|Year : 2013 | Volume
| Issue : 2 | Page : 240-244
The histological characteristics of clinically normal mucosa adjacent to oral cancer
Milos Cankovic1, Miroslav P Ilic2, Nada Vuckovic3, Marija Bokor-Bratic1
1 Clinic for Dentistry, Faculty of Medicine, University of Novi Sad, Serbia
2 Clinic for Maxillofacial and Oral Surgery, Clinical Centre of Vojvodina, Serbia
3 Centre for Pathology and Histology, Clinical Centre of Vojvodina, Serbia
|Date of Web Publication||13-Jun-2013|
Clinic for Dentistry, Faculty of Medicine, Hajduk Veljkova 12, Novi Sad
Source of Support: This study was supported by the Faculty of Medicine
Novi Sad, Serbia., Conflict of Interest: None
Background: The 'field cancerization' theory tries to explain the risk of local recurrences and development of second primary tumors in oral sqamous cell carcinoma (OSCC) patients. According to this theory it is assumed that clinically normal mucosa adjacent to oral cancer, except molecular, has already developed certain premalignant histopathological changes.
Aims: The aim of this study was to determine histological characteristics of clinically normal-looking mucosa at different distances from the apparent tumor lesion margins in OSCC patients.
Materials and Methods: Normal-appearing oral mucosa biopsy specimens were obtained from 30 new (untreated) oral cancer patients from sites at a distance of 10 mm and 20 mm from the tumor lesion margins and were compared with normal oral mucosa from 30 control patients with benign oral lesions.
Results: A total of 21 patients (70%) in the OSCC group demonstrated histological abnormalities under microscopic examination versus 7 (23.3%) control patients (P<0.01). Seventeen oral cancer patients (57%) showed significant difference in incidence and type of histological changes of normal-looking mucosa at a distance of 10 mm from the tumor lesion; 8 (27%) demonstrated reactive changes, 6 (20%) mild dysplasia and 3 (10%) squamous cell carcinoma, compared to histological abnormalities registered in 11 (OSCC) patients (36%) at a distance of 20 mm from the tumor; 10 (33%) displayed reactive changes and 1 (3%) mild dysplasia.
Conclusions: Histological abnormalities of clinically normal-looking oral mucosa taken at different distances from the tumor lesion indicated the existence of subclinical field change and represent an important parameter during the assessment of the adequacy of surgical resection margins in oral cancer management.
Keywords: Dysplasia, field change, histopathology, oral cancer, squamous cell carcinoma
|How to cite this article:|
Cankovic M, Ilic MP, Vuckovic N, Bokor-Bratic M. The histological characteristics of clinically normal mucosa adjacent to oral cancer. J Can Res Ther 2013;9:240-4
|How to cite this URL:|
Cankovic M, Ilic MP, Vuckovic N, Bokor-Bratic M. The histological characteristics of clinically normal mucosa adjacent to oral cancer. J Can Res Ther [serial online] 2013 [cited 2015 Nov 27];9:240-4. Available from: http://www.cancerjournal.net/text.asp?2013/9/2/240/113365
| > Introduction|| |
It is known that oral squamous cell carcinoma (OSCC) starts with multiple cumulative epigenetic and genetic changes caused by carcinogens, ultimately leading to clinical and microscopic visible changes called invasive neoplasm.  Some studies suggest that precursor lesions may appear at any part of the oral mucosa exposed to carcinogens. , The field of genetically altered cells in patients with squamous cell carcinoma, called 'field cancerization', has been documented with numerous clinical, histopathological and molecular studies. , Therefore, the patients with head and neck squamous cell carcinoma are subject to risk of developing local recurrences or second primary tumors as a consequence of the field cancerization, which is considered to be a bad prognostic sign.  Local recurrences occur in 10-30% of patients with surgical margins diagnosed as tumor-free by histopathology  while the incidence rate of second primary tumors is 10-35%, depending on both the localization of the first primary tumor and the age of patients with oral squamous cell carcinoma. , On the other hand, attempts of surgeons to resect a 10-mm margin of normal tissue around a tumor during excision of oral cancer, due to the need for preservation of vital anatomic structures and complete clearance without need for revision is not always possible. , Therefore, according to the concept of the field cancerization, it is assumed that clinically normal mucosa adjacent to oral cancer has suffered certain histopathological changes, as well as molecular, which can be the cause for the development of multiple premalignant lesions. Apart from western and northern European literature, there is a lack of evidence regarding this subject of oral pathology in the Balkan population.
