|Year : 2022 | Volume
| Issue : 2 | Page : 168-175
Co-expression of P53 and Ki67 in premalignant and malignant oral/oropharyngeal biopsies in Bundelkhand region, India
Swati Raj1, Nidhi Vachher2, Durre Aden3, Amit Srivastava4, Dwijendra Nath2
1 Department of Pathology, GDMC, Dehradun, Uttarakhand; Department of Pathology, Maharani Laxmi Bai Medical College, Jhansi, Uttar Pradesh, India
2 Department of Pathology, Maharani Laxmi Bai Medical College, Jhansi, Uttar Pradesh, India
3 Department of Pathology, HIMSR, New Delhi, India
4 Department of Surgery, Maharani Laxmi Bai Medical College, Jhansi, Uttar Pradesh, India
|Date of Submission||04-Jul-2022|
|Date of Acceptance||23-Sep-2022|
|Date of Web Publication||23-Dec-2022|
Dr. Swati Raj
316/14 Vijay Park Extension, Dehradun, Uttarakhand
Source of Support: None, Conflict of Interest: None
Background and Aim: India is the third most common country being entitled as “The Oral Cancer Capital of the World.” Around one-third cancer deaths are due to tobacco use. In India, oral cancer ranks first among all cancers in men and second among all cancers in women. p53 over-expression has been widely demonstrated to be a reliable predictor of progression to oral squamous cell carcinoma (OSCC). Ki67 expression is strictly associated with cell proliferation and used as a proliferation marker. Oral cancer was studied in Bundelkhand region with special reference to Gutkha (a form of tobacco) chewing and relative risk. p53 and Ki 67 expression was assessed in premalignant and malignant oral and oropharyngeal lesions and their correlation was evaluated. Materials and Methods: A descriptive study including both prospective and retrospective studies carried over a period of 18 months (April 2019–September 2020) at Department of Pathology, Maharani Laxmi Bai Medical College, Jhansi, India. Immunohistochemical evaluation was done by using markers: anti p53 and anti K67 markers with positive control, i.e. p53 – colon and Ki 67 – tonsil) sections stained omitting primary antibody were taken as negative control. Results: OSCC and premalignant oral lesions are at high incidence in Bundelkhand region, India. Tobacco is found to be most identifiable risk factor with risk ratio of 0.45. Conclusion: The over/co-expression of p53 and Ki67 plays a pivotal role in labeling and predictive marker for malignant transformation as their intensity and pattern of staining increases with increasing grade (highly statistically significant; Pearson's correlation applied).
Keywords: Immunohistochemistry, oral human papillomavirus, oral squamous cell carcinoma
|How to cite this article:|
Raj S, Vachher N, Aden D, Srivastava A, Nath D. Co-expression of P53 and Ki67 in premalignant and malignant oral/oropharyngeal biopsies in Bundelkhand region, India. Arch Med Health Sci 2022;10:168-75
|How to cite this URL:|
Raj S, Vachher N, Aden D, Srivastava A, Nath D. Co-expression of P53 and Ki67 in premalignant and malignant oral/oropharyngeal biopsies in Bundelkhand region, India. Arch Med Health Sci [serial online] 2022 [cited 2023 Jan 28];10:168-75. Available from: https://www.amhsjournal.org/text.asp?2022/10/2/168/364952
| Introduction|| |
As per the WHO 2020 global consensus, oral squamous cell carcinoma (OSCC) is at the 16th place in cancer worldwide, comprising 2.1% of global cancer burden in both sexes, with 377,713 new cases. However, in India, it is the third-most common cancer being entitled “The Oral Cancer Capital of the World.” Around one-third cancer deaths are due to tobacco use. In India, oral cancer ranks first among all cancers in men and second among all cancers in women. The sex ratio is 2:1; male predominance. In males, oral and lung cancers contribute >25% cancer deaths in India, whereas in females, breast cancer and oral cancers contribute 25% cancers in India. Among all regions in India, the incidence of oral cancer for males and females is the highest in the central region. For males, it is 64.8%, and for females, it is 37.2% at 70 years of age. The next highest magnitude is observed in the west and northeast regions (58.4%) at 60 years of age.
There is the poor survival rate of 5 years (overall) mainly due to large proportion of advanced cases. The survival rate in India is only 27% for patients with the advanced stage of oral cancer. Since cancer registration is not mandatory in India, a large number of cases go unrecorded or are lost to follow-up and the true incidence and deaths may vary widely.
More than five persons die from oral cancer every hour daily in India and nearly same numbers of individuals die from malignancies of the oropharynx and hypopharynx.
