“Mirror Image Biopsy:” An Emerging Tool for the Identification of Field Cancerization

Abstract

Background:

The most dreaded word - “Cancer” - comprises a wide category of diseases that can attack and conquer any part of the body, metastasis being the primary cause of death around the world. Early detection, appropriate care, and treatment of patients play a major role in bringing down the cancer burden leading to a higher chance of cure. Patients debilitated by oral cancer carry a high probability of developing multiple primary tumors due to repeated exposure to carcinogens and this concept is known by field cancerization.

Aims and Objectives:

This study was carried out to better define the incidence of field cancerization in patients diagnosed with carcinoma in the oral cavity, by histopathological examination of the so-called “mirror image biopsies” and its immunohistochemical evaluation using P53 expression.

Materials and Methods:

Incisional biopsies were taken from the contralateral oral cavity site of 30 patients histologically diagnosed with OSCC in D Y Patil Dental College and Hospital, Navi Mumbai. The H and E stained OSCC tissues of these patients were histologically graded and tabulated into well-differentiated, moderately differentiated, and poorly differentiated squamous cell carcinoma. The H and E stained “Mirror image” tissues were analyzed histologically to observe for dysplastic changes by three observers. Finally, the p53 stained “Mirror image” tissues were visually analyzed and scored for staining intensity and precentage of positive cells.

Results:

The OSCC cases were histologically graded and divided using Broder’s classification, among which 90% showed well-differentiated squamous cell carcinoma and 10% showed moderately differentiated squamous cell carcinoma. There were no poorly differentiated OSCC cases. The “mirror image” biopsy sites were histologically graded using the WHO classification of oral epithelial dysplasia (2017) which revealed that eighteen patients(60%) showed mild dysplasia, 11 patients (37%) had moderate dysplasia, and 1 patient (3%) revealed severe dysplasia. The p53 stained MIB tissues were visually analyzed out of which 10 were scored as negative, 12 cases as mild, 7 as moderate, and 1 as strongly positive. The histopathological diagnosis of H and E-stained MIB tissues was correlated with the IHC diagnosis of P53-stained MIB tissues. Of 18 mild dysplasia cases, 8 were scored as negative, 7 as mild and 3 as moderate. Similarly, out of 11 moderate dysplasia cases, 1 was scored as negative, 5 as mild, 4 as moderate and 1 as strongly positive. However, the correlation between these two, showed no statistical significance (P > 0.05).

Conclusion:

all efforts should be made to exclude simultaneously developing multiple primaries at the initial evaluation, as the consequences of missing such a precancerous lesion can be disastrous. OSCC patients must be subjected to a long-term and careful follow-up. Identifying those sites with increased risk for second primary tumors effectively improves early diagnosis and prognosis.

INTRODUCTION

The most dreaded word - “Cancer” - comprises a large group of diseases that can affect any part of the body, metastasis being the primary cause of death around the world and accounting for nearly 10 million deaths in 2020.[1] Between 30% and 50% of cancers can currently be prevented by avoiding the possible risk factors and executing the existing evidence-based prevention strategies. Early detection, appropriate care, and treatment of patients play a major role in bringing down the cancer burden leading to a higher chance of cure.[1,2]

Patients debilitated by oral cancer carry a high probability of developing multiple primary tumors due to repeated exposure to carcinogens and this concept is known by field cancerization.[3,4] In Oral Squamous Cell Carcinoma (OSCC) patients, the incidence rate of the second primary tumor was found to be 14%.[3,5] In another study, a 5-year incidence rate was 13% and a 10-year incidence rate was 21%.[3,6] The detection of multifocal cancerous processes at an earlier stage with appropriate diagnostic procedures for better treatment should improve survival in these patients.

In view of this probability, this study was carried out to better define the incidence of field change in patients diagnosed with oral cancer by histopathological examination of the incised biopsy sections from the apparently normal contralateral site, also known as “mirror image biopsy” and its immunohistochemical (IHC) evaluation using P53 expression.

