Rom J Morphol Embryol 2015, 56(3):1027–1033 R J M E RIGINAL APER Romanian Journal of O P Morphology & Embryology http://www.rjme.ro/

Evidence based demonstration of the concept of ‘field cancerization’ by p53 expression in mirror image biopsies of patients with oral squamous carcinoma – an immunohistochemical study

ALKA H. HANDE1), DEEPALI P. MOHITE2), MINAL S. CHAUDHARY1), MIMANSHA PATEL1), PRIYANKA AGARWAL1), SHRUTI BOHRA1)

1)Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences (DU), Sawangi (Meghe), Wardha, Maharashtra, India 2)Department of Oral Pathology and Microbiology, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Hingna, Nagpur, Maharashtra, India

Abstract Objective: The main rationale for treatment failure and death of the patients with oral squamous cell carcinoma (OSCC) is loco-regional recurrence, development of second primary tumor (SPT) and , which could be well explained by concept of field cancerization. Identification of patients at high risk for development of SPT is an important part of research for management. This study was designed keeping this aspect in mind and utilizing the increased expression of p53 as an indicator of existence of altered fields in mirror image biopsies of OSCC patients. Design: Forty clinically diagnosed oral cancer patients were included in the study. Biopsy tissue samples from clinically diagnosed oral cancer patients (Group A) and the mirror image, clinically normal looking mucosa at corresponding contralateral anatomical site (Group B) were studied for histopathological evaluation and p53 immunoexpression. Results: Tissue alterations were observed in Groups A and B. There was statistically significant (chi-square value – 126.6, p=0.0001) difference in grades of epithelial and p53 immunoexpression in Group B. Spearman’s Rank Correlation Coefficient shows non-significant positive correlation between epithelial dysplasia and p53 (r=0.28, p=0.05) in Group B. Conclusions: Evidence of presence of field cancerization, evaluated by histopathological alterations and enhanced p53 expression was observed in mirror image biopsies of OSCC patients. This could predict the altered state of oral mucosa secondary to carcinogen exposure. The realization of a genetically altered field as a cancer risk factor provides a new paradigm. It would be prudent to keep these patients under close observation and to advice them chemotherapeutic regimes. Keywords: oral squamous cell carcinoma, mirror image biopsies, p53, field cancerization.

 Introduction primary tumor (SPT). The incidence rate of SPT is 10– 35%, depending on both the location of first primary includes originating in and age of the patient [6]. The term SPT may be used in the oral cavity, the oropharynx, the hypopharynx, and connotation with ‘field cancerization’, the term and the larynx [1]. Oral squamous cell carcinoma (OSCC) is concept first put forth by Slaughter et al. in 1953 [7]. the sixth most common cancer for both sexes in the The principle of field cancerization encompasses the general population, and the third most common cancer carcinogen induced early genetic changes in the mucosa in developing nations with high incidence of mortality and of oral cavity, which leads to development of precancerous morbidity rate [2]. OSCC is a heterogeneous disease with lesions and further multifocal tumors. Further, it is complex molecular abnormalities. It may arise from a frequently used to explain the persistence of abnormal precancerous progenitor followed by outgrowth of clonal tissue after surgical removal of tumor, which may result populations associated with cumulative genetic alterations in local recurrence and or SPT. The concept of field and phenotypic progression to invasive malignancy. During cancerization in context to SPT is further elaborated by this process of , principal etiological factors many researchers [8–12]. The phenotypic expression of like tobacco, in its various forms (chewing, snuff, smoking), field cancerization could be attributed to independent and alcohol are responsible for genetic alterations, which molecular events affecting multiple cells independently in result in inactivation of multiple tumor suppressor genes the whole tissue region subsequent to carcinogen exposure and activation of proto-oncogenes [3]. or to molecular events occurring in a single progenitor Survival of OSCC patients is related to the site and with widespread expansion or lateral spread across the pathological stage of the primary tumor as well as the mucosa. Two types of migration may be involved that is histological grade and pattern of invasion [4]. Despite migration of tumor cells via saliva – micrometastasis or advances in treatment, the five-year relative survival intraepithelial migration of the progeny of the initially rate remains less than 50% and has not improved much transformed cells throughout the mucosa. There is also a over the past three decades [5]. The prognosis of OSCC possibility that the tumor itself exercises a regional is adversely influenced by the development of second effect on the oral mucosa [13].

