatively high and no significant difference was found between them, so this hypothesis that the LCH type of PG has similar biologic behavior to hemangioma and differs from the non-LCH type of PG with a reactive nature, will be propounded. [2],[8],[9]

This study aims to compare the proliferation rate of LCH and non-LCH form of PG by an immunohistochemical method using anti-Ki-67 antibody as a proliferative marker and CD31 as an endothelial cell marker.


   Materials and Methods Top


Twenty cases of surgically resected specimens of PG (10 LCH, 10 non-LCH) were selected at various sites from archival paraffin embedded blocks of the Department of Oral and Maxillofacial Pathology in our Dental School, from 1990 to 2007. The criteria for selection were adequacy of tissue, minimal hemorrhage and inflammation and proper fixation. Clinical data such as age, gender, location, duration of the lesion, microscopic appearances and treatment for each lesion were collected from the clinical chart. Sections were cut from the paraffin-embedded blocks for hematoxylin and eosin (H and E) staining and simultaneous immunohistochemical detection of CD31 and Ki-67.

Formalin-fixed paraffin-embedded tissue in 4΅m sections were dewaxed by xylene and hydrated with ethanol then pretreated with a sodium citrate buffer (10 m mol, pH=6.6) for 30 minutes for antigenic retrieval. To block endogenous enzymes, 3% hydrogen peroxide was applied, then sections were incubated overnight with mouse monoclonal antibody against Ki-67 antigen (MIB-1, 1:100, DAKO, Denmark) and mouse monoclonal antibody against CD31 antigen (clone JC70A, prediluted, DAKO, Denmark) as primary antibodies. The slides were then rinsed gently with phosphate-buffered saline (PBS) and Envision Dual link system-HRP (ready to use, DAKO, Denmark) was used as secondary antibody. Incubation with 3-3 diaminobenzidine tetrahydrochloride (DAB) was performed for 10 minutes as a substrate chromogen solution to produce a brown color. After that, sections were counterstained with Mayer's hematoxylin. All steps were carried out at room temperature. Appropriate positive and negative control sections were processed in parallel.

In order to determine and compare the angiogenic and proliferative indices between the two histological types of PG, all slides were examined independently by two observers.

To determine the proliferative status, the labeling index for Ki-67 immunostaining (Ki-67 LI) was calculated by counting the positively stained nuclei in more than 1000 endothelial cells of both vascular component and stromal spindle-shaped cells. CD31 labeling index (CD31 LI) was also calculated with the same method for the cells that had positively stained cytoplasm, irrespective of labeling intensity. Finally, labeling index was determined for both markers by counting the cells which showed both nuclear and cytoplasmic staining.

The angiogenic index of each lesion was determined based on micro vessel count (MVC) which is the mean number of micro vessels in five areas of highest vascular density at ΄200 magnification, as described by Maeda et al. [10]

In order to compare the two groups, immunohistochemical results were analyzed using the Mann-Whitney U-test with the level of significance set at 95% (a=0.05) by SPSS software version 11.5 .


   Results Top


LCH PG was diagnosed in two male and eight female patients between the ages of four to 55 years (26.4 ± 16.2). Most common sites were gingival (80%) and lips. The duration of the lesions in LCH was reported from three to 48 weeks and the maximum diameter of the lesions ranged from 0.1 to 2.5 cm, most of them being pedunculated (80%).

In the non-LCH group, all patients were female between the ages of 10 to 65 (35.9 ± 19.2). The gingiva was the only site of involvement. Duration of the lesions ranged from 3 to 36 weeks and the size of the lesions ranged from 0.25 to 3 cm. Two parts were seen in both groups: an ulcerated surface and a deep portion. The ulcerated surface was similar in the two groups and consisted of an inflamed granulation tissue covered by a fibrinopurulent membrane.

