Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contact Us Login 
An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists

  Table of Contents    
Year : 2013  |  Volume : 17  |  Issue : 2  |  Page : 207-211

Evaluation of stromal myofibroblasts expression in keratocystic odontogenic tumor and orthokeratinized odontogenic cysts: A comparative study

1 Department of Oral and Maxillofacial Pathology, Himachal Institute of Dental Sciences, Paonta Sahib, Himachal Pradesh, India
2 Department of Oral and Maxillofacial Surgery, Himachal Institute of Dental Sciences, Paonta Sahib, Himachal Pradesh, India

Date of Web Publication11-Oct-2013

Correspondence Address:
Swati Roy
Department of Oral and Maxillofacial Pathology, Himachal Institute of Dental Sciences, Paonta Sahib, Himachal Pradesh - 173 025
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-029X.119789

Rights and Permissions



Objective: Keratocystic odontogenic tumor (KCOT) has an aggressive clinical course and a high tendency of recurrence, while orthokeratinized odontogenic cyst (OOC) has different characteristics and does not show aggressive behaviour. Even the treatment of these two lesions varies considerably. A large number of epithelial molecules have been studied in order to differentiate odontogenic keratocyst (OKC) from OOC, but stromal factors have not been adequately studied. Recently, tumor stroma has evolved as a particular field of interest. In the present study, we aim to evaluate and compare the expression of stromal myofibroblasts (MFs) in these entities and correlate it to its aggressive behavior. The term 'keratocystic odontogenic tumor' has been introduced by WHO in 2005 for odontogenic keratocyst keeping in mind its aggressive behavior, however still many pathologists and clinicians use the term OKC synonymously. Materials and Methods: A total of 10 cases of KCOT and 10 cases of OOC were stained for alpha-smooth muscle actin (αSMA) for demonstration of stromal MFs. MF frequency was assessed as the number of αSMA-positive stromal cells in 10 high power fields, presented as the mean number of positive cells per field. Results: Counts showed that the mean number of positive cells in KCOT (20.6 ± 2.05) was significantly higher than that seen in OOC (10.4 ± 1.06) (P < 0.05). Conclusion: The different behaviors of these lesions are compatible with the finding of the present study. The increased number of stromal MFs in KCOT in comparison to OOC correlates with its aggressive behavior and increased tendency towards recurrence.

Keywords: Keratocystic odontogenic tumor, orthokeratinized odontogenic cyst, stromal myofibroblasts, αSMA

How to cite this article:
Roy S, Garg V. Evaluation of stromal myofibroblasts expression in keratocystic odontogenic tumor and orthokeratinized odontogenic cysts: A comparative study. J Oral Maxillofac Pathol 2013;17:207-11

How to cite this URL:
Roy S, Garg V. Evaluation of stromal myofibroblasts expression in keratocystic odontogenic tumor and orthokeratinized odontogenic cysts: A comparative study. J Oral Maxillofac Pathol [serial online] 2013 [cited 2022 Aug 15];17:207-11. Available from: https://www.jomfp.in/text.asp?2013/17/2/207/119789

   Introduction Top

Keratocystic odontogenic tumor (KCOT), a developmental abnormality from derivatives of the dental lamina, has an aggressive clinical behavior and a propensity towards recurrence. It arises sporadically or in association with the nevoid basal cell carcinoma syndrome (NBCCS). [1]

It was first described by Philipsen in 1956, [2] and is now designated by the World Health Organization (WHO) as KCOT and is defined as "a benign uni- or multicystic, intraosseous tumor of odontogenic origin, with a characteristic lining of parakeratinized stratified squamous epithelium and potential for aggressive, infiltrative behavior." [3] WHO "recommends the term keratocystic odontogenic tumor as it better reflects its neoplastic nature." [3] Orthokeratinized odontogenic cyst (OOC), has originally been described by Philipsen [2] as a possible type of OKC; but is now considered as a distinct entity. [4]

The typical histology of KCOT includes lesions whose epithelial lining is uniformly thin, ranging from 8 to 10 cell layers. The basal layer exhibits a characteristic palisaded pattern with uniform nuclei and the luminal epithelial cells are often parakeratinized. Orthokeratinized foci, however, can also be found. Occasionally, budding of the basal cell layer into surrounding connective tissue and the formation of microcysts can be seen. The fibrous cyst wall is relatively thin and usually lacks inflammatory cell infiltrate. While OOC demonstrates a orthokeratinized surface and a prominent granular layer lying immediately below the flat surface. [5]