ISSN (print) 1220–0522 ISSN (on-line) 2066–8279 1028 Alka H. Hande et al. The realization of a genetically altered field as a cancer obtaining written informed consent, 40 patients were risk factor provides a new paradigm. The existence of enrolled in the study. Detailed intraoral examination was field with genetically altered cells is a distinct biological done including both the pathological lesion and the contra- stage with continued risk for SPT [5]. Identification lateral mirror image biopsy site from clinically normal of patients at high risk for development of SPT is an looking mucosa. The pathological lesions were designated important part of management of cancer. Advances in as Group A lesions, whereas contralateral mirror image molecular biology have made it possible to investigate biopsy site from clinically normal looking mucosa were tumor growth and field at molecular level with some designated as Group B lesions. Under all aseptic precautions degree of accuracy. The process of carcinogenesis might and surgical preparation, a clean and defined elliptical involve not only increased cell proliferation but also incision was given producing a “V”-shaped wedge that decreased cell apoptosis and failure to undergo apoptosis includes both the lesion and healthy margin and the soft is a major contributor to the uncontrolled growth of tumors tissue specimen was excised. Biopsy from mirror image [14]. The carcinogens might induce a “fingerprint”-like site was performed from clinically normal looking mucosa pattern of mutations at the p53 gene. The p53 gene at corresponding contralateral anatomical site. Biopsy inactivation by means of carcinogen induced mutations, samples were fixed in 10% formalin and forwarded for deletions, and binding to viral proteins has been demon- conventional tissue processing in the histopathology strated in oral cancers [15]. laboratory. Two sections of 4-μm thickness were obtained Although a visible lesion precedes the development of from both the groups (Groups A and B) tissue. One section majority of OSCC, sometimes it may disguise itself and of each was stained with routine Hematoxylin–Eosin precancerous or cancerous changes are not always be (HE) stain for evaluation of the histopathological changes. discernible clinically or histologically. However, several The other section from both the groups (Groups A and B) studies support the fact that clinically normal appearing tissue was subjected to immunohistochemistry (IHC), mucosa of OSCC patients may be genetically altered due which was carried out by using monoclonal mouse anti- to disordered epithelial maturation and differentiation human antibody against p53 antibody. resultant to carcinogen exposure [5, 16, 17]. The genetic Histopathological evaluation changes present in normal appearing cells can be used for identification and recruitment of individuals at increased Histopathological evaluation of the clinically diagnosed risk of developing SPT for primary chemoprevention. oral cancer and mirror image tissue sections (Groups A Keeping this in mind, this study was aimed at and B) was done after staining with HE, by two oral demonstration of the concept of field cancerization by pathologists in double-blinded manner. Cases with discor- expression of structural and molecular alterations in mirror dant results underwent a consensus review with a third oral image biopsies of patients with OSCC. pathologist. The inclusion criteria used for the clinically diagnosed oral cancer tissue sections was in accordance  Materials and Methods with the histological typing of cancer and precancer of oral mucosa [18]. The cases, which fulfill the definition of Study subjects squamous cell carcinoma that is, “A malignant epithelial The present study was carried out at the Department of neoplasm exhibiting squamous differentiation characterized Oral Pathology and Microbiology, Sharad Pawar Dental by the formation of keratin and/the presence of intercellular College and Hospital, Datta Meghe Institute of Medical bridges”, were included in the study. The histopathological Sciences (DU), Sawangi (Meghe), Wardha, Maharashtra, grading of oral cancer is generally based on the subjective India. The duration of the study was six months, from assessment of the degree of keratinization, cellular and 01/05/2012 to 30/10/12. The outpatients who reported nuclear pleomorphism, mitotic activity and lymphocytic to the Department of Oral Pathology and Microbiology infiltration. Thus, OSCC was graded as well, moderately, presenting with unilateral, clinically diagnosed oral cancer, and poorly differentiated OSCC (WDOSCC, MDOSCC, irrespective of their age, gender and race were enrolled and PDOSCC, respectively) and verrucous carcinoma [18]. in the study. Other criteria set for inclusion were that: The mirror image tissue sections, which is clinically normal all patients presenting with oral cancer of less than 12 looking, were analyzed histopathologically for abnormal months duration and with the habit of betel nut chewing, features of hyperkeratosis with or without, mild, moderate, tobacco chewing, smoking, and alcohol consumption. The and severe epithelial dysplasia (dysplastic changes affecting exclusion criteria were: patients with extensive midline the lower third of the epithelium, up to two-thirds of the tumors, bilateral tumors, and metastatic disease in the epithelium, or greater than two-thirds of the epithelium, oral cavity, previously treated OSCC, clinical evidence of respectively) or (CIS), which shows widespread precancerous lesions including contralateral dysplastic changes affecting entire thickness of the epi- site, history of another prior cancerous disease in the body. thelium [19]. Subsequently, the patients were referred to the oncology clinic for further clinical management. Study protocol Immunohistochemical method for the detec- The study was approved by the Ethical Committee of tion of p53 antigen the Datta Meghe Institute of Medical Sciences (DU). A pre-enrolment screening questionnaire was used to record For immunohistochemistry, universal immunoenzyme the history regarding the patients complaints associated polymer method was employed. The tissue sections from with the lesion, duration of its presence, information Groups A and B were deparaffinized with xylene and regarding habits, and patient’s general health. After hydrated. Antigen retrieval for p53 was carried out by

