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ORIGINAL ARTICLE  
Year : 2019  |  Volume : 62  |  Issue : 1  |  Page : 3-6
Assessment of fragile histidine triad expression in ameloblastoma, odontogenic keratocyst and dentigerous cyst


1 Department of Oral Pathology and Microbiology, Faculty of Dental Sciences, M. S. Ramaiah University of Applied Sciences, Bengaluru, Karnataka, India
2 Department of Oral Pathology and Microbiology, Faculty of Dentistry, Malaysia
3 Department of Oral Pathology and Microbiology, Coorg Institute of Dental Sciences, Virajpet, Karnataka, India
4 Department of Oral Pathology and Microbiology, Srinivas Dental College and Hospital, Rajiv Gandhi University of Health Sciences, Mangalore, Karnataka, India
5 Department of Oral Pathology and Microbiology, Educare Institute of Dental Sciences, Malappuram, Kerala, India
6 Department of Oral Pathology and Microbiology, Subbaiah Institute of Dental Sciences, Shimoga, Karnataka, India

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Date of Web Publication31-Jan-2019
 

   Abstract 


Background: FHIT (Fragile histidine triad) a member of tumor suppressor family, has been extensively studied in many solid tumors including head and neck squamous cell carcinoma. Among all head and neck cyst and tumors odontogenic lesions account approximately 3%-9%. The molecular pathogenesis of these lesions is less explored. Defects in cell cycle regulators and tumor suppressor genes could result in the development of odontogenic cyst and tumors. Hence, we aimed to determine the significant role of a tumor suppressor gene FHIT in most commonly occurring odontogenic lesions mainly ameloblastoma, odontogenic keratocyst and dentigerous cyst. Subjects and Methods: Immunohistochemical analysis of FHIT was done in ameloblastoma, odontogenic keratocyst, dentigerous cyst and dental follicle. Interpretation of the stained slides were done using standard scoring criteria by two pathologist. The results were subjected for statistical analysis. Results: Expression of FHIT varied among the groups, with highest negative expression in ameloblastoma 44.4% followed by odontogenic keratocyst 14% and 100%positive expression was seen in dentigerous cyst. The expression levels between the groups were statistically insignificant. Conclusion: The varied expression or negative expression of FHIT could be considered as an indicator for aggressive behavior and transformation of preneoplastic/cystic epithelium.

Keywords: Ameloblastoma, dental follicle, dentigerous cyst, fragile histidine triad, odontogenic keratocyst

How to cite this article:
Haragannavar VC, Tegginamani AS, Raju S, Kudva S, Peter CD, Shruthi D K. Assessment of fragile histidine triad expression in ameloblastoma, odontogenic keratocyst and dentigerous cyst. Indian J Pathol Microbiol 2019;62:3-6

How to cite this URL:
Haragannavar VC, Tegginamani AS, Raju S, Kudva S, Peter CD, Shruthi D K. Assessment of fragile histidine triad expression in ameloblastoma, odontogenic keratocyst and dentigerous cyst. Indian J Pathol Microbiol [serial online] 2019 [cited 2019 Feb 17];62:3-6. Available from: http://www.ijpmonline.org/text.asp?2019/62/1/3/251253





   Introduction Top


Odontogenic cysts and tumors arise from cell rests of dental lamina or root sheath/structures associated with tooth apparatus.[1] Odontogenic lesions arising from odontogenic tissue comprises diverse set of lesions. This diversity reflects the complex development of the tooth apparatus, as these set of lesions arise from any of the changes occurring during the normal odontogenesis.[2]

It is important to understand the biological behavior of the Odontogenic lesions to achieve a better treatment strategies and prognosis. It is well-known that the cell cycle is controlled by many regulators and genes such as oncogenes, proto-oncogenes, cyclin dependent kinases, cyclin dependent kinase inhibitors and tumor suppressor genes.[3] Presently these regulators of cell cycle have gained the attention of researchers to know the exact role in the Odontogenic lesions.

