|Year : 2022 | Volume
| Issue : 4 | Page : 598
Apoptotic and mitotic indices in oral epithelial dysplasia and squamous cell carcinoma: A comparative study
PD Suhasini, Shaila Mulki, H Supriya
Department of Oral Pathology and Microbiology, KVG Dental College and Hospital, Sullia, Karnataka, India
|Date of Submission||18-Dec-2021|
|Date of Decision||11-Feb-2022|
|Date of Acceptance||28-Feb-2022|
|Date of Web Publication||22-Dec-2022|
Department of Oral Pathology and Microbiology, KVG Dental College and Hospital, Sullia, Karnataka - 574 327
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Assessment of apoptotic cells and mitotic figures using light microscopy is an easy and viable alternative to assess tumour behaviour.
Aims: To evaluate apoptotic index (AI), mitotic index (MI) and apoptotic to mitotic index ratio (AI: MI) in different grades of oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC) in haematoxylin and eosin-stained (H&E) sections.
Settings and Design: The study included 45 cases each of OED and OSCC cases which were further subgrouped into groups of 15 each based on their grades. AI, MI and AI/MI were assessed and compared with 15 cases of normal mucosa.
Methods and Material: Apoptotic cells and mitotic figures were counted using a binocular light microscope equipped with an oculometer grid (20 × 20 squares) on the eyepiece. Cells were counted in 15 grid fields under oil immersion lenses (×100) in a stepladder fashion. AI/MI ratio was calculated.
Statistical Analysis: The results obtained were statistically analysed using Analysis of variance and Tukey Honestly Significant Difference tests with SPSS 20 software at a 0.05 significance level.
Results: AI increased with increasing grades of dysplasia and decreased with increasing grades of OSCC. MI increased with increasing grades of OED and OSCC. AI/MI increased with increasing grades of OED but decreased with increasing grades of OSCC.
Conclusion: In the light of the current observations, AI, MI and AI: MI can be considered as valuable parameters to assess the biological behaviour of OED and OSCC.
Keywords: Apoptosis, mitosis, oral epithelial dysplasia, oral squamous cell carcinoma
|How to cite this article:|
Suhasini P D, Mulki S, Supriya H. Apoptotic and mitotic indices in oral epithelial dysplasia and squamous cell carcinoma: A comparative study. J Oral Maxillofac Pathol 2022;26:598
|How to cite this URL:|
Suhasini P D, Mulki S, Supriya H. Apoptotic and mitotic indices in oral epithelial dysplasia and squamous cell carcinoma: A comparative study. J Oral Maxillofac Pathol [serial online] 2022 [cited 2023 Jan 27];26:598. Available from: https://www.jomfp.in/text.asp?2022/26/4/598/364818
| Introduction|| |
Squamous cell carcinoma accounts for 90% of all oral cancers. It usually arises from a pre-existing potentially malignant lesion, and infrequently de novo, but in either case from within a field of precancerized epithelium.
Mitosis and apoptosis are two processes that oppose each other but are essential for the survival of an organism. The cell count is tightly regulated not simply by controlling the rate of cell division but also cell death. Oral carcinogenesis is correlated with a progressive accumulation of genetic alterations in molecules that play crucial roles during apoptosis. The relationship between cell growth in the form of mitosis and cell death in the form of apoptosis in cancer will predict the growth rate of the tumour.
As counting of apoptotic bodies using light microscopy is possible, the technique has been used and described by several authors, thus making it a putative prognostic marker.
Mitosis is the fundamental biological process because of its role in the growth and maintenance of tissue homeostasis, and its phases can be observed in tissue sections.,
Apoptotic index (AI) to mitotic index (MI) ratio (AI: MI) provides a conceptual framework to link cancer genetics, tumour initiation progression or metastasis with cancer therapy and prognosis. Therefore, research continues to focus on the cell cycle machinery and signalling pathways that control cell cycle arrest and apoptosis. The present study was undertaken to evaluate AI, MI and AI: MI in different grades of OED and OSCC.
| Aim of the Study|| |
To evaluate AI, MI and AI: MI in different grades of OED and OSCC on H&E stained sections.
Objectives of the study
- To assess and compare the AI in normal oral mucosa, different grades of OED and OSCC.
- To assess and compare the MI in normal oral mucosa, different grades of OED and OSCC.
