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ORIGINAL ARTICLE
Year : 2021  |  Volume : 13  |  Issue : 2  |  Page : 148-154

Assessment of Inflammatory Domain on the Proliferative Activity of Odontogenic Keratocyst in Comparison with Dentigerous Cyst and Perapical Cyst


1 Department of Oral and Maxillofacial Pathology and Oral Microbiology, Nitte (Deemed to be University), AB Shetty Memorial Institute of Dental Sciences, Deralakatte, Mangalore, Karnataka, India
2 Department of Public Health Dentistry, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka, India

Date of Submission11-Nov-2021
Date of Acceptance15-Dec-2021
Date of Web Publication14-Jan-2022

Correspondence Address:
Dr. Reshma Amin
Department of Oral and Maxillofacial Pathology and Oral Microbiology, Nitte (Deemed to be University), AB Shetty Memorial Institute of Dental Sciences, Deralakatte 575018, Mangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jofs.jofs_257_21

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  Abstract 


Introduction: Ki67 is a proven marker in assessing the aggressiveness of various neoplasms expressed in proliferating cells. The recognized role of p53 in a stress-induced cell represents gene mutation or disturbance of growth regulation. Therefore, a comparative analysis of p53 can give a better picture of proliferation in odontogenic keratocyst (OKC). In recent years, minichromosome maintenance (MCM) proteins are frequently in use for the application and evaluating cell proliferation. Our study plan was to compare the inter-relationship of p53, Ki67, minimicrosome (MCM2) markers in OKC, and other odontogenic cysts in inflammation, further checking the markers as a valuable tool in the prognosis and treatment of OKC. Materials and methods: Selected cases of 40 OKCs, 10 cases each of dentigerous cyst (DC) and periapical (PA) cyst showing mild to moderate inflammation were chosen from the department archives. Immunohistochemical procedure was carried out on all cases; interexaminer reliability was checked with Cronbach Alfa; and Chi-square was applied to check the association between the data. Results: Immunoexpression was significantly higher in OKC among p53, Ki67, and MCM2. The positivity of cells observed in OKC in the three markers did not show much difference (P = 0.666), though the intensity was statistically significant (P = 0.010). Comparative analysis among OKC, DC, and PA in all the three markers indicated statistical significance in the percentage of the positive cells and intensity. Conclusion: Considering the proportion of cycling cells relative to the expression of three markers, OKC imparts valuable information on proliferation. We speculate that the biological potential of OKC’s histopathogenesis lies within the lining epithelium, inflammatory cytokines contributing to the changes.

Keywords: Cell proliferation, inflammation, Ki67, MCM2, odontogenic keratocyst, p53


How to cite this article:
Amin R, Shenoy R. Assessment of Inflammatory Domain on the Proliferative Activity of Odontogenic Keratocyst in Comparison with Dentigerous Cyst and Perapical Cyst. J Orofac Sci 2021;13:148-54

How to cite this URL:
Amin R, Shenoy R. Assessment of Inflammatory Domain on the Proliferative Activity of Odontogenic Keratocyst in Comparison with Dentigerous Cyst and Perapical Cyst. J Orofac Sci [serial online] 2021 [cited 2022 Jan 24];13:148-54. Available from: https://www.jofs.in/text.asp?2021/13/2/148/335846




  Introduction Top


Proteins executing molecular cell functions are necessary for normal growth and proliferation. Disturbances in coordination of cell regulatory mechanism lead to various lesions. Studies on the proliferation provide information on pathologic behavior of various lesions. A great deal of progress on immunohistochemical (IHC) studies in research, including routine histopathology, has impacted prediction and prognosis. Odontogenic cysts are either developmental or inflammatory in origin; they derive from tissue remnants of tooth formation. The dental lamina residues within the jaws form the origin of the controversial odontogenic keratocysts (OKCs). The typical trait of the cyst is due to the proliferative potential of the epithelial lining. OKCs intrigue researchers from time and again for the reason of the unusual recurrences. Patients with OKC majority range from 5% to 62% at risk for recurrences, henceforth undergo multiple surgeries. Cell proliferation marker studies in odontogenic cysts and tumors aid in assessing likely biologic growth potentials. The IHC studies of OKC point toward upgrading the treatment and the surgery-related impediment among patients.[1],[2]