OSCC may also result from many risk factors, where tobacco is the most important cause in India, especially in UP, MP, and Bihar, where Gutka, khaini, betel nut, paan, etc., are practiced in majority that too 5–20 times/day. The tobacco in the form of quid in mouth causes change in pH and local normal flora slowly causing ulcer and leads to cancer at last. Other factors are smoking which is found to be the most common cause in European countries. Other factors include chronic irritation, such as dental caries, overuse of mouthwash, poor nutrition, and poor oral hygiene. Oral human papillomavirus (HPV), typically acquired through oral-genital contact, may have a role in the etiology of some oral cancers; however, HPV is identified in oral cancer much less often than it is in oropharyngeal cancer.
About 40% of OSCC begin on the floor of the mouth or on the lateral and ventral surfaces of the tongue. About 38% of all OSCCs occurs on the lower lip; these are usually solar-related cancers on the external surface.
Histologically, squamous cell carcinoma is the most common cancer in the oral cavity (90%) and oropharynx. Among the premalignant lesions are leukoplakia, erythroplakia, and oral submucous fibrosis.
A significant proportion of OSCC develops from premalignant lesions. Any suspicious areas should be biopsied. Incisional or brush biopsy can be done depending on the surgeon's preference. Histological examination of the tissue remains the “gold standard” for the diagnosis and identification of OSCC. However, molecular level changes in the lesion occur before the clinical and histopathological changes.
p53 over-expression has been widely demonstrated to be a reliable predictor of progression to OSCC, either if we consider the over-expression as a physiologic response to induce cell cycle arrest in genetically altered hyper-proliferating cells or as a primary genetic defect leading to the production of high levels of mutated nonfunctional p53 protein; this is why the most utilized antibodies in the literature stain the total form of p53 protein and cannot discriminate between wild or mutated form.
P53 is one of the surrogate markers to identify HPV-positive OSCC. The chemoradiosensitivity of HPV-positive tumors is thought to be because of the preserved wild-type p53 gene with functional apoptotic pathways. The most frequent molecular alteration carried by HPV-negative OSCCs is the p53 mutation which correlates with poor response to chemotherapy and radiotherapy.,, They also have a poor prognosis. In a study, multivariate analysis showed that p53 positivity was significantly associated with higher risk of disease-specific survival and recurrence-free mortality of HNC. The presence of nonfunctional p53 in pretreatment biopsy was predictive of incomplete histopathological response to neoadjuvant cisplatin/fluorouracil chemotherapy as assessed in surgical specimens of locally advanced OSCC.
Ki67 expression is strictly associated with cell proliferation and used as a proliferation marker.
Aims and objectives
Our aims and objectives of the study are as follows:
- To study oral cancer in Bundelkhand region with special reference to Gutkha (a form of tobacco) chewing and its relative risk
- To assess the p53 and Ki 67 expression in premalignant and malignant oral and oropharyngeal lesions and to evaluate their correlation.
Many epidemiological studies have been done in OSCC, but literature testifies the rarity of such kind of study ever been conducted in Bundelkhand region, where the incidence is as high as found in UP, AP, and Bihar. Therefore, we performed this study.
| Materials and Methods|| |
Study design and site
A descriptive study including both prospective and retrospective studies carried over a period of 18 months (April 2019–September 2020) at Department of Pathology, Maharani Laxmi Bai Medical College, Jhansi, India. A total of 3828 biopsies submitted in this time duration comprising of total 302 biopsies of head-and-neck region from ENT ward/outpatient department. Out of it, 84 cases diagnosed by two histopathologists showed 68 cases as oral and oropharyngeal carcinomas and 16 cases as premalignant lesions of the oral cavity and oropharynx.
The clinical details of personal habit of tobacco (including type, duration of intake, and frequency), symptoms, treatment history was taken with brief physical examination and clinical staging of patients were recorded on a detailed pretested structured pro forma, by interviewing the prospective patients. Ethical clearance from the Institutional Ethical Committee and informed and written consents from all patients taken.
Inclusion and exclusion criteria
Only biopsy-proven cases were included. Inadequate biopsies were excluded from the study.
The grading of dysplasia into mild, moderate, and severe dysplasia was done, taking into account the cellular and tissue changes according to the criteria proposed by Speight et al.
The malignant lesions were classified as per the WHO as well as Broder's criteria into well, moderate, and poorly differentiated.
Immunohistochemical evaluation was done by using markers: anti-p53 and anti-K67 markers with positive control, i.e. p53 – colon and Ki 67 – tonsil).
Sections-stained omitting primary antibodies were taken as negative control.
Anti p53 and Ki67 immunohistochemistry
Nuclear staining was taken as positive in the epithelium. The pattern and intensity of staining were done.
