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 »  Abstract
 » Introduction
 » Subjects and Methods
 » Results
 » Discussion
 » Acknowledgment
 »  References
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  Table of Contents  
Year : 2015  |  Volume : 52  |  Issue : 4  |  Page : 498-502

The role of Ki-67, p16, CD34, Bcl-2, cyclooxygenase-2 in the pathogenesis of proliferative verrucous leukoplakia

Department of Oral Pathology, Manipal College of Dental Sciences, Manipal University, Manipal, Karnataka, India

Date of Web Publication10-Mar-2016

Correspondence Address:
R A Radhakrishnan
Department of Oral Pathology, Manipal College of Dental Sciences, Manipal University, Manipal, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0019-509X.178424

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 » Abstract 

CONTEXT: Proliferative verrucous leukoplakia (PVL) is a highly persistent and aggressive oral pre-malignant lesion with an obscure etiopathogenesis and a malignant transformation rate of 85-100%. AIMS: The aim of the present study is to assess the role of Ki-67, p16, CD34, Bcl-2, cyclooxygenase-2 (COX-2) in the spectrum of PVL to ascertain their role in its etiopathogenesis. SETTINGS AND DESIGN: A retrospective chart analysis was carried out on a series of seven confirmed cases of PVL, which were followed-up for 2 years. SUBJECTS AND METHODS: Immunohistochemical appraisal of these cases was carried out by a panel of markers, related to cell proliferation, cell cycle regulation, angiogenesis, apoptosis and inflammation. The expression of these markers was correlated with patients' clinicopathological profile. STATISTICAL ANALYSIS USED: The frequency distribution of the group data was analyzed. RESULTS: The latest labeling index of Ki-67 in our cases ranged from 8.18 to 12.6. p16 was positive in 3/7 cases. Bcl-2 expression was moderately positive in 2/7 cases. All cases were intensely positive for COX-2 staining. Microvascular density assessed by CD34 staining ranged from 11 to 20/high power fields. One case which transformed into squamous cell carcinoma demonstrated increased Ki-67, Bcl-2, COX-2, CD34 expression, but negative p16 and Bcl-2 expression. CONCLUSIONS: Application of these markers in understanding the behavior of PVL suggests that an imbalance between the proliferation apoptosis dynamics of the lesion accompanied by an increase in inflammation and angiogenesis underlie the molecular pathogenesis of the PVL spectrum.

Keywords: Bcl-2, cyclooxygenase-2, immunohistochemistry, Ki-67, p16, proliferative verrucous leukoplakia

How to cite this article:
Thennavan A, Byatnal A A, Solomon M C, Radhakrishnan R A. The role of Ki-67, p16, CD34, Bcl-2, cyclooxygenase-2 in the pathogenesis of proliferative verrucous leukoplakia. Indian J Cancer 2015;52:498-502

How to cite this URL:
Thennavan A, Byatnal A A, Solomon M C, Radhakrishnan R A. The role of Ki-67, p16, CD34, Bcl-2, cyclooxygenase-2 in the pathogenesis of proliferative verrucous leukoplakia. Indian J Cancer [serial online] 2015 [cited 2021 Jun 12];52:498-502. Available from:

 » Introduction Top

The genesis of the term “proliferative verrucous leukoplakia” (PVL), is attributed to the work of Hansen in the year 1985 that recognized this entity as a highly aggressive form of oral leukoplakia.[1] Although the diagnosis of PVL has been disputed, discredited, debated and defended over the years, our understanding of this inscrutable disease continuum remains more or less the same. Its etiopathogenesis still remains unsolved. Clinically, the usual presentation is in elderly women commonly involving the gingiva, buccal mucosa and tongue, manifesting initially as a white elevated plaque, eventually progressing into a warty exophytic and verrucous mass with a multifocal distribution.[2],[3] Histologically, a spectrum of lesions ranging from simple hyperplasia or hyperkeratosis without dysplasia to areas of severe dysplasia (D), verrucous hyperplasia (VH), verrucous carcinoma and oral squamous cell carcinoma (OSCC) encompass PVL.[1],[3] The present diagnostic requisite is a white lesion that over a period of time (1-20 years) becomes warty and recurs after treatment involving more than one site in the oral cavity.[3] A definitive diagnosis of PVL thus presents a challenge as a long term follow-up period is required. Moreover, the diagnosis must be made swiftly as 85-100% of PVL cases reportedly turn malignant. The process of malignant transformation and its duration has remained contentious as some cases transform early while others lie dormant for many years.[2],[3]