The Ki67, as a proliferation marker, is used to study cell cycle, it increases in the second half of the S phase, high at G2 and M phases, then degrades after mitosis. It is applicable in investigating the immunoreactivity in the epithelial cells of OKC as well as to estimate the prognosis with p53 protein. P53, a transcription integrator of genome stability, upturns in response to DNA damage or any stress related to the cell function. The expression is more at the G1 phase of the cell cycle, participates in the growth arrest, initiates repair, or induces apoptosis. P53 and Ki67 marker expressions in OKC are directly proportional to its biologic potentiality, as many studies confirm. To avoid recurrences, the prognostic IHC marker analysis can prove supportive in treatment terms. Recently, minichromosome maintenance (MCM) of MCM2 to MCM7 complex proteins crucial in DNA synthesis is used for cell-growth assessment. Many studies indicate MCM, a better prognostic marker besides its expression in the early G1 phase, can be advantageous to assess the prognosis together with Ki67.[3],[4]

Inflammation involves cellular interactions with a range of local and systemic factors of host immune protective mechanism. Chronic inflammation eventually promotes tissue destruction with more number of immunocyte recruitment. Consequently, inflammation existing for long time triggers cell proliferation process with molecular events in action already set up. Research on OKC specifies the role of inflammation in proliferation activity of the epithelial lining along the increased intrinsic growth potential of the cystic lining. Consistent change from classic keratinized epithelial lining to nonkeratinized epithelial lining in inflammation is quite typical finding.[5],[6]

Our study aim was to investigate the changes in the proliferative activity in OKC due to inflammation based on the IHC expression of p53, Ki67, and MCM2. Also to estimate the possible correlation of marker expression between OKC with other odontogenic cysts such as periapical (PA) and dentigerous cysts (DCs).


  Materials and Methods Top


The study was conducted from December 2018 to April 2020, approved by the Review Board of the Ethics Committee of AB Shetty Memorial Institute of Dental Sciences, Mangalore on 4/6/2018 (protocol No.: ABSM/EC19/2018). The study design consisted of two groups. Group 1: OKC − 40 cases with inflammation, and group 2 other odontogenic cysts − 10 cases each of PA and DC. The formalin-fixed, paraffin-embedded tissue blocks were chosen from archives of the Department of Oral Pathology. Hematoxylin and Eosin-stained sections were used to confirm the diagnosis. All 60 cases were treated with three different nuclear markers p53, Ki67, and MCM2. IHC procedure was performed on tissues fixed in 10% neutral buffered formalin, paraffin-embedded tissue. The protocol for each marker was followed from the manufacturer’s procedure guide (PathnSitu, Bangalore, Karnataka-560043, India).

Image interpretation

To determine the positivity and intensity of protein expression in the area of staining was analyzed at 40× magnification. The staining intensity was graded on a 4-point scale as “0” for negative staining, “1” for mild staining, “2” for moderate staining, and “3” for intense staining. Percentage of cells was noted under light microscope. The area of positive cells was counted in the entire depth of the epithelium in OKC, PA, and DC, from four high power fields. Percentage of cells were graded on a 5-point scale “0” no positive cells, “1” for <10% of positive cells, “2” for 10% to 50% positive cells, “3” for 51% to 80% positive cells, and “4” for >80% positive cells. Examination was carried out by the two oral pathologists. The following parameters were used to evaluate the staining results: nuclei with brown color regardless of staining intensity were considered as positive, in case of intensity negative if any extranuclear staining was found.

Statistical analysis

Statistical analysis is carried out using SPSS (17.0) version (IBM SPSS® Statistics))-Java™ platform SE binary-IBM Corp: London: UK (Trail version). The descriptive statistics was tabulated. Interexaminer reliability was checked with Cronbach Alfa; there was a substantial agreement with p53 and Ki67, whereas MCM2 in total agreement. Chi-square is applied to check the association between the data. P-value ≤0.05 was considered as statistically significant.


  Results Top


The results of IHC for OKC, PA, and DC were studied and compared with each other.