MATERIALS AND METHODS

After obtaining institutional ethical approval on 08/01/2020 (IREB/ 2019/ OPATH /10) and informed consent from the participating patients, incisional biopsies were taken from the contralateral oral cavity site of 30 patients histologically diagnosed with OSCC in D Y Patil Dental College and Hospital, Navi Mumbai. Patients with unilateral, untreated, histologically confirmed OSCC were included in the study, and patients with tumors extending beyond the midline, bilateral tumors, metastatic disease in the oral cavity, previously treated OSCC, long-term review for dysplasia or clinically evident premalignancy including contralateral site were not included in the study [Figure 1].

Figure 1.

(a) Extraoral photograph (b) Clinical photo of the lesion site (c) Clinical photo of the mirror image site

The “Mirror image biopsy” tissue-embedded blocks were sectioned and stained with Hematoxylin and Eosin (H and E). The OSCC tissues of these patients were histologically graded and tabulated into well-differentiated, moderately differentiated, and poorly differentiated squamous cell carcinoma. The “Mirror image” tissues were analyzed histologically by three observers, as they inspected and noted down the presence of any dysplasia using standardized pathological criteria to eliminate interobserver bias.[7,8] In addition, IHC staining of mirror image biopsy tissues was done using p53 IHC marker (Clone: D07, Company: Dako). Finally, these tissues were visually analyzed and scored for staining intensity and percentage of positive cells by 3 observers using Leica Research Microscope [Figure 2].[9,10]

Figure 2.

(a) H and E-stained section of lesional tissue (×100 magnification) showing a well differentiated squamous cell carcinoma. (b) H and E-stained section of Mirror Image Biopsy (×100 magnification), confirmed as mild dysplasia with an interobserver agreement. (c) Photomicrograph of Mirror Image Biopsy stained with p53 (IHC– ×400)-Mainly basal cell layer staining with light brown staining of the suprabasal layer observed. H and E: Haematoxylin and Eosin

RESULTS

The present study included 30 OSCC patients out of which, 24 (80%) were male and 6 (20%) female patients with a male are to female ratio being 4:1 with an age range of 34 years to 72 years [Graph 1]. All patients enrolled in this study had the habit of indulging in smoking tobacco (bidis, cigarettes), chewing tobacco (pan, gutka, mawa), or in a combination of both. Among chewing tobacco patients, the frequency of habit ranged from twice a day to about 8 times per day and those with both smoking and chewing habits, the range was from five to eight times per day. In decreasing order of frequency, the site of occurrence of OSCC included buccal mucosa (40%), alveolus (33.3%), retromolar trigone (13.3%), labial mucosa (6.7%), lingual mucosa (3.3%), and lingual vestibule (3.3%), among which buccal mucosa was the most common site involved [Graph 2].

Graph 1.

Bar graph representing 80% male and 20 and female patients with a male is to female ratio of 4:1

Graph 2.

Bar graph representing buccal mucosa being the most common site of OSCC in male patients. OSCC: Oral squamous cell carcinoma

The OSCC cases were histologically graded and divided using Broder’s classification, among which 90% showed well-differentiated squamous cell carcinoma and 10% showed moderately differentiated squamous cell carcinoma. There were no poorly differentiated OSCC cases [Graph 3].

Graph 3.

Bar graph representing distribution of well differentiated squamous cell carcinoma cases and moderately differentiated squamous cell carcinoma cases. There were no cases of poorly differentiated squamous cell carcinoma

