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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 30  |  Issue : 3  |  Page : 201-206

Assessing field change through the expression of cytokeratin 18 and 19 in areas of erosive oral lichen planus and adjoining normal mucosa


Department of Oral Medicine and Radiology, Saveetha Dental College and Hospital, Chennai, Tamil Nadu, India

Date of Submission10-Nov-2017
Date of Acceptance04-Jul-2018
Date of Web Publication18-Oct-2018

Correspondence Address:
Dr. Sam Prasad Prabakaran
Department of Oral Medicine and Radiology, Saveetha Dental College and Hospital, Chennai - 600 077, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_117_17

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   Abstract 


Introduction: Oral lichen planus (OLP) is a potentially malignant disorder. Of its seven different clinical forms, erosive is most prone for malignant transformation. The concept of field cancerization postulates that the mucosa of upper aero-digestive tract has a potential for developing malignancy due to genetic abnormalities and migration of transformed cells. Cytokeratins (CKs) are intermediate filament proteins that are specifically expressed in epithelial tissues and used as tools in diagnostic pathology, particularly in the detection of tumors. Aim: To identify field change through the expression of CK 18 and 19 in areas of erosive OLP and adjoining normal mucosa (ANM) and assess whether CK 18 and 19 could predict malignant transformation. Materials and Methods: Tissue samples from 19 patients with erosive OLP were subjected to immunohistochemical analysis for CK 18 and 19. Results: CK 18 was expressed in 11 out of the 19 samples of erosive OLP, of which nine samples showed expression in ANM as well. CK 19 was expressed in 10 out of the 19 samples of erosive OLP, of which eight samples showed expression in ANM as well. Conclusion: Expression of CK 18 and 19 could be used to predict malignant transformation of erosive OLP and also aids in assessing field change.

Keywords: Cytokeratin, erosive oral lichen planus, field cancerization, malignant transformation, potentially malignant disorder


How to cite this article:
Prabakaran SP, Muthukrishnan A. Assessing field change through the expression of cytokeratin 18 and 19 in areas of erosive oral lichen planus and adjoining normal mucosa. J Indian Acad Oral Med Radiol 2018;30:201-6

How to cite this URL:
Prabakaran SP, Muthukrishnan A. Assessing field change through the expression of cytokeratin 18 and 19 in areas of erosive oral lichen planus and adjoining normal mucosa. J Indian Acad Oral Med Radiol [serial online] 2018 [cited 2018 Nov 14];30:201-6. Available from: http://www.jiaomr.in/text.asp?2018/30/3/201/243649




   Introduction Top


Oral lichen planus (OLP) is a chronic immune-mediated inflammatory disorder affecting the skin and oral mucosa.[1] First described by British Physician Wilson Erasmus in 1869, lichens are primitive plant that consists of symbiotic algae and fungi, and the word “planus” in Latin means flat. Lichen planus has varied clinical manifestations affecting the skin, oral mucosa, nail, genital mucosa, nail, and scalp.

Previously considered as a precancerous condition, World Health Organization (WHO) Collaboration Centre for Oral Cancer and Precancer in 2005 termed OLP as a potentially malignant disorder. The prevalence of OLP is approximately about 1–2% of the general population.[2] It is mostly seen in the fourth and fifth decades of life and is twice as common in women as in men.

OLP is considered to be a cell-mediated autoimmune disease where the accumulation of T-lymphocytes beneath the oral epithelium triggers the epithelial proliferation, differentiation, or apoptosis of keratinocytes.[1] The WHO recognizes seven clinical forms of OLP: reticular, papular, annular, plaque-like, erosive, bullous, and ulcerated.

Malignant transformation of OLP has been reported in the Western literature at the rate of about 1.09% ranging between 1 and 5%.[3] Erosive type of OLP is most prone to malignant transformation. The risk of malignant transformation associated with OLP essentiates careful examination, early diagnosis, established treatment protocol, and regular follow-up sessions.

