|Year : 2020 | Volume
| Issue : 1 | Page : 52-60
Predicting the Malignant Transformation of Oral Submucous Fibrosis Using Quantitative Biomarkers p63 and CD31
Radhika Manoj Bavle1, Konda Paremala2, Makarala Sowmya Reader 1, Muniswamappa Sudhakara1, Venugopal Reshma1, Sreelatha Hosthor1
1 Department of Oral and Maxillofacial Pathology, Krishnadevaraya College of Dental Sciences & Hospital, Bangalore, Karnataka, India
2 Department of Oral Pathology, Govt Dental College and Hospital, Afzalgunj, Hyderabad, Telangana, India
|Date of Submission||09-Jan-2020|
|Date of Acceptance||10-Jan-2020|
|Date of Web Publication||12-Jun-2020|
Dr Makarala Sowmya
Department of Oral Pathology, Krishnadevaraya College of Dental Sciences, MVIT Campus, Via Yelahanka, Hunusamaranahalli, Bangalore-562157, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Oral submucous fibrosis (OSMF) is a pre-malignant condition highly prevalent in India with a malignant transformation rate of 2–8%. Its incidence amongst younger population has risen due to increased consumption of commercial preparations of areca. p63, a homolog of p53, has a role in epithelial proliferation and is frequently altered in dysplasia and associated with tumorigenesis. CD31 is a highly specific endothelial marker with varied expression in epithelial dysplasia and carcinoma. Epithelial proliferation and underlying angiogenic support are vital processes for malignant transformation. Therefore the present study aims to determine if p63 and CD31 expression is associated with increased malignant transformation of OSMF. Materials and Methods: A cross-sectional study wherein n = 36 OSMF samples were histologically graded into Group I (Early-Ea), II (Moderately advanced-MA) and III (Advanced-Ad) and analysed for anti-p63 and anti-CD31 antibodies. The expression was evaluated quantitatively and by pattern of distribution across the groups and statistically analysed with Chi-Square and Kruskal-Wallis tests (SPSS v2.0). Results: p63 expression of epithelium in Group I ∼100% (basal, suprabasal), II − 60.9% (basal, spinous), III − 62.5% (basal, spinous, granular) with a significant P-value < 0.001*. Predominant pattern of CD31 positive vessels in Group I ∼60% (constricted), II > 50% (normal diameter) and III ∼ 75% (dilated) with a significant P-value of 0.02*. Conclusion: A significant linear increase in nuclear staining of p63 and involvement of epithelial strata observed from Ea → MA → Ad grade of OSMF. CD31 expression exhibited more dilated vessels as OSMF grade increased from Ea → Ad. Therefore p63 and CD31 could be used as quantitative predictive biomarkers of malignant transformation of OSMF.
Keywords: CD31, oral submucous fibrosis, p63, quantitative biomarkers, malignant transformation
|How to cite this article:|
Bavle RM, Paremala K, Sowmya M, Sudhakara M, Reshma V, Hosthor S. Predicting the Malignant Transformation of Oral Submucous Fibrosis Using Quantitative Biomarkers p63 and CD31. J Orofac Sci 2020;12:52-60
|How to cite this URL:|
Bavle RM, Paremala K, Sowmya M, Sudhakara M, Reshma V, Hosthor S. Predicting the Malignant Transformation of Oral Submucous Fibrosis Using Quantitative Biomarkers p63 and CD31. J Orofac Sci [serial online] 2020 [cited 2020 Dec 1];12:52-60. Available from: https://www.jofs.in/text.asp?2020/12/1/52/286484
| Introduction|| |
Oral submucous fibrosis is a chronic progressive disorder of the connective tissue with a malignant transformation rate of 2–8%.  Recently, it has been suggested that oral cancers arising in OSMF comprise a clinico-pathologically discrete disease, due to various mechanisms in play with the association of arecoline as the major carcinogen. 
Though, OSMF is a connective tissue disorder, epithelial changes leading to dysplasia precede its malignant transformation. Due to excessive fibrosis, the existing vessels undergo constriction with restricted nutrient supply to the epithelium.  Moreover, the epithelium is exposed to constant insult from the various carcinogens. This results in compromised healing and/or repair of the epithelium, which in turn leads to accumulation of cellular and nuclear defects manifesting as epithelial changes in cell proliferation, maturation and metabolism., To compensate for the ischemia, secretion of angiogenic factors by the inflammatory component induces vascular dilatation in the underlying connective tissue.
p63 is a transcriptional regulator of the tumour suppressor p53 family with a major role in regulating keratinocyte cell proliferation, differentiation and maturation. It is found in chromosome 3q27-28, a region, frequently altered in epithelial dysplasia, amplified in squamous cell carcinomas, a target for overexpression in more than 75% of Head neck SCCs (HNSCC).
CD31 is one the most specific markers to detect endothelial cells lining vascular channels and a member of the immunoglobin super family with major roles in inflammation and angiogenesis. It is present in chromosome 17q23 and has varied expression across grades of dysplasia and in Oral squamous cell carcinoma (OSCC).