The “mirror image” biopsy sites were histologically graded using the WHO classification of oral epithelial dysplasia (2017) which revealed the following findings. Eighteen patients (60%) showed mild dysplasia, 11 patients (37%) had moderate dysplasia, and 1 patient (3%) revealed severe dysplasia. These abnormal dysplastic changes in the mirror image sites were noted more frequently in males as compared to females with no statistically significant difference (P > 0.05) [Graph 4 and Table 1]. The years of habit had no correlation with the grade of OSCC on the lesion site as well as the grades of dysplasia on the mirror image biopsy site. The “mirror image” biopsy site revealed maximum histopathological changes of dysplasia in the buccal mucosa (40%) and alveolus (33%), followed by retromolar trigone (13%), labial mucosa (6%), buccal vestibule (3%), and lingual vestibule (3%) which is statistically significant (P < 0.05) [Graph 5 and Table 2]. The p53 IHC staining of Mirror Image Biopsy tissues was performed and was visually analyzed for staining intensity and percentage of positive cells by three observers out of which 10 were scored as negative, 12 cases as mild, 7 as moderate, and 1 as strongly positive.

Graph 4.

Bar graph representing the distribution of mild dysplasia, moderate dysplasia and severe dysplasia cases in the apparently normal mucosa site/Mirror Image Biopsy site. Note that most number of cases with these abnormal dysplastic changes on the mirror image site were seen in males compared to females. There was no significant correlation (P > 0.05)

Table 1.

Correlation of histopathological diagnosis given to the biopsies taken from the mirror image biopsy site with the gender of the patient

Gender	MIB site final diagnosis			Total
	Mild	Moderate	Severe
Male	15	9	0	24
Female	3	2	1	6
Total	18	11	1	30
Chi-square tests	Value	df	Asymptomatic significance (two-sided)
Pearson Chi-square	4.148a	2	0.126
Likelihood ratio	3.373	2	0.185
Linear-by-linear association	1.264	1	0.261
Number of valid cases	30

^a4 cells (66.7%) have an expected count <5. The minimum expected count is 0.20. This correlation was not statistically significant (P>0.05). MIB: Mirror image biopsy

Graph 5.

Bar graph representing the correlation of grades of dysplasia with the mirror image biopsy site which showed a significant statistical correlation (P < 0.05)

Table 2.

Maximum histopathological changes of dysplasia seen in the mirror image biopsy site was in the buccal mucosa

MIB site	MIB site final diagnosis
	Mild	Moderate	Severe	Total
Buccal mucosa	9	3	0	12
Buccal vestibule	0	0	1	1
Alveolus	7	3	0	10
Retromolar trigone	1	3	0	4
Labial mucosa	0	2	0	2
Lingual vestibule	1	0	0	1
Total	18	11	1	30
Chi-square tests	Value	df	Asymptomatic significance (two-sided)
Pearson Chi-square	37.591a	10	0.000
Likelihood ratio	17.053	10	0.073
Linear-by-linear association	1.252	1	0.263
Number of valid cases	30

^a16 cells (88.9%) have an expected count <5. The minimum expected count is 0.03. This correlation was statistically significant (P<0.05). MIB: Mirror image biopsy

The histopathological diagnosis of H and E-stained MIB tissues was correlated with the IHC diagnosis of P53-stained MIB tissues. Of 18 mild dysplasia cases, 8 were scored as negative, 7 as mild and 3 as moderate. Similarly, out of 11 moderate dysplasia cases, 1 was scored as negative, 5 as mild, 4 as moderate and 1 as strongly positive. Interestingly, 1 case of severe dysplasia was given a negative immunoreactive score (IRS) score. However, the correlation between these two, showed no statistical significance (P > 0.05) [Graph 6 and Table 3].

Graph 6.

Bar graph representing the correlation of H and E-stained histopathological diagnosis with p53 immunohistochemical diagnosis of the mirror image biopsy tissue. There was no significant correlation (P > 0.05). H and E: Haematoxylin and Eosin

Table 3.