Cytokeratins (CKs) are a group of intermediate filament proteins dispersed in the cytoplasm of eukaryotic cells that contribute to the maintenance of the cytoskeletal framework of these cells and are specifically expressed in the epithelial tissues.[4] CKs are broadly classified, based on their molecular weight and iso-electric points, into Type 1 – acidic with low molecular weight CKs 9-23, and Type 2 – basic or neutral with high molecular weight CKs 1-8.[5]

Each type of epithelium expresses two or more specific pairs of CKs based on their differentiation status. The specific nature of these heterodimers serves to distinguish different epithelial cells, in which they are expressed, and has also become important in the classification of tumor cells besides other protein tumor markers.[6] Presence of antibodies to CKs is important markers of tissue differentiation. Upon release from proliferating or apoptotic cells, CKs provide useful markers for epithelial malignancies, distinctly reflecting ongoing cell activity. Patterns in certain CKs make them likely to be substrates for caspase degradation, and their subsequent release occurs during the intermediate events in apoptosis.[7]

CKs are used as tools in diagnostic pathology; most importantly in the detection of tumors. Primary tumors and metastases of a given carcinoma share the same pattern of CKs that distinguish them from other types of carcinomas, thereby allowing differentiation between different tumors.[8] Alterations in CK pattern have been reported in oral potentially malignant and malignant lesions. CK 8, 18, and 19 are most abundant in simple epithelial cells.[5] Both CK 18 and 19 have been demonstrated in oral carcinoma.

Field cancerization is a concept proposed by Slaughter et al. in 1953. The concept postulates that the mucosa of upper aero-digestive tract has the potential for development of malignancy due to genetic abnormalities resulting from exposure to several carcinogens and existence of satellites of dysplastic-looking epithelium away from the main lesion and occurrence of multiple lesions due to migration of transformed cells either by micro-metastasis through saliva or by intraepithelial migration.[9] This concept attempts to explain the occurrence of another tumor following complete excision of the original lesion. Studies have shown that alteration in CK expression could be considered as evidence of field change in oral carcinoma.

Thus, we hypothesized that the assessment of CK 18 and 19 – a proven tumor marker – in an oral potentially malignant disorder such as erosive OLP could aid in possible prediction of a malignant transformation of the lesion.

Aim

To assess whether CK 18 and 19 are expressed in areas of erosive OLP and adjoining clinically normal mucosa.

Objectives

  • To assess whether a positive expression of CK 18 and 19 can be used to predict definitive malignant transformation from erosive OLP
  • To identify field change through the expression of CK 18 and 19 in areas of erosive OLP and adjoining clinically normal mucosa.



   Materials and Methods Top


This is an in vivo, observational study done in Saveetha Dental College, Chennai, Tamil Nadu, India. Ethical approval was obtained from Saveetha University Institutional Human Ethical Committee in December 2013. Patients reporting to Department of Oral Medicine in Saveetha Dental College were examined for signs and symptoms of erosive OLP.

Inclusion criteria

  • Patients with erosive OLP were diagnosed clinically based on Kobkan Thongprasom's criteria for clinical diagnosis of OLP and confirmed histopathologically based on Eisenberg's criteria for histopathological diagnosis of OLP.


Exclusion criteria

  • Patients with other clinical varieties of OLP
  • Patients with other white lesions or ulcerative lesions
  • Patients who had been or currently under any systemic steroids or immunosuppressive drugs or nonsteroidal antiinflammatory drugs
  • Patient with any systemic or dermatological disease affecting immune system or any malignancy
  • Patients already diagnosed with erosive OLP and under treatment.


Sample selection

This study included 19 samples (male: 7 and female: 12) of erosive OLP. The age range of the study population was 20–75 years with eight patients between 21 and 40 years of age, nine patients between 41 and 60 years of age, and two patients above 60 years. All patients were clinically evaluated for erosive type of OLP. Informed consent was taken. The patients were subjected to biopsy for confirmatory diagnosis of OLP. Tissue samples of the lesion and adjoining clinically normal appearing mucosa (NM) were incised independently under local anesthesia. The specimens were transferred to sterile bottles containing saline and submitted to Department of Oral Pathology for histopathological confirmation of OLP followed by immunohistochemical evaluation of CK 18 and 19.