The deep portion in the LCH group showed lobular accumulation of inconspicuous small capillaries and clusters of plump ovoid cells which were separated by fibrous stroma with mild or no inflammation [Figure 1]. The deep portion of the non-LCH type revealed numerous dilated capillaries with a scattered pattern exactly similar to the granulation tissue that was more infiltrated by chronic inflammatory cells compared to the LCH group [Figure 2].
Figure 1 :Lobular patt ern in the deep porti on of LCH PG. (H and E, ×100)

Click here to view
Figure 2 :Scatt ered patt ern of capillaries in the deep porti on of non-LCH PG. (H and E, ×100)

Click here to view


In both groups, most of the luminal cells showed positive immunoreactivity for CD31 [Figure 3] but only a few stromal ovoid cells expressed the CD31 marker [Figure 4]. However, positive immunoreactivity for Ki-67 was observed in fewer luminal cells [Figure 5] and certain ovoid cells in stroma [Figure 6], some of which were stained simultaneously with both Ki-67 and CD31 markers [Figure 7]. The results of counting immunoreactive cells for Ki-67, CD31 and both markers are summarized in [Table 1].
Table 1 :Percentages of staining cells by Ki-6 and CD31 anti bodies separately and simultaneously in each subtype of pyogenic granuloma

Click here to view
Figure 3 :CD31 positi ve luminal cells. (DAB, ×400)

Click here to view
Figure 4 :CD31 positi ve stromal cells. (DAB, ×400)

Click here to view
Figure 5 :Ki-67 positi ve luminal cells. (DAB, ×400)

Click here to view
Figure 6 :Ki-67 positi ve stromal cells. (DAB, ×400)

Click here to view
Figure 7 :Positive immunoreacti on with both Ki-67 and CD31 markers in luminal cells (long arrow) and stromal cells (short arrow). (DAB, ×400)

Click here to view


As the table shows, the mean number of Ki-67 positive cells in total, luminal and stromal cells were higher in the LCH group than the non-LCH. The percentage of CD31 positive cells in total (irrespective of location) and in luminal location was lower in the LCH group but it was higher in stromal cells compared to the non-LCH and the mean numbers of cells with positive immunoreactivity for both antibodies in the whole and in each location were higher in LCH than non-LCH. However, these differences were not statistically significant.

In each group, Ki-67 LI was higher in ovoid cells without lumen of stroma than capillary endothelial cells. This difference was statistically significant in the non-LCH type (P = 0.008) but not significant in LCH. CD31 positive cells in the endothelial cells of capillary were significantly higher than ovoid plump cells in stroma, (P (LCH) = 0.047, P (non-LCH) = 0.005).

In the LCH type, the density of cells in reacting to both markers in stroma was higher than luminal cells, (P (LCH) = 0.514). Conversely, in the non-LCH type, capillary endothelial cells show higher immunoreactivity for both Ki-67 and CD31 antibodies simultaneously than stromal ovoid cells, (P (non-LCH) = 0.362).

The mean numbers of blood vessels, determined by Micro vessel count (MVC), were 77.35 ± 34.6 and 82.58 ± 42.69 in LCH and non-LCH PG respectively, with no significant difference.


   Discussion Top


The biological behavior of any lesion is highly dependent on its proliferative activity. The present study aimed to determine proliferative activity of endothelial cells in two subtypes of PG. Detection of Ki-67 positive endothelial cells with the presence of inflammatory cells which have grate affinity to the Ki-67 antibody, would be problematic. In order to overcome this problem, a specific marker for recognizing endothelial cells was necessary.

CD3, also known as PECAM-1 (platelet endotelial cell adhesion molecule-1) is a 130 KDa integral membrane protein, a member of the immunoglobin super family, which mediates cell-to-cell adhesion. It is expressed constitutively on the surface of adult and embryonic endothelial cells and is expressed weakly on many peripheral leukocytes and platelets. [11] CD31 is involved in the transendothelial emigration of neutrophils and neutrophil PECAM-1 appears to be down-regulated after extravasation into inflamed tissues. [12] According to some studies, CD31 is the antibody of choice in the assessment of angiogenesis. [13]

The number of proliferating endothelial cells (both marker positive cells) in the LCH group was greater than the non-LCH and stromal endothelial cells in the LCH-type showed more proliferative activity than luminal endothelial cells, while in the non-LCH group proliferative activity in luminal endothelial cells was higher than stromal endothelial cells. Although these differences were not statistically significant, it can indicate the development of new capillary vessels in non-LCH type by angiogenesis in which preexisting vessels send out capillary sprouts to produce new vessels. [14]

However, in the LCH type, blood vessels are assembled by vasculogenesis in which the primitive vascular network is assembled from angioblasts. Such a pathway is seen during embryogenesis and in the cellular phase of juvenile hemangioma. [14] In our study, the density of CD31 positive cells in non-LCH PG was higher than the LCH. Regarding the role of CD31 in diapedesis, the high percentage of CD31 positive cells in the non-LCH type can represent the inflammatory or reactive nature of this type of PG. Lower density of CD31 positive endothelial cells can be due to lower differentiation in the endothelial cells of the LCH type compared to the non-LCH PG.