Ki67

In cases of OKC, 38 (95%) were detected with positivity, 28 out of 40 OKC cases with intense staining, 9 cases of PA(90%) showed cell positivity, 5 out of 10 with moderate, and 1 case with intense reaction [Figure 1]e. Among DC, 7 (70%) cases showed positivity, 4 cases with moderate reaction, and 1 case with intense reaction [Figure 1]f, [Table 1]. Majority of the positive cells were distributed in the suprabasal layers and few in the basal layer [Figure 1]a–d. The comparison between all the three cysts and the comparison between cell positivity (P = 0.025) and intensity (P = 0.002) were statistically significant [Table 2].
Figure 1 Photomicrographs showing (a, b) odontogenic keratocyst (OKC), Ki67 immunopositive nuclei where majority are noted in the suprabasal cell layers and few in the basal cell layer of lining. (c) Ki67 in OKC immunopositive cells of the proliferating epithelial lining (magnification ×40). (d) Ki67 in OKC proliferating epithelial lining with underlying connective tissue capsule showing dense inflammation (×10). (e) In the periapical cyst, majority of Ki67 immunopositive nuclei are in the basal layer. (f) Ki67-positive nuclei observed in dentigerous cyst (×40).

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Table 1 Depicting comparison of OKC, PA, and DC with three biomarkers

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Table 2 Depicting comparison of MCM2, Ki67, and p53 biomarkers with three cysts

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p53

Thirty-seven (92.5%) cases of OKC were positive, where most of them were detected above the basal cell layer [Figure 2]a–d, 8 (80%) cases of periapical cyst, and 5 (50%) cases of DC showed positivity to p53. Intensity of stained cells in p53: 24 cases showed moderate reaction, and 15 cases showed intense reaction. The PA cases out of 10, 5 cases showed moderate reaction [Figure 2]e and DC 3 cases showed mild reaction [Figure 2]f, [Table 1].Cell positivity (P = 0.025) and intensity (P = 0.000) were statistically significant [Table 2].
Figure 2 (a,b) P53 immunopositive cells in suprabasal cell layers of the proliferating lining epithelium of odontogenic keratocyst (OKC) (magnification ×40). (c) Distribution of p53-positive nuclei in OKC. (d) P53 immunopositive cells in OKC with underlying connective tissue capsule with inflammation (×10). (e,f) P53 immunopositive cell distribution in periapical and dentigerous cysts (×40).

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MCM2

In OKC, 37 cases (92.5%) were positive, 15 cases with intense reaction, 24 cases showed moderate intensity distributed suprabasally [Figure 3]a–d. MCM2 in DC showed positivity in seven cases (70%), showed three cases with mild reaction, and one each showed the moderate and intense reactions [Figure 3]e. As for PA, MCM2 was positive in nine cases (90%), where the five PA cases showed moderate reaction, and two cases with mild reaction [Figure 3]f. As there was cytoplasmic staining present in few cells in one case each of OKC and PA, it was not considered for intensity [Table 1]. Both the comparison between all the three cysts, between cell positivity(P = 0.021) and intensity (P = 0.018) were statistically significant [Table 2].
Figure 3 (a,b) In odontogenic keratocyst (OKC), MCM2 immunopositive cells showing dense and intense staining in the suprabasal cell layers (magnification ×40). (c) In OKC, MCM2 immunopositive cells, predominantly in the suprabasal cell layers (×10). (d) MCM2 in OKC with underlying connective tissue capsule showing dense inflammation. (e,f) MCM2 immunopositive cells in periapical and dentigerous cysts (×40).

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  Discussion Top


The cause for OKC’s unusual behavior and the influence of inflammation are researched wide. Kaplan and Hirshberg have suggested that inflammation is the leading cause for OKC proliferation besides the change in epithelial phenotype to nonkeratinized type. The results of their study verify that inflammation has a localized effect causing a focal increase in the expression of Ki67. Güler et al. described that the inflammation stimulates the proliferation of the epithelial cells causing higher expression of MCM2 in PA than in OKC and statistically significant results for Ki67 expression in Dental follicle and OKC.[6],[7] Our data showed a considerable increase in Ki67, MCM2, and p53 expression in OKC than in the PA and DC.

The Ki67 is helpful to evaluate the changes in tissue for research and prognostic purposes, and the primary marker of cell proliferation is visible during cell division. This nuclear protein is present in all active parts of the cell cycle, rises during the second half of the S phase, reaches a peak in the G2 and M phases, and rapidly degrades after mitosis. It is expressed more intensely in proliferating cells. A positive expression of Ki67 is considerably significant during the mitotic division and in other active phases of the cell cycle. Hence, it is possible to score a more significant number of cells using IHC results than the classic method of counting. Our study indicates intense reaction in Ki67 higher in OKC compared to the other two cysts. In many studies, correlation was noted in the expression of Ki67 and p53.[8],[9] Likewise, in our study, it was noted with Ki67, p53, and MCM2. Ki67 was actively expressed in the suprabasal and the basal cells. P53 and MCM2 are staining mainly in the suprabasal cells of the OKC cases. The cell cycle-related proliferating markers detectable more in the suprabasal layers suggest an increase in the cell activity at this level; it may be mimicking its intrinsic potential of cell origin in proliferation.[10]

