|
 
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| ORIGINAL ARTICLE |
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| Year : 2018 | Volume
: 7
| Issue : 2 | Page : 120-128 |
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A Semi-quantitative analysis of immunohistochemical expression of p63, Ki-67, Cyclin-D1, and p16 in common oral potentially malignant disorders and oral squamous cell carcinoma
P Sharada, Uma Swaminathan, BR Nagamalini, K Vinodkumar, BK Ashwini, V Lavanya
Department of Oral and Maxillofacial Pathology, AECS Maaruti College of Dental Sciences and Research Centre, Bengaluru, Karnataka, India
| Date of Web Publication | 6-Jun-2018 |
Correspondence Address:
Dr. P Sharada
Department of Oral and Maxillofacial Pathology, AECS Maaruti College of Dental Sciences and Research Centre, Bengaluru - 560 076, Karnataka
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_13_18
Background: Immunohistochemical (IHC) expressions of p63, Ki-67, cyclin-D1, and p16 have been evaluated to assess the malignant potential of oral potentially malignant disorders (OPMDs); most of these studies are subjective and lack a definite quantitative approach. Further, H score of IHC expression has produced promising results and this has not been explored in any marker to predict malignant transformation in OPMDs.
Aim: To evaluate the IHC expressions of p63, Ki-67, cyclin-D1, and p16INK4a in predicting the malignant transformation of the most common OPMDs.
Materials and Methods: The IHC expressions of p63, Ki-67, cyclin-D1, and p16INK4a on paraffin-embedded sections of 10 cases each in normal oral mucosa, mild, moderate, and severe oral epithelial dysplasia, oral submucous fibrosis (OSMF), and oral squamous cell carcinoma (OSSC) were evaluated using mean H score for every group. Mean H score was compared within and between the groups using analysis of variance. Pairwise comparison was made using Tukey's multiple post hoc procedure and strength of association was assessed using Karl's Pearson's correlation.
Results: Mean H scores of p63, Ki-67, and cyclin-D1 increased significantly (P = 0.0001) with increasing grades of dysplasia to OSCC, whereas mean H score of p16 significantly (0.041) decreased with increasing grades of dysplasia to malignancy. OSMF indicated a variation in its expression pattern. Only mean H scores of Ki-67 (0.0239) and cyclin-D1 (0.0044) significantly differentiated severe dysplasia from OSCC.
Conclusion: Combination of mean H score of Ki-67 and mean H score of cyclin-D1 could be effectively used to identify transformation potential of OPMDs.
Keywords: Cyclin-D1, H score, Ki-67, p16INK4a, p63
How to cite this article:
Sharada P, Swaminathan U, Nagamalini B R, Vinodkumar K, Ashwini B K, Lavanya V. A Semi-quantitative analysis of immunohistochemical expression of p63, Ki-67, Cyclin-D1, and p16 in common oral potentially malignant disorders and oral squamous cell carcinoma. J NTR Univ Health Sci 2018;7:120-8 |
How to cite this URL:
Sharada P, Swaminathan U, Nagamalini B R, Vinodkumar K, Ashwini B K, Lavanya V. A Semi-quantitative analysis of immunohistochemical expression of p63, Ki-67, Cyclin-D1, and p16 in common oral potentially malignant disorders and oral squamous cell carcinoma. J NTR Univ Health Sci [serial online] 2018 [cited 2023 Mar 27];7:120-8. Available from: https://www.jdrntruhs.org/text.asp?2018/7/2/120/233841 |
| Introduction | |  |
Oral cancer (OC) is the 11th most common cancer in the world [1] and 12th most common cancer in women, and 6th in men,[2] with a very significant incidence in developing countries.1 Recently, it has come to light that the incidence of OC is increasing in several parts of the world, particularly in Australia, Japan, and parts of Europe. In India, OC is the third most common cancer among women.[1] Thus, OC projects as a significant global burden. The age standardized incidence rate of OC in India is 12.6 per 100,000 people.[1] International Agency for Research on Cancer has predicted that India's incidence of cancer will be approximately 1.7 million in 2035.[3] This high incidence of OC in India has been attributed to smoking and other smokeless tobacco habits, alcohol, spicy food, and neglected overall oral health. However, tobacco and alcohol are regarded as the major causes for OC exerting a strong synergistic effect. Further, tobacco-related cancers are seen only in a small fraction of the individuals exposed to tobacco smoke; it is therefore rational to hypothesize that genetic factors may render some smokers more susceptible to cancer. If oral squamous cell carcinoma (OSCC) is diagnosed in early stages, survival rate is 80% but falls to 30–50% in the later stages.[4]
