Assessment of apoptotic index in potentially malignant pathologies and squamours cell carcinoma of oral cavity- an in vitro study

Introduction

Oral Squamous Cell Carcinoma(OSCC) spreads locally and metastasizes in the different routes of lymph nodes. This chronic disease is a public health problem both in developing as well as developed countries. The burden of Oral squamous cell carcinoma is great because of the associated high cost of treatment, permanent impairment and high mortality. The prevalence of oral squamous cell carcinoma is high in Asian countries especially Southeast Asia.¹

The disfigurement consequential of the disease and treatment is permanent affecting the quality of life. The annual estimated incidenceis around 2,75,000 for oral and 1,30,300 for pharyngeal cancer excluding nasopharynx, two-thirds of these cases occurring in developingcountries.² Considering all the age groups, men are more affected than women. The site of occurrence depends on the predominant risk factors in that particular geographical region.

In recent years many countries report the tongue as the most frequently affected site. Prognostic evaluation for oral squamous cell carcinoma (OSCC) is mainly based on clinical TNM classification, but this staging system is not sufficient for optimal prognostication and must be supplemented by other reliable methods. The biological activity of OSCC is evaluated and descriptively categorized as highly, moderately and poorly differentiated. Broder primarily developed this quantitative grading of cancer in 1920. Broder primarily developed this quantitative grading of cancer in 1920.

Lack of correlation between Broders’ grades and the prognosis of Oral squamous cell carcinoma has been explained by the fact that SCC’s usually exhibit a heterogeneous cell population with probable differences in invasiveness and metastasis behavior. Carcinogenesis is a multistep process at both the phenotypic and genetic levels, resulting from the accumulation of multiple mutations. These nonlethal genetic damage may be acquired by the action of environmental agents such as chemicals, radiation or viruses or it may be inherited in the germ line.Apoptosis is an efficient method of preventing malignant transformation because it removes cells with genetic damage.

An inefficient apoptotic mechanism can promote cancer development, both by allowing accumulation of dividing cells and by obstructing removal of genetic variants with enhanced malignant potential.³ Various means of detecting apoptotic cells have been explored and made available over the time.⁴ They include electron microscopy, flow cytometry, gel electrophoresis, in-situ labelling methods, immunohistochemistry etc. However, these procedures are time consuming, expensive and not easily applied in routine practices.

Conventional light microscopic examination with routine hematoxylin and eosin stained can identify the various morphological changes that occur during apoptosis. The same H&E stained sections which are used for the diagnosis of oral squamous cell carcinomas may be analyzed for quantifying the apoptosis, by means of the Apoptotic Index (AI). This would provide a relatively inexpensive and fairly fast and accurate means of evaluating the apoptosis in oral squamous cell carcinoma.

The present study was therefore designed to evaluate the apoptotic index in H&E stained sections of oral epithelial dysplasia and different grades of oral squamous cell carcinoma and to elucidate the prognostic relevance of the same, if any.

Aims & Objectives

The present study was conducted with the following Aims and Objectives:

• To identify Apoptotic bodies in H&E stained tissue sections.
• To quantify the Apoptotic Index in Hyperkeratosis with varying degree of dysplasia and normal mucosa.
• To quantify Apoptotic Index among well, moderate and poorly differentiated oral squamous cell carcinoma and normal mucosa.
• To compare the Apoptotic Index among normal mucosa, dysplastic lesions and various grades of squamous cell carcinoma.

Materials and Method

The study was conducted in the Department of Oral Pathology and Microbiology, Kothiwal Dental College and Research Centre, Moradabad. Armamentarium used are Microtome – Rotary semiautomatic microtome- Leica (RM 2245), Water bath, Coplin jars, Slide rack, Filter paper,Cover slips (Blue star – 22X40mm, thickness 0.13 to 0.16),Microscope Slides (lkon – 25X75 mm, thickness 1.3 to 1.45mm),Microscope – Binocular light microscope (Olympus CX-31).

Methodology included experimental and control groups. The control group included 10 cases and experimental cases included 10 cases each. Total number of cases included was 80. The cases were selected on histopathologically diagnosed cases of hyperkeratosis with varying degree of dysplasia and different grades of oral squamous cell carcinomas which were retrieved from the archives of the Department of Oral and Maxillofacial Pathology and Microbiology, Kothiwal Dental College. The case details and other relevant data were retrieved from the case records of the patients and tabulated as follows: (Table 1)

The formalin- fixed paraffin embedded tissues for the study were sectioned using Rotary Semiautomatic Microtome- Leica (RM 2245). Standard 5µm sections were obtained and placed onto adhesive coated slides followed the sections were then subjected to routine h&e staining viewed under light microscope.

Apoptotic cells/bodies in the stroma around the tumor were not counted. Apoptotic cells/bodies were morphologically identified as shrunken cells with compact, segregated and sharply delineated mass of chromatin with a deeply eosinophilic cytoplasm. Cells showing fragmented nuclei were also included in the counting. For calculating apoptotic index, 5 high power fields were analysed per case and in each case field apoptotic cell/bodies were counted against total number of tumor cells by using Image-Pro Express software (Version 6.0).

