|Year : 2016 | Volume
| Issue : 7 | Page : 7-12
Immunohistochemical evaluation and biological role of stromal myofibroblasts in odontogenic keratocyst, dentigerous cyst, and ameloblastoma: A comparative study
Swati Roy1, Satevanthan Hemavathy2, Vipul Garg3
1 Department of Oral and Maxillofacial Pathology, Yamuna Institute of Dental Sciences and Research, Yamunanagar, Gadholi, Haryana, India
2 Department of Oral and Maxillofacial Pathology, Government Dental College and Research Institute, Bengaluru, Karnataka, India
3 Department of Oral and Maxillofacial Surgery, Uttaranchal Dental and Medical Research Institute, Dehradun, Uttarakhand, India
|Date of Web Publication||28-Jul-2016|
Department of Oral and Maxillofacial Pathology, Yamuna Institute of Dental Sciences and Research, Yamunanagar, Gadholi, Haryana
Source of Support: None, Conflict of Interest: None
Context: Stromal myofibroblasts (MFs) are key cells for connective tissue remodeling and interact with epithelial cells and other connective tissue cells to control phenomena as tumor invasion and angiogenesis thereby contributing to their biological behavior. Aims: The study assesses the frequency of stromal MF in solid ameloblastoma (SA), odontogenic keratocyst (OKC), and dentigerous cyst (DC) and relates it to their biological behavior. Settings and Design: Ten cases of each lesion were taken and stained immunohistochemically for alpha-smooth muscle actin (α-SMA) which is a marker for stromal MF. Materials and Methods: Ten cases each of SA, OKC, and DC were included in the study. Cases of oral squamous cell carcinoma (SCC, n = 5) served as the baseline for comparison as they are aggressive lesions expressing increased number of stromal MFs. The frequency of MFs was assessed as the number of α-SMA-positive stromal cells in 10 high-power fields and presented as the mean number of positive cells per field. Statistical Analysis Used: Differences in the mean number of α-SMA-positive cell per field among SA, OKC, DC, and SCC were analyzed using one-way ANOVA test. Results: Counts showed that mean number of α-SMA-positive MFs in SA, OKC, and DC were 24.56 (±4.63), 21.37 (±4.17), and 8.03 (±2.15), respectively. Results showed that the mean number of stromal MFs in SA and OKC was significantly higher than that in DC (8.03 ± 2.15) (P < 0.05). The count of MFs in SA and OKC was not significantly different from that of SCC (25.06 ± 4.61) (P > 0.05). Conclusion: Activated MF participates in the matrix degradation process which is considered to be one of the main forces in tumor growth and invasion. Among odontogenic lesions, ameloblastoma and OKC (presently termed as keratocystic odontogenic tumor) are well known for their higher growth and recurrence potential. They tend to show burrowing growth pattern. Various studies have evaluated the epithelial factors responsible for their growth potential; we in our study have tried to relate the emergence of stromal MF to the biological behavior of these lesions. The frequency of stromal MF in OKC and ameloblastoma was almost similar to that in SCC, thereby implying that MF can contribute to the biological behavior of these odontogenic lesions.
Keywords: Dentigerous cyst, immunohistochemical, odontogenic keratocyst, solid ameloblastoma, stromal myofibroblasts
|How to cite this article:|
Roy S, Hemavathy S, Garg V. Immunohistochemical evaluation and biological role of stromal myofibroblasts in odontogenic keratocyst, dentigerous cyst, and ameloblastoma: A comparative study. N Niger J Clin Res 2016;5:7-12
|How to cite this URL:|
Roy S, Hemavathy S, Garg V. Immunohistochemical evaluation and biological role of stromal myofibroblasts in odontogenic keratocyst, dentigerous cyst, and ameloblastoma: A comparative study. N Niger J Clin Res [serial online] 2016 [cited 2022 May 23];5:7-12. Available from: https://www.mdcan-uath.org/text.asp?2016/5/7/7/187181
| Introduction|| |
Tissue integrity is maintained by the stroma under physiological conditions.  Coordinated activity of the epithelial cells with their stroma is fundamental in controlling growth and differentiation in normal and pathological situations. 