- To calculate and compare the AI: MI in normal oral mucosa, different grades of OED and OSCC.
Source of the data
The study design was approved by the Institutional Research Ethics committee. Study sample included 105 formalin-fixed paraffin-embedded tissue sections of the normal oral mucosa (15), histopathologically diagnosed cases OED (45) and OSCC (45) from the archives of Department of Oral Pathology and Microbiology, KVG Dental College and Hospital, Sullia. Specimen of normal oral mucosa was obtained during surgical removal of impacted teeth.
Method of collection of data:
They study sample were grouped and subgrouped as following:
- Group 1: Fifteen normal mucosa
- Group 2: Forty-five cases of OED with 15 cases each of Mild, Moderate and Severe dysplasia.
- Group 3: Forty-five cases of OSCC with 15 cases each of Well, Moderate and Poor
| Methodology|| |
Preparation of the slides and staining
A uniform section of 3–4 μm thickness was cut from the selected blocks and routinely stained with H&E stain.
Quantification of Apoptotic cells and Mitotic figures
Apoptotic cells and Mitotic figures were counted using a binocular light microscope equipped with an oculometer grid (20 × 20 squares) on the eyepiece. A minimum of 1000 cells were counted in 15 grid fields under oil immersion lenses (×100) in a stepladder fashion. The area selected for counting apoptotic cells and mitotic figures included the most invasive and the most cellular part of the epithelial tissue. Apoptotic cells and mitotic figures were counted according to the morphological criteria proposed by Kerr et al. and Van Diest et al., Liu et al. respectively. respectively.
Finally, AI, MI and AI to MI ratio were calculated as follows,
AI = Number of apoptotic cells X 100/Total number of cells counted (1000)
MI = Number of mitotic figures X 100/Total number of cells counted (1000)
AI:MI = AI/MI
The results obtained were statistically analysed using ANOVA and Tukey HSD tests with SPSS 20 software at P < 0.05.
| Results|| |
The study group of 105 cases comprised 68 male patients and 37 female patients with ages ranging from 30 to 80 years. The majority of the cases were seen in the buccal mucosa. Descriptive analysis of the parameters is tabulated in [Table 1], [Table 4] and [Table 7]. The differences between and within the group are tabulated in [Table 2] and intragroup comparisons are noted in [Table 3]. The differences between and within each grade of OED and OSCC were analysed using ANOVA. [Table 5] The mean AI, MI and AI: MI between the grades of OED and OSCC were compared by using the Tukey HSD test [Table 6]. AI within OED groups was found to be significant whereas MI was significant only between mild and severe dysplasia and AI: MI within the OED groups was not significant [Table 6]. Photomicrograph of mitotic figures and apoptotic cell are featured in [Figure 1] and [Figure 2] respectively.
|Table 1: Descriptive analysis of AI, MI and AI: MI in normal mucosa, OED and OSCC|
Click here to view
|Table 3: Intergroup comparison of AI, MI and AI: MI between Normal mucosa, OED and OSCC groups using Tukey HSD|
Click here to view
|Table 4: Descriptive analysis of AI, MI and AI: MI within the different grades of OED|
Click here to view
|Table 6: Intergroup comparison of AI, MI and AI: MI in subgroups of OED and OSCC using Tukey HSD|
Click here to view
|Table 7: Descriptive analysis of AI, MI and AI: MI within different grades of OSCC|
Click here to view
| Discussion|| |
Oral lesions with epithelial dysplasia may be a morphological phenotype of the various steps within the progression from normal to malignant tissue. Apoptosis is linked to the elimination of potentially malignant cells, hyperplasia and tumour progression. Apoptosis prevents the development of aneuploidy and other genetic aberrations that are associated with the development and progression of OPMD. A malignant cell can acquire a reduction in apoptosis or apoptosis resistance.
Apoptosis plays a great role in cancer dynamics, hence, induction of apoptosis as a treatment mode in cancer is initiated.
Assessment of the number of apoptotic cells and mitotic figures and formulation of apoptotic to mitosis ratio provides a figure which reflects tumour dynamics at the light microscopic level and may provide a numerical index of biological behaviour.
A statistically significant increase in AI from normal mucosa to OED to OSCC was observed in the present study [Table 6], which is in accordance but a higher mean AI than those reported by Jain et al. Yet another study showed a progressive increase in AI from normal to carcinoma in situ but decreased in OSCC. However the study did not analyse AI in different grades of OSCC. Our study evidenced a gradual increase in AI from group 2 to group 3, showing a gradual decrease in advancing grades of OSCC.