Evidence based demonstration of the concept of ‘field cancerization’ by p53 expression in mirror image biopsies… 1029 heating tissue sections in microwave oven for 10 minutes the TBS washing three times for 5 minutes each, freshly in 0.01 M sodium citrate buffer (pH 6.0) and bench cooled prepared substrate⁄chromogen solution of 3,3’-Diamino- for 20 minutes, and again the same cycle was repeated benzidine (DAB) in provided buffer (mixing 25 μL of once. Endogenous peroxidase activity was blocked by concentrated DAB in 1 mL of substrate buffer) was incubating the section with 3% H2O2 in methanol for used to visualize the antigen–antibody reaction. Finally, 20 minutes. To prevent non-specific reactions, sections the sections were counterstained in Mayer’s Hematoxylin. were incubated with 10% serum for 10 minutes. p53 Immunohistochemical evaluation antibody (concentrated (1:100 dilution) anti-human p53 protein clone – DO-7, code M7001, Dako, Denmark) was Two oral pathologists evaluated immunohistochemically- incubated at room temperature in humidifying chamber stained tissues independently in double-blinded manner. for 90 minutes. From archives of Department of Oral Differences in interpretation were resolved by consensus Pathology, sections of normal oral mucosa samples review with a third oral pathologist. Cells were considered showing p53 expression positivity were used as a positive positive for the p53 when they showed nuclear staining control. One section from each positive control was used (brown color), regardless of intensity of staining, in basal, as the negative control by omitting the primary antibody parabasal and above cell layers, in clusters or discrete and by incubating with serum. After the primary antibody manner (Figure 1). and antigen reaction, the sections were rinsed in Tris A semi-quantitative analysis was performed on immuno- Buffer Saline (TBS) three times for 5 minutes each. The histochemically-stained sections. Scores 1–4 (1: 1–10%, HRP Labeled Polymer Anti-mouse (Dako EnVision+ 2: 11–30%, 3: 31–50%, 4: 51–100% of positive cells) were System HRP Labeled Polymer Anti-mouse, product code calculated after inspection of multiple neoplastic fields SM 802, Dako North America Inc.) was incubated at room at higher magnification [2] (Figure 2). temperature in humidifying chamber for 30 minutes. After

Figure 1 – Photomicrograph showing enhanced immuno- Figure 2 – Photomicrograph showing enhanced immuno- histochemical reactivity of p53 antigen in Group A (clinically histochemical reactivity of p53 antigen in Group B (mirror diagnosed oral cancer tissue), ×100. image, clinically normal looking tissue), ×400.