Myofibroblast (MF), a modulated fibroblast which has acquired the capacity to neoexpress alpha-smooth muscle actin (α-SMA), is one of the cellular components of the stroma. These cells play an important role in remodeling connective tissue and also interact with epithelial cells and other connective tissue cells and thereby control tumor invasion. 
In humans and animals, MFs are found in normal tissues (lymph nodes and blood vessels) and pathological conditions (reactive lesions, benign tumors, locally aggressive borderline fibromatoses, and sarcomas). Transdifferentiation of fibroblast to MFs marks the stromal change during tumorigenesis and wound healing. 
Many studies have stated the presence of stromal MFs in different neoplasms including squamous cell carcinoma (SCC) while there is less of literature and evidence-based studies in relation to odontogenic lesions. Although odontogenic lesions share a common cell of origin, they demonstrate different biological behavior. Odontogenic epithelium is responsible for tooth development under physiologic condition but may give rise to diverse group of pathologies exhibiting different degrees of aggressiveness. ,, This discrepancy in biological behavior has been attributed to differences in the specific features that the epithelial component acquires during the lesion development such as increased proliferative potential as reflected by various proliferative markers, for example, Ki-67,  IPO-38,  Argyrophilic nucleolar organizer region (AgNOR);  impaired expression of tumor suppressor genes and their protein products, for example, PTCH,  p53, and MDM2; , and abnormal activity of cell-cycle-related pathways, for example, inducible nitric oxide synthase, HSP30, and telomerase. However, only a few studies have investigated nonepithelial factors that could contribute to the variable behavior of the different types of odontogenic cysts and tumors. 
MFs are known to act at the tumor front and may facilitate the growth by dissolution of the ground substance or restrict by causing fibrosis. Immunohistochemical studies and co-culture systems of MFs with malignant cells showed elevated production of extracellular matrix proteins such as tenascin and fibronectin and altered secretion of proteolytic enzymes such as stromelysin-3 and matrix metalloproteinase-2 (MMP-2), thereby suggesting that the MFs might support cancer-invasive properties through facilitation of the initial attachment and also through infiltrative movement of cancer epithelial cells. ,
Ameloblastoma and odontogenic keratocyst (OKC), although defined as benign, demonstrate locally aggressive behavior. The purpose of this study was to evaluate and compare the presence of MFs in solid ameloblastoma (SA), OKC, and dentigerous cyst (DC) and relate it to their biological behavior.
| Materials and Methods|| |
A total of 30 archival specimens which were formalin fixed, processed, and embedded tissues of prediagnosed 10 cases of SA (Group A), 10 cases of OKC (Group B), 10 cases of DC (Group C), and 5 cases of SCC (Group X) were retrieved from the Department of Oral Pathology and Microbiology by random sampling. Cases of SCC were also included in our study to facilitate the comparison of odontogenic lesion with a well-known aggressive lesion that expresses increased number of stromal MFs.
All the archival specimens that had been previously diagnosed as SA (only solid multicystic variant, no unicystic variant), OKC, and DC with no history of malignancy either orally or systemically were included in the study group.
All the cases of SA, OKC, and DC diagnosed with other odontogenic cysts, tumors, or systemic malignancies were excluded from the study.
The 3 μm thick sections were mounted on positively charged microscope slides (BioGenex, USA). After dewaxing in xylene, they were rehydrated in graded solutions of ethanol, rinsed with distilled water, placed in 3% H 2 O 2 for 10 min, and then rinsed with distilled water for 15 min. For antigen retrieval, the 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, the slides were exposed to primary α-SMA mouse anti-human antibody (clone 1A4, Dako A/S, Glostrup, Denmark), dilution 1:100, for 60 min at room temperature and then rinsed with phosphate-buffered saline (PBS) for 10 min. A universal immune peroxidase polymer anti-mouse rabbit Histofine R (multi) kit (Nichirei, Tokyo, Japan) was used for antibody detection. The sections were rinsed with PBS for 10 min, reacted with an AEC substrate-chromogen kit (Zymed, San Francisco, CA, USA), rinsed with PBS for 2 min, counterstained in Mayer's hematoxylin, and then wee mounted using Dibutyle phthalate xylene (DPX).