Many OSCCs develop from oral potentially malignant disorders (OPMDs). Sir James Paget was the first to describe the malignant transformation of an oral lesion in 1870.[2] Early diagnosis and treatment of OPMDs helps prevent malignant transformation.[2] Incidence of OPMDs in the Indian scenario ranges between 6/1000 and 30.2/1000.[5] Among OPMDs, oral leukoplakia appears as the most common OPMD in India followed by oral submucous fibrosis (OSMF) and oral lichen planus.[1] The malignant transformation rate for oral leukoplakia ranges from 15 to 20% and OSMF approximately 7.6% over a 10-year period incidence in India.[6]
Biopsy is regarded as the standard operating procedure to assess risk of malignant transformation.[5] The histological criteria to assess cellular and tissue changes have been put forward by World Health Organization (WHO, 2005),[7] but the histopathological diagnosis is subjective and lacks sensitivity.[8] Further, histological changes that characterize epithelial dysplasia appear as latter events to molecular alterations. Therefore, early identification of malignant transformation in OED is based on early molecular events rather than histological changes.
A number of molecular markers have been identified over the years to detect the transition from normal epithelium to premalignancy to OSCC, which is a resultant of genetic and epigenetic alterations in a multistep process.[9] Various proliferative markers have enabled the detection of the hyperactive state of the epithelium and have been suggested to be of prognostic significance.[9] One such proliferative marker is p63. Human p63 gene is located on chromosome 3q28.[10] p63 gene encodes N-terminal Transactivation Domain (TAP63 with amino acids 1-59), a core DNA binding domain (amino acids 142-321), and a carboxy-oligomerization domain (amino acids 353-397). p63 has two transcriptional sites that generate transcripts encoding proteins with or without an N-terminal TA. Proteins with TA are termed TAP63 and proteins without it are termed ΔNP63.[11] TAP63 isoforms transactivate P53 downstream targets, induce apoptosis, and mediate cell cycle control. But ΔNP63 isoforms have functions opposite to TAP63 by acting as oncoproteins.[12] In normal oral mucosa (NOM) and reactive epithelial hyperplasia, ΔNP63 expression has been reported in the basal layer, decreasing towards the middle-third of the epithelium. But, in the dysplastic lesions, expression has been observed up to spinous layer or sometimes the entire thickness of the epithelium.[13]
Ki-67, also a proliferative marker, was identified at the University of Kiel, Germany. Ki-67 consists of a nuclear nonhistone protein, with a molecular weight between 345 to 395 kDa. It is encoded by a gene on chromosome 10q25. The expression of Ki-67 occurs in all phases of the cell cycle except G0 phase and early G1 phase. It increases in S phase reaching a peak in G2 and M phase, and is degraded rapidly after mitosis.[14] It has been reported that Ki-67 is overexpressed in the suprabasal epithelium, increasing with the severity of dysplasia.[9] In OSCC, the increase in Ki-67 expression has been correlated with poor survival.[14]
Cyclin-D1 is the first cyclin to increase in the cell cycle, it is increased in the G1 phase but not seen in the S phase. In G1 phase, it activates CDK4 and cyclin-D–CDK4 complex, phosphorylates the Rb protein promoting cell replication after the release of E2F. Overexpression of cyclin-D1 is correlated with a shorter G phase, and abnormal cell proliferation. A sequential increase in cyclin-D1 expression has been observed from normal oral tissues to dysplastic lesions and OSCCs, by Soni et al. and Izzo et al.[9]
The tumor suppressor gene p16INK4a is present on chromosome 9P21 and inactivation of p16 gene causes altered expression of p161NK4a (p16) and is considered to be a significant event in the development of many tumors including OC.[15] p16 binds to CDK4 and CDK6 and this inhibits the catalytic activity of CDK4/6–cyclin-D complex and also blocks Rb gene phosphorylation, preventing cell cycle progression through G1 to S phase.[9],[15] A significant correlation has been reported by Gologen et al. between the degree of dysplasia and expression of p16 in dysplastic epithelium, but Bradley et al. reported that p16 is not a useful marker for differentiating between dysplastic and nondysplastic lesions.[9]
Although the IHC expressions of p63, Ki-67, cyclin-D1, and p16 have been studied in OPMDs, most of these studies appear to be subjective, lacking systematic and quantitative approach. Hence, the present study evaluated H score, a semi-quantitative analysis to assess immunoreactivity of p63, Ki-67, cyclin-D1, and p16 in OPMDs and OSMF to predict malignant changes.