Fragile histidine triad (FHIT) is a tumor suppressor gene. This was first determined and duplicated by Ohta et al. using Excision acquisition method in 1996.[4] This gene belongs to the histidine triad gene family. FHIT is first tumor suppressor gene connected to the human fragile chromosomal site FRA 3B at 3p14.2 in human genome, FHIT is thought to play an important role in the cell cycle regulation and apoptosis.[4],[5] This gene is considered as tumor suppressor gene, which controls the process of carcinogenesis. But the mechanisms causing the reduced expression of FHIT and the manner in which FHIT promotes tumorogenesis remains obscure. The hypothesized reason for loss of FHIT protein expression is thought to be either of these scenario - point mutation or promoter methylation resulting in inactivation of FHIT.[4] Loss of expression or complete absence of FHIT expression immunohistochemically has been found in about 50-90% cases of esophageal cancers suggesting it as tumor suppressor gene. There are various studies in breast, cervical cancers and Barrett's esophagus suggesting that the loss of FHIT may be correlated to the poor prognosis and enhancement of the carcinogenesis.[5],[6],[7]

Recently FHIT and its products have been studied by many researchers in regards to its role in tumor formation and progression. According to the literature its role in various human cancers (bronchial, esophageal, colorectal, breast, and cervical cancers)[4],[5],[6],[7],[8] have been studied, but in Odontogenic lesions is sparse and only one study has been documented.[4] Hence, our aim was to determine whether the loss of expression of FHIT occurs specifically to the aggressiveness of the Odontogenic lesions mainly in dentigerous cyst, Odontogenic keratocyst, ameloblastoma.


   Subjects and Methods Top


Study sample consisted of 40 diagnosed cases of Odontogenic lesions which were sub grouped into ameloblastoma, Odontogenic keratocyst, and dentigerous cyst 10 each with 10 cases of dental follicle. The above mentioned samples were confirmed histopathologically under routine stains and Formalin Fixed Paraffin Embedded (FFPE) tissue blocks were retrieved from the departmental archives of Oral Pathology CIDS-Virajpet [Figure 1]. The cases not satisfying the histopathological criteria outlined by the World Health Organization were excluded along with the cases showing intense inflammation, OKC associated with Nevoid Basal cell syndrome.
Figure 1: Photomicrograph of hematoxylin and eosin (H and E) stained sections and immunohistochemcial expression of FHIT under x40 magnification. (a) H and E of ameloblastoma, (b) H and E of OKC, (c) H and E of dentigerous cyst, (d) H and E of dental follicle, (e) FHIT negative expression in ameloblastoma, (f) FHIT negative expression in OKC, (g) FHIT positive expression in dentigerous cyst and (h) FHIT positive expression in dental follicle

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The study was approved by an Institutional ethical committee of institute CIDS (Ref No: CIDS/ADM/4318/2012).

FHIT immunohistochemical staining

The selected cases were subjected to immunohistochemical staining with FHIT antibody using standard protocol of immunohistochemistry.

The expression of FHIT was determined immunohistochemically by using monoclonal antibody. (ABCAM life sciences.) With Biogenex polymer HRP detection system. The sections were mounted on charged slides (commercially available Biogenex Life Sciences Pvt. Ltd) were incubated at 58°C overnight. The sections were subjected for the standard immunohistochemical staining procedure according to the data sheet provided with kit. Antigen retrieval was performed by use of the microwave method under citrate buffer (pH-6.0) for 20 minutes. The slides which were treated with antigen retrieval solution were allowed to cool to room temperature. The slides were rinsed in distilled water for 5 min and transferred into TBS buffer (pH-7.5) for 10 minutes.

Post power block the sections were treated with primary antibody FHIT for one and half hour. This was followed by 2 changes of TBS buffer wash for 5 minutes each to remove unbound antibodies. Super enhancer was used for duration of 30 minutes to enhance the reaction between primary and secondary antibodies.TBS buffer was ensured for removal of any unbound antibodies. The sections were stained by the modified labeled avidin – biotin technique with Biogenex polymer HRP for 30 minutes to elongate chain and label the enzyme. This was followed with a TBS buffer wash. Chromogen and DAB buffer were added to stain the tissue antigen for around 8-10 minutes than the reaction was arrested by distilled water and counter stained with Harris's Hematoxylin, dried and mounted using DPX. During each batch suitable positive (breast carcinoma) and negative control (without primary antibody) were used. Oral squamous cell carcinoma and breast carcinoma were used as a positive control.