Statistical analysis Regarding histopathological evaluation, in Groups A and B, there was presence of tissue alterations. These Histopathological evaluation and p53 immunoreactivity were in the form of verrucous carcinoma, WDOSCC were analyzed. Differences in p53 immunoreactivity and and MDOSCC in Group A. There were no cases of histopathological evaluation between and within Groups PDOSCC included in the study. In Group B, the tissue A and B were done by using descriptive and inferential alterations were in the form of hyperkeratosis with or statistics using chi-square test and Spearman’s Rank without mild epithelial dysplasia (Table 2). Correlation Coefficient. Statistical significance was set We observed correlation in histopathological alterations at p<0.05. All statistical analyses were performed using between Groups A and B. On correlation in histopatho- SPSS 17.0 ver. 8 software package. logical evaluation between Groups A and B, 12.5%, 35% and 5% patients of MDOSCC, WDOSCC and verrucous  Results carcinoma, respectively, shows hyperkeratosis with mild We evaluated 80 tissue samples of 40 OSCC patients, epithelial dysplasia in the mirror image biopsies. Whereas to assess the possible correlation of p53 immunohisto- only 7.5%, and 2.5% patients of WDOSCC and verrucous chemical expression with histopathological observations carcinoma, respectively, shows hyperkeratosis without amongst Groups A and B. The demographic and clinico- epithelial dysplasia (Table 3). pathological characteristics of the patients are described There was enhanced p53 immunoexpression in Group A in Table 1. as well as in Group B (Table 4).

1030 Alka H. Hande et al. Table 1 – Demographic and clinicopathological charac- Demographic and clinico- No. of Percentage teristics of the patients pathological characteristics patients [%] Demographic and clinico- No. of Percentage Duration of the lesions pathological characteristics patients [%] <3 months 17 42.5 Gender <6 months 11 27.5 Males 34 85 <9 months 12 30 Females 6 15 Age Table 2 – Comparison of histopathological diagnosis amongst Groups A and B Mean age – male [years] 51.81±10.31 Histopathological Group Group χ2- p- % % Mean age – females [years] 53±10.58 diagnosis A B value value Mean age – total patients [years] 52±10.21 Normal 0 0 15 37.5 TNM staging of pathological lesions Hyperkeratosis 0 0 4 10 Stage I 9 22.5 Hyperkeratosis with mild epithelial 0 0 21 52.5 <0.0001 86.15 Stage II 17 42.5 dysplasia (sig.) Stage III 12 30 VC 7 17.5 0 0 Stage IV 2 5 WDOSCC 26 65 0 0 Site of pathological lesions MDOSCC 7 17.5 0 0 Left buccal mucosa 26 65 VC: Verrucous carcinoma; WDOSCC: Well-differentiated oral squamous cell carcinoma; MDOSCC: Moderately differentiated oral squamous cell Right buccal mucosa 14 35 carcinoma; sig.: Significant. Table 3 – Correlation in histopathological evaluation amongst Groups A and B Group B / No. of cases (percentage) Correlation in histopathological Total / No. of cases Hyperkeratosis with mild alterations Normal Hyperkeratosis (percentage) epithelial dysplasia VC 4 (10%) 1 (2.5%) 2 (5%) 7 (17.5%) Group A MDOSCC 2 (5%) 0 (0%) 5 (12.5%) 7 (17.5%) WDOSCC 9 (22.5%) 3 (7.5%) 14 (35%) 26 (65%) Total / No. of cases (percentage) 15 (37.5%) 4 (10%) 21 (52.5%) 40 (100%) VC: Verrucous carcinoma; MDOSCC: Moderately differentiated oral squamous cell carcinoma; WDOSCC: Well-differentiated oral squamous cell carcinoma.

Table 4 – Comparison of p53 immunoexpression We further assessed Group B for comparison in amongst Groups A and B alterations at structural level by evaluating grades of p53 Group A % Group B % χ2-value p-value epithelial dysplasia and at molecular level by evaluating Score 0 0 0 6 15 p53 immunoexpression (Figure 3). Alterations at molecular Score 1 9 22.5 14 35 level as evaluated by p53 immunoexpression in clinically Score 2 13 32.5 15 37.5 normal looking mucosa showed alterations in 85% patients 0.0004 Score 3 10 25 5 12.5 18.25 (Figure 3). At structural level, we observed 62.5% patients (sig.) Score 4 8 20 0 0 showed tissue alterations, which were in the form of Total 40 100 40 100 hyperkeratosis and hyperkeratosis with mild epithelial dysplasia. 37.5% of patients showed no changes. Increase Score 0: Absence of p53 expression; Score 1: <10% cells positive for p53 expression; Score 2: 1–30% cells positive for p53 expression; in p53 immunoexpression was reflected as increase in Score 3: 31–50% cells positive for p53 expression; Score 4: >51% degree of epithelial dysplasia in 92% patients (out of cells positive for p53 expression; sig.: Significant. 62.5% patients with epithelial dysplasia).