Ki-67 is a proliferative marker that has been used frequently to assess the aggressiveness of oral dysplasias and OSCC.[4] p16, a cyclin-dependent kinase inhibitor (CDK-INK4A) is considered a tumor suppressor protein having been implicated in oral carcinogenesis.[5] Bcl-2, a proto-oncogene is an anti-apoptotic molecule that has been implicated in prolonging cell survival and acquisition of mutations.[6] The role of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids in carcinogenesis by inducing neo-angiogenesis, inhibiting apoptosis, and altering cell adhesion molecules [7],[8] has been implicated. Finally, the role of angiogenesis for tumor induction and continued growth has been established.[9]

In an attempt to understand the progression of the PVL spectrum a series of seven confirmed cases of PVL was investigated for proliferation-apoptosis dynamics using Ki-67 labeling index (LI), p16 and Bcl-2 immunoexpression. In addition, the role of angiogenesis and inflammation in PVL was studied by determining the microvascular density (MVD) through CD34 and COX-2 immunostaining respectively.

 » Subjects and Methods Top

Inclusion criterion and tissue blocks

Diagnosed cases of PVL that recurred and turned warty over a period of time after treatment were included. All the cases had multifocal presentation and a minimum of two recurrences following treatment. Formalin fixed paraffin embedded tissue blocks of these cases with latest recurrence were employed for the study. In addition, one tissue block of well differentiated SCC was included.

Immunohistochemical staining

Briefly, paraffin sections of 4 μm were mounted on 3-aminopropyl triethoxysilane coated slides. Sections were incubated for 30 min each with anti-Ki-67 (clone BGX-Ki-67, Biogenex) diluted 1:30; anti-p16 (clone G175-405, Biogenex) diluted 1:50; anti-CD34 (clone QBend/10, Biogenex) diluted 1:50; anti-Bcl-2 (clone E17, Biogenex) diluted 1:50 and anti-COX-2 (clone 4H12, Novocastra) diluted 1:100. Immunohistochemistry was carried out using SuperSensitive™ Link-Label IHC Detection System, Biogenex, USA. Color reaction was developed using diaminobenzidine, counterstained with Mayer's hematoxylin and mounted.

Immunohistochemical staining analysis

The LI of Ki-67 was calculated as percentage of stained nuclei per 5000 epithelial nuclei under × 20 magnifications. The expression of p16 was defined by nuclear and cytoplasmic staining in the epithelial cells.[5] Positive cases were defined as having at least 10% stained cells when compared with the positive control tissue of cervical cancer. The immunohistochemical expression of Bcl-2 was defined by cytoplasmic granular staining in the epithelial cells less than × 20 magnification. It was then graded based on staining intensity and staining topography as none (0), weak (1+), moderate (2+), or strong (3+).[6] Lymphocytes were used as the internal positive controls. For COX-2, the presence of cytoplasmic staining was considered positive and the immunopositivity was graded as none (0), weak (1+), moderate (2+), or strong (3+) according to the staining intensity.[7] Microvessels were highlighted by staining endothelial cells for CD34.[9] The sections were then scanned at low magnification (×4) to locate areas of highest density of highlighted microvessels (hot-spots). Minimum of three such hotspots were captured at × 20 and counted. The MVD was expressed as the number of vessels/3 high power fields (HPF).

Ki-67 LI and MVD was assessed using an image analysis software Fiji, an open source image processing package based on ImageJ while p16, Bcl-2 and COX-2 were evaluated by semi-quantitative analysis. Frequency and distribution of the clinical and immunohistochemical observation was assessed using Statistical Package for the Social Sciences (SPSS) version 15.