In contrast to KCOTs, the OOC has no tendency to recur and it is not associated with the NBCCS. [6] The higher recurrence rate, aggressive behavior, and neoplastic potential of KCOT suggest the importance of distinguishing KCOT and OOC. Recent investigations have demonstrated significant differences in the histological features and clinical behavior of these entities. [1] A wide range of epithelial-associated factors are implicated in the relative aggressive biological behavior of the odontogenic epithelium, but only a few studies have investigated nonepithelial factors that could contribute to the variable biological behavior of different types of odontogenic cysts and tumors. [7] It is now well-accepted that the coordinated activity of epithelial cells with their stroma is fundamental in controlling growth and differentiation in normal and pathological situations. [8] The role of tumor stroma in tumor progression is an important area of current research and has become a potential target for therapeutic intervention. [9] Presence of stromal MFs, an important component of tumor stroma has been linked to the biological behavior of both benign and malignant tumors. [10],[11] Vered et al., in a immunohistochemical (IHC) study assessed the frequency of MF in different odontogenic cysts and tumors and correlated it to their aggressive biological behavior. [7]

The aim of the present study was to quantitatively assess and compare the number of stromal MFs in KCOT and OOC.

   Materials and Methods Top

Study cases

Formalin-fixed, paraffin-embedded blocks of 10 cases each of KCOT and OOC were retrieved from the archives of the Department of Oral Pathology. Diagnoses were established on the hematoxylin and eosin (H and E) stained-slides [Figure 1] and [Figure 2]. Included cases were devoid of considerable inflammatory infiltrate in cystic walls.
Figure 1: Lining of KCOT showing palisaded basal layer of tall columnar cells with reversal of nuclear polarity. The surface exhibits parakeratinization. Subepithelial connective tissue shows radially arranged collagen fibers (H&E stain, ×200)

Click here to view
Figure 2: Orthokeratinized odontogenic cyst: Stratified squamous epithelial lining with surface thick layer of orthokeratin (H&E stain, ×100)

Click here to view

Staining procedure

Sections, 3 μm thick, were mounted on silane coated slides. After dewaxing in xylene, sections were dehydrated in ethanol, rinsed in distilled water, placed in 3% H 2 O 2 for 10 min, and rinsed in distilled water for 15 min. For antigen retrieval procedure, slides were placed in citrate buffer solution, pH = 6, in a microwave at 92°C for 10 min. After cooling at room temperature for 20 min, slides were exposed to primary alpha-smooth muscle actin (αSMA; marker for MFs) mouse anti-human antibody (Biogenex Ltd.) of dilution 1:100, for 60 min at room temperature. Slides were rinsed in phosphate buffer solution for 10 min. For antibody detection, universal immune peroxidase polymer anti-mouse rabbit HistofineR (Multi) kit (Nichirei, Tokyo, Japan) was used. Sections were rinsed in phosphate buffered saline (PBS) for 10 min, reacted with 3-Amino-9-Ethylcarbazole (AEC) substrate-chromagen kit, rinsed in PBS for 2 min, counterstained in Mayer's hematoxylin, and mounted with nonaqueous mounting agent.

Histomorphometric evaluation of αSMA in stained sections

Representative fields were randomly selected in each immunohistochemically stained section.

Counts were performed with a BH-2 Olympus microscope ×10 ocular, ×40 objective, and a counting grid containing 100 squares that determined the perimeter of the chosen field. Ten fields were chosen for each section. The grid was placed immediately beneath the cystic epithelial lining. Each αSMA-positive cell, excluding those surrounding blood vessels, was counted and the total number of positive cells for all 10 examined fields per case was calculated. This allowed calculation of the mean number of αSMA-positive cells per field.

Statistical analysis

Difference in the mean number of αSMA-positive cells per field between KCOT and OOC were analyzed using independent Student's t-test. Statistical significance was at P < 0.05. The Statistical Package for the Social Sciences (SPSS 14) software was used for computations.