Figure 3 – Comparison of epithelial dysplasia with p53 immunoexpression in Group B. Score 0: Absence of p53 expression; Score 1: <10% cells positive for p53 expression; Score 2: 1–30% cells positive for p53 expression; Score 3: 31–50% cells positive for p53 expression; Score 4: >51% cells positive for p53 expression.

Evidence based demonstration of the concept of ‘field cancerization’ by p53 expression in mirror image biopsies… 1031

The other finding of interest in this study is that 73.33% of SPT in apparently unaffected mucosa of OSCC patients (out of 37.5% patients without epithelial dysplasia) patients [34]. Focal p53 positivity was detected more often in showed increase in p53 immunoexpression in clinically normal mucosa adjacent to cancer than healthy control normal looking mucosa, without presence of epithelial epithelium from HNSCC (head and neck squamous cell dysplasia on histopathological evaluation (Figures 3 and 4). carcinoma) patients [21, 29, 35]. The frequency of p53 positive cells gradually increases as oral epithelium progresses from normal to hyperplasia to dysplasia to carcinoma [21]. This study was designed keeping this aspect in mind and utilizing the increased expression of p53 as an indicator of existence of altered fields in mirror image biopsies of patients with OSCC. The concept of field cancerization in oral cavity is based on the fact that carcinogens via the saliva affect the entire mucosa. As entire mucosa is affected by exposure to carcinogens, it can be assumed that genetic aberrations would not be limited to one particular area but an entire exposed mucosa. The proof to this concept was sought by Figure 4 – Correlation of epithelial dysplasia with p53 evaluating mirror image biopsies from clinically normal immunoexpression in Group B. appearing oral mucosa from contralateral sites of establi- shed OSCC patients. This evaluation was carried out both  Discussion by histopathology and p53 immunoexpression. The present study results demonstrated histological evidence of high The main rationale for treatment failure and death incidence of tissue instability in the mirror image biopsies of the patients with OSCC is loco-regional recurrence, of clinically normal looking oral mucosa. The immuno- development of SPT and metastasis. The loco-regional histochemical expression of p53 protein could be because recurrence and development of SPT could be well of altered p53 protein metabolism, which is either caused explained by the concept of field cancerization. The oral by mutation or altered turnover of the wild p53 protein. epithelium is inevitably exposed to environmental subs- This indicates that the molecular alteration is an early tances, including carcinogens. The accumulation of genetic event in the process of field cancerization and it occurs alterations due to carcinogen exposure can create a large even before there is histological evidence in the form of area of genetically altered precancerous fields, which forms epithelial dysplasia. All these alterations are suggestive of the basis for the expanding fields in carcinogenesis. The the concept of field cancerization that, there is compre- need of the hour is to identify the specific biological hensive change in entire mucosa exposed to carcinogen. markers, which will help identify an altered state of tissue The proof lies in architectural, cellular as well as molecular that is field. The altered state of tissue is rarely clinically level changes seen in the mirror image biopsies. apparent and it is difficult for a clinician to identify the Our results are in agreement with Thomson [35], presence of field based on clinical examination only. This who demonstrated histologically abnormal tissue, varied is supported by the finding that as many as 50% of OSCC from reactive change/cellular atypia, dysplasia, CIS, or arise from apparently clinically normal mucosa [20]. microinvasive SCC from mirror image biopsies of As suggested by current theories of carcinogenesis, the OSCC patients. Partridge et al. reported that two-third of transition from normal oral epithelium to oral dysplasia and diagnosed cases of oral precancer subsequently shown cancer results from accumulated genetic and epigenetic development of invasive cancer at sites distinct from alterations due to exposure to carcinogens [21]. However, their original dysplasia [9]. the precise nature of genetic or structural alterations pre- To predict the recurrence and development of second ceding cancerous change is unclear. To support the concept primary tumors in HNSCC, Shin et al. [31] examined p53 of field cancerization in routine histological tissues various protein expression in primary HNSCC. They believed that molecular techniques like microsatellite analysis [8–10, p53 protein has the prognostic significance for identifying 22, 23], electron microscopy [24], exfoliative cytology individuals at high risk of developing a recurrence of [25, 26], PCR (polymerase chain reaction) [27], IHC primary disease and also SPT. Nees et al. [34] noted the (immunohistochemistry) [13, 28–31] and frozen IHC expression of mutated p53 protein in histologically normal [32] have been used which also confirm the presence of appearing tumor distant epithelial cells in HNSCC patients. cellular alterations. These findings were confirmed by Waridel et al. [29], who The first molecular indicator of disturbance in cell observed the presence of p53 mutations in histologically division is enhanced expression of p53 protein. Mutations normal mucosa of aero digestive cancer patients. They in the p53 gene frequently result in the synthesis of a believed that this observation coincides with the likelihood mutant protein that has a longer half-life than that of the of development of SPT. To check the predictive power wild-type protein, causing an accumulation of altered p53 of p53, Homann et al. [32] investigated p53 expression protein within the nuclei of affected cells and thereby in tumor distant epithelia and in the corresponding primary increases its expression [33]. Loss of function of p53 tumors by frozen IHC. They observed that p53 over tumor suppressor gene results in uncontrolled cell division expression was specifically associated with SPT but not and progressive genomic instability. Therefore, over- with recurrences. Our findings seem to find agreement expression of p53 may be indicative of an increased risk with all of these studies. However, our observations were