To the best of our knowledge, only a few studies have been done to investigate the quantitive assessment of CD31 marker in PG. Freitas et al, [13] qualitatively analyzed the immunohistochemical labeling patterns obtained with anti-CD31 antibody in PG and hemangioma. They found that cellular reactivity in PG was restricted to the cytoplasm of lining endothelial cells whereas in oral hemangioma cellular reactivity was observed in the cytoplasm of lining endothelial cells and clusters of proliferating endothelial cells without a lumen which is similar to the CD31 reactivity pattern in both two subtypes of PG in our study.

The mean number of CD31 positive cells at the luminal endothelial cells was significantly higher than stromal ovoid cells in both subtypes of PG. These findings with respect to the function of CD31 in the adhesion of endothelial cells are acceptable and logical.

In this study, proliferative activity in LCH PG was higher than non-LCH. Our results regarding Ki-67 LI are similar to Ki-67 LI of infantile capillary hemangioma (ICH) in the study of Iwata et al. [9] They did not find any significant differences between the proliferative activity of ICH and LCH. On the other hand, Nakamura et al, [2] showed a variable proliferative activity in PG from case to case and did not show any significant differences between pyogenic granuloma, granulation tissue and capillary hemangioma due to difference of developmental stage at the point of lesion resection. Ki-67 LI for LCH in a study by Dyduch et al, [8] was 14.13 ± 10.05. They counted positive cells in 10 high power fields, which is equivalent to approximately 2000 cells, and selected the lesions in active phase, [8] which was not feasible in our study due to the retrospective case selection.

In this study, proliferative activity in plump ovoid cells in stroma was higher than capillary endothelial cells. Since the luminal capillary endothelial cells are more differentiated than plump ovoid cells in stroma and the fact that differentiation usually has an inverse relation with proliferation, it is acceptable that luminal cells show lower proliferative activity than ovoid stromal cells which are probably immature endothelial cells.

The angiogenic index or micro vessel count (MVC) in the LCH group was lower than the non-LCH. Since the endothelial cells in the LCH type were mostly seen as clusters of endothelial cells without a distinct lumen, they showed lower MVC than the non-LCH PG which consisted of large vessels with distinct lumens. Our results about MVC for LCH- type were consistent with MVC for hemangioma in Feritas's study. [13]


   Conclusion Top


In summary, this study confirms that the LCH type of PG has more proliferative activity than the non-LCH type, so it can behave more aggressively and seems to be histologically more similar to a benign vascular neoplasm such as hemangioma rather than a reactive lesion such as pyogenic granuloma.


   Acknowledgment Top


The authors thank the "Shiraz Transplant Research Center" for their technical assistance.