| Materials and Methods | | |
An in vitro case–control study was performed on 60 specimens obtained from the archives of the Department of Oral and Maxillofacial Pathology. Ethical clearance has been obtained by Ethical Committee of the institution headed by Principal, AECS Maaruti College of Dental Sciences and Research Centre.
The study group was divided into six groups with 10 specimens in each group on the basis of clinicopathology as Group I (n = 10) – mild epithelial dysplasia, Group II (n = 10) – moderate epithelial dysplasia, Group III (n = 10) – severe epithelial dysplasia, Group IV (n = 10) – OSMF, Group V (n = 10) – normal oral mucosa, Group VI (n = 10) – OSCC. All the archival specimens were sectioned and stained with hematoxylin and eosin stain to re-confirm the diagnosis. All the slides in Groups I, II, and III were graded using WHO criteria (2005) to reconfirm the grade of dysplasia. Ethical clearance was taken from the Ethical Committee of the institution headed by Principal, AECS Maaruti College of Dental Sciences and Research Centre.
Immunohistochemical technique
3 μm thick sections from tissue blocks of samples and control (lymph node – Ki-67, Skin – p63, mantle cell lymphoma – cyclin-D1, and squamous cell carcinoma – p16) were obtained and mounted on Amino Propyl Triethoxy silane-coated slides. These slides were incubated at 33°C overnight on the previous day of staining, and at 60°C for 1 hour on the day of staining. The slides were deparaffinized using three changes of xylene, each of 5 min duration and were then hydrated through decreasing grades of isopropyl alcohol (100%, 90%, 70%) and sections were brought to water. The tissues were then incubated with peroxide block for 20 min at room temperature to block endogenous peroxide activity and washed in distilled water and Tris buffer for 5 min. The slides were then subjected to antigen retrieval using Tris ethylenediaminetetraacetic acid buffer. The antigen retrieval was carried out in a pressure cooker at 150°C for 55 min for Ki-67 and p63, whereas the antigen retrieval time for p16INK4a and cyclin-D1 was 65 and 75 min, respectively. After the retrieval, slides were allowed to cool down to the room temperature. The sections were subjected to two washes of Tris buffer for 10 min each and were subsequently incubated for 15 min with protein block to eliminate background staining. The sections required for Ki-67 immunostain were then incubated with Ki-67 primary mouse monoclonal antibody (Pathnsitu Lot. no. R02096UA) and sections required for p63 were incubated with p63 primary mouse monoclonal antibody (Pathnsitu Lot. no. R02105UA) for 30 min. Sections that required immunostaining for cyclin-D1, p16INK4a were incubated with cyclin-D1 primary rabbit monoclonal antibody (Pathnsitu Lot. no. PRO36AP14AX) and p16INK4a primary mouse monoclonal antibody (Biogenex. Lot. no. AM5400315), respectively. The primary antibody incubation period for cyclin-D1 and p16INK4a was 45 and 50 min, respectively. The sections were washed with Tris buffer twice for 5 min each. Subsequently, the slides were incubated with poly horseradish peroxidase for 30 min. The slides were then washed as before and incubated with fresh diaminobenzidine (DAB) chromogen for 2 min. The DAB chromogen was prepared by adding DAB to the buffer at the ratio of 1:20. The slides were then washed in water to stop the chromogen reaction and excess DAB and counterstained with Mayer's hematoxylin for 6 min. The slides were then dehydrated through graded isopropyl alcohol (70%, 90%, 100%) cleared using xylene and mounted with Dibutyl pthalate xylene. The stained sections were viewed under binocular Olympus research microscope (BX41).
