Immunohistochemical Evaluation of HPV, Proliferation and Apoptosis in Oral Squamous Cell Carcinoma among Young and Old Patients: Comparative Study

Bashar H Abdullah

^*Correspondence: Alaa S Saeed, Department of Oral Diagnosis, College of Dentistry, University of Baghdad, Iraq, Email:

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Introduction

Oral Squamous Cell Carcinoma (OSCC) is an epithelial malignancy that commonly metastasis for the adjacent lymph node. Even though the incidence of this malignancy is rare in young individuals ranging from 1 % to 6 % [1], however its rate has been increased in the last few years [2]. Developing of this malignancy in young patients is mostly associated with different etiology, different prognosis, and biological behavior [1]. The potentially attributing risk factors in young patients may include viruses, especially a high-risk human papillomavirus HPV, genetic predisposition, feeding habits, and immunosuppressive conditions [3]. The tumor appears more aggressive in this subset of patients, so many research and studies have been made to define the nature of the aggressiveness of this malignancy, and the possible associated risk factors using a different molecular markers [4]. As previous studies suggest high risk HPV, especially HPV 16 and 18, may play a role in oral carcinogenesis, particularly in young individuals [5,6]. Research reported that the HPV can promote tumorigenesis by transforming the infected epithelial cells in to malignant cells, and may cause a defect in the genes that control the apoptosis, cell cycle, and DNA repair [7]. Many studies pointed to the role of p16, which is a tumor suppressor gene in OSCC. Studies suggested, a high expression rate for p16 in HPV infected tumor due to the transforming activity of E7.

It’s well known that one of the significant biological mechanisms in oncogenesis is the disturbance in cells proliferation. Ki-67 is an important protein in cell cycle regulation and can be used to assess the growth fraction [8]. Cell cycle proteins dysregulation is evident in oral cancers , hence it can play a role in OSCC biological behavior [9].

P53 is a molecular marker that can be detected immunohistochemically in a nearly half of all oral cancers, and it is one of the most remarkable cell cycle regulatory proteins ,where its activation can prompt the growth arrest, as well as the cell death. However, when this gene gets mutated, it will lose its function, hens its regulatory action on normal cell cycle control and cell proliferation will inhibit [10,11]. P21 protein, which is a cyclindependent kinase inhibitor regulated by p53-dependent and independent pathways is frequently expressed in OSCC [12].

Hypothetically, different molecular mechanisms may be seen in OSCC of young versus old patients; accordingly, different patterns of cell cycle proteins expression may be seen. This study intended to compare the expression of HPV, proliferative and apoptotic markers in OSCC from patients above and below the age of 40, and correlate the expression with the Clinocopathological characters including the tumor staging and grading using the following markers (HPV E6/16,18, p16, Ki-67, p53, p21). It is worth mentioning that young age categorization is difficult to establish, however most of the studies used the age of 40 as a cut-off point between young and old patients [13,14].

Materials and Methods

This study was conducted on 35case of formalin-fixed paraffin-embedded tissue blocks of OSCC. From each case, five sections of a four-micron thickness were subjected to standard immunohistochemical protocol using a Leica staining kit. The cases were divided into two age groups including 20 cases with patients >40 and 15 ≤ 40. Two investigators evaluated the staining. The scoring was considered positive according to the following scale: score 1: 10-25% cells score 2:25-50% cells score 3: 50- 75% cells, Score 4: >75% cells positive. Both intensity and proportion have been evaluated, where the intensity is graded as a weak, moderate, and strong. For the statistical analysis of the data, multivariate analysis was used for evaluating the expression of each protein in relation to other variables.

Results

This study did not reveal significant association between the markers expression and the Clinocopathological parameters including: gender of the patient, tumor anatomic site, perineural invasion, tumor grading and staging where p-value > 0.05 (Figure1).

Figure 1. Positive immune expression x 100. (A) HPV16/18, (B) p16.(C) p53 ,(D) Ki-67, (E) p21.