Histomorphometric evaluation of alpha-smooth muscle actin stained sections
The presence of brown-colored end product at the site of target antigen was indicative of positive reactivity. Representative fields were randomly selected in each immunohistochemically stained section. Counts were performed with Olympus compound microscope fitted with an eyepiece ×10 magnification and objective ×40 magnification. Ten fields were chosen for each section.
For cystic lesions, the fields were selected immediately beneath the cystic epithelium lining and for SA, it was selected immediately adjacent to the tumor islands/nests/cords.
Each α-SMA 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 α-SMA-positive cell per field.
Results are represented as the mean number of α-SMA-positive cell per high-power field for each type of lesions.
To eliminate observer bias, the count was performed by three different observers and kappa index was found to be >0.7, thus showing good interobserver agreement.
Differences in the mean number of α-SMA-positive cell per field among all the four type of lesions (SA, OKC, DC, and SCC) were analyzed using one-way ANOVA test.
The statistical significance was at P < 0.05.
The statistical package for social sciences (version 14, IBM Corporation) software was used for computations.
| Results|| |
In SA, islands of odontogenic epithelium were surrounded by layers of α-SMA-positive cell. Spindle cells showing fine α-SMA positivity were located beneath and parallel to the basement membrane of the odontogenic epithelium of the cystic lesions [Figure 1] [Figure 2] [Figure 3]. Additional small aggregates and short, delicate bundles of similar cells were found within the fibrous wall. These findings were remarkable in OKC. Sections of SCC showed that malignant islands were surrounded by abundant α-SMA-positive stromal cells [Figure 4]. Cases of SCC were used for comparison as studies have shown that the malignant epithelial islands are surrounded by increased number of stromal MFs. The count of α-SMA-positive cells as for SA, OKC, DC, and SCC is as shown in [Table 1].
|Figure 1: Alpha-smooth muscle actin-positive myofibroblasts adjacent to the epithelial islands (×200)|
Click here to view
|Figure 2: Considerable alpha-smooth muscle actin expression by myofibroblasts in odontogenic keratocyst-P in the connective tissue adjacent to the epithelium (×200)|
Click here to view
|Figure 3: Few myofibroblasts in the connective tissue wall of dentigerous cyst|
Click here to view
|Figure 4: Islands of squamous cell carcinoma surrounded by alpha-smooth muscle actin-positive myofibroblasts (×100)|
Click here to view
|Table 1: Mean number of alpha - smooth muscle actin positive cells per 10 high-power field per slide for the four groups |
Click here to view
The mean number of α-SMA-positive cells per field in all examined cases of odontogenic lesions and SCC is shown in [Table 2].
|Table 2: Mean number of alpha - smooth muscle actin-positive myofibroblasts per field (±standard deviation) in solid ameloblastoma, odontogenic keratocyst, dentigerous cyst and squamous cell carcinoma |
Click here to view
SA and OKC demonstrated a higher number of α-SMA-positive cells per field with a value of 24.56 (±4.63) and 21.37 (±4.17), respectively while DC demonstrated a lower mean of 8.03 (±2.15).
There was no statistically significant difference between SA and OKC (P > 0.05). The difference between SA and DC and between OKC and DC was statistically significant (P < 0.05).
When SA and OKC were compared to SCC, the mean number of α-SMA-positive cells per field in SCC was not significantly different from that of SA and OKC (P > 0.05).
| Discussion|| |
Emergence of a tumor is a multistep process accompanied by genetic alterations of precancerous cells and simultaneously by building up the microenvironment that promotes transformation. Epigenetic contributions from the stroma also play important roles for formation of progressive neoplasm.  The "seed and soil hypothesis" in cancer as proposed by Paget (1889) over a century ago also emphasized the role of tumor microenvironment and tumor-host crosstalk in organ-specific cancer metastasis and the "seed-soil" compatibility contributing to the establishment of metastasis and colonization of cancer cells. However, he stated that the molecular features of the "soil" are less well understood than those of the "seed," and understanding the "soil" is now an important issue in cancer research and therapy. 