Defining positivity of immunohistochemical stain
Presence of brown-colored end product (DAB positivity) was indicative of positive immunoreactivity. The intensity of IHC expression of positive Controls [Figure 1] was utilized as a reference to grade the intensity of IHC expression. | Figure 1: Controls for IHC; a: Skin- P63, b: Lymph node - Ki-67, c: Mantle cell Lymphoma – Cyclin D1, d: Squamous Cell Carcinoma – p16
Click here to view |
Interpretation of immunohistochemistry expression
The intensity of IHC expression was determined using the intensity score criteria as described by Allred et al. (1993)[16] and the scores were interpreted as: 0 – No positive cells, 1+ – Mild intensity, 2+ – Moderate intensity, and 3+ – Strong Intensity. For semi-quantitative analysis of immunohistochemistry, H score as described by Hinsch et al.[17] was used. The H score was calculated for every sample and tabulated using the formula as shown below:
1× (% cells 1+) +2 × (% of cells 2+) +3× (% of cells 3+)
Statistical analysis
Mean H score for each group was calculated and was used to compare and correlate within and between the groups. Kolmogorov–Smirnov (KS) test performed to assess the distribution of samples within the six groups for all the four parameters (p63 H score, Ki-67 H score, cyclin-D1 H score, and p16 H score) showed a normal distribution. Therefore, parametric test was applied to compare and correlate the findings within and between the groups. The parametric test considered was one-way analysis of variance (ANOVA) to compare mean H score of each parameter between the six groups and within the groups and pairwise comparison with Tukey's multiple post hoc was used to identify any statistically significant difference between them. Strength of association of all the parameters in every group was analyzed using Karl's Pearson's co-relation.
| Results | | |
The mean H score of p63 expression in groups – NOM, mild epithelial dysplasia, moderate epithelial dysplasia, severe epithelial dysplasia, OSMF, and OSCC was 130 ± 15.09 (Mean ± SD), 172 ± 15.49 (Mean ± SD), 200.5 ± 21.79 (Mean ± SD), 227.5 ± 9.5 (Mean ± SD), 168.5 ± 14.5 (Mean ± SD), and 247 ± 9.19 (Mean ± SD), respectively. ANOVA was applied to compare these findings and a statistically significant difference was observed between and within the groups with a P value of 0.00001, as indicated in [Table 1] (Photomicrograph 2). The Tukey's multiple post hoc procedure indicated a statistical significant difference between mild and moderate dysplasia (P = 0.0011), mild and severe dysplasia (P = 0.0001), mild dysplasia, and NOM (P = 0.0001), mild dysplasia, and OSCC (P = 0.0001), moderate and severe dysplasia (0.0022), moderate dysplasia, and OSMF (P = 0.0003), moderate and NOM (P = 0.0001), moderate dysplasia and OSCC (P = 0.0001), severe dysplasia and OSMF (P = 0.0001), severe dysplasia and NOM (P = 0.0001), severe dysplasia and OSCC (0.0532), OSMF and NOM (P = 0.0001), OSMF and OSCC (P = 0.0001), and OSCC and NOM (P = 0.0001) ([Table 1], [Graph 1] & [Figure 2]). | Table 1: Comparison of p63, Ki67, cyclin D1 and p16 INK4a IHC expression within and between the six groups
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 | Figure 2: IHC expression of P63 in all the six groups (20x); OSMF: Oral Submucous Fibrosis; OSCC: Oral Squamous Cell Carcinoma
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The mean score of Ki-67 expression in NOM, mild epithelial dysplasia, moderate epithelial dysplasia, severe epithelial dysplasia, OSMF, OSCC groups was 102 ± 20.98 (mean ± SD), 115.5 ± 17.39 (mean ± SD), 143 ± 19.16 (mean ± SD), 151.5 ± 21.35 (mean ± SD) 108 ± 23.38 (mean ± SD) and 181 ± 18.8 (mean ± SD), respectively. ANOVA when applied to compare these findings showed a statistically significant difference between and within the groups with a P value of 0.00001, as indicated in [Table 1] (Photomicrograph 3). Tukey's multiple post hoc procedure showed a statistical significant difference between mild and moderate dysplasia (P = 0.0369), mild and severe dysplasia (P = 0.0031), mild dysplasia and OSCC (P = 0.0001), moderate dysplasia and OSMF (P = 0.0036), moderate dysplasia and NOM (P = 0.0005), moderate dysplasia and OSCC (P = 0.0019), severe dysplasia and OSMF (P = 0.0003), severe dysplasia and NOM (P = 0.0002), severe dysplasia and OSCC (P = 0.0239), OSMF and OSCC (P = 0.0001), OSCC and NOM (P = 0.0001) as indicated in [Table 1], [Graph 2], [Figure 3].