Also, the mean AI showed an ascending increase from mild to moderate and to severe dysplasia. The increase in mean AI with the increasing grades of dysplasia was found to be statistically significant [Table 6]. This was as per another study that acknowledged an increased number of apoptotic bodies from mild-to-severe dysplasia suggesting a mechanism whereby apoptosis aids in eliminating those cells which have proliferated due to increased or abnormal mitosis. Thus, it may be assumed that epithelium is trying to maintain balance by removing cells with damaged genomes that are induced by adjacent normal or infiltrating inflammatory cells. It has been suggested that factors responsible for dysplastic change may themselves be converting increased situations of apoptosis.
The mean AI showed an inverse relation with the histological grades of OSCC. A significant difference was found between well, moderate and poorly differentiated carcinoma [Table 7], which is harmonious with the findings of another study. An additional study also showed a drop in mean AI with progression towards advanced grades, still, their study showed a significant difference only between WDSCC and PDSCC.
Yet another study that assessed the impact of the AI, MI and turnover index in OED and OSCC showed no significant difference among the grades of oral dysplasia, whereas premalignant and malignant cases showed a significant difference.
An immunohistochemical study conducted in OSCC showed that the ratio of bcl-2/bax mRNA was advanced in carcinomas than in the adjacent normal oral epithelium, and higher ratios were seen in most of PDSCCs. In both normal and tumour tissues, the distribution of bax are inversely related to that of bcl-2 and bax plays a role as a dominant inhibitor of bcl-2. When bcl-2 is present in redundant, cells are defended, and when bax is in redundant, cells are susceptible to apoptosis. The results showed that inhibition of apoptosis had an upper hand in carcinoma and PDSCC in particular. The present study shows reduced apoptosis with increasing grades of OSCC and the inference of the above study justifies the same using IHC, where a decrease in bax/bcl2 (proapoptotic/antiapoptotic) suggests higher grade of OSCC.
Mitotic figures were readily identifiable in prophase, metaphase, anaphase and telophase among OED and OSCC cases. Mitotic numbers increased incrementally from OED to OSCC and a statistically significant difference was observed between groups [Table 3]. This is consistent with other studies which showed a significant increase in MI from normal through OED to OSCC.,, Statistical significance noted in the present study might be due to a considerable increase in the sample size when compared to other studies.
The mean MI increased with advancing grades of dysplasia but statistically, a significant difference was not found between mild to moderate dysplasia and moderate to severe dysplasia. But there was a significant difference between mild and severe dysplasia [Table 6]. This may be due to inter-observer variability in grading dysplasia of epithelial lesions. Analogous results were observed in several studies., The mean MI increased with the histological grades of OSCC. The difference between WDSCC and PDSCC, MDSCC and PDSCC were found to be significant [Table 6].
A statistically significant difference in AI to MI ratio was found between OED and OSCC. A1: MI dropped significantly from OED to OSCC [Table 3]. Similar results were observed in a former study. The high AI: MI attained in OED indicates the increased rate of apoptosis in OED when compared to mitosis. This may be related to the vulnerable immune system of the body to counteract the tumour progression. The process involved in the robotic regression of tumours is substantially related to the process of apoptosis and the activity of the immune system, as well as to conditions in the tumour microenvironment. Kurita et al. reported a case of squamous cell carcinoma undergoing spontaneous regression in which enhanced apoptosis was demonstrated quantitatively.
The mean AI: MI was found to be increased with advancing grades of dysplasia but a statistically significant difference was not found [Table 6], [Graph 1]. The results of the present study showed a drop in the mean AI: MI with increasing grades of OSCC [Table 6], [Graph 1]. There was a decrease in apoptosis and an increase in mitosis as the tumour progressed to higher grades. This suggests that evasion of apoptosis and increase in mitosis play an important role in tumour progression and hence the ratio can be used to assess tumour progression.