1032 Alka H. Hande et al. not in agreement with that of Bongers et al. [30] and [3] Perez-Ordoñez B, Beauchemin M, Jordan RCK. Molecular Ogden et al. [36] who found that p53 over expression in biology of squamous cell carcinoma of the head and neck. J Clin Pathol, 2006, 59(5):445–453. normal mucosa of oral cancer patients, could not predict [4] Woolgar JA, Rogers S, West CR, Errington RD, Brown JS, the likelihood of SPT. Vaughan SD. Survival and patterns of recurrence in 200 oral The field cancerization concept has always emphasized cancer patients treated by radical surgery and neck dissection. the fact that, carcinogens do not act in isolation on limited Oral Oncol, 1999, 35(3):257–265. [5] Kale AD, Mane DR, Babji D, Gupta K. Establishment of field fields but conversely act over a large generalized area. change by expression of cytokeratins 8/18, 19, and MMP-9 Due to interaction with additional factors, certain fields in an apparently normal oral mucosa adjacent to squamous get converted into malignancy. This, however, does not cell carcinoma: a immunohistochemical study. J Oral Maxillofac rule out the fact that a SPT could develop in a geogra- Pathol, 2012, 16(1):10–15. [6] van Oijen MG, Slootweg PJ. Oral field cancerization: carcinogen- phically independent location later. The increased expression induced independent events or micrometastatic deposits? of p53 in mirror image tissue is seen in geographically Cancer Epidemiol Biomarkers Prev, 2000, 9(3):249–256. altered cells supporting the concept of field cancerization. [7] Slaughter DP, Southwick HW, Smejkal W. Field cancerization Thompson evaluated mirror image biopsies of OSCC in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer, 1953, 6(5):963–968. patients, to quantify the incidence and type of field change [8] Bedi GC, Westra WH, Gabrielson E, Koch W, Sidransky D. in oral mucosa. This is to our knowledge is the first study Multiple head and neck tumors: evidence for a common clonal providing evidence of p53 overexpression in clinically origin. Cancer Res, 1996, 56(11):2484–2487. normal looking mucosa (mirror image) of OSCC patients, [9] Partridge M, Emilion G, Pateromichelakis S, Phillips E, Langdon J. Field cancerisation of the oral cavity: comparison which could be predictable to an altered state of oral of the spectrum of molecular alterations in cases presenting mucosa secondary to carcinogen exposure. The realization with both dysplastic and malignant lesions. Oral Oncol, 1997, of a genetically altered field as a cancer risk factor 33(5):332–337. provides a new paradigm. We are in agreement with [10] Califano J, van der Riet P, Westra W, Nawroz H, Clayman G, Piantadosi S, Corio R, Lee D, Greenberg B, Koch W, van Oijen & Slootweg [6] that the field changes appear Sidransky D. Genetic progression model for head and neck to be induced by continuous carcinogenetic influence cancer: implications for field cancerization. Cancer Res, 1996, rather than field cancerization due to migrated transformed 56(11):2488–2492. cells. The development of field at contralateral site of [11] Dakubo GD, Jakupciak JP, Birch-Machin MA, Parr RL. Clinical implications and utility of field cancerization. Cancer Cell Int, OSCC may be due to carcinogen exposure rather than 2007, 7:2. migrated transformed cells. [12] Braakhuis BJ, Tabor MP, Leemans CR, van der Waal I, Snow GB, Brakenhoff RH. Second primary tumors and field cancerization in oral and oropharyngeal cancer: molecular  Conclusions