 | Figure 3: IHC expression of Ki 67 in all the six groups (20x); OSMF: Oral Submucous Fibrosis; OSCC: Oral Squamous Cell Carcinoma
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The mean H score of cyclin-D1 expression in NOM, mild epithelial dysplasia, moderate epithelial dysplasia, severe epithelial dysplasia, OSMF and OSCC groups was 13.5 ± 4.12 (Mean ± SD), 16 ± 8.76 (Mean ± SD), 29.5 ± 10.1(Mean ± SD), 46.5 ± 14.15 (Mean ± SD), 20.5 ± 7.62 (Mean ± SD), and 63 ± 10.06 respectively. The findings of ANOVA indicated a statistically significant difference between and within the groups with a P value of 0.0001, as indicated in [Table 1] (Photomicrograph 4). The Tukey's multiple post hoc procedure indicated a statistical significant difference between mild and moderate dysplasia (P = 0.0314), mild and severe dysplasia (P = 0.0001), mild dysplasia and OSCC (P = 0.0001), moderate dysplasia and severe dysplasia (P = 0.0031), moderate dysplasia and NOM (P = 0.0062), moderate dysplasia and OSCC (P = 0.0001), severe dysplasia and OSMF (P = 0.0001), severe dysplasia and NOM (P = 0.0001), severe dysplasia and OSCC (P = 0.0044), OSCC and OSMF (P = 0.0001) and OSCC and NOM (P = 0.0001) as indicated in [Table 1], [Graph 3], [Figure 4].
 | Figure 4: IHC expression of Cyclin D1 in all the six groups (20x); OSMF: Oral Submucous Fibrosis; OSCC: Oral Squamous Cell Carcinoma
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The mean H score of p16INK4a expression in NOM, mild epithelial dysplasia, moderate epithelial dysplasia, severe epithelial dysplasia, OSMF, and OSCC groups was 39 ± 13.5 (mean ± SD), 26 ± 10.49 (mean ± SD), 24 ± 11.74 (mean ± SD), 22 ± 19.32 (mean ± SD), 30 ± 12.25 (mean ± SD), and 20.50 ± 12.35, respectively. ANOVA was used to compare the findings and a statistically significant difference was observed between and within the groups with a P value of 0.0001, as indicated in [Table 1] (Photomicrograph 5). The Tukey's multiple post hoc procedure indicated a statistical significant difference between NOM and OSCC group (P = 0.0396) as indicated in [Table 1], [Graph 4], [Figure 5].
 | Figure 5: IHC expression of p16 in all the six groups (20x); OSMF: Oral Submucous Fibrosis; OSCC: Oral Squamous Cell Carcinoma
Click here to view |
Further, Karl's Pearson's co-relation indicated a statistically significant and very strong positive correlation between p63 and Ki-67 ((r = 0.7852) (P = 0.0001)), p63, and cyclin-D1 ((r = 0.7786) (P = 0.0001)) and strong positive correlation between cyclin-D1 and Ki-67 ((r = 0.6959) (P = 0.0001)). A statistically significant weak negative correlation was seen between p16INK4a and p63 ((r = −0.3702) (P = 0.0041) and statistically significant weak negative correlation between p16INK4a and Ki-67 (r = −0.293), (P = 0.0231)).