The epithelial cells expressing brown color in the cytoplasm were considered as immunohistochemically positive for FHIT [Figure 1]. Interpretation of the slides were done by two pathologists by selecting 10 random high power fields. The interpretation of the immunostaining was classified as either positive (>10% cells stained brown) or negative (<10% of cells stained brown) as mentioned in previous studies done by Lee JI, et al. 2001.[8] The percentage of cells expressing the FHIT immunostain were calculated by using the below mentioned formula. Also the intensity of the staining was considered as low, moderate and strong. The results were subjected for statistical test using SPSS Statistics software 21.0.




   Results Top


According to our scoring criteria the negative expression or complete absence of FHIT expression was observed in ameloblastoma cases 44.4% (4 out of 9) mainly in the peripheral cells of follicles than the central part, the staining intensity was almost low to moderate in these cases. Following this Odontogenic keratocyst (OKC) 14% of cases showed negative expression (1 out of 6) and the staining intensity was moderate throughout the epithelial lining, all the cases of dentigerous cyst showed positive expression 100% (5 out of 5) with moderate to strong staining intensity and among 4 cases of dental follicle one case showed negative expression and three showed strong positive expression. There was no statistical differences between the groups by Chi-square test with P value =0.261. The distribution of expression levels of FHIT has been shown in [Graph 1] and [Table 1].
Table 1: cross tabulation of expression of FHIT in study groups

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   Discussion Top


The development and progression of odontogenic lesions are affected by alterations in genes and molecules responsible for odontogenesis. In particularly, the features of odontogenic lesions usually depends upon mechanisms involved in the development of tooth apparatus at the molecular level.[9] So, the studies have focused on its intrinsic growth potential and related behavior to understand the exact role of various types of genes in the development of odontogenic lesions.[10],[11],[12],[13]

It is well documented in the literature that FHIT is an important tumor suppressor gene, which was isolated and identified by collaborating researchers in the USA and Japan in 1996 and it showed to extent the most common human fragile chromosomal site FRA 3B at 3p14.2.[4],[14],[15] The role of FHIT has also been studied in head and neck cancer, along with this, reduced or absent expression of FHIT has been reported in primary cancers, in cancer cell lines, and in precancerous lesions. Along with deletion mechanism of FHIT, there are other modes like DNA methylation and histone modification which can be together termed as epigenetic changes support the concept of FHIT as a tumor suppressor.[14]

Absence or reduced expression of FHIT can indicate the aggressiveness of the lesion or transformation into higher grade of the neoplasia and pre-neoplasia.[14],[15] Recent studies on immunohistochemical assessment of FHIT expression in head and neck squamous cell carcinoma have shown substantial reduction in the expression suggesting the poorer prognosis in cases of poorly differentiated cases.[8],[13] On literature search the role of FHIT gene has not be explored in odontogenic lesions in Indian scenario. Hence, we attempted to study its role in odontogenic lesions.

The present results are in accordance with study done by Malcic A et al., in 2008, where they found that the negative reaction was more in odontogenic keratocyst than the radicular and dentigerous cyst (74%, 49% and 31%) respectively.[4] In our study ameloblastoma showed higher negative expression suggesting its local aggressive behavior. Thus, it can be differentiated from cystic lesion and dental follicle.

On the other hand among 5 cases of dental follicle one case has shown negative expression, suggesting it might undergo either cystic or neoplastic changes. Therefore it is important to clinically evaluate further to observe the changes occurring in same case. Inference of our study proposes that there is strong differences in FHIT negative expression between the ameloblastoma, OKC, dentigerous cyst and dental follicle. Whereas DC has positive expression than that of OKC and ameloblastoma supporting its cystic behavior. It was stated in the study of Tanimoto et al. that unusual expression of FHIT could be seen in pre-neoplastic/non-neoplastic epithelium.[16] Few of the leukoplakic cases in his study showed negative expression and one of the leukoplakic case has undergone malignant transformation within three years of duration.[16] Thus, it suggests that the aberrant expression of FHIT in the pre-neoplastic and normal epithelium could undergo malignant transformation in future. Hence, it is important to follow up the patients diagnosed for odontogenic lesions to know the transformation and reoccurrence.

Due to the lack of clinical data of the cases considered in our study and we couldn't stain the entire samples (1-ameloblastoma, 3- OKC,5-DC and 6-DF) mentioned in the study as the FHIT antibody material was lost during the process of standardization, because of which we couldn't draw a proper conclusion.