Histopathological examination

The affected mucosa and clinically normal appearing mucosa were subjected to histological examination. Samples were received by the Department of Oral Pathology stored in bottles containing saline. They were then transferred to 37% formalin for 24 h. The tissues were processed by storing in alcohol for 30 min followed by acetone-1 and acetone-2 for 30 min each. The tissue samples were then stored in xylene for 1 h. Then the samples were embedded in paraffin wax overnight; later 25 micron sections were made and stained with eosin and hematoxylin. The processed samples were viewed under microscope in 4×, 10×, and 40× magnifications.

Histological confirmation of OLP was arrived based on Eisenberg's histopathological diagnostic criteria and WHO histopathological diagnostic criteria for OLP. The normal mucosa tissue samples were also subjected to histological examination for any dysplastic changes.

Immunohistochemical analysis

Once the histopathological diagnosis of OLP was confirmed, further sections were cut using microtome and stored in gelatin-coated slides for immunohistochemical analysis. Overnight incubation was done. The slides were deparaffinized by washing with xylene for 15 min twice, then washed with alcohol for 5 min twice and finally washed with distilled water for 10 min.

Antigen retrieval buffer – Tris–ethylenediaminetetraacetic acid was prepared with 500 ml water and sodium hydroxide was used to bring the pH to 9. The slides were washed with Tris buffer – 1000 ml water + 8 g sodium chloride + 0.68 Tris buffer. Hydrochloric acid was used to bring the pH to 7.6. Using HIER – Heat-Induced Epitome Retrieval technique, antigen retrieval was done, followed by bench cooling for 15 min.

Peroxidase activity blocking was done using peroxidase block for 5 min and washed with Tris buffer for 3 min. Protein blocking was done using protein block for 3 min and washed with Tris buffer.

Primary antibodies of flex monoclonal cytokeratin 18 and 19 for 1 h and 15 min were washed with TRIS buffer. Post primary or secondary antibody was added for half-an-hour followed by novolin polymer.

Diaminobenzidine (DAB) 1:20 dilution (30 μl) was added and washed with Tris buffer. Haemotoxylin was used as a counter stain. The slides were mounted and viewed in 4×, 10×, and 40× magnifications.

Two different oral pathologists independent of each other observed the expressions of CK 18 and 19 through a brown positive staining-DAB in a hematoxylin background. Samples were considered positive and accounted for the study only if both the observers agreed on the expression of CK 18 and 19.


   Results Top


The expressions of CK 18 and 19 in areas of erosive OLP and ANM in terms of age are as follows: (P-value < 0.005).

Expression of cytokeratin based on age

21–40 years:

  • CK 18 showed an expression of 50% in erosive OLP and 37.5% in ANM
  • CK 19 showed an expression of 37.5% in erosive OLP and 25% in ANM.


41–60 years:

  • CK 18 showed an expression of 66% in erosive OLP and 55% in ANM
  • CK 19 showed an expression of 66% in erosive OLP and 55% in ANM.


>60 years:

  • CK 18 showed an expression of 50% in both erosive OLP and ANM
  • CK 19 showed an expression of 50% in both erosive OLP and ANM [Figure 1] and [Figure 2].
Figure 1: Positivity of cytokeratin 18 in erosive OLP and adjoining normal mucosa based on age

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Figure 2: Positivity of cytokeratin 19 in erosive OLP and adjoining normal mucosa based on age

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Expression of cytokeratin based on gender

Male:

  • CK 18 showed an expression of 42.8% in erosive OLP and 28.5% in ANM
  • CK 19 showed an expression of 42.8% in erosive OLP and 14.2% in ANM.


Female:

  • CK 18 showed an expression of 66% in erosive OLP and 58.3% in ANM
  • CK 19 showed an expression of 58.3% in erosive OLP and 58.3% in ANM [Figure 3] and [Figure 4].
Figure 3: Positivity of cytokeratin 18 in erosive OLP and adjoining normal mucosa based on sex

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Figure 4: Positivity of cytokeratin 19 in erosive OLP and adjoining normal mucosa based on sex

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Expression of cytokeratin in erosive OLP and adjoining normal mucosa

In 19 samples, 11 cases expressed CK 18 (57.8%) in erosive OLP. Out of the 11 samples that showed positive expression of CK 18 in erosive OLP, 9 cases (81.8%) expressed CK 18 in ANM as well.