The aim of this study was to determine the histological characteristics of clinically normal-looking mucosa at different distances from the apparent tumor lesion margins in oral squamous cell carcinoma patients.
| > Materials and Methods|| |
Research was performed as a prospective study of clinical type. Following approval from the Local Ethics Committee, the study was conducted on 30 consecutive, newly diagnosed primary oral squamous cell carcinoma patients, with clinically and histologically determined stage T1, T2 or T3; N0 or N1. The group with the cancer consisted of 24 men and 6 women, of average age of 61.5 years (41-81). The patients with extensive tumors, previously treated for oral cancer as well as with multiple lesions were not included in the study. The control (non-cancer) group consisted of 30 patients (16 men and 14 women), of average age of 54.5 years (16-83) with clinically visible and histologically confirmed benign lesion of oral mucous membrane: mucosal fibroma 8 patients (26.6%), pyogenic granuloma 4 patients (13.3%), mucocele 4 patients (13.3%), capillar mucous hemangioma 3 patients (10.0%), mucosal polyp 3 patients (10.0%), retention cyst 3 patients (10.0%), chronic nonspecific sialoadenitis 2 patients (6.7%), mucosal papilloma 2 patients (6.7%), and subacute nonspecific inflammation 1 patient (3.3%). In the cancer group there were 24 (80.0%) smokers and 20 (66.7%) patients who consumed alcohol on a daily basis (5 of them were just smokers, 1 only consumed alcohol, 19 were smokers and consumed alcohol), while in the control group there were 7 (23.3%) smokers and 6 (20.0%) patients who consumed alcohol on daily basis (6 were just smokers, 5 only consumed alcohol and 1 was both smoker and consumed alcohol). The patients of both groups were selected in the order of applying for the treatment. The information of the contents of the research and the purpose of the usage of obtained results were distributed to the patients in written form. After the procedure was introduced, the patients gave their written consent.
Four biopsy specimens were taken from every patient with OSCC, 2-4 mm in size, from the clinically normal-looking oral mucosa. Two specimens were taken from two different directions from each other (i.e. on 12 o'clock and 6 o'clock or on 12 o'clock and 3 o'clock depending on the localization), two at a distance of 10 mm from the apparent tumor lesion margin, and the other two at a distance of 20 mm from the tumor margin the same way. The highest degree of histological abnormality was considered as the representative for that patient, and for the certain distance from the tumor lesion margin. During the planned oral-surgical intervention, one specimen of the normal-looking mucosa adjacent to the clinically benign lesion, of size 2-4 mm, was taken from the patients in the control group. Mucosa samples were taken from all the patients in the same manner, with appropriate instruments in local infiltration anesthesia given by the same person each time. Lydocain with adrenaline (Galenika) was used as local anesthetic. The biopsy specimens taken from both groups were fixed in formalin solution and subjected to conventional histopathological examination and analyzed according to the standard pathological criteria by one pathologist.
The commercial statistical program "SPSS 14 for Windows" was used for statistical analysis. Chi-square test was applied for the testing of the differences between the two groups for categorical data, Student's t-test for parametric and Mann-Whitney for nonparametric data. The value P<0.05 was considered to be statistically significant.
| > Results|| |
In the cancer group, there were four times more males while in the control group the representation of both genders was almost equal. In relation to the gender, the difference found was significant (P<0.05). Comparing the average age of the patients there was no significant difference (P>0.05).
OSCC was most frequently localized at the floor of the mouth (33.3%), on mandibular gingiva (20.0%) and tongue (13.3%). In the control group, lesions were most frequently localized on the tongue (43.3%) and lower lip (33.3%). There was a significant difference (P<0.01) related to incidence of smokers between the cancer group (80%) and the control group (23.3%). In the cancer group 66.7% of the patients regularly consumed alcohol, while in the control group 20% of patients did so. There was a significant difference in the alcohol consumption between the groups (P<0.01).