techniques provide new insights and definitions. Head Neck, Presence of field cancerization, in mirror image biopsies 2002, 24(2):198–206. [13] Ogden GR, Lane EB, Hopwood DV, Chisholm DM. Evidence of OSCC patients could predict the altered state of oral for field change in oral cancer based on cytokeratin expression. mucosa secondary to carcinogen exposure and shows Br J Cancer, 1993, 67(6):1324–1330. increased predisposition or susceptibility of the mucosa [14] Miyazaki T, Kato H, Shitara Y, Yoshikawa M, Tajima K, to progression to second primary tumor. Masuda N, Shouji H, Tsukada K, Nakajima T, Kuwano H. Mutation and expression of the metastasis suppressor gene Conflict of interests KAI1 in esophageal squamous cell carcinoma. Cancer, 2000, 89(5):955–962. The authors declare that they have no conflict of [15] Fabbrocini G, Russo N, Pagliuca MC, Delfino M, Staibano S, interests. Molea G, Mancini A, Virgili A, Valente MG, Bratina G, Galbiati S, Marzaduri A, Orlandi K, Bottoni U, Calvieri S, Di Landro A, Acknowledgments Cainelli T, Delfino S, De Rosa G. p53, cyclin-D1, PCNA, The authors extend their sincere thanks to AgNOR expression in squamous cell cancer of the lip: a (1) Dr. Ravikant Sune (MDS), Assistant Professor, multicenter study. Photodermatol Photoimmunol Photomed, Department of Oral Diagnosis Medicine and Radiology, 2000, 16(4):172–177. [16] Dhooge IJ, De Vos M, Van Cauwenberge PB. Multiple primary Sharad Pawar Dental College and Hospital, Datta Meghe malignant tumors in patients with head and neck cancer: Institute of Medical Sciences (DU), Sawangi (Meghe), results of a prospective study and future perspectives. Laryn- Wardha, Maharashtra, India. goscope, 1998, 108(2):250–256. (2) Mr. Rupesh Maladhari, Laboratory Technician, [17] Brickley MR, Cowpe JG, Shepherd JP. Performance of a computer simulated neural network trained to categorise Department of Oral and Maxillofacial Pathology, Sharad normal, premalignant and malignant oral smears. J Oral Pathol Pawar Dental College and Hospital, Datta Meghe Institute Med, 1996, 25(8):424–428. of Medical Sciences (DU), Sawangi (Meghe), Wardha, [18] Pindborg JJ, Reichart PA, Smith CJ, van der Waal I. Histolo- nd Maharashtra, India. gical typing of cancer and precancer of oral mucosa. 2 edition, World Health Organization (WHO) International Histological Classification of Tumors, Spinger-Verlag, Berlin– References Heidelberg–New York, 1997, 11–14. [1] Conway DI, Hashibe M, Boffetta P; INHANCE consortium, [19] Warnakulasuriya S, Reibel J, Bouquot J, Dabelsteen E. Oral Wunsch-Filho V, Muscat J, La Vecchia C, Winn DM. Enhancing epithelial dysplasia classification systems: predictive value, epidemiologic research on head and neck cancer: INHANCE utility, weaknesses and scope for improvement. J Oral Pathol – The International Head and Neck Cancer Epidemiology Med, 2008, 37(3):127–133. Consortium. Oral Oncol, 2009, 45(9):743–746. [20] Speight PM. Update on oral epithelial dysplasia and progression [2] Farhadieh RD, Smee R, Ow K, Yang JL, Russell PJ, Crouch R, to cancer. Head Neck Pathol, 2007, 1(1):61–66. Jackson P, Jacobson IV. Down-regulation of KAI1/CD82 protein [21] Shin DM, Kim J, Ro JY, Hittelman J, Roth JA, Hong WK, expression in oral cancer correlates with reduced disease free Hittelman WN. Activation of p53 gene expression in prema- survival and overall patient survival. Cancer Letters, 2004, lignant lesions during head and neck tumorigenesis. Cancer 213(1):91–98. Res, 1994, 54(2):321–326.