Malignant tumors such as those originating in the colon, breast, liver, lung, prostate, and pancreas, as well as in oral carcinoma have shown the presence of MFs at the tumor invasion front. Cytokines such as transforming growth factor-β and platelet-derived growth factor released from the cancer cells play an important role in emergence of MFs. When involved in physiological process such as wound healing, these cells undergo apoptosis, quite in contrast to tumors where they persist as in a wound that does not close (malignancies). ,
The presence of stromal MFs has been linked to the biological behavior of both benign and malignant tumors.  Powell et al. found global MF activation in precancerous tubular and villous adenomas, thereby stating that they are present at an earlier stage of this process, i.e., the transition from normal colonic tissue to adenomatous polyp.  These cells have also been reported in a number of pathological states involving the oral tissues: Nodular fasciitis, giant cell fibroma, malignant fibrous histiocytoma, gingival hyperplasia, central and peripheral giant cell granulomas, and adult and infantile fibromatosis. 
Although fundamental studies suggest that MFs may either facilitate or inhibit cancer progression, cumulative evidence supports their role in promoting tumor progression.  High frequency of stromal MFs in cases of oral cancer is significantly associated with poor survival. Recent findings have suggested that the tumor is not acting on its own but is rather surrounded by a "milieu" of malignancy made up by the tumor-associated stroma, which is created by and acts for the tumor itself. 
Fregnani et al.,  Souza Freitas et al.,  and Kumamoto  reported abundant presence of MFs and high levels of MMPs in ameloblastoma and mentioned their role in degradation of the extracellular matrix and the basement membrane components. SA and OKC are characterized by a benign but locally invasive behavior with a high risk of recurrence, both capable of showing involvement of adjacent cancellous bone, and destructive growth. OKC that has been renamed as keratocystic odontogenic tumor is now classified under the list of odontogenic tumor as per the new WHO classification (2005). , Hirshberg et al found that the staining of the collagen fibers in OKCs was similar to that reported in the odontogenic neoplasms, suggesting that the stroma of OKCs not only as a structural support of the cyst wall but also plays a part in neoplastic behavior of the cyst. 
The presence of MF in odontogenic lesions has not been thoroughly investigated. Some of the previous studies were case reports identifying the presence MFs in different odontogenic lesions while others assessed the frequency and intensity of these cells to establish a correlation between the appearance of MFs and the biological behavior of the respective odontogenic cyst or tumor. However, no unified results have yet been obtained. 
Rothouse et al. in 1980 for the first described the presence of MFs in the stromal component with a unique and previously unreported demonstration of intracellular septate junctions.  Vered et al. found that among the odontogenic cysts, OKC had the highest mean number of MF and DC had the lowest. Furthermore, among the odontogenic tumors, MF in ameloblastoma was significantly higher than unicystic ameloblastoma, and they concluded that MFs in the stroma of odontogenic cysts and tumors can contribute to variations in the biological behavior of these lesions.  While on the other hand, Lombardi and Morgan, in spite of confirming the presence of MF in odontogenic cysts wall, suggested that they might be part of a homeostatic response to the distension caused by cyst enlargement.  Mashhadiabbas et al. also demonstrated the presence of MF in the stroma of the odontogenic lesions but did not report any positive relationship with the lesion's aggressive behavior. 