Microscopically visible changes of the surrounding clinically normal-looking epithelium of the oral cavity were found in 21 (70%) patients with cancer and 7 (23.3%) patients from the control group. There was significant difference between the groups (P<0.01). Reactive changes of the epithelium were noticed in 12 (40%) patients [Figure 1], mild dysplasia in 6 (20%) [Figure 2] and squamous cell carcinoma in 3 (10%) patients in the cancer group [Figure 3] while in the control group there were only reactive changes of the epithelium in 7 (23.3%) patients. A statistically significant difference between the groups was also found in the presence of lamina propria inflammatory cell infiltrate (P<0.01) [Table 1].
|Table 1: Histological findings of clinically normal-looking tissue adjacent to lesions in OSCC and control group|
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|Figure 1: Microphotograph of oral mucosa with reactive changes— basal cell epithelial hyperplasia without inflammatory infiltrate in the lamina propria (H and E, x 200)|
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|Figure 2: Microphotograph of oral mucosa with reactive and dysplastic changes and inflammatory infiltrate in the lamina propria— focal epithelial hyperplasia, parakeratosis and mild dysplasia (H and E, x 200)|
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|Figure 3: Microphotograph of oral mucosa with infiltrative squamous cell keratinized carcinoma (H and E, x 200)|
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A significantly greater number of histological abnormalities in clinically normal-looking epithelium was found at a distance of 10 mm from the clinically apparent tumor lesion compared to the biopsy specimens taken at a distance of 20 mm from the tumor lesion margins (P<0.01) [Table 2]. It is interesting data that histological changes like dysplasia and squamous cell carcinoma of clinically normal-looking mucosa were diagnosed in 8 (40%) of 19 patients with cancer who both smoked and consumed alcohol whereas such abnormalities were not registered in non-smokers and people who did not consume alcohol, with no significant difference (P>0.05).
|Table 2: Histological findings of clinically normal-looking epithelium at different distances from the tumor lesion margins in OSCC group|
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| > Discussion|| |
The results of this study indicate that clinically normal-looking mucosa adjacent to tumor lesion in patients with oral squamous cell carcinoma had suffered certain histomorphologic changes. These histological abnormalities manifested with signs of chronic mucosal irritation, cellular atypia, mild epithelium dysplasia or even squamous cell carcinoma are caused by the effects of the carcinogens (smoking, alcohol, radiation) on the entire oral mucosa.
Similar results to ours were obtained by Thomson et al.,  in patients with a unilateral OSCC or a premalignant lesion who underwent "mirror image" biopsies from clinically normal-looking mucosa at the corresponding contralateral anatomic site, using standard microscopic examination. In the sample of 26 consecutive new patients, 15 of them (58%) showed histologically abnormal tissue; 6 (23%) with reactive changes, 7 (27%) with dysplasia and 2 (8%) with early squamous cell carcinoma.
Inzeet al.,  state the role of chronic mucosal irritation induced by tobacco carcinogens in the multistep process of sequential neoplastic development in the upper aerodigestive tracts. On the biopsy specimen of the normal-looking mucosa, cellular pleomorphism, disorder in cellular maturation and the presence of inflammatory cells within the epithelium can be seen with the electronic microscope. Histologically similar changes were reported in this study, characterized as reactive, with significantly increased presence of lamina propria peritumoral inflammatory infiltrate in patients with oral cancer compared to control ones. Independently from the histological gradation of tumor Vieira et al.,  was found the dominance of mononuclear inflammatory cells (T lymphocytes, B lymphocytes and macrophages) using immunohistochemical analysis of peritumoral inflammatory infiltrate in oral squamous cell carcinoma, and a positive correlation between the highest degree of malignancy and intensity of inflammation. These results suggest that cellular immune response has an important role in the defense mechanism of the oral mucosa in patients with oral squamous cell carcinoma.
The studies of exfoliative cytology also show the existence of field change within normal-looking mucosa in patients with oral cancer. Boloching et al.,  confirmed the concept of field cancerization by the significantly increasing micronuclei (MN) count in patients with OSCC, especially in heavy smokers, as a result of the cytogenetic damage of the buccal mucosa cells.
Comparing histological and molecular characteristics of the field with genetically altered cells based on the loss of the heterozygosity (LOH), Tabor et al., , suggest that precursor lesions are macroscopically not visible to the surgeon, however pathologists can recognize most of these lesions as mild dysplasia, which was confirmed in 6 (20%) patients in our study.
It has been previously described that altered expression of p53 genes in the histologically tumor-free surgical resection margins adjacent to the primary tumor may have indicated an early stage of cancer development.  The high incidence of TP53 mutation in histologically tumor-free surgical margins in oral squamous cell carcinoma patients that has been reported in some studies is associated with an increased risk of developing local recurrences and multiple primary tumors. , On the other hand, some authors have previously shown no or small p53 activity, as well as rare TP53 mutation on the biopsy material in histologically normal epithelium adjacent to OSCC. ,, This contradiction of results suggests that even with the lack of conventional histological examination, the application of the sophisticated diagnostic methods is not able to show a clear image of molecular events in clinically and histologically normal mucosa adjacent to oral squamous cell carcinoma, which indicated the complex nature of the field cancerization concept.