The apoptotic cells/bodies ratio for one case was evaluated as:

Apoptotic Index (AI) = Total no. of apoptotic cells

                                     Total no. of cells in 10 HPF

 1. Epithelial Dysplasia: The Apoptotic Index (AI) was seen to increase within the dysplasia group, with higher AI seen with high grades of dysplasia. This is shown by Graph 1. The mean AI observed in case of Normal mucosa was (0.0013±0.13). In the study group comprising of cases with varying grades of dysplasia, the AI was in the range of (0-0.0120) Comparing the two, we saw that the AI was less in case of normal mucosa. At the same time, the AI was also seen to increase within the dysplasia group, with higher AI seen with higher grades of dysplasia (Table 2).

Comparing the various degrees of Epithelial Dysplasia with normal cells, the apoptotic index (AI) is found to be insignificant. Slides were viewed under 40X magnification.

1. Oral Squamous Cell Carcinoma (OSCC): The Apoptotic Index (AI)was also seen to increase in Well Differentiated Squamous Cell Carcinoma and decrease towards Poorly differentiated Squamous Cell Carcinoma.

Apoptotic Index as observed for the varying grades of Squamous cell carcinoma is shown in Table 3. As can be seen, the AI showed a steady decrease from well differentiated SCC (0.019±.036) to Poorly differentiated SCC (.001±.001). Comparing the results with that for normal mucosa showed that the overall AI was higher for SCC group; however, the AI for PDSCC was seen to be even lower than that of normal mucosa.

2. Comparing the various grades of OSCC with normal cells, the apoptotic index (AI) is found to be insignificant. Slides below are viewed under 40X magnification:(Fig 5,6,7).

Discussion

Oral cancer is the sixth most common cancer worldwide and oral squamous cell carcinoma accounts for 90% of all oral cancers. The most common sites affected are tongue and floor of the mouth. 5 Oral cancer though uncommon in developed countries is a serious and growing problem in many parts of the globe. Oral and pharyngeal cancer, grouped together is the sixth leading cancer in the world and ranks in the top three in high incidence areas. The annual estimated incidence is around 275,000 for oral and 130,300 for pharyngeal cancers excluding nasopharynx; two thirds of these cases occurring in developing countries.⁶

Oral squamous cell carcinoma may arise spontaneously or following a precancerous lesion with dysplasia; severe dysplasia carry a very high risk of malignant transformation. Investigation into the segmental expression of apoptosis and related oncoprotein in precancerous lesions of the oral mucosa has been studied but the possible role in the progression of oral tumor is still known.

Approximately 70,000 new cases and more than 48,000 oral cancer-related deaths occur yearly. In most regions of India, oral cancer is the second most common malignancy diagnosed in men, accounting for upto 20% of cancers and the fourth most common in women.⁷

Most OSCC is seen in older males: factors, especially tobacco and alcohol are the most important mutagens but betel chewing, radiation exposure, infections or immune -incompetence are relevant in some cases.⁸ Since apoptosis play an important role in carcinogenesis, its potential clinical application such as predicting the prognosis and monitoring reactions to chemotherapy has been studied. Expression of apoptotic oncoproteins is related to the prognosis.^9,11 In oral squamous cell carcinoma, expression of anti-apoptotic oncoproteins has been correlated with a more aggressive disease lymph node metastasis and poor prognosis.^12-14

Dysregulated apoptosis is implicated in pathogenesis of a variety of diseases including oral pathologies. Reactive lesions like pyogenic granuloma shows more frequent Bcl-2/Bax expression than granulation tissue and capillary hemangioma suggested that the low apoptotic index rate in Pyogenic granuloma is closely related to its characteristic rapid growth (15). Epithelium of benign odontogenic cyst like radicular and residual cysts showed expression of apoptosis related factors like ssDNA, p53, Bax, Bcl-2, caspase-3, Fas, Fas-L and Ki-67 antigen suggesting their role in the pathophysiology activity of periapical inflammatory lesions.¹⁶

The present study was therefore designed to evaluate the apoptotic index in H&E stained sections of oral epithelial dysplasia and different grades of oral squamous cell carcinoma and to elucidate the prognostic relevance of the same, if any. The study consisted of 80 cases where 10 cases of normal mucosa acted as a control group, 40 cases of hyperkeratosis showed varying degree of dysplasia including cases of no, mild, moderate, severe dysplasia and 30 cases of OSCC with 10 cases each of well, moderately and poorly differentiated carcinomas.

Mean apoptotic bodies of each group was analyzed and intergroup statistical comparison was done. Result showed a statistically significant increase of apoptotic index count from normal mucosa to cases of severe dysplasia. However mean apoptotic bodies were increased in hyperkeratosis with no dysplasia, mild dysplasia, severe dysplasia but with a decrease of apoptotic bodies in moderate dysplasia. There is statistically significant difference among the groups when compared together.