There was a close association between the Ki67 and MCM3 in DC from a study carried out by Cosarc AS.[8] In addition, stronger p53 expression in the two layers in DCs with statistically significant results for p53, Ki67, and MCM3 in OKC and DC. In contrast, our data did not show statistically significant results for all the three markers for the percentage of the cell positivity in OKC. Nevertheless, there was a difference in the intensity. Comparison between OKC, DC, and PA among three markers was statistically significant. Ogden et al. suggest that the increased p53 expression by OKCs but not in other cystic lesions can be due to the mutation of the p53 gene or overexpression of the wild-type product.[11],[12]

P53 expression in the G1 phase of the cell cycle is due to various causes, from DNA damage to cell hypoxia. It forces the cell to undergo repair, apoptosis, and cell cycle arrest. The expression of different p53 isoforms is responsible for the proliferation of normal or mutated cells due to the disturbances in tumor suppressor activity. P53 protein accumulation in the nucleus is due to the inhibition in its degradation process not directly related to its transcriptional activity. P53, the primary tumor suppressor protein function, produces apoptosis or growth arrest to bring back the biologic clock to maintain tissue homeostasis.[13] Slootweg showed densely stained p53-positive cell equivalent to the positive cells of Ki67.[14] Our study showed similar results in three markers for OKC with variation in the grading percentage of cell positivity. In addition, they found OKC cases with more positive cells than the PA and DC, likewise in our cases.

Lombardi et al. observed no correlation in cell population expression stained positive in OKC cases with basal budding or satellite cysts. Piattelli et al. found that 9.1% of DCs, 8.3% of PAs, and 45% of OKCs expressed the p53 protein. In OKC cases, the p53 positivity was located in parabasal layers in DC and PA cysts were located in the basal layer.[15],[16] It is found by El-Hajj and Anneroth that most of the OKC cases (72.5%) with inflammation in the capsule, 30% recurrence was observed among them. de Oliveira et al. in their study of p53 distribution was similar in DC and PA much like our study and noted high number of positive cells in suprabasal layer of OKC. They emphasized the role of growth factors and cytokines produced in inflammation for increase in the cell proliferation.[17],[18]

As early mediators of inflammation, the macrophage inhibitory factors take action during inflammation as p53 expression increases. The implication of p53 tumor suppressor gene expression was uncertain to estimate as it is related to increase in protein stabilization. In addition, the reactive oxidants produced by the phagocytes are responsible for further proliferation or DNA damage. P53 overexpression in OKC explains the innate nature of dental lamina rests. Factors that add aggressiveness also include angiogenesis and expression of podoplanin, as previous studies have proved.[19],[20],[21]Studies carried out by Guven et al. on dental lamina cells show a decrease in Ki67 during the later stage of odontogenesis. However, it is interesting to observe that in the early developmental stages, all parts of the developing tooth are positive for Ki67, indicating normal proliferative activity of the dental lamina cells. The proper disruption of dental lamina has clinical significance, as the clusters of epithelial cells from dental lamina remnants give rise to cysts. Thus, it explains the rationale for the expression in proliferating cells in OKC. Differences in expression may also be related to the cell cycle phase. In addition, some cells enter the S phase quickly, for example, the embryonic cells. The MCM2 proteins participate in the early genome replication sequence, which influences marker detection.[22],[23] Hence with Ki67, MCM2 proves an impressive prognostic marker.


  Conclusion Top


Considering the proportion of cycling cells relative to the expression of three markers, OKC imparts valuable information on proliferation. We speculate that the biologic potential of OKC’s histopathogenesis lies within the lining epithelium, inflammatory cytokines contributing to the changes. Future studies for the MCM2 hold creditable as a prognostic indicator with other clinical variables to predict the recurrences and treatment options.

Acknowledgment

The authors are grateful for the grant given by Nitte Deemed to be University and for all the technical assistance received from the laboratory of Department of Oral and Maxillofacial Pathology and Oral Microbiology, ABSMIDS.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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