Hence, the role of MF has yet not been established in the odontogenic lesions as there still exists divergence of opinion. In the present study, which quantitatively evaluated, the presence of MFs in the stroma of SA, OKC, and DC has provided persuasive evidence that the stroma of these lesions harbor MF as reflected by α-SMA-positive cell. Results have clearly shown that the mean number of MF in SA and OKC was high and did not differ significantly from that in SCC (P > 0.05). In contrast, DC showed significantly lower number of MF in the adjacent stroma as compared to SA and OKC (P < 0.05). Ameloblastoma demonstrated the presence of MFs adjacent to the odontogenic islands similar to that seen in SCC, where these cells were seen in concentric layers surrounding the dysplastic epithelial islands present in the connective tissue. In odontogenic cyst, the cells were oriented parallel and were present adjacent to the lining epithelium. Deeper connective tissue showed haphazardly arranged α-SMA positive cells. The findings of the present study were in accordance with the study done by Vered et al.  evaluating the role of MFs in different odontogenic cysts and tumors.
Based on the findings of the present study, the variable clinical behavior of an odontogenic cyst and tumor can be attributed to the emergence of α-SMA-positive MFs in the supportive connective tissue. These cells constantly interact with the tumor cells and create a favorable environment for their infiltrative growth. It can be suggested that odontogenic epithelium, mainly in SA and OKC, can act and modulate the stromal MF in the same manner as that of SCC, thereby confirming a positive link stating that when more MF are present in the stroma, a more extensive growth and recurrence of the respective odontogenic lesion can be anticipated.
Stromal MFs are so now recognized to be the main effectors of tumor needs in terms of angiogenesis, production of metalloproteinases for collagen breakdown, and further invasion and suppression of the host immune response. Local recurrence and overall survival are negatively influenced by abundance of stromal MFs. 
Therefore, recently, greater emphasis has been given to the advantages of therapeutic targeting of the tumor-associated stroma; first, because the ongoing functioning of stromal cells is presumably critical to the growth of nearby neoplastic cells and second, as the stromal cells are stable genetically in contrast to carcinoma cells, which are genetically unstable and accumulate adaptive mutations during the course of therapy to acquire drug resistance. ,
| Conclusion|| |
The current investigation demonstrated α-SMA-positive MFs in the stroma of SA, OKC, and DC. The mean number of MFs was higher in OKC and ameloblastoma and the value obtained was comparable to that of SCC with no statistically significant difference while the mean number of MFs in DC was quite low and significantly different from that of SA and OKC.
The findings of the present study indicated that the locally invasive growth pattern of ameloblastoma and OKC can be positively correlated to the emergence of MF in the adjacent stroma.
Further investigation and studies in this field with a note on the treatment perspective will help in establishing the role of MFs in the biological behavior of odontogenic lesions.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
De Wever O, Demetter P, Mareel M, Bracke M. Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer 2008;123:2229-38.
Desmoulière 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.
Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI, West AB. Myofibroblasts. I. Paracrine cells important in health and disease. Am J Physiol 1999;277(1 Pt 1):C1-9.
Kumamoto H, Yoshida M, Ooya K. Immunohistochemical detection of hepatocyte growth factor, transforming growth factor-beta and their receptors in epithelial odontogenic tumors. J Oral Pathol Med 2002;31:539-48.
Kumamoto H. Molecular pathology of odontogenic tumors. J Oral Pathol Med 2006;35:65-74.
Gnepp DR. Diagnostic Surgical Pathology of Head and Neck. 1 st
ed. WB Saunder Company; 2001. p. 605.
Shear M. The aggressive nature of the odontogenic keratocyst: Is it a benign cystic neoplasm? Part 2. Proliferation and genetic studies. Oral Oncol 2002;38:323-31.
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.
Eslami B, Yaghmaei M, Firoozi M, Saffar AS. Nucleolar organizer regions in selected odontogenic lesions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:187-92.
Agaram NP, Collins BM, Barnes L, Lomago D, Aldeeb D, Swalsky P, et al.
Molecular analysis to demonstrate that odontogenic keratocysts are neoplastic. Arch Pathol Lab Med 2004;128:313-7.
Malcic A, Jukic S, Anic I, Pavelic B, Kapitanovic S, Kruslin B, et al.
Alterations of FHIT and P53 genes in keratocystic odontogenic tumor, dentigerous and radicular cyst. J Oral Pathol Med 2008;37:294-301.