The field change can be large, a diameter of >70 mm in the oral cavity and oropharynx has been reported.  We have found histologically abnormal tissue of clinically normal-looking mucosa at a distance of 10 mm from the tumor lesion in 17 (57%) patients; 8 (27%) demonstrated reactive changes; 6 (20%) mild dysplasia and 3 (10%) squamous cell carcinoma, while histological abnormalities at a distance of 20 mm from tumor margins were registered in 11 (36%) patients; 10 (33%) displayed reactive changes and 1 (3%) mild dysplasia. These results show that increasing a distance between biopsy samples for 10 mm was significantly related with the reduced degree and number of histological abnormalities of the oral mucosa adjacent to oral cancer.
Examining angiogenesis of normal oral mucosa and its potential role as an indicator of field cancerization in patients with oral squamous cell carcinoma El-Gazzaret al.,  demonstrated results that support our findings. At a distance of 1 cm from the primary tumor, normal oral mucosa showed significantly higher vascularity, especially in patients who both smoked and consumed alcohol compared to normal mucosa from non-cancer patients.
It was previously reported by Poh et al.,  that direct visual fluorescence can identify subclinical high-risk fields with cancerous and precancerous changes in the operating room setting. The loss of fluorescence was found in almost all the 20 patients with oral cancer as a sign of mucosal alterations in the area of 4-25 mm from the clinically apparent lesion in one or more directions. These subclinical changes were found in 10 tumors at a distance greater than 10 mm, 6 of them showed histological changes of high-grade dysplasia in the biopsies taken from these regions.
Some authors describe that a high proliferative status of oral mucosa epithelium, which has been found using Ki-67 expression in areas very distant from the primary tumor, is associated with the existence of premalignant fields, therefore it is a bad prognostic sign and indicator of the risk of development of new tumors. ,
| > Conclusion|| |
To summarize, the current study shows that histological abnormalities of clinically normal-looking oral mucosa taken at different distances from the tumor lesion indicated the existence of subclinical field change and represent an important parameter during the assessment of the adequacy of surgical resection margins in oral cancer management. Conventional clinical and histological examinations are not sufficient for the evaluation of epithelial behavior adjacent to oral cancer. Therefore it is necessary to give greater importance to field cancerization in oral squamous cell carcinoma patients for the successful treatment and prevention of local recurrences and second primary tumors.
| > Acknowledgments|| |
The authors would like to express their gratitude to the Clinic for Maxillofacial and Oral Surgery Clinical Centre of Vojvodina and their chief DMD, PhD Aleksandar Kiralj for supporting this study.
| > References|| |
|1.||Dakubo GD, Jakupciak JP, Birch-Machin MA, Parr RL. Clinical implications and utility of field cancerization. Cancer Cell Int 2007;7:2. |
|2.||Hittelmann WN. Genetic instability in epithelial tissues at risk for cancer. Ann NY Acad Sci 2001;952:1-12. |
|3.||Tabor MP, Brakenhoff RH, van Houten VM, Kummer JA, Snel MH, Snijders PJ, et al. Persistence of genetically altered fields in head and neck cancer patients: Biological and clinical implications. Clin Cancer Res 2001;7:1523-32. |
|4.||Van Oijen MG, Slootweg PJ. Oral field cancerisation: Carcinogeninducedindependet events or micrometastasis deposits? Cancer Epidemiol Biomarkers Prev 2000;9:249-56. |
|5.||Mashberg A. Diagnosis of early oral and oropharyngeal squamous carcinoma: Obstacles and their amelioration. Oral Oncol 2000;36:253-5. |
|6.||Sutton DN, Brown JS, Rogers SN, Vaughan ED, Woolgar JA. The prognostic implications of the surgical margin in oral squamous cell carcinoma. Int J Oral Maxillofac Surg 2003;32:30-4. |