The observations in our study indicate a gradual increase in IHC expression of p63, Ki-67, and cyclin-D1 as the disease progresses from NOM to OED to OSCC. Reverse pattern of staining in all the groups was seen with respect to p161NK4a. A proportionate increase in the expression of Ki-67 and cyclin-D1 was noted with increase in p63, whereas the expression of p16INK4a was inversely proportional to expression of p63 and Ki-67.
| Discussion | | |
p63 is known to be secreted in the embryonic ectoderm from 7th to 11th day.[18] Primary function of p63 is to regulate epithelial stratification [19] by maintaining the proliferative undifferentiated state of basal keratinocytes,[19] thereby establishing the basal status of epithelium.[19] Therefore, p63 is postulated as the first gene product that differentiates between stem cell and transient amplifying cells in stratified squamous epithelium.[18] The present study showed intense p63 staining in the nuclei of basal cells of NOM, which was in accordance with the studies by Varun et al.,[6] Sinha et al.,[18] and Ramasubramanium et al.[19] The expression of p63 in the present study increased significantly with the increasing grades of dysplasia. This increased expression of p63 was observed predominantly in the suprabasal cells of mild, moderate, and severe OED groups, which was in par with the findings of Varun et al.,[6] Chen et al.,[20] Sinha et al.,[18] Ramasubramanium et al.,[19] Shetty et al.,[12] Haniffa et al.[21] and Bortoluzzi et al.,[22] In the present study, the expression of p63 in OSMF group was almost similar to that seen in groups of NOM and mild epithelial dysplasia which is in accordance with the studies by Varun et al.[6] and Anju Sinha et al.[18] The unique finding in our study was that the p63 H score significantly differentiated (P = 0.001) grades of OED from NOM, OSMF, and OSCC groups. Well-differentiated SCCs showed p63 positive cells at the periphery of the tumor islands. Minimal expression was observed in the center of the tumor islands, suggesting that the cells at the periphery are less differentiated than those at the center. This may be resultant of disrupted normal p53 function leading to compensatory upregulation of p63, such that excessive production of mutant p53 or reduction in p21 may trigger the overexpression of p63.[20] This overexpression of p63 may in reflux upregulate HSP 70 known to be associated with metastasis.[18]
Ki-67 is another proliferative marker very much involved in nuclear disassembly and reassembly at either side of mitosis and localization of nucleolar granular components to mitotic chromosomes and heterochromatin organization. Hence, it plays a vital role in nuclear segregation between daughter cells.[23] The present study showed basal and parabasal staining pattern of Ki-67 in NOM, in the suprabasal layers of the epithelium in dysplasia, and this expression of Ki-67 increased with increase in the severity of dysplasia, which was consistent with the studies conducted by Bortoluzzi et al.,[22] Birajdar et al.,[24] and Torres et al.[4] Furthermore, Birajdar et al.[24] observed basal, parabasal, and lower spinous layer expression in low-risk groups, whereas the expression extended to superficial layer in high-risk groups. Reddy et al. have reported Ki67 expression throughout the epithelial thickness in premalignant lesions. These findings may be attributed to asymmetric cell division in stem cells component of basal layer giving rise to transient amplifying cells in parabasal layer.[25],[26] In addition to this, in the present study, Ki-67 H score significantly differentiated (P = 0.001) grades of OED from NOM, OSMF, and OSCC groups. In OSMF, the H score was almost similar to that of mild epithelial dysplasia. In OSCC group, Ki-67 expression was seen at the periphery of cell nests, similar to the staining pattern of p63, suggestive of less differentiated cells at the periphery. Similar observations have been reported by Birajdar et al.[24] and Bhattacharya et al.,[27] which may be due to decrease in cells undergoing apoptosis with an increase in tumor growth at the periphery of the tumor.[27] The tumor cells may also bypass the tumor suppressive action of p53. p53 appears very early in carcinogenesis and cells show increased proliferation without involving immunohistochemically detectable alterations in p53.[28]