Tumour growth is a summation of cell production and cell death. Although, it is not possible to determine which particular dysplastic lesion will progress to carcinoma, the AI: MI may provide a good model of tumour progression. Therefore, a high AI to MI ratio obtained in OED compared to OSCC in the present study conceivably suggests lesions exhibiting an increase in apoptosis may be slower growing and thus be biologically less aggressive.
| Conclusion|| |
In the light of the current observations, AI, MI and AI: MI can be considered as valuable parameters to assess the biological behaviour of OED and OSCC. In future, histopathological reports can include AI/MI as an insight into tumour behavior, which will invariably aid in treatment planning.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Feller L, Lemmer J. Oral squamous cell carcinoma: Epidemiology, clinical presentation and treatment. J Cancer Ther 2012;3:263-8.
Ghatage DD, Gosavi SR, Ganvir SM, Hazarey VK. Apoptosis: Molecular mechanism. J Orofac Sci 2012;4:103-7. [Full text]
Wong RS. Apoptosis in cancer: From pathogenesis to treatment. J Exp Clin Cancer Res 2011;30:87.
Jain A, Maheshwari V, Alam K, Mehdi G, Sharma SC. Apoptosis in premalignant and malignant squamous cell lesions of the oral cavity: A light microscopic study. Indian J Pathol Microbiol 2009;52:164-6.
] [Full text]
Van Diest PJ, Brugal G, Baak JPA. Proliferation markers in tumours: Interpretation and clinical value. Clin Pathol 1998;51:716-24.
Ankle MR, Kale AD, Charantimath S. Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma. Indian J Dent Res 2007;18;101-5.
Elmore S. Apoptosis: A review of programmed cell death. Toxicol Pathol 2007;35:495-516.
Kerr JFR, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239-57.
Liu S, Edgerton SM, Moore II DH, Thor AD. Measures of cell turnover (proliferation and apoptosis) and their association with survival in breast cancer. Clin Cancer Res 2001;7:1716-23.
Kujan O, Oliver RJ, Khattab A, Roberts SA, Thakker N, Sloan P. Evaluation of a new binary system of grading oral epithelial dysplasia for prediction of malignant transformation. Oral Oncol 2006;42:987-93.
Pawar RB, Palaskar SJ, Kalavankar SS. Assessment of apoptotic index in various grades of oral epithelial dysplasia: A cross-sectional study. Indian J Pathol Microbiol 2020;63:534-7.
] [Full text]
Mooney EE, Peris JMR, O'Neill A, Sweeney EC. Apoptotic and mitotic indices in malignant melanoma and basal cell carcinoma. J Clin Pathol 1995;48:242-4.
Birchall MA, Winterford CM, Allan DJ, Harmon BV. Apoptosis in normal epithelium, premalignant and malignant lesions of the oropharynx and oral cavity: A preliminary study. Eur J Cancer B Oral Oncol 1995;31:380-3.
Mandal AK, Verma D, Mohanta PK, Saha R, Maeda T, Sugino T, et al
. Prognostic significance of apoptosis in squamous cell carcinoma of oral cavity with special reference to TNM stage, histological grade and survival. Indian J Pathol Microbiol 2001;44:257-9.
] [Full text]
Singh P, Kumar V, Singh G, Singh A. Significance of the apoptotic index, mitotic index, and turnover index in premalignant and malignant squamous cell lesion of the oral cavity. How much is their diagnostic and prognostic role? J Sci Soc 2019;46:49-52. [Full text]
Chen Y, Kayano T, Takagi M. Dysregulated expression of bcl-2 and bax in oral carcinomas: Evidence of post-transcriptional control. J Oral Pathol Med 2000;29:63-9.
Birchall MA, Winterford CM, Tripconi L, Gob G, Harmon BV. Apoptosis and mitosis in oral and oropharyngeal epithelia: Evidence for a topographical switch in premalignant lesions. Cell Prolif 1996;29:441-56.
Jadhav KB, Mujib ABR, Gupta N. Crystal violet stain as a selective stain for the assessment of mitotic figures in oral epithelial dsplasia and oral squamous cell carcinoma. Indian J Pathol Microbiol 2012;55:283-7. [Full text]
Ricci SB, Cerchiari U. Spontaneous regression of malignant tumors: Importance of the immune system and other factors. Oncol Lett 2010;1:941-5.
Kurita M, Hirano K, Ebihara S, Takushima A, Harii K, Fujino T, et al
. Spontaneous regression of cervical lymph node metastasis in a patient with nasopharyngeal squamous cell carcinoma of the tongue: Possible association between apoptosis and tumor regression. Int J Clin Oncol 2007;12:448-54.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]