 » Results Top

In the present case series, 6 out of 7 were women (85.7%) with an average age of presentation being 63.7 years (54-76 years). 3/7 patients reported with a history of tobacco use (42.8%) in the form of betel quid or beedi smoking. Clinically, patients presented irregularly whitish, warty proliferations, which were multifocal in all cases. Buccal mucosa and gingiva were the most frequently involved areas. Histologically, these lesions were signed out as either hyperplasia, VH and VH with D. All the cases in the series had a minimum of two recurrences over a period of 2 years follow-up. During the follow-up period one case progressed to SCC in about 6 months [Table 1].
Table 1: The clinicopathological profile of patients in the case series

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The Ki-67 LI in PVL patients was in a range of 8.18-12.6% with an average of 10.74% [Figure 1]a. The Ki-67 LI of the PVL sample that transformed into SCC was 12.6% [Figure 1]b and the primary SCC sample used as a positive control was 14.02% [Figure 1]c. Immunopositivity to p16 was observed in four cases (57.14%) of PVL [Figure 2]a, whereas the case of PVL that transformed into SCC showed negative p16 expression [Figure 2]b. However, p16 was positive as well in the primary SCC [Figure 2]c. Bcl-2 expression exhibited moderate staining intensity (2+) in two cases of PVL extending up to the upper spinous layer of epithelium [Figure 3]a including the PVL that transformed to SCC [Figure 3]b. However, the Bcl-2 expression in SCC was noted in the peripheral cells of the tumor islands with weak staining intensity (1+) [Figure 3]c. MVD was consistently high (>10/3 HPF) in all PVL samples [Figure 4]a as well as the one that progressed to SCC [Figure 4]b. MVD was however lower in primary SCC [Figure 4]c. COX-2 expression showed intense diffuse positivity (3+) [Figure 5]a in all but one (2+) case throughout the entire thickness of epithelium in all PVL cases and the one that progressed to SCC [Figure 5]b and also in primary SCC [Figure 5]c. The immunohistochemical profiling of all cases in our series is summarized in [Table 2].
Figure 1: (a) Representative image of proliferative verrucous leukoplakia stained with Ki-67 (×20) (b) Proliferative verrucous leukoplakia progressed to squamous cell carcinoma stained with Ki-67 (×20)

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Figure 2: (a) Representative image of proliferative verrucous leukoplakia stained with p16 (×20) (b) Proliferative verrucous leukoplakia having progressed to squamous cell carcinoma stained with p16 (×20) (c) A primary squamous cell carcinoma stained with p16 (×20)

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Figure 3: (a) Representative image of proliferative verrucous leukoplakia stained with Bcl-2 (×20) (b) Proliferative verrucous leukoplakia having progressed to squamous cell carcinoma stained with Bcl-2 (×20) (c) a primary squamous cell carcinoma stained with Bcl-2 (×20)

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Figure 4: (a) Representative image of proliferative verrucous leukoplakia stained with CD34 (×20) (b) Proliferative verrucous leukoplakia having progressed to squamous cell carcinoma stained with CD34 (×20) (c) A primary squamous cell carcinoma stained with CD34 (×20)

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Figure 5: (a) Representative image of proliferative verrucous leukoplakia stained with cyclooxygenase-2 (×20) (b) Proliferative verrucous leukoplakia having progressed to squamous cell carcinoma stained with cyclooxygenase-2 (×20)

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Table 2: The immunohistochemical profile of the PVL in our case series

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 » Discussion Top

The diagnosis of PVL is primarily retrospective based on the clinicopathological features and the history of recurrence. Any leukoplakic lesion that becomes warty or exophytic over a period of time, exhibiting hyperkeratosis, acanthosis, and basilar hyperplasia with or without dysplasia microscopically and that has recurred following treatment usually involving more than one site should arouse a clinical suspicion of PVL. Buccal mucosa, gingiva, alveolar mucosa and tongue being the most preferred sites in the oral cavity [1],[2] were observed in our cases too.

Ki-67 antigen, a non-histone protein, expressed during the cell cycle phases G1, S, G2/M is considered as a specific marker for cell proliferation.[4] Fettig et al.[10] in their series of 10 cases of PVL reported a higher LI compared with normal mucosa. Similarly, Gouvea et al.[11] correlated higher Ki-67 LI to epithelial alterations and reported that Ki-67 LI increased with an increase in the grade of epithelial dysplasia in PVL. In the present study, we found an average Ki-67 LI of 10.74% in the PVL cases. Significantly, Ki-67 LI in some cases of PVL (case 2, 3, 4, and 7) were nearly as high as those observed in SCC (14.02%), which is similar to the findings of Gouvea et al.[11]