Correlation of diagnosis obtained from H and E stained mirror image biopsy tissues with the immunoreactive score obtained from P53 stained mirror image biopsy tissue

MIB site final diagnosis	P53 IRS				Total
	Negative	Mild	Moderate	Strongly positive
Mild	8	7	3	0	18
Moderate	1	5	4	1	11
Severe	1	0	0	0	1
Total	10	12	7	1	30
Chi-square tests	Value	df	Asymptomatic significance (two-sided)
Pearson Chi-square	7.531a	6	0.275
Likelihood ratio	8.623	6	0.196
Linear-by-linear association	1.741	1	0.187
Number of valid cases	30

^a10 cells (83.3%) have expected count <5. The minimum expected count is 0.03. This correlation was not statistically significant (P>0.05). MIB: Mirror image biopsy, IRS: Immunoreactive score

DISCUSSION

Theodore Billroth (1882) was first among the researchers, to recognize that a cancer patient can develop simultaneous multiple cancerous lesions that are independent of each other. This finding was followed by numerous studies documenting multiple primary cancers in literature [Table 4]. Clinically significant multicentric neoplasia has been recognized in recent years due to early detection at the time of initial examination.[11] “Field cancerization” is a concept which suggests that the entirety of the epithelial surface, which is repeatedly exposed and insulted by carcinogens, increases the likelihood of developing multiple, independent premalignant lesions and malignant tumors.[12] On this basis, in 1953, Slaughter et al. paved the way for modern research on the subject of field cancerization. Organ systems in which field cancerization has been described since then includes the head and neck (oral cavity, oropharynx, and larynx), lung, vulva, esophagus, cervix, breast, skin, colon, and bladder.[12,13]

Table 4.

Various studies on field cancerization, second primary tumors, and mirror image biopsy