   Results Top

The mean number of αSMA-positive cells per field in all examined cases of KCOT and OOC is shown in [Table 1]. Spindle cells showing fine αSMA-positivity were located beneath and parallel to the basement membrane of the odontogenic epithelium of these cystic lesions [Figure 3] and [Figure 4]. Additional small aggregates and short, delicate bundles of similar cells were found within the fibrous wall. αSMA positive cells within blood vessel walls served as positive control for the specificity of the stain.
Figure 3: KCOT: Subepithelial connective tissue shows alpha-smooth muscle positive cells radially arranged parallel to each other (IHC stain, ×200)

Click here to view
Figure 4: αSMA positive cells present only in the wall of blood vessels, while subepithelial connective tissue shows very few scattered cells. (IHC stain ×200)

Click here to view
Table 1: Mean number of alpha-smooth muscle actin positive cells per case

Click here to view

The value of αSMA positive cells in KCOT (20.6 ± 2.05) was significantly higher than that seen in OOC (10.4 ± 1.06) (P < 0.05) [Table 2].
Table 2: Mean±standard deviation for KCOT and OOC

Click here to view

   Discussion Top

The purpose of this study was to evaluate and compare the stromal MFs in KCOT and OOC. We found that the mean number of stromal MFs per high power field was considerably higher in the KCOTs.

KCOT is an aggressive cystic lesion that has a tendency to recur if not adequately removed. The recurrence rates have been documented with variable results from 3 to 60%. The recurrence rates of KCOT and OOC were studied and the results showed that KCOT recurred in at least 42.6%, compared with only 2.2% for the OOC. These data suggested the importance of distinguishing KCOT and OOC. [6]

Several attempts have been made to clinically, histologically, and biologically distinguish KCOT from OOC. A large series of the study revealed that OOC was more often associated with an impacted tooth than KCOT. [6] However, there were no significant differences between OOC and KCOT when age, race, sex, presenting symptoms, and the clinical impression were compared. Histologically, KCOT has a thin wall unless there is inflammation. [12]

The basal layer of the epithelium is well-defined and is composed of either columnar or cuboidal cells arranged in a palisaded pattern. The luminal surface, often corrugated, is typically covered with parakeratin. The OOC has a thin, uniform epithelial lining with a luminal surface of orthokeratin and well-developed granular layer. [4] Basal cells of the OOC are much less developed than those in the KCOT. They tend to be cuboidal or squamous and show little tendency to polarize or palisade. It was suggested that care must be taken to distinguish between keratin metaplasia in otherwise non-keratinized odontogenic cysts and the OOC. [4]

The proliferative activity of the lining epithelium of KCOT has been the subject of various investigations aiming at the expression of p53, proliferating cell nuclear antigen (PCNA), and Ki67. Such studies concluded that p53, PCNA, and Ki67 are more strongly expressed in KCOTs than in other types of odontogenic cysts. [13] Thosaporn et al., indicated that the proliferation index of IPO-38 was useful + in predicting the different biological behavior of the odontogenic lesions. Moreover, the KCOT should be regarded as benign tumor, while the OOC as a nonaggressive cystic lesion. [14]

Rangiani and Motahhary in their study showed that there is a significant difference between the two cysts regarding bcl-2 proapoptotic protein expression that is compatible with their different clinical behaviors. [1] Recently, the IHC profiles of cytokeratins 10, 13, and 14 and extracellular matrix proteins, fibronectin, types I and III collagen and tenascin, indicated that the OOC presented a well-formed cystic enveloping, whereas the KCOT profile was compatible with a more aggressive biological behavior. [15] However, till date there are only few studies evaluating the role of the stromal cells in the aggressiveness of these cystic lesions.

Stromal involvement in tumor progression was examined by extracellular matrix components in syndrome and non-syndrome OKC [16] and showed differences in expression of tenascin, fibronectin, and collagen IV, explaining, in part, the different degrees of aggressiveness of biological behavior of these entities. In an additional study, collagen in the walls of OKCs was analyzed histochemically by staining the sections with picrosirius red and examining them with polarizing microscopy. [17] It was found that the staining of the collagen fibers in the KCOTs was similar to that reported in the odontogenic neoplasms, [18] suggesting that the stroma of KCOT could be regarded not just a structural support of the cyst wall, but as the one playing a part in the neoplastic behavior of the cyst as well.