Similar observations were seen in studies by Birchallet al.¹⁷ Their results showed an increase in apoptosis from normal epithelium to carcinoma-in-situ, suggesting that a change of apoptosis accompanies the onset of invasion in a premalignant lesion of the human oral cavity and oropharynx. In addition, they also suggested that there was a topographical shift in the pattern of apoptosis with peak apoptosis occurring in superficial layers than basal cell layer in higher grades of dysplasia. They interpreted reduction in the efficiency of the apoptotic system, whereby programmed cell death became more remote from stem cells with defective genome.¹⁷

Birchall et al in 1996 analyzed apoptotic index in 72 cases of normal, dysplasia and OSCC. They found that apoptotic index was static in cases of dysplasia and SCC while the mitotic index increased from normal to SCC. Therefore, the apoptotic by mitotic index ratio increased significantly from normal to SCC. They suggested that low apoptotic index represents a failure of intra or intercellular signaling process, despite the increased prevalence of disordered genomes under dysplastic conditions.¹⁷

In the present study analysis of mean apoptotic index in cases of well, moderate and poorly differentiated squamous cell carcinoma showed a statistically significant increase as compared to normal mucosa. Maximum apoptotic bodies were observed in cases of well differentiated SCC and a decrease in value was observed towards the higher grades i.e-moderate and poorly differentiated cases.

However, conducted a similar study and showed a significant increase of apoptotic index progressively from dysplasia to SCC. They postulated that apoptosis may have a role in preventing the development of aneuploidy and other genetic abnormalities that are commonly associated with cancer cells and progression of neoplasia.¹⁸

Similar findings was also observed by Ribeiro et al in 2013 where hestated that apoptosis was significantly more frequent in well differentiated carcinomas and in papillomas, and a higher growth fraction of expressed nucleolus organizer regions and cells that expressed a greater than average number of nucleolus organizer regions were more frequently noted in undifferentiated carcinomas.¹⁹

In other study, Vishwanathan et al in 2014 found AI increased gradually from normal to dysplasia to carcinoma and the highest AI was seen in welldifferentiated squamous cell carcinomas (SCCs) and it was mentioned in Apoptotic Index and Proliferative Index in Premalignant and Malignant Squamous Cell Lesions of the Oral Cavity.²⁰

To summarize, in our study the Apoptotic Index (AI) was found to be the highest in Well differentiated Squamous Cell Carcinoma and least in Poorly differentiated Squamous Cell Carcinoma. This implies that tumor exhibiting more apoptosis may be slow growing and therefore may be less biologically aggressive. On the other hand tumor exhibiting less apoptosis is rapidly growing and therefore have more chance for metastasis. This in turn could contribute to the relatively poorer prognosis associated with poorer grades of SCC. This suggests that apoptotic index is a significant prognostic marker, it is relatively easy to use and can be performed in routine H&E sections as an adjunct to TNM staging and Broder’s grading.

Secondly, the Apoptotic Index (AI) was found to increase with increasing grades of Oral Epithelial Dysplasia i.e, Apoptotic Index (AI) was highest in Severe Epithelial Dysplasia and least in normal tissue sections. There was also a topographical shift in the pattern of apoptosis with peak apoptosis occurring in superficial layers than basal cell layer in higher grades of dysplasia. Apoptotic bodies were seen in the suprabasal and basal regions of the normal oral mucosa and early dysplastic lesions, but as the severity of the premalignant or malignant lesion increases the apoptosis becomes generalized. Thus, evaluating the apoptotic index in potentially malignant disorders might help in better predicting their biologic behavior and better guide the treatment strategy for them. At the same time, development of newer anticancer therapies and intermediate biomarkers utilizing apoptosis as a target for the drugs should be the aim of future studies in this field.

Conclusion

Apoptosis occurs in several pathological situations in multicellular organism and constitutes part of a common mechanism of cell replacement, tissue remodeling and the removal of damaged cells. It is a genetically regulated cell death involved in the deletion of cells in both normal and malignant tissues. The features of apoptotic cells under light microscopy are condensed(eosinophilic) cytoplasm, condensation of chromatin at the nuclear membrane (crescent formation) and the presence of cell fragments (apoptotic bodies).

Two important group of proteins involved in apoptotic cell death are the members of the bcl-2 family and a class of cysteine proteases known as caspases. The Bcl-2 family can be classified into two functionally distinct groups: antiapoptotic protein and pro-apoptotic protein. Bcl-2, an apoptotic protein of the same family, is expressed selectively during apoptosis and promotes cell death.

Present study was conducted to quantitatively assess the apoptotic index (AI) at light microscopic level in hyperkeratosis with varying degree of dysplasia and Oral squamous cell carcinoma (OSCC).

The following conclusions are drawn from the present study:

1. The apoptotic cell can be seen using routine light microscopy technique with H&E staining as it is feasible and can be readily be used.

2. An overall analysis revealed that apoptotic count significantly increases in cases of dysplasia and squamous cell carcinoma when compared to normal mucosa. In cases of hyperkeratosis with dysplasia including no dysplasia, mild, moderate and severe dysplasia a significant increased value was seen.