Authors	Year	Number of patients used for the study	Result
Frank C. Marchetta, Kumao Sako, and Frank Camp	1965	1919	Found multiple malignancies in 144 patients
			Most frequently seen in head, neck, and skin cancer patients
Sol Silverman and Michael Griffith	1972	174	Confirmed an association between smoking and oral cancer
			Stated that the risk of developing a second oral primary cancer was greatly increased by the continued use of tobacco
Kathryn Ann Weichert and Donald Schumrick	1979	825	Found that the frequency of second neoplasms in embryologically similar tissues is higher due to this influence
			Concluded that with continued carcinogenic influence, patients will continue to be affected
G. J. Hordijk and J. M. A. De Jong	1981	1194	Revealed the increased frequency of incidence of both synchronous and metachronous SPTs
			Most were found in the head and neck region and in the lung
H. Shibuya et al.	1982	339	Evaluated patients with esophageal carcinoma patients
			Found one or more primary neoplasms other than the carcinoma of the esophagus in 28 patients
			All esophageal tumors were SCC
			The most frequent sites of additional tumors were the oral cavity and pharynx
W. Frederick et al.	1982	81	Carried out pan-endoscopic study of SCC patients to check for synchronous SPTs
			Revealed 14 patients having multiple primary lesions in the lung, esophagus, oral cavity, and larynx
W. Fred McGuirt et al.	1982	100	Carried out pan-endoscopic study of untreated head and neck primary tumor patients to check for synchronous SPTs
			Revealed 16 patients having multiple primary lesions, out of which 11 were silent lesions found only by endoscopy and 6 were found only due to full pan endoscopic protocol
			Suggested an intensive, full pan-endoscopic work-up as a screening test in all patients with primary head-and-neck tumors
Alain H Shikhani et al.	1986	1961	SPTs were detected in 190 patients
			89 patients revealed synchronous and 101 patients revealed metachronous SPTs
			The risk of development of SPT was seen in the esophagus, followed by pharynx, oral cavity, and larynx
N. De Vries, I. Van Der Waal and G.B. Snow	1986	210	Conducted a retrospective analysis of OSCC cases to check for the incidence of multiple primary tumors
			38 patients showed SPTs, with higher risk noted in males than in females
			A higher risk of second primaries was observed in the floor of the mouth followed by retromolar trigone and alveolar process
W.F. Lau et al.	1986	105	Prospectively screened patients with head and neck primary SCC using pan endoscopy
			9 patients were found to have simultaneous SPTs
			2 patients with triple primary tumors
			1 patient with quadruple primary tumors
			Additional tumors were found in the esophagus, tonsil, floor of the mouth, nasopharynx, bronchus, palate, and oropharynx
Hitoshi Shibuya et al.	1987	1429	136 new cancerous lesions were seen in 117 OSCC patients and stated that the mucosa of the upper aerodigestive tract was at a higher risk for development of cancer
Jay S. Cooper et al.	1989	928	Prospectively evaluated patients with head and neck cancers who underwent radiation therapy alone as the treatment for the initial primary tumor
			The risk of development of the SPTs in these patients within 3 years of radiotherapy was evaluated to be 10%, within 5 years 15% and within 8 years 23%
Ogden G.R et al.	1990	34	Performed quantitative cytomorphometric analysis of 34 apparently normal buccal mucosa biopsies in oral cancer and cancer-free patients
			Expression of keratin K8/K7 was revealed in 12 of these patients revealing the abnormal epithelial changes
Waun Ki Hong et al.	1990	103	Prospectively analyzed already treated primary tumor patients by administering isotretinoin (49 patients) and placebo (51 patients) to be taken for 12 months
			The isotretinoin-treated patients showed significantly fewer SPTs
A. Sirokawa et al.	1992	535	Retrospectively analyzed patients for incidence of SPTs and found this development in 61 patients out of which 45 were male patients and 16 patients were female
			Most common site of SPT was oral cavity, with most of it occurring 5 years of treatment of primary tumor
Bruce Gaughey et al.	1992	40,287	Conducted meta-analysis of data received from Washington University Department of Otolaryngology-head-and-neck cancer patients
			Metachronous SPTs were noted in 5721 patients
			A higher risk of SPT was observed in the respiratory or digestive tract axis
			The most common site of SPT was the oral cavity followed by the oropharynx and hypopharynx
Gina L Day and William Blot	1992	21,371	Conducted analysis of data on oral and pharyngeal cancers from nine population-based registries in US
			The risk of SPT was observed to be more in the oral and oesophageal cancers.
			This observation was seen equally in males and females
			Mostly seen in patients 60 years of younger
Gina L Day et al.	1994	1090	Evaluated the relation of smoking and alcohol consumption to the development of SPTs
			Smoking was seen to be a higher risk contributing factor for the development of SPTs
			The duration and intensity of smoking were strongest for the tumors of aerodigestive tract mainly the oral cavity followed by pharynx, esophagus, larynx, and lungs
			Cessation of smoking did not reduce the risk of development of SPTs
Harry Lumermann et al.	1995	308	Clinical and histopathological features of oral epithelial dysplasia cases were studied
			44 of these were retrospectively analyzed for transformation to SCC from the follow-up data which revealed that
			20 cases were diseases free
			15 cases had a recurrence of dysplasia
			2 cases recurred as CIS
			7 cases developed invasive SCC within 3 years
Yukihiro Nakanishi et al.	1995	42	The nondysplastic squamous epithelia surrounding 20 cases of multicentric SCC and 22 cases of unicentric SCC of the upper aerodigestive tract were subjected to immunohistochemical examination using p53