Evidence based demonstration of the concept of ‘field cancerization’ by p53 expression in mirror image biopsies… 1033 [22] Piccinin S, Gasparotto D, Vukosavljevic T, Barzan L, Sulfaro S, [30] Bongers V, Snow GB, van der Waal I, Braakhuis BJ. Value of Maestro R, Boiocchi M. Microsatellite instability in squamous p53 expression in oral cancer and adjacent normal mucosa cell carcinomas of the head and neck related to field cance- in relation to the occurrence of multiple primary carcinomas. rization phenomena. Br J Cancer, 1998, 78(9):1147–1151. Eur J Cancer B Oral Oncol, 1995, 31B(6):392–395. [23] Allegra E, Baudi F, La Boria A, Fagiani F, Garozzo A, [31] Shin DM, Lee JS, Lippman SM, Lee JJ, Tu ZN, Choi G, Costanzo FS. Multiple head and neck tumours and their Heyne K, Shin HJ, Ro JY, Goepfert H, Hong WK, Hittelman WN. genetic relationship. Acta Otorhinolaryngol Ital, 2009, 29(5): p53 expressions: predicting recurrence and second primary 237–241. tumors in head and neck squamous cell carcinoma. J Natl [24] Incze J, Vaughan CW Jr, Lui P, Strong MS, Kulapaditharom B. Cancer Inst, 1996, 88(8):519–529. Premalignant changes in normal appearing epithelium in [32] Homann N, Nees M, Conradt C, Dietz A, Weidauer H, Maier H, patients with squamous cell carcinoma of the upper aero- Bosch FX. Overexpression of p53 in tumor-distant epithelia of digestive tract. Am J Surg, 1982, 144(4):401–405. head and neck cancer patients is associated with an increased [25] Ogden GR, Cowpe JG, Green MW. Evidence of field change incidence of second primary carcinoma. Clin Cancer Res, in oral cancer. Br J Oral Maxillofac Surg, 1990, 28(6):390– 2001, 7(2):290–296. 392. [33] Tian D, Feng Z, Hanley NM, Setzer RW, Mumford JL, [26] Siar CH, Mah MC, Gill PP, Nagatsuka H, Tsujigiwa H, DeMarini DM. Multifocal accumulation of p53 protein in eso- Gunduz M, Nagai M, Ng KH. Oral potentially malignant oral phageal carcinoma: evidence for field cancerization. Int J lesions and associated risk in the contralateral side. J Hard Cancer, 1998, 78(5):568–575. Tissue Biol Proc, 2005, 14(2):183–184. [34] Nees M, Homann N, Discher H, Andl T, Enders C, Herold- [27] Lydiatt WM, Anderson PE, Bazzana T, Casale M, Hughes CJ, Mende C, Schuhmann A, Bosch FX. Expression of mutated Huvos AG, Lydiatt DD, Schantz SP. Molecular support for p53 occurs in tumor distant epithelia of head and neck cancer field cancerization in the head and neck. Cancer, 1998, 82(7): patients; a possible molecular basis for the development of 1376–1380. multiple tumors. Cancer Res, 1993, 53(18):4189–4196. [28] van Oijen MG, Gilsing MM, Rijksen G, Hordijk GJ, Slootweg PJ. [35] Thomson PJ. Field change and oral cancer: new evidence Increased number of proliferating cells in oral epithelium from for widespread carcinogenesis? Int J Oral Maxillofac Surg, smokers and ex-smokers. Oral Oncol, 1998, 34(4):297–303. 2002, 31(3):262–266. [29] Waridel F, Estreicher A, Bron L, Flaman JM, Fontolliet C, [36] Ogden GR, Chisholm DM, Morris AM, Stevenson JH. Over- Monnier P, Frebourg T, Iggo R. Field cancerisation and poly- expression of p53 in normal oral mucosa of oral cancer clonal p53 mutation in the upper aero-digestive tract. Onco- patients does not necessarily predict further malignant disease. gene, 1997, 14(2):163–169. J Pathol, 1997, 182(2):180–184.

Corresponding author Alka H. Hande, Associate Professor, MDS, PhD, Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences (DU), Sawangi (Meghe), Wardha–442001, Maharashtra, India; Fax 07152–287731, e-mail: [email protected]

Received: November 21, 2014

Accepted: November 30, 2015