The pattern of staining in accordance of positivity in following epithelial layer: basal, suprabasal, and all layers.
The intensity of staining in accordance of positivity in following:
No staining-Negative, 1+ Mild Staining, 2+ Moderate staining, and 3+ Maximal staining.
All the statistical analyses were performed using the IBM SPSS Statistics for Windows, Version 25.0. Armonk, New York, IBM Corp. Frequency, cumulative percentage, mean, and standard deviation were calculated. Difference in means was evaluated using the Student's t-test. Correlation was analyzed using Pearson's correlation test and relative risk was calculated.
| Results|| |
The following results were observed and calculated in the present study: [Table 1]
|Table 1: Comparative analysis done on clinicohistological parameters in recent studies (last 15 years) in India|
Click here to view
- The relative incidence of premalignant and malignant lesions of the oral cavity in all oral biopsies received in the department is found to be 27.81% (among the patients at risk)
- Male predominance was seen in ratio of M:F = 4:1. Majority of the patients visiting our hospital belonged to the rural areas, so as our study also showed a rural urban ratio of 1.5:1. 84% patients were Hindu in our study and rest 16% belonged to the Muslim community
- Majority cases were encountered in their 4th and 5th decades of life, comprising of 40% of total cases. The common risk factors in patients with oral and oropharyngeal lesions observed were tobacco chewing comprising 88% of total cases, especially gutkha (52% among all tobacco habitual), smoking alone (10%), and chewing pan and pan masala alone (2%) [Figure 1]
- Majority (36%) of cases revealed 20–30 years of persistent exposure to tobacco, following 24% occurred after 10–20 years of exposure and 24% after 30–40 year of exposure [Figure 2]
- In the present study, only seven cases had a history of occasional alcohol intake combined with the habit of tobacco use. None of the cases had exclusive history of alcohol intake; thus, no association was found between alcohol intake and cancers of these sites
- The average duration of presentation was 6 months form the onset of symptoms
- Malignant oral lesions included Grade I lesions − well-differentiated (47.06%); Grade II lesions − moderately differentiated (35.30%); Grade III lesions – poorly differentiated (17.64%)
- Most common site of OSCC observed was buccal mucosa (41.17%), followed by tongue (23.52%), as far as sex predisposition was concerned OSCC was more common in males (76.47%) as compared to females (23.52%). The most common site of oropharyngeal SCC found to be base of the tongue (83.3%)
- Localized growth/ulcer was the main presenting symptom in malignant lesions that showed 90% with ulceroproliferative growth and 10% with ulcer in OSCC. However, all (100%) cases of oropharyngeal SCC presented with dysphagia and ulceroproliferative growth.
- Premalignant lesions (16/84 cases) accounted total 19% and included most commonly leukoplakia (62.5%) followed by leukoplakia with dysplasia
- All premalignant lesions presented with white plaque/patch or ulcer. Most of the cases (60%) of leukoplakia were observed in buccal mucosa; leukoplakia with dysplasia were well spread over the buccal mucosa, tongue, and lower lip
- P 53 and Ki 67 expression was in concordance and both found in 87.5% of premalignant lesions and 94.11% of malignant lesions [Figure 3]
- Among premalignant lesions, leukoplakia showed only basal layer positivity in 50% for p53and 43.75% for Ki 67. Leukoplakia with mild and moderate dysplasia shows basal and suprabasal layer positivity in 31.3% for both markers. Rest of severe dysplasia showed expression in all the layers for both markers [Table 2] and [Table 3]
- Almost all cases of OSCC showed P 53 as well as Ki 67 staining in all layers (92%) except six cases which were p53 and Ki 67 negative [Table 2] and [Table 3]
- All cases of oropharyngeal SCC (100%) showed p53 and Ki67 expression in all layers [Table 2] and [Table 3]
- Among premalignant lesion, p53 staining was moderate in 62.5% cases and intense in 25% of leukoplakia with and without dysplasia. Ki 67 staining was moderate in 37.05% cases of leukoplakia and 12.5% with mild staining and 12.5% with no staining [Table 2] and [Table 3]
- P53 expression in 46.7% of OSCC showed intense staining, 43.5% showed moderate staining and rest showed no staining. However, Ki 67 staining was intense in 58% cases and moderate in 35.5% OSCC. Rest 6.5% showed no staining [Table 2] and [Table 3]
- Majority of oropharyngeal carcinoma showed intense p53 and Ki 67 staining in 66.66% and 50% cases, respectively. 33.34% cases showed mild and moderate staining equally for p53. 50% showed moderate Ki 67 positivity. No mild positivity noted for Ki 67 for oropharyngeal SCC [Table 2] and [Table 3]
- A positive significant correlation was observed between the positivity index of p53 and Ki67 which indicates that there is increase in p53 and Ki67 co-expression in malignant lesions, P value is highly significant (P = 0.000), Pearson correlation performed [Table 1], [Table 2], [Table 3], [Table 4]
- A statistical correlation was found in p53 expression with increasing grade (P = 0.000), highly significant
- A statistical correlation was found in Ki 67 expression with increasing grade (P = 0.000), highly significant
- A highly significant statistical correlation was found with respect to pattern and staining of p53 and Ki 67 staining in premalignant and malignant oral and Oropharyngeal SCC (Pearson correlation test applied) [Table 2] and [Table 3]
- Risk ratio is calculated in our study, found to be RR = 0.45.