P16, a CDK-INK4A, located on chromosome 9p21 controls the cell cycle during the G1 phase.[5] Both, over expression [12] and decreased expression [13] have been reported. Overexpression of p16 in premalignant and malignant oral lesions has been found to be associated with high-risk of HPV infection and seemingly better prognosis.[12] In the present study, 4/7 cases of PVL showing p16 immunopositivity suggested that p16 protein accumulation may be an early indicator of malignant transformation. However, loss of p16 expression, which was observed in a case of PVL that progressed to SCC, may foretell poor prognosis and rapid malignant transformation. In our observation, it was noted that cases with higher Ki-67 LI had stronger p16 expression. Although Ki-67 is used as a marker of cell proliferation, simultaneous increase in the expression of p16 as was noted some of our cases suggest a subset of PVL with better prognosis. The unusually high proliferation rate of PVL with the absence of cytoplasmic p16 expression in one of our cases which progressed to malignancy seems to indicate an underlying defect of the p16 gene.[14]

In the present case series, only 2/7 cases of PVL showed Bcl-2 positivity. The non-expression of Bcl-2 in the remaining cases could be explained on the basis of a high Ki-67 LI. An increase in anti-apoptotic marker, Bcl-2 resulting in the down regulation of proliferation marker, Ki-67 has been reported by Piattelli et al.[15] They reported a higher Ki-67 LI in cases of severe dysplasia and carcinoma in situ, where the Bcl-2 protein expression was lower or even absent. Increased expression of Bcl-2 with increase in grades of dysplasia and a decrease in its expression in SCC reported by Singh et al.[6] is consistent with our finding as well. The expression of Bcl-2 restricted to peripheral cells of the tumor islands noted in the primary SCC was also noted in the case of PVL that progressed to SCC suggesting that Bcl-2 alterations may play a role in tumorigenesis.[6]

Chronic inflammation is a well-established risk factor for several types of cancer, particularly the gastrointestinal tract. The most extensively investigated bridge molecule between chronic inflammation and carcinogenesis is COX-2.[7] COX-2 overexpression has been detected in inflamed, non-dysplastic mucosa in inflammatory bowel disease and Barrett esophagus, and this change reflected with its carcinomatous progression.[16] In the present case series, all cases of PVL showed strong diffuse COX-2 immunopositivity similar to primary SCC indicating a possible role of inflammation in the malignant transformation of PVL.

All the cases of PVL showed a consistently increased MVD than normal mucosa. The individual samples exhibited substantial heterogeneity in vascularity. The MVD of PVL was more when compared with the sample of primary SCC. This could be explained by the model of angiogenesis put forth by Shieh et al.[9] Accordingly, as epithelial cells transition from normal to dysplasia, angiogenesis increases in the peripheral stromal areas whereas when early localized tumor transform into advanced tumor, intra-tumoral angiogenesis increases. As the present study dealt with studying the MVD in the stroma, there was an increased MVD by default in the PVL samples than the primary SCC, which showed more intra-tumoral than peritumoral angiogenesis. Thus MVD as an independent finding was constantly increased in PVL. However, the exact role that angiogenesis may play in its malignant transformation needs to be further validated.

In lieu of the findings of the present study, we propose a model for the malignant transformation of PVL [Figure 6]. PVL consists of cells exhibiting a high proliferative ability. Certain cell populations in PVL exhibit the anti-apoptotic ability along with a high proliferative capacity. Subsequently, a second mutation causes the loss of p16 tumor suppressor protein allowing these cell populations to pass through the cell cycle unchecked may ultimately lead to cancer formation. This pathway of proliferative-apoptotic imbalance may be further augmented by inflammation and angiogenesis at the microenvironment level, increasing the risk of malignant transformation.
Figure 6: A progression model of malignant transformation of proliferative verrucous leukoplakia

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Current treatment of PVL is surgical excision through scalpel or laser with an extensive follow-up schedule of an average of 6.7 years.[2] Non-surgical therapeutic approaches for PVL have been considered, but none has proven to be beneficial. In recent times, selective inhibitors of COX-2 have been developed, which possess anticancer properties with low side effects. Based on the present findings, the use of selective inhibitors of COX-2 may prove to be useful in treating PVL, for which further studies assessing its efficacy is warranted.

 » Acknowledgment Top

Authors recognize the efforts of Dr. Rekha V Kumar, Professor, Department of Surgical Pathology, Kidwai Memorial Institute of Oncology, Bangalore for timely help and guidance in carrying out this study.