In 19 samples, 10 cases expressed CK 19 (52.6%) in erosive OLP. Out of the 10 samples that showed positive expression of CK 19 in erosive OLP, 8 cases (80%) expressed CK 19 in ANM as well [Figure 5].
Figure 5: Expression of cytokeratin 18 and 19 in both erosive OLP and adjoining normal mucosa

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Inter observer agreement

The inter-observer agreement was calculated using the Cohen's kappa. The average inter-observer agreement, both CK 18 and 19 together, is 0.805, which is interpreted as very good agreement between the observers on the expressions [Table 1].
Table 1: Kappa statistics-inter-observer agreement using Cohen's kappa

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   Discussion Top


Expressions of CK 18 and 19 in OLP have been assessed by various researchers. Boisnic et al. (1995) found an increased expression of cytokeratin 19 in OLP.[10] Kittipong et al. (2007) found the expression of cytokeratin 18 and 19 in OLP were 16.7 and 30%, respectively.[11] Jacques et al. (2009) had a 19% positive expression of cytokeratin 19 in OLP.[12]

Studies have assessed the expression of cytokeratin 18 and 19 in oral squamous cell carcinoma and adjoining normal mucosa to establish field change. Kale et al. (2012) attempted to identify the change in expression of cytokeratin 8, 18 and 19 to visualize field change in clinically normal mucosa adjacent to oral squamous cell carcinoma. The authors concluded that an enhanced expression of cytokeratin 18 (80%) and cytokeratin 19 (70%) could suggest field cancerization.[13]

This is the first study to assess the expression of cytokeratin 18 and 19 in erosive OLP and ANM.

Expression of cytokeratin based on gender

Axéll et al. and several other studies suggest that OLP has a female predilection with a female/male ratio of about 2:1.[14] The sample distribution of this study corresponds to the same and out of 19 samples, 12 (63.15%) were women. Expression of CK 18 (66%) and CK 19 (58.3%) is relatively high in females than in males – CK 18 (42.8%) and CK 19 (42.8%). This also supports the already existing evidence that the disease severity is more in females than in males.

Expression of cytokeratin based on age

Mccartan and Healy (2008) in his review stated that OLP is more prevalent in the fourth to sixth decade of life.[15] The age distribution of this study samples supports the already existing evidence as 9 out of 19 samples fall between the ages of 41 and 60 years (47.36%). The expressions of CK 18 as well as 19 reached a maximum of 66% in patients within 41–60 years of age, suggesting an increased disease prevalence and severity in the particular age group.

Expression of cytokeratin in erosive OLP and adjoining normal mucosa

CK 18 is expressed more in areas of erosive OLP (57.8%) as well as the ANM (47.3%) than CK 19, which is expressed as 52.6% in erosive OLP and 42.1% in ANM.

This study has a higher expression of CK 18 and 19 than studies reported earlier. Moreover, this is the only study to assess cytokeratin 18 and 19 in specifically erosive form of OLP, which is reported to be the most potentially malignant variety. The increase in expression could be attributed to the samples of the study being confined to erosive form alone.

Prediction of malignant transformation

The aim of the study was to determine the potential of CK 18 and 19 to predict malignant transformation in erosive OLP. Holmstrup et al. (1988) reported that patients with OLP have a 50 times higher risk of developing oral cancer than did the general population. For women the risk of malignancy is 70 times higher and for men 14 times higher.[16]

A review of literature by Mattsson et al. (2002) involving 11 patients revealed that females are more prone to malignant transformation of erosive OLP[17] and our study results also revealed that expression of CK 18 and 19 is more in females. Increased expression of CK 18 and 19 in females with erosive lichen planus implies a correlation between its expression and malignant transformation.

A systematic review that included 16 studies by Fitzpatrick et al. (2014) determined the average onset of malignancy from OLP to be 60.8 years.[3] This study revealed the maximum expression of CK 18 and 19 of 66% in patients in the age group of 41–60 years of age. The level of expression in patients above 60 years of age was 50%. With age, the probability of malignant transformation increases, therefore an increased expression of CK 18 and 19 in patients above fourth decade of life suggests that CK 18 and 19 could also be a predictor of malignant transformation of erosive lichen planus.