   Conclusion Top


To conclude the varied expression of FHIT in our study cases may add knowledge to the existing literature on FHIT as a tumor suppressor gene and its role in odontogenic lesions Therefore, it is important to understand the biological behavior of odontogenic lesions and lining epithelia at the earliest to achieve better treatment plan, prognosis and low recurrence rate. Future studies with more sample, clinical data and advanced techniques are necessary to understand the exact mechanism of FHIT.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Nanci A. Ten Cate's Textbook of Oral Histology and Embryology. 7th ed. St. Louis: Mosby Elsevier Publishers; 2008.  Back to cited text no. 1
    
2.
Shear M. Cysts of the Oral Regions. 4th ed. Blackwell: Munksgaard Publishers; 2007.  Back to cited text no. 2
    
3.
Ruddon RW. Cancer Biology. 4th ed. England: Oxford University Press; 2000.  Back to cited text no. 3
    
4.
Malcić A, Jukić S, Anić I, Pavelić B, Kapitanović 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.  Back to cited text no. 4
    
5.
Matthews CP, Shera K, Kiviat N, McDougall JK. Expression of truncated FHIT transcripts in cervical cancers and in normal human cells. Oncogene 2001;20:4665-75.  Back to cited text no. 5
    
6.
Zhao P, Liu W, Lu YL. Clinicopathological significance of FHIT protein expression in gastric adenocarcinoma patients. World J Gastroenterol 2005;11:5735-8.  Back to cited text no. 6
    
7.
Ingvarsson S, Agnarsson BA, Sigbjornsdottir BI, Kononen J, Kallioniemi OP, Barkardottir RB, et al. Reduced fhit expression in sporadic and BRCA2-linked breast carcinomas. Cancer Res 1999;59:2682-9.  Back to cited text no. 7
    
8.
Lee JI, Soria JC, Hassan K, Liu D, Tang X, El-Naggar A, et al. Loss of fhit expression is a predictor of poor outcome in tongue cancer. Cancer Res 2001;61:837-41.  Back to cited text no. 8
    
9.
Reichart PA, Philipsen HP. Odontogenic Tumors and Allied Lesions. London: Quintessence Publishing Co. Ltd.; 2004.  Back to cited text no. 9
    
10.
Mosqueda-Taylor A. New findings and controversies in odontogenic tumors. Med Oral Patol Oral Cir Bucal 2008;13:E555-8.  Back to cited text no. 10
    
11.
Gaitán-Cepeda LA, Quezada-Rivera D, Tenorio-Rocha F, Leyva-Huerta ER. Reclassification of odontogenic keratocyst as tumour. Impact on the odontogenic tumours prevalence. Oral Dis 2010;16:185-7.  Back to cited text no. 11
    
12.
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. 12
    
13.
Kujan O, Oliver R, Roz L, Sozzi G, Ribeiro N, Woodwards R, et al. Fragile histidine triad expression in oral squamous cell carcinoma and precursor lesions. Clin Cancer Res 2006;12:6723-9.  Back to cited text no. 13
    
14.
Kitamura K, Hayashi K, Kobayashi R, Ishii H, Matsubayashi J, Matsumoto T, et al. Clinicopathological significance of the fragile histidine triad transcription protein expression in laryngeal carcinogenesis. Oncol Rep 2008;19:847-52.  Back to cited text no. 14
    
15.
Druck T, Hadaczek P, Fu TB, Ohta M, Siprashvili Z, Baffa R, et al. Structure and expression of the human FHIT gene in normal and tumor cells. Cancer Res 1997;57:504-12.  Back to cited text no. 15
    
16.
Tanimoto K, Hayashi S, Tsuchiya E, Tokuchi Y, Kobayashi Y, Yoshiga K, et al. Abnormalities of the FHIT gene in human oral carcinogenesis. Br J Cancer 2000;82:838-43.  Back to cited text no. 16
    

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Correspondence Address:
Vanishri Chandrashekhar Haragannavar
Department of Oral Pathology and Microbiology, Faculty of Dental Sciences, M. S. Ramaiah University of Applied Sciences, 2nd Floor, New Bel Road, Bengaluru - 560 054, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_403_18

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