Concluding; “Those who take tobacco regularly have 0.45 times the risk of premalignant and malignant oral and oropharyngeal lesions compared to those who do not take tobacco.”
| Discussion|| |
During the course of this study, it was found that in majority of the cases, there was undue delay in the presentation due to earlier ignorance by the patient or the lack of awareness (degree of suspicion) of the possibility of malignancy on the part of the first medical attendant while examining the patient.
In present study [Table 1], the relative incidence of premalignant and malignant lesions of oral cavity is 27.81% and is concordance with Bahadur et al. study i.e. 23.3%.However Gangadharan found 63.5% incidence.
Present study showed peak age of presentation is 40-50yrs which is one decade earlier then other studies, however in concordance with Gupta et al., Babu et al., Bhayekar et al., Patel et al.,,, mentioned range of 40–60 years our mean age of prsesentation was 46.8 years, almost similar to Maheshwari et al. (45.4 years). A study in Uttar Pradesh, Mehrotra et al. mentioned mean age 55 years. Average duration of presentation in our study was 6 months form the onset of symptoms. The reasons for earlier occurrence of carcinoma among Indian people might be habit of tobacco smoking started early in the life and prevailing poor socio-economic conditions which affect the general nutritional status of the individual.
Male predominance observed in our study which is in concordance with other studies [Table 1]. Majority of the patients visiting our hospital belonged to rural areas, so as our study also showed a rural urban ratio of 1.5:1. As such these findings could not be compared with the findings of others due to paucity of such work available in literature.
Proportion of premalignant lesions in our series is 19%.Most common is leukoplakia which accounted for 62.5% and are 2.5 times mentioned in Mehrotra et al. study Considering age relationships, leukoplakia was observed from 20 to 70 years of age and mostly patients were males in a ratio of M: F =2.1:1. Leukoplakia with dysplasia similarly showed a male preponderance.
Proportion of malignant lesions (OSCC and Oropharyngeal SCC) in our series was 81%, we found well differentiated (47.03%) to be most common, followed by moderately differentiated (35.30%) and poorly differentiated (17.64%). There are similar incidence rate found in Verma et al., Maheshwari et al. and Yadav et al. studies,,, reported moderately differentiated SCC most commonly.
Tobacco intake in the form of reverse smoking/paan is the most common frequent personal habit practiced in India since an ancient times by both sexes. Our study showed most common risk factor found to be tobacco chewing comprising of 88% of total cases specially gutkha (52% amongst all tobacco habitual), smoking alone (10%) and chewing pan and pan masala alone (2%) [Figure 1] Majority (36%) of cases revealed 20–30 years of persistent exposure to tobacco, following 24% occurred after 10-20yrs of exposure and 24% after 30–40 year of exposure [Figure 2].Our study are in concordance with various studies; Gupta et al., Babu et al., Mehrotra et al., Patel et al. and Maheshwari et al.,,,,
An attempt has been done to calculate Risk ratio which is found to be RR = 0.45 signifying, the dominant role of tobacco in oral/oropharyngeal carcinoma as well as premalignant lesions of oral cavity. The prevalence rate was observed to be closely associated with age at which the patient started chewing and the frequency of tobacco chewing per day.
Smoking was most common risk factor found by Wahi in Mainpuri, an Uttar Pradesh study, Agarwal et al. and Reddy an Andhra Pradesh study, mentioned reverse smoking of “chutta”.
Smokeless tobacco has a stronger effect than a smoking type because of the direct contact of the tobacco carcinogens with the oral epithelium as the chewing tobacco products are chewed or kept in the mouth. However, the etiologic role of these factors is not well understood and further methods for modifying them need to be developed Weinberg and Estefan.
Khuri et al., Nandakumar et al., and Balaram et al., showed paan-tobacco chewing a major risk factor for OSCC.