In general stromal reaction to epithelial neoplasm is marked by the appearance of MFs. [19] Approximately 30 years ago, MF were shown to be present in the stroma of various invasive and metastatic malignant tumors characterized by hard consistency and retraction (e.g., ductal mammary carcinoma associated with skin or nipple retraction and metastatic carcinoma to lymph nodes fixed to surrounding tissue and overlying skin). At that time it was assumed that this phenomenon was part of the host reaction to prevent invasion of malignant cells, since MF were numerous, particularly at the invasive front. However, over the past 10 years, there has been an abundance of evidence that the presence of MF at the invasion front is not part of the host defence mechanism against tumor invasion, but actually promotes it. [7]

These cells have also been reported in a number of pathological states involving the oral tissues: Nodular fascitis, giant cell fibroma, malignant fibrous histiocytoma, gingival hyperplasia, central and peripheral giant cell granulomas, and adult and infantile fibromatosis. [20] Stromal MFs are known to remodel the extracellular matrix and helps in its degradation by secretion of matrix metalloproteinases thereby promoting the invasive growth of epithelial lesions. [8],[21]

The presence of MF in odontogenic lesions has not been thoroughly investigated. Vered et al., evaluated quantitavely the expression of MFs in different odontogenic cysts and tumors, and the results showed that the mean number of MF, in well-recognized aggressive odontogenic lesions (ameloblastoma and OKC) was high and did not differ significantly from that in SCC. In contrast, known nonaggressive lesions (unicystic ameloblastoma, ameloblastic fibro-odontoma, OOC, and dentigerous cyst) showed significantly lower results compared to ameloblastoma and OKC. They suggested a positive link, that is when more MFs are present in the stroma, a more aggressive behavior of the odontogenic cyst/tumor can be anticipated. [7]

Our findings showed that the mean number of αSMA positive cells in the connective tissue wall of KCOT was significantly higher than that in OOC and stated that the two cysts not only differ in the epithelial characteristics, but also in the stromal wall component. This study is also an evidence for the distinction between the two cysts as separate lesions, and categorizing KCOT as a cystic tumor; while the OOC as a nonaggressive cystic lesion.

   Acknowledgement Top

We would like to acknowledge Dr. S Hemavathy, Retd. Professor and Head, Department of Oral and Maxillofacial Pathology, Government Dental College and Research Institute, Bangalore for her constant support and guidance throughout the study. We would also like to extend a heartfelt thanks to Dr. G Girraddi, Professor and Head, Dept of Oral Surgery and Dr. S Srinath, Dr.Chandrakala and Dr. T Suresh, faculty of Department of Oral Pathology, GDCRI, Bangalore for their invaluable help in completion of the study.