|7.||Cianfrigkia F, Di Gregorio DA, Manieri A. Multiple primary tumors in patients with oral squamous cell carcinoma. Oral Oncol 1999;35:157-63. |
|8.||De Vries N, van dr Waal I, Snow GB. Multiple primary tumors in oral cancer. Int J Oral Maxillofac Surg 1986;15:85-7. |
|9.||Priya SR, D'Cruz AK, Pai PS. Cut margins and disease control in oral cancers. J Cancer Res Ther 2012;8:74-9. |
|10.||Thomson PJ. Field change and oral cancer: New evidence for widespread carcinogenesis? Int J Oral Maxillofac Surg 2002;31:262-6. |
|11.||Incze J, Vaughan CW, Lui P, Strong MS, Kulapaditharom B. Premalignant changes in normal appearing epithelium in patients with squamouse cell carcinom of the upper aerodigestiv tract. Am J Surg 1982;144:401-5. |
|12.||Vieira FL, Vieira BJ, Guimaraes MA, Aarestrup FM. Cellular profile of the peritumoral inflammatory infiltrate in squamouse cell carcinoma of the oral mucose: Correlation with the expression of Ki67 and histologic grading. BMC Oral Health 2008;8:25. |
|13.||Bloching M, Hofmann A, Lautenschlager CH, Berghaus A, Grummt T. Exfoliative cytology of normal buccal mucosa to predict the relative risk of cancer in the upper aerodigestive tract using the MN-assay. Oral Oncol 2000;36:550-5. |
|14.||Tabor MP, Braakhuis BJ, Van Der Wal JE, van Diest PJ, Leemans CR, Brakenhoff RH, et al. Comparative molecular and histological grading of epithelial dyspalsia of the oral cavity and the oropharynx. J Pathol 2003;199:354-60. |
|15.||Tabor MP, Brakenhoff RH, Ruijter-Schippers HJ, Kummer JA, Leemans CR, Braakhuis BJ. Genetically altered fields as origin of locally reccurent head and neck cancer: A retrospective study. Clin Cancer Res 2004;10:3607-13. |
|16.||van Houten VM, Leemans CR, Kummer JA, Dijkstra J, Kuik DJ, van der Brekel MW, et al. Molecular diagnosis of surgical margins and local recurrence in head and neck cancer patients: A prospective study. Clin Cancer Res 2004;10:3614-20. |
|17.||Huang X, Pateromichelakis S, Hills A, Sherriff M, Lyons A, Langdon J, et al. p53 mutations in deep tissues are more strongly associated with recurrence than mutation-positive mucosal margins. Clin Cancer Res 2007;13:6099-106. |
|18.||vanHouten VM, Tabor MP, van den Brekel MW, Kummer JA, Denkers F, Dijkstra J, et al. Mutated p53 as a molecular marker for the diagnosis of haed and neck cancer. J Pathol 2002;198:476-86. |
|19.||Bongers V, Snow GB, van der Waal I, Braakhuis BJ. Value of p53 expression in oral cancer and adjacent normal mucosa in relation to the occurrence of multiple primary carcinomas. Eur J Cancer B Oral Oncol 1995;31B:392-5. |
|20.||Bilde A, von Buchwald C, Dabelsteen E, Therkildsen MH, Dabelsteen S. Molecular markers in the surgical margin of oral carcinomas. J Oral Pathol Med 2009;38:72-8. |
|21.||Thode C, Bilde A, von Buchwald C, Dabelsteen E. TP53 mutations in clinically normal mucosa adjacent to oral carcinomas. J Oral Pathol Med 2010;39:662-6. |
|22.||Tabor MP, Brakenhoff RH, Ruijter-Schippers HJ, van der Wal JE, Snow GB, Leemans CR, et al. Multiple head and neck tumors frequently originate from a single preneoplastic lesion. Am J Pathol 2002;161:1051-60. |
|23.||El-Gazzar R, Macluskey M, Ogden GR. Evidence for a field change effect based on angiogenesis in the oral mucosa? A brief report. Oral Oncol 2005;41:25-30. |
|24.||Poh CF, Zhang L, Anderson DW, Durham JS, Williams PM, Priddy RW, et al. Fluorescence visualization detection of field alterations in tumor margins of oral cancer patients. Clin Cancer Res 2006;12:6716-22. |
|25.||Montebugnoli L, Gissi DB, Badiali G, Marchetti C, Cervellati F, Farnedi A, et al. Ki-67 from clinically and histologically "normal" distant mucosa as prognostic marker in early-stage (T1-T2N0) oral squamous cell carcinoma: Aprospective study. J Oral Maxillofac Surg 2011;69:2579-84. |
|26.||Gonzales-Moles MA, Bravo M, Ruiz-Avila I, Acebal F, Gil-Montoya JA, Brener S, et al. Ki-67 expression in non-tumour epithelium adjacent to oral cancer as risk marker for multiple oral tumors. Oral Dis 2010;16:68-75. |
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]