However, the rate of positive expression for p53, p21, ki-67 were significantly higher in elderly than younger patients where p-value <0.05 (Tables 1-3).

Table 1: Multivariate analysis of HPV and p16 in relation to other variables

Table 2: Multivariate analysis of p53and p21 in relation to other variables

Table 3: Multivariate analysis of Ki-67 in relation to other variables

Discussion

High-risk human papillomaviruses have an important role in the etiology of OSCC, however the literatures vary in the prevalence rate ranging from 20-50% , and this variation may be related to the technique of identification, the geographic or the ethnic distribution, the sample size, the method of preparation, whether it is formalinfixed or fresh frozen , and the tumor anatomical site [15]. The present study did not reveal significant difference between HPV expression and the Clinocopathological parameters of the compared groups , which was in agreement with other studies [15-18]. HPV can induce carcinogenesis alone or with other well-known risk factors including tobacco and/or alcohol. The present study did not show a significant difference between p16 expression and the clinicopathological parameters. This is again in agreement with other studies [19-21]. The relation between HPV and p16 is still a matter of discussion. Many authors mentioned that overexpression of p16INK4A protein by immunohistochemistry can act as a biomarker for HPV-induced oral carcinomas [21,22]. The current study revealed a significant correlation between HPV and p16 INK4A. However, the studies were variable regarding this aspect, and this variation may be related to variation in methodologies or the techniques that were used for HPV and p16 identification ,in addition to variation in the scoring system, or variation in the types of HPV that were used in the studies [23].

There are many regulators for the cell cycle such as: cyclins, cyclin dependent kinases, oncogenes, and tumor suppressor genes. These regulators play a crucial role in the cell cycle maintenance, and control the balance between growth and death of the cell. One of these important regulators is p21 [24]. According to many literatures this new emerging marker has a valuable role in predicting the prognosis of HNSCC. The current study showed a significant correlation for p21 expression and age category on multivariate analysis. Where p21 is highly expressed in adult patients. This is in agreement with other studies [12,25]. The present study did not show any significant correlation for p21 expression and other Clinocopathological parameters in both age groups [26]. In a fact, there are not many previous reports on the study of p21 expression in OSCC, and most of the literatures studied the gene expression in the tumor from different sites of the oral cavity. Tumors developing at a different site of the head and neck region have different biologic behaviors, so the clinical features, risks of lymphatic invasion, treatment modalities, and prognoses are highly variable [25].

Regarding the proliferative aspect, this study did not reveal a significant correlation for the proliferative marker and the Clinocopathological parameters of the compared groups, however the rate of expression was significantly higher in young than old patients, which was in agreement with other studies [8,27]. The role of ki-67 in evaluating the outcome of OSCC is still vague, and finding from the previous studies were conflicting. This may be due to variation in the methods used to assess Ki-67expression, or variations in the process applied for counting the positive tumor cells among other cells.

Concerning apoptosis, the current study did not show a significant difference in p53 expression and the Clinocopathological parameters in both age groups, however the expression rate was higher in old patients. This finding is similar to other studies [28,29]. The high apoptotic rate in the old patients could be attributed to many causes such as: a prolonged duration of carcinogen exposure with a low immune response, or delay in a tumor diagnosis. The result of the study about p53 expression and its relation to other parameters is highly variable, and affected by different parameters such as techniques that were used, and the sample size.

Conclusion

Proliferative and replicative potential, apoptosis, and viral expression did not show any significant effect in determining the biological behavior of OSCC considering the age groups, namely young vs. elderly.

Ethical Approval

All experimental protocols were approved by the College of Dentistry, University of Baghdad. All experiments were carried out following the approved guidelines. (Ref no.167719 on 31/12/2019).

Financial Support

There was no financial disclosure.

Conflict of Interest

The authors declare that there are no conflicts of interest.

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