			Both groups showed positive p53 staining
			P53 expression was higher in the nondysplstic epithelia surrounding multicentric SCC (60%) than unicentric SCC (22%)
Kannan et al.	1996	15	The light microscopic and electron microscopic features of the immediate adjacent epithelium of OSCC were compared
			Ultrastructurally, similarities in malignant features were seen in the lower half of the epithelium
			Pointed out the need for ultrastructural evaluation of adjacent mucosa of OSCC using electron microscopy
M. Partridge et al.	1997	15	Analyzed cases included either the distant erythroplakia or leukoplakia at the margins of frank OSCCs or those on the opposite side of the mouth from the tumor
			The analysis was done using restriction fragment length polymorphism and microsatellite assays
			Four out of 15 cases showed the same abnormalities as in the OSCC lesion (identical novel microsatellite alleles)
			Eight out of 15 cases showed alterations which were different from the OSCC lesion (evolution of subclones, fusion of multiple clones)
F. Cianfriglia et al.	1999	200	Retrospective analysis of the risk factors in patients for SPTs, their impact on survival, and the effective measures
			With a median follow-up of 3.2 years, the incidence rate of SPTs was seen more in the oral cavity (39%) followed by oropharynx (18%), lung (10%), lips (7%), and larynx (7%)
			40% were synchronous and 60% were metachronous lesions
			Heavy tobacco consumption was the statistically significant risk associated with the development of these tumors
Nils Homann et al.	2001	105	Investigated for p53 immunohistochemical expression in frozen sections of primary tumor epithelia and the tumor distant epithelia
			Overexpression of p53 was seen in 49 tissue sections taken from tumor distant epithelia
			This expression was independent of the protein status of the primary tumor, site, stage, and grading
			Concluded that IHC staining of tumor distant epithelia is a simple and rapid tool to identify SPTs
Maarten P. Tabor et al.	2002	10	Studied the primary tumor, the SPT >3 cm away from the primary tumor, and the mucosa from the tumor-free margins to see if they are clonally related and originate from the primary tumor
			TP53 mutation and loss of heterozygosity were observed
			Common clonal origin was seen in 6 out of 10 cases, which implicated that the major proportion originated from the primary tumor itself
Thomson P.J	2002	26	18 patients presenting with untreated unilateral OSCCs and 8 premalignant lesions underwent MIBs from clinically normal-looking mucosa contralateral to the lesion site
			15 cases showed changes, out of which
			6 showed cellular atypia due to chronic irritation
			7 exhibited dysplasia
			2 displayed severe dysplasia or microinvasive SCC
A Hebbale et al.	2012	50	Patients with unilateral, single, untreated, histologically confirmed OSCC cases underwent MIB from apparently normal-looking mucosa at the contralateral site and were subjected to histopathological examination
			Out of 50 cases
			12 displayed normal oral mucosa
			6 showed hyperkeratosis
			8 showed OSMF
			24 cases revealed dysplasia
Ramani et al.	2016	15	Conducted a pilot study on patients clinically diagnosed with carcinoma of the oral cavity
			The MIB sections were stained using CK, Ki-67, and p53
			Out of 15 MIBs
			7 cases showed strong CK expression
			8 cases showed moderate CK expression
			2 cases showed mild positive KI 67 expression
			13 cases showed negative KI 67 expression
			1 case showed mild positive expression with p53
			14 cases showed mild positive expression with p53
			Suggested CK as a useful marker to predict epithelial differentiation, KI 67 and p53 as weal indicators of malignant disease progression
Sune et al.	2017	38	MIB sections were taken from 38 newly diagnosed patients with unilateral OSCC
			The mirror image tissue was analyzed histologically for abnormal features of dysplasia by two oral pathologists
			Observer 1: 22 cases showed dysplasia
			19 showed mild dysplasia
			3 showed moderate dysplasia
			Observer 2: 17 cases showed dysplasia
			16 showed mild dysplasia
			1 showed moderate dysplasia
			Concluded from the study that oral mucosa chronically exposed to carcinogens is vulnerable to development of SPTs
Dr. Rahman QB and Bajgai DP	2017	44	44 patients diagnosed with unilateral and untreated OSCC were subjected to MIB procedure
			Out of 44 cases: 16 showed dysplasia
			12 showed mild dysplasia
			4 showed moderate dysplasia
			Buccal mucosa and GBS were the most common sites that displayed dysplastic changes
Pereira et al.	2019	45	Analyzed cases to understand the involvement of miRNAs in gastric field cancerization by taking samples from
			15 noncancerous patients as controls
			15 samples adjacent to a cancerous lesion
			15 samples from the adenocarcinoma lesion site
			Results showed that
			14 miRNAs exclusively deregulated in adjacent tissue of the lesion site, of which 6 have potential diagnostic value for advanced diseases
			9 miRNAs with known TS-miRs were deregulated exclusively in adjacent tissue, of which 5 have potential diagnostic value for the early stages of gastric cancer
			This indicated that miRNAs are intimately associated with field cancerization in gastric cancer
Mohammed et al.	2021	7	Patients diagnosed with OSCC of the tongue underwent reflect copy biopsies from contralateral apparently normal-looking site
			Out of 7 cases
			1 showed severe dysplasia
			6 showed hyperplasia
			Concluded MIB as useful tool to detect early field change
Alhatab et al.	2021	7	Patients diagnosed with OSCC of the tongue underwent reflect copy biopsies from contralateral apparently normal-looking site
			Out of 7 cases
			1 showed severe dysplasia
			6 showed hyperplasia
			Concluded MIB as a useful tool to detect early field change