 » References Top

Hansen LS, Olson JA, Silverman S Jr. Proliferative verrucous leukoplakia. A long-term study of thirty patients. Oral Surg Oral Med Oral Pathol 1985;60:285-98.  Back to cited text no. 1
Cabay RJ, Morton TH Jr, Epstein JB. Proliferative verrucous leukoplakia and its progression to oral carcinoma: A review of the literature. J Oral Pathol Med 2007;36:255-61.  Back to cited text no. 2
Batsakis JG, Suarez P, el-Naggar AK. Proliferative verrucous leukoplakia and its related lesions. Oral Oncol 1999;35:354-9.  Back to cited text no. 3
Girod SC, Pfeiffer P, Ries J, Pape HD. Proliferative activity and loss of function of tumour suppressor genes as 'biomarkers' in diagnosis and prognosis of benign and preneoplastic oral lesions and oral squamous cell carcinoma. Br J Oral Maxillofac Surg 1998;36:252-60.  Back to cited text no. 4
Dong Y, Wang J, Dong F, Wang X, Zhang Y. The correlations between alteration of p16 gene and clinicopathological factors and prognosis in squamous cell carcinomas of the buccal mucosa. J Oral Pathol Med 2012;41:463-9.  Back to cited text no. 5
Singh BB, Chandler FW Jr, Whitaker SB, Forbes-Nelson AE. Immunohistochemical evaluation of bcl-2 oncoprotein in oral dysplasia and carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:692-8.  Back to cited text no. 6
Renkonen J, Wolff H, Paavonen T. Expression of cyclo-oxygenase-2 in human tongue carcinoma and its precursor lesions. Virchows Arch 2002;440:594-7.  Back to cited text no. 7
Zha S, Yegnasubramanian V, Nelson WG, Isaacs WB, De Marzo AM. Cyclooxygenases in cancer: Progress and perspective. Cancer Lett 2004;215:1-20.  Back to cited text no. 8
Shieh YS, Lee HS, Shiah SG, Chu YW, Wu CW, Chang LC. Role of angiogenic and non-angiogenic mechanisms in oral squamous cell carcinoma: Correlation with histologic differentiation and tumor progression. J Oral Pathol Med 2004;33:601-6.  Back to cited text no. 9
Fettig A, Pogrel MA, Silverman S Jr, Bramanti TE, Da Costa M, Regezi JA. Proliferative verrucous leukoplakia of the gingiva. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;90:723-30.  Back to cited text no. 10
Gouvêa AF, Vargas PA, Coletta RD, Jorge J, Lopes MA. Clinicopathological features and immunohistochemical expression of p53, Ki-67, Mcm-2 and Mcm-5 in proliferative verrucous leukoplakia. J Oral Pathol Med 2010;39:447-52.  Back to cited text no. 11
Buajeeb W, Poomsawat S, Punyasingh J, Sanguansin S. Expression of p16 in oral cancer and premalignant lesions. J Oral Pathol Med 2009;38:104-8.  Back to cited text no. 12
Greer RO Jr, Meyers A, Said SM, Shroyer KR. Is p16 (INK4a) protein expression in oral ST lesions a reliable precancerous marker? Int J Oral Maxillofac Surg 2008;37:840-6.  Back to cited text no. 13
Kresty LA, Mallery SR, Knobloch TJ, Li J, Lloyd M, Casto BC, et al. Frequent alterations of p16INK4a and p14ARF in oral proliferative verrucous leukoplakia. Cancer Epidemiol Biomarkers Prev 2008;17:3179-87.  Back to cited text no. 14
Piattelli A, Rubini C, Fioroni M, Iezzi G, Santinelli A. Prevalence of p53, bcl-2, and Ki-67 immunoreactivity and of apoptosis in normal oral epithelium and in premalignant and malignant lesions of the oral cavity. J Oral Maxillofac Surg 2002;60:532-40.  Back to cited text no. 15
Lagorce C, Paraf F, Vidaud D, Couvelard A, Wendum D, Martin A, et al. Cyclooxygenase-2 is expressed frequently and early in Barrett's oesophagus and associated adenocarcinoma. Histopathology 2003;42:457-65.  Back to cited text no. 16


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2]

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