In the present study only 10/84 cases had history of occasional alcohol intake combined with the habit of tobacco use. None of the cases had exclusive history of alcohol intake thus no association was found between alcohol intake and cancers of these sites. This was in contrast to the findings of Keller and Terris, Wynder and Stellman, found association of alcohol with carcinoma or oral cavity.
Our study showed buccal mucosa (60%) followed by tongue the most common site found in premalignant and OSCC. The findings are in concordance with other studies.,, The reason is the habit of gutka in a form of quid has been kept in buccal mucosa for very long period, changing pH and forming an ulcer followed by growth. The most common presentation found in OSCC in our study was ulceroproliferative growth (90%) followed by ulceroinfiltrative (7.6%) and ulcerative (2.4%) and in concordance with Bhayekar et al. study. The commonest presentation in oropharyngeal cancers was dysphagia and ulceroproliferative growth at base of tongue (83.3%). Similar to the finding in the study of Agarwal et al.
This study conforms and extends previous findings that the pattern of p53 and Ki67 over expression are similar and predominant in precancerous lesions and Squamous cell carcinoma. They reached their maximum values in severe dysplasia and high grade carcinomas.
In present study, p53 and Ki 67 expression was in concordance and both found in 87.5% of premalignant lesions and 94.11% of malignant lesions. The result are almost similar to Patel et al., Kerdpon et al. and Kannan et al.
A positive significant correlation was observed between positivity index of p53 and Ki67 which indicates that there is increase in p53 immunoexpression with increase in Ki67 immunoexpression in malignant lesions. Motta Rda et al. also found significant coexpression of these two markers. Lavertu et al. 2001 stated that the coexpression of p53 and Ki67 markers is associated with a lower time of survival free from disease attributed to both neoplasia recurrence and/or an appearance of a second growth early in primary site.
In our study, the staining patterns of p53 and Ki67 were found to be almost similar. Various studies showed similar findings Kerdpon et al. and Kannan et al.
In our study the p53 and Ki67 staining was restricted to the basal layer in nondysplastic leukoplakia, with an increase in suprabasal positivity as the grade of dysplasia is increased. Such a pattern of staining was also observed by Kerdpon et al., Nasser et al.,
In OSCC and oropharyngeal carcinoma all positive cases showed p53 and Ki67 immunostanining in all layers.
Our study is in accordance with Hirayama who reported p53 and Ki 67 staining was confined to basal layer in most of the cases of leukoplakia except OSCC in which it was seen in all layers.
Among 16 cases of leukoplakia, majority of them have moderate intensity (62.5%) while 4 cases have maximal intensity for p53. 46.7% of OSCC showed maximal intensity and 43.5% cases showed moderate intensity for p53.
Our findings are comparable with Hirayama who reported that 3/4 cases of leukoplakia with moderate intensity. Intensity was moderate to intense in OSCC.
The previous studies on p53 expression in Oral premalignant lesions and OSCC showed a range of positivity of 35%–90% and this finding also observed in the present study.
The wide range values may be due to the substantial differences in detection techniques applied, the difference in clones of p53 used for immunohistochemical studies, oral hygiene, nutritional as well as the varied oral habits practiced in different geographical regions and races Iamaroon et al.
Similarly, Ki67 staining was intense and moderate in 37.05% cases each and mild in 12.5% cases of leukoplakia. Among OSCC, 58% cases have maximal intensity and 35.5% cases have moderate intensity for Ki 67. This is in concordance with the study of Hirayama Yadav et al.,
All cases of oropharyngeal carcinomas showed positive p53 and Ki 67 staining ranged from mild to intense. These findings could not be compared with the findings of others due to paucity of such work available in the literature.
The present study also shows a statistical correlation of p53 and Ki 67 co-expression with increasing grade (P = 0.000), highly significant. The statistics are in concordance with Gupta et al. and Yadav et al. studies.
Furthermore, highly significant statistical correlation is found with respect to pattern and staining of p53 and Ki 67 staining in premalignant and oral and oropharyngeal SCC.
| Conclusion|| |
OSCC and premalignant oral lesions are at the high incidence in Bundelkhand region, India. Tobacco is found to be most identifiable risk factor with the risk ratio of 0.45. The over/co-expression and immunostaining patterns of p53 increase cell proliferation by Ki67 plays a pivotal role in labeling and predictive marker for malignant transformation. Their co-expression with respect to the intensity and pattern of staining increases with increasing grade. They also have prognostic value especially in premalignant oral lesions to see the survival rates if caught early by these two labelling markers.
Carry home message: Say no to tobacco, it causes oral cancer irrespective of age and sex and can ruin the quality of life.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]