SPTs: Second primary tumors, MIB: Mirror image biopsy, SCC: Squamous cell carcinoma, CK: Cytokeratin, TS: Tumor suppressor activities, TP: Tumor protein, miRNAs: MicroRNAs, OSCC: Oral SCC, CIS: Carcinoma in situ, OSMF: Oral submucous fibrosis, GBS: Gingivobuccal sulcus

Two main theories have been penned in literature to better understand the development of cancer in particular sites. One states that vulnerability of the entire oral cavity surface to carcinogens at the same time, leads to multitudinous areas of genetic abnormalities, eventually resulting in multiple squamous cell carcinomas occurring independently of each other. Other states that micro-metastasis through saliva or the intra-epithelial migration of transformed malignant cells results in multiple lesions.[4,14]

Positivity to Cytokeratin 7, 8, 13, 16, and 19 at levels above the normal limit within the epithelial component and foci of cyclin D1 expression, have been detected in the normal mucosa adjacent to the cancer-affected areas.[15,16] Vascular markers such as Von Willebrand factor and CD31 have also been proven to be significantly high in the apparently normal mucosal areas adjacent to carcinomatous areas, owing to the gradual rise of angiogenic stimulators or fall of angiogenic inhibitors. A rise in the expression of Ki-67 was also noted, indicating a surge in the number of proliferating cells. An optimistic tumor marker to detect field cancerization is p53, which complements with the progression of the tumor from benign to malignant state.[17,18]

The present study was carried out to evaluate histologically the “mirror image” biopsy taken from the apparently normal-looking mucosa at corresponding contralateral anatomical sites in patients with single, unilateral clinically confirmed OSCC to know the incidence of occurrence of multiple premalignant and malignant lesions.

The predilection of OSCC in males is consistent with many other studies which can be correlated with the population of lower socioeconomic groups taking up tobacco-related habits from a young age due to ease of availability. The type of work taken up by such a population also plays a major role in this prevalence. For example, taxi drivers and security guards, who are required to stay awake at night, indulge in the practice of tobacco smoking and chewing habits to keep them awake during their work hours.[19] A survey conducted by Corsi and Subramanian in 2014, assessing the socioeconomic inequalities in smoking behavior among males in India and reported that people who were wealthier, more educated, and having jobs were more exposed to the culture of cigarette smoking, and on the other side, people of poor socioeconomic status, being less educated indulge into the habit of bidi smoking.[20]

All patients enrolled in this study had the habit of indulging in smoking tobacco, chewing tobacco or both combined. Among chewing tobacco patients, the frequency of habit ranged from twice a day to about 8 times per day and those with both smoking and chewing habits, the range was from five to eight times per day.