The other proliferative marker studied in the group was cyclin-D1 – an initiator involved in transition between G1 and S phases of the cell cycle. The marker is well known to be susceptible to the influence of various mitogenic stimuli and hence known to be increased in various neoplasm.[29] Studies of Wato et al.[30] and Ramakrishna et al.[31] have demonstrated a substantial increase of cyclin-D1 expression with increasing grades of dysplasia and malignancy. Its expression in OSMF has, however, been varied with limited studies.[32],[33] Similar results were observed in our study with significant increase in the expression from NOM to mild-to-moderate-to-severe dysplasia and OSCC. The cyclin-D1 expression significantly (P = 0.0001) differentiated mild dysplasia and moderate, severe dysplasia and OSCC (P = 0.0044). Compared to NOM, expression increased in all the groups significantly and perceptibly in mild dysplasia. Expression in OSMF was comparable to moderate dysplasia. Also expression increased from basal and parabasal level to higher level with increasing grade of dysplasia. The results indicate that the concentration and action of cyclin-D1 increased with increase in carcinogenic hits and cell abnormalities and also away from the germinative pool, leading to a mitotic shift. This may be more pronounced when the lesions are associated with patient adverse habits, some of which may have significant mitogenic potential.[29],[31]
In the present study, p16INK4a expression demonstrated increased H score in NOM group. However, the expression was restricted to basilar and parabasilar cells, whereas the expression reduced as the disease progressed from OED to OSCC. The expression pattern in OSMF was intermediate between NOM and mild epithelial dysplasia. The findings in our study were in accordance with the observations put forth by Bradley et al.,[15] demonstrating a decreased p16INK4a expression in OPMDs and malignancies and attributed the same to gene inactivating mechanisms. Muniswamy et al.[34] also showed that p16INK4a positivity decreased from NOM to OED, however, with a slight increase in OSCC. Yet another study by Cuevas et al.[35] showed reduction in p16INK4a expression from mild to moderate OED with increased expression in severe epithelial dysplasia. In addition to this, Karl's Pearson's correlation demonstrated strong positive association between p63, Ki-67, and cyclin-D1 and a significant inverse relation with respect to p16INK4a versus Ki-67 and p63 versus p16INK4a in all the groups considered. These findings of the present study are indicative of a proportionate increase of Ki-67 and cyclin-D1 expressions with respect to increase in p63 expression, and a decreased expression of p16INK4a with respect to p63 expression, which may be attributed to loss of function of tumor suppressor genes, a vital factor responsible for tumor progression.[15]
| Conclusion | | |
The findings of our study indicate an increased expression of p63, Ki-67, and cyclin-D1 and reduced expression of p16INK4a with increasing grades of OED to malignancy. However, only Ki-67 mean H score and cyclin-D1 mean H score could statistically significantly differentiate severe epithelial dysplasia from OSCC, whereas p63 and p16INK4a failed to differentiate the same. Due to lack of ideal markers that could predict malignant transformation in OED, we propose for application of combination of Ki-67 and cyclin-D1 to be used effectively to predict malignant transformation in OEDs. The limitation of the present study was that H score was not evaluated for different layers of epithelium for all the parameters considered in all the groups, as most of the proliferative markers considered increase in suprabasilar layers in cases of dysplasias, specifically p63. This opens up a new insight for consideration of coalition of suprabasilar H scores of p63, Ki-67, and cyclin-D1 expression to predict malignant potential of OEDs. However, these group of markers in future should be evaluated with larger sample size along with designs to overcome the limitation of the present study, and further confirmation with gene knock down of studies of these markers becomes necessary.
Acknowledgement
We are grateful to Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka for providing grant for this Original research work.
Financial support and sponsorship
This original research work has been funded by Rajiv Gandhi University of Health Sciences, Bangalore.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1]
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