Vered M, Shohat I, Buchner A, Dayan D. Myofibroblasts in stroma of odontogenic cysts and tumors can contribute to variations in the biological behavior of lesions. Oral Oncol 2005;41:1028-33.
Orimo A, Tomioka Y, Shimizu Y, Sato M, Oigawa S, Kamata K, et al.
Cancer-associated myofibroblasts possess various factors to promote endometrial tumor progression. Clin Cancer Res 2001;7:3097-105.
Nakagawa H, Liyanarachchi S, Davuluri RV, Auer H, Martin EW Jr., de la Chapelle A, et al.
Role of cancer-associated stromal fibroblasts in metastatic colon cancer to the liver and their expression profiles. Oncogene 2004;23:7366-77.
Mareel M, Leroy A. Clinical, cellular, and molecular aspects of cancer invasion. Physiol Rev 2003;83:337-76.
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.
Powell DW, Adegboyega PA, Di Mari JF, Mifflin RC. Epithelial cells and their neighbors I. Role of intestinal myofibroblasts in development, repair, and cancer. Am J Physiol Gastrointest Liver Physiol 2005;289:G2-7.
Lombardi T, Morgan PR. Immunohistochemical characterisation of odontogenic cysts with mesenchymal and myofilament markers. J Oral Pathol Med 1995;24:170-6.
Tsujino T, Seshimo I, Yamamoto H, Ngan CY, Ezumi K, Takemasa I, et al.
Stromal myofibroblasts predict disease recurrence for colorectal cancer. Clin Cancer Res 2007;13:2082-90.
Vered M, Dobriyan A, Dayan D, Yahalom R, Talmi YP, Bedrin L, et al.
Tumor-host histopathologic variables, stromal myofibroblasts and risk score, are significantly associated with recurrent disease in tongue cancer. Cancer Sci 2010;101:274-80.
Fregnani ER, Sobral LM, Alves FA, Soares FA, Kowalski LP, Coletta RD. Presence of myofibroblasts and expression of matrix metalloproteinase-2 (MMP-2) in ameloblastomas correlate with rupture of the osseous cortical. Pathol Oncol Res 2009;15:231-40.
Souza Freitas V, Ferreira de Araújo CR, Alves PM, de Souza LB, Galvão HC, de Almeida Freitas R. Immunohistochemical expression of matrilysins (MMP-7 and MMP-26) in ameloblastomas and adenomatoid odontogenic tumors. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:417-24.
Barnes L, Eveson JW, Reichart P, Sidransky D, editors. Pathology and Genetics of Head and Neck Tumors. WHO Classification of Tumours. Lyon: International Agency for Research on Cancer (IARC); 2005. p. 306-7.
Madras J, Lapointe H. Keratocystic odontogenic tumour: Reclassification of the odontogenic keratocyst from cyst to tumour. J Can Dent Assoc 2008;74:165a-165h.
Hirshberg A, Sherman S, Buchner A, Dayan D. Collagen fibres in the wall of odontogenic keratocysts: A study with picrosirius red and polarizing microscopy. J Oral Pathol Med 1999;28:410-2.
Rothouse LS, Majack RA, Fay JT. An ameloblastoma with myofibroblasts and intracellular septate junctions. Cancer 1980;45:2858-63.
Mashhadiabbas F, Moghadam SA, Moshref M, Elahi M. Immunohistochemical detection and ultrastructure of myofibroblasts in the stroma of odontogenic cysts and ameloblastoma. Iran Red Crescent Med J 2010;12:453-7.
Orimo A, Weinberg RA. Stromal fibroblasts in cancer: A novel tumor-promoting cell type. Cell Cycle 2006;5:1597-601.
Cat B, Stuhlmann D, Steinbrenner H, Alili L, Holtkötter O, Sies H, et al.
Enhancement of tumor invasion depends on transdifferentiation of skin fibroblasts mediated by reactive oxygen species. J Cell Sci 2006;119(Pt 13):2727-38.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]