   References Top

1.Rangiani A, Motahhary P. Evaluation of bax and bcl-2 expression in odontogenic keratocysts and orthokeratinized odontogenic cysts: A comparison of two cysts. Oral Oncol 2009;45:e41-4.  Back to cited text no. 1
2.Philipsen HP. Om keratocyster (Kolesteatom) I kaeberne. Tandlaegebladet 1956;60:963-71.  Back to cited text no. 2
3.Barnes L, Eveson JW, Reichart P, Sidransky D, editor. World Health Organization classification of tumors. Pathology and Genetics of Head and Neck Tumours. Lyon: IARC Press; 2005. series.  Back to cited text no. 3
4.Wright JM. The odontogenic keratocyst: Orthokeratinized variant. Oral Surg Oral Med Oral Pathol 1981;51:609-18.  Back to cited text no. 4
5.Kola ZK, Geierova M, Bouchal J, Pazderaz J, Zobril V, Tvrdy P. Immunohistochemical analysis of the biological potential of odontogenic keratocysts. J Oral Pathol Med 2006;35:75-80.  Back to cited text no. 5
6.Crowley TE, Kaugars GE, Gunsolley JC. Odontogenic keratocysts: A clinical and histologic comparison of the parakeratin and orthokeratin variants. J Oral Maxillofac Surg 1992;50:22-6.  Back to cited text no. 6
7.Vered M, Shohat I, Buchner A, Dayan D. Myofibroblasts in stroma of odontogenic cysts and tumors can contribute to variations in the biologic behavior of the lesions. Oral Oncol 2005;41:1028-33.  Back to cited text no. 7
8.Desmouliere A, Guyot C, Gabbiani G. The stroma reaction myofibroblast: A key player in the control of tumor cell behavior. Int J Dev Biol 2004;48:509-17.  Back to cited text no. 8
9.Walter-Yohrling J, Pratt BM, Ledbetter S, Teacher BA. Myofibroblasts enable invasion of endothelial cells into three-dimensional tumor cell clusters: A novel in vitro tumor model. Cancer Chemother Pharmacol 2003;52:263-9.  Back to cited text no. 9
10.Sobral AP, Loducca SV, Nunes FD, De Araujo NS, Kowalski LP, De Araujo VC. Relationship between major and minor salivary gland mucoepidermoid carcinoma Malignancy grading and presence of stromal myofibroblasts: Immunohistochemical study. J Oral Pathol Med 2004;33:335-9.  Back to cited text no. 10
11.Vered M, Nasrallah W, Buchner A, Dayan D. Stromal myofibroblasts in central giant cell granuloma of the jaws cannot distinguish between non-aggressive and Aggressive lesions. J Oral Pathol Med 2007;36:495-500.  Back to cited text no. 11
12.Kramer IR, Pindborg JJ, Shear M. World health Organization. International histological classification of tumors. Histological typing of odontogenic tumors. Berlin: Spinger-Verlag; 1992.  Back to cited text no. 12
13.Mendes RA, Carvalho JF, van der Waal I. Biological pathways involved in the aggressive behavior of the Keratocystic Odontogenic tumor and possible implications for molecular oriented treatment - An overview. Oral Oncol 2010;46:19-24.  Back to cited text no. 13
14.Thosaporn W, Iamaroon A, Pongsiriwet S, Ng KH. A comparative study of epithelial cell proliferation between the odontogenic keratocyst, orthokeratinized odontogenic cyst, dentigerous cyst, and ameloblastoma. Oral Dis 2004;10:22-6.  Back to cited text no. 14
15.da Silva MJ, de Sousa SO, Correa L, Carvalhosa AA, De Araujo VC. Immunohistochemical study of the orthokeratinized odontogenic cysts: A comparison with the Odontogenic keratocyst. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:732-7.  Back to cited text no. 15
16.Amorim RF, Godoy GP, Galvao HC, Souza LB, Freitas RA. Immunohisto-chemical assessment of extracellular matrix components in syndrome and non-syndrome odontogenic Keratocysts. Oral Dis 2004;10:265-70.  Back to cited text no. 16
17.Hirshberg A, Sherman S, Buchner A, Dayan D. Collagen fibers in the wall of odontogenic keratocysts: A study with picrosirius red and polarizing microscopy. J Oral Pathol Med 1999;28:410-2.  Back to cited text no. 17
18.Hirshberg A, Buchner A, Dayan D. The central odontogenic fibroma and the hyperplastic dental follicle: Study with picrosirius red and polarizing microscopy. J Oral Pathol Med 1996;25:125-7.  Back to cited text no. 18
19.De Wever O, Mareel M. Role of tissue stroma in cancer cell invasion. J Pathol 2003;200:429-47.  Back to cited text no. 19
20.Lombardi T, Morgan PR. Immunohistochemical characterisation of odontogenic cysts with mesenchymal and myofilament markers. J Oral Pathol Med 1995;24:170-6.  Back to cited text no. 20
21.Orimo A, Weinberg RA. Stromal fibroblasts in cancer: A novel tumor-promoting cell type. Cell Cycle 2006;5:1597-601.  Back to cited text no. 21


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]

This article has been cited by
1 Multiple Orthokeratinized Odontogenic Cysts: A Case Report
Yi-Shing Lisa Cheng,Hui Liang,John Wright,Tom Teenier
Head and Neck Pathology. 2014;
[Pubmed] | [DOI]


Print this article  Email this article


    Similar in PUBMED
    Search Pubmed for
    Search in Google Scholar for
  Related articles
    Article in PDF (1,392 KB)
    Citation Manager
    Access Statistics
    Reader Comments
    Email Alert *
    Add to My List *
* Registration required (free)  

    Materials and Me...
    Article Figures
    Article Tables

 Article Access Statistics
    PDF Downloaded352    
    Comments [Add]    
    Cited by others 1    

Recommend this journal

Journal of Oral and Maxillofacial Pathology | Published by Wolters Kluwer - Medknow
Online since 15th Aug, 2007