In a 3-year retrospective study by Garg et al. in 2013, involving 109 patients who consumed tobacco more than five times per day reported with high-risk lesions.[21] Another study conducted by Aiswarya et al. in 2017, showed that the highest risk was reported for habit frequency of >15 times/day for both smoking and mixed habits.[22]

Buccal mucosa, being the most common site of OSCC in males, was consistent with other studies.[23,24] With supported evidence from many institutional retrospective studies recorded in literature featuring buccal mucosa as the most common site of OSCC, due to years of exposure to tobacco habits, the dysplastic changes in the “mirror image” site reflect this effect on clinically normal looking mucosa as well.[25,26]

In the present study, the “mirror image” biopsy site revealed maximum histopathological changes of dysplasia in the buccal mucosa (40%) and alveolus (33%) which is statistically significant (P < 0.05). A published study, done by Cankovic et al. in 2013 revealed microscopically visible changes of the surrounding clinically normal-looking mucosa at dissimilar span from the lesion margins in OSCC patients.[27] Similar results were recorded by Thomson et al., in patients with a unilateral OSCC who underwent “mirror image” biopsies from clinically normal-looking mucosa contralateral to the corresponding anatomic site, using the standard microscopic examination. From the sample of 26 consecutive subjects, 11 tissues revealed normal histology, 6 showed reactive changes, 2 conveyed features of early SCC and 7 cases showed dysplasia.[28] Electron microscopic studies have shown alterations in the maturation of the epithelial components, cellular pleomorphism, increased distance between cells, and the existence of many inflammatory cells within the epithelial layers. On the other hand, exfoliative cytological studies have evidently shown an increase in nuclear-cytoplasmic ratios from the smears taken from normal mucosa contralateral to oral cancers or tissue adjacent to the cancer affected lesions.[28,29]

The histopathological diagnosis of H and E stained MIB tissues was correlated with the IRS obtained from P53 staining of the same, which showed no statistical significance (P > 0.05). The histopathological diagnosis of H and E stained MIB tissues was correlated with the p53 IHC diagnosis of the same. Out of 18 mild dysplasia cases, 8 were scored as negative, 7 as mild, and 3 as moderate. Similarly, Out of 11 moderate dysplasia cases, 1 was scored as negative, 5 as mild, 4 as moderate, and 1 as strongly positive. Interestingly, 1 case of severe dysplasia was given a negative IRS score.

However, the correlation between these two showed no statistical significance (P > 0.05).

As discussed earlier, p53, commonly referred to as the “guardian of the genome,” functions as a tumor suppressor. It responds to various environmental stressors such as DNA damage, hypoxia, and tumor formation.[18] It is found at low levels in the normal state, but when the DNA is damaged, the level rises and initiates a protective mechanism. If the damage exceeds this mechanism, p53 enforces the affected cells towards a programmed cell death, or apoptosis, permanently getting rid of the damage.[18,30] P53 mutations usually undergo missense mutations, by changing the information in the DNA at one position and allowing the cell to build a faulty p53. Hence, the normal functioning of p53 is blocked and is restricted to stop the damaged cells from multiplying and will develop into a tumor.[18] Bongers et al. did not observe p53 protein expression in normal-looking mucosa, adjacent to the primary tumor[31] Frameshift mutations and stop mutations, may result in the production of abnormal shorter forms of p53 and can lead to altered p53 protein which remains undetected by the monoclonal antibodies and in turn absence its expression through IHC techniques.[32,33]

CONCLUSION

This study was conducted to evaluate the theory of field cancerization, which states that “the entire epithelial surface of the upper aerodigestive tract has an increased risk for the development of malignant and premalignant lesions because of multiple genetic abnormalities in the whole tissue exposed to the same carcinogen.” To our disadvantage, neither the specific genetic nature nor the stepwise order of structural alterations that precede the development of malignancy has been clearly understood.[28] Hence, all efforts should be made to exclude simultaneously developing multiple primaries at the initial evaluation, as the consequences of missing such a precancerous lesion can be disastrous. OSCC patients must be subjected to a long-term and careful follow-up. Identifying those sites with increased risk for second primary tumors effectively improves early diagnosis and prognosis.

The examination of the oral cavity by conventional means and the resultant histopathological diagnosis are limited purely by its observational nature. Large-sample size investigations and long-term follow-ups are hence required to authenticate the importance of field cancer changes in modern clinical management. More dependent, accurate, and predictive evaluation of epithelial behavior will be required for the prevention of multiple primary tumors and long-term management of OSCC patients.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.