 
   References Top

1.Neville BW, Damm DD, Allen CM, Bouquot JE. Oral and maxillofacial pathology, 2 nd ed. Philadelphia: WB Saunders; 2002. p. 447-9.  Back to cited text no. 1      
2.Nakamura T. Apoptosis and expression of Bax/Bcl-2 proteins in pyogenic granuloma: A comparative study with granulation tissue and capillary hemangioma. J Cutan Pathol 2000;27:400-5.   Back to cited text no. 2  [PUBMED]  [FULLTEXT]  
3.Mills SE, Cooper PH, Fechner RE. Lobular capillary hemangiom: the underlying lesion of pyogenic granuloma: A study of 73 cases from the oral and nasal mucous membranes. Am J Surg Pathol 1980:4:470-9.  Back to cited text no. 3      
4.Toida M, Hasegawa T, Watanabe F, Kato K, Makita H, Fujitsuka H, et al. Lobular capillary hemangioma of the oral mucosa: Clinicopathological study of 43 cases with a special reference to immunohistochemical characterization of the vascular elements. Pathol Int 2003;53:1-7.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]  
5.Epivatianos A, Antoniades D, Zaraboukas T, Zairi E, Poulopoulos A, Kiziridou A, et al. Pyogenic granuloma of the oral cavity: Comparative study of its clinicopathological and immunohistochemical features. Pathol Int 2005;55:391-7.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]  
6.AL-Khateeb T, Ababneh K. Oral pyogenic granuloma in Jordanians: A retrospective analysis of 108 Cases. J Oral Maxillofac Surg 2003;61:1285-8.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]  
7.Jafarzadeh H, Sanatkhani M, Mohtasham N. Oral pyogenic granuloma: A review. J Oral Sci 2006;48:167-75.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]  
8.Dyduch G, Okoρ K, Mierzyρski W. Benign vascular proliferations an immunohistochemical and comparative study. Pol J Pathol 2004;55:59-64.  Back to cited text no. 8      
9.Iwata J, Sonobe H, Furihata M, Ido E, Ohtsuki Y. High Frequency of Apoptosis in infantile capillary hemangioma. J Pathol 1996;179:403-8.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]  
10.Maeda K, Chung YS, Takatsuka S, Ogawa Y, Onoda N, Sawada T, et al. Tumour angiogenesis and tumour cell proliferation as prognostic indicators in gastric carcinoma. Br J Cancer 1995;72:319-23.   Back to cited text no. 10  [PUBMED]  [FULLTEXT]  
11.Newman PJ. The biology of PECAM-1. J Clin Invest 1997,99:3-8.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]  
12.Muller WA, Weigl SA, Deng X, Phillips DM. PECAM-1 is required for transendothelial migration of leukocytes. J Exp Med 1993,178:449-60.   Back to cited text no. 12  [PUBMED]  [FULLTEXT]  
13.Freitas TM, Miguel MC, Silveira EJ, Freitas RA, Galvγo HC. Assessment of angiogenic markers in oral hemangiomas and pyogenic granulomas. Exp Mol Pathol 2005;79:79-85.  Back to cited text no. 13      
14.Kumar V, Abbas AK, Fausto N. Robbins and Cotran pathologic basis of disease, 7 th ed. Philadelphia: WB Saunders; 2005. p. 107-8.  Back to cited text no. 14      

Top
Correspondence Address:
Gita Rezvani
Department of Oral and Maxillofacial Pathology, Dental School of Shiraz University of Medical Sciences, Shiraz
Iran
Login to access the Email id

Source of Support: Shiraz Transplant Research Center, Conflict of Interest: None


DOI: 10.4103/0377-4929.68242

Rights and Permissions


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1]

This article has been cited by
1 Association study between clinicopathological variables and periodontal breakdown in gingival pyogenic granuloma
Leonor V. González-Pérez,Diana M. Isaza-Guzmán,Sergio I. Tobón-Arroyave
Clinical Oral Investigations. 2014;
[Pubmed] | [DOI]
2 Assessment of clinicopathological characteristics and immunoexpression of COX-2 and IL-10 in oral pyogenic granuloma
Isaza-Guzmán, D.M., Teller-Carrero, C.B., Laberry-Bermúdez, M.P., González-Pérez, L.V., Tobón-Arroyave, S.I.
Archives of Oral Biology. 2012; 57(5): 503-512
[Pubmed]
3 Assessment of clinicopathological characteristics and immunoexpression of COX-2 and IL-10 in oral pyogenic granuloma
Diana M. Isaza-Guzmán,Clara B. Teller-Carrero,María P. Laberry-Bermúdez,Leonor V. González-Pérez,Sergio I. Tobón-Arroyave
Archives of Oral Biology. 2012; 57(5): 503
[Pubmed] | [DOI]
4 An epiglottic pyogenic granuloma presenting with spontaneous hemoptysis
Wang, H., Cerda, S., Hwang, H.S.
Laryngoscope. 2011; 121(sup 5): 309
[Pubmed]



 

Top
 
  Search
 
  
  
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
    Introduction
    Materials and Me...
    Results
    Discussion
    Conclusion
    Acknowledgment
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed4947    
    Printed161    
    Emailed11    
    PDF Downloaded331    
    Comments [Add]    
    Cited by others 4    

Recommend this journal