Field cancerization

Mignogna et al. (2007) reported that patients with OLP and subsequent development of dysplasia/oral squamous cell carcinoma are at risk of having multiple and multifocal neoplastic events of the oral cavity, a phenomenon which parallels the concept of field cancerization of traditional head and neck cancers.[18] Tamgadge et al. (2012) reported a case of field cancerization in OLP.[19] Kale et al. (2012) stated that CK 18 and 19 could be considered as a marker of altered cells and concluded that their expression in ANM to oral squamous cell carcinoma can suggest an altered mucosa with the field change.[13]

CK 18 was expressed in 11 out of the 19 samples of erosive OLP, of which nine samples showed expression in ANM as well. CK 19 was expressed in 10 out of the 19 samples of erosive OLP of which eight samples showed expression in ANM as well.

Such high expression of CK 18 (81.8%) and CK 19 (80%) in erosive OLP and ANM could point toward a possible field change.


   Conclusion Top


Various markers are being used to predict a possible malignant transformation from OLP. The peculiarity of the carcinogenesis in the upper aero-digestive tract region is the concept of field cancerization. CKs, a group of intermediate filament proteins, are a set of tumor markers whose presence has been well established in oral squamous cell carcinoma,[20] but their presence is not fully studied in oral potentially malignant disorders.

More prevalence and incidence of oral carcinoma because of erosive OLP is reported in the Western literature but very few reports are available from the Indian scientific community. This study aimed to assess the expression of cytokeratin 18 and 19 in areas of erosive OLP and adjoining clinically normal appearing mucosa.

There is a significant presence of CK 18 and 19 in erosive OLP that could suggest a possible malignant transformation. An interesting outcome of this study is the presence of CK 18 and 19 in adjoining clinically normal oral mucosa also, which further validates the concept of field cancerization.

In conclusion, CK 18 and 19 can be definitely used as a diagnostic marker to predict a possible malignant transformation. Further studies with larger sample size also need to done to firmly establish the diagnostic and prognostic potentiality of CK 18 and 19.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Lavanya N, Jayanthi P, Rao UK, Ranganathan K. Oral lichen planus: An update on pathogenesis and treatment. J Oral Maxillofac Pathol 2011;15:127-32.  Back to cited text no. 1
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Prabakaran SP, Muthukrishnan A. Expression of cytokeratin 18 and 19 in oral potentially malignant disorders: A systematic review. J Indian Acad Oral Med Radiol 2014;26:173-7.  Back to cited text no. 5
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Boisnic S, Ouhayoun JP, Branchet MC, Frances C, Béranger JY, Le Charpentier Y, et al. Alteration of cytokeratin expression in oral lichen planus. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;79:207-15.  Back to cited text no. 10
    
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Jacques CM, Pereira AL, Maia V, Cuzzi T, Ramos-e-Silva M. Expression of cytokeratins 10, 13, 14 and 19 in oral lichen planus. J Oral Sci 2009;51:355-65.  Back to cited text no. 12
    
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Kale AD, Mane DR, Babji D, Gupta K. Establishment of field change by expression of cytokeratins 8/18, 19, and MMP-9 in an apparently normal oral mucosa adjacent to squamous cell carcinoma: A immunohistochemical study. J Oral Maxillofac Pathol 2012;16:10-5.  Back to cited text no. 13
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Axéll T, Rundquist L. Oral lichen planus – A demographic study. Community Dent Oral Epidemiol 1987;15:52-6.  Back to cited text no. 14
    
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Mattsson U, Jontell M, Holmstrup P. Oral lichen planus and malignant transformation: Is a recall of patients justified? Crit Rev Oral Biol Med 2002;13:390-6.  Back to cited text no. 17
    
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Mignogna MD, Fedele S, Lo Russo L, Mignogna C, de Rosa G, Porter SR, et al. Field cancerization in oral lichen planus. Eur J Surg Oncol 2007;33:383-9.  Back to cited text no. 18
    
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van Dalen A. Significance of cytokeratin markers TPA, TPA (cyk), TPS and CYFRA 21.1 in metastatic disease. Anticancer Res 1996;16:2345-9.  Back to cited text no. 20
    


    Figures

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