Examining epithelial alterations with underlying vascular support is an important step in determining conversion into OSCC. Therefore understanding the behaviour of the overlying epithelium using markers like p63, in conjunction with vascular markers like CD31 in different grades of OSMF will provide a clue in predicting their malignant transformation.
| Aims and objectives|| |
- To assess epithelial cell proliferation and differentiation in various grades of oral submucous fibrosis by immunohistochemical evaluation of p63.
- To examine endothelial expression of CD31 across different grades of oral submucous fibrosis.
- To determine if expression pattern of p63 and CD31 is associated with increased malignant transformation of oral submucous fibrosis.
| Materials and methods|| |
Ethical approval for this study (KCDS/984/2015-16) was provided by the Institutional Ethical Committee of Krishnadevaraya College of Dental Sciences and Hospital, Bangalore on 11th November, 2015.
- n = 36 archival cases of oral submucous fibrosis were categorized into early, moderate and advanced grade (Khanna et al., 1995) on histologic examination of Hematoxylin and eosin-stained sections [Figure 1: A1, A2, A3].
- The cases were divided into three categories:
- Group I—Early cases of OSMF (n = 5).
- Group II—Moderately advanced OSMF (n = 23).
- Group III—Advanced OSMF cases (n = 8) using Khanna JN and Andrade NN (1995) grading system.
|Figure 1 Comparison of the staining patterns of p63 and CD31 with corresponding H&E sections of the OSMF cases divided into Groups I (A1, B1, C1), II (A2, B2, C2) and III (A3, B3, C3) (H&E, p63, CD31 × 100).|
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- The sections were stained with immunohistochemical (IHC) markers and compared with their corresponding hematoxylin and eosin-stained sections.
Immunohistochemical staining was performed using the HRP detection system (BioGenex Life Sciences Pvt Ltd.). 4–5µ thick sections from archival OSMF cases were taken and subject to primary monoclonal anti-mouse anti-p63 antibody (prediluted, AM418-5M, BioGenex Inc.) and monoclonal anti-mouse anti-CD31 antibody (prediluted, AM241-10M, BioGenex Inc., San Ramon, CA, USA) for 1 hour each, followed by secondary antibody (poly-HRP reagent).
The stained sections were observed under Olympus research microscope and photomicrographs obtained using Procapture ProgRes 3.0 imaging software. The cases that exhibited consistent brown colour of the nuclei due to 3, 3’-diaminobenzidine-tetrahydrochloride (DAB) chromogen were considered as positive for p63. The cells lining the vessels highlighted with DAB were considered positive for CD31 [[Figure 1] B and C].
1. Assessment of the p63 immunostained slides were carried out as follows:
The sections were scored by quantitative method.
- The p63 stained sections were assigned three scores
- + denotes nuclear positivity of the entire length of basal layer and few suprabasal cells.
- ++ denotes nuclear positivity of basal and spinous layer with few granular cells.
- +++ denotes nuclear positivity of basal, spinous and granular layers with/without involvement of Str. Corneum.
- Predominant pattern of p63 staining throughout the epithelium (full length) was assessed on 4x magnification
- Pattern I—Uniform staining of lower one-third of epithelium.
- Pattern II—Uniform staining of two-third of epithelium.
- Pattern III—Uniform staining of all the layers with few corneal cells showing positivity.
2. Assessment of the CD31 immunostained slides were carried out as follows:
Cytoplasmic staining of the cells was considered as immunopositive for CD31 antibody
- Counting score
- The number of vessels present adjacent to a fixed length of the epithelium was assigned to each case (at x100 magnification).
- The vessels present juxtaepithelially in the connective tissue within an area of 0.1 mm from the basement membrane and present within a length of 0.4 mm of the epithelium were considered [Figure 2].
|Figure 2 The number of CD31 positive vessels were calculated across a fixed length of epithelium that is 0.4 × 0.1 mm (IHC CD31 × 100).|
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- Evaluation of the predominant pattern (diameter) of the positive blood vessels were scored.
- c—Constricted—Pattern I
- n—Normal—Pattern II
- d—Dilated—Pattern III
Chi-square test was used to compare p63 expression for nuclear staining intensity and epithelial staining pattern and also CD31 expression for predominant pattern of blood vessel staining between different grades of OSMF cases. Kruskal Wallis test was used to compare the mean number of Blood vessels stained using CD31 between different grades of OSMF. The level of significance (P-value) was set at P < 0.05. The expression of the study variables was studied with categorical data in terms of number and percentage whereas in mean and SD for continuous data. Statistical Package for Social Sciences (SPSS) for Windows, Version 22.0. Released 2013 (Armonk, NY: IBM Corp.) was used to perform statistical analyses.
Observation and results
A quantitative assessment was carried out by marking out the immunopositive nuclei in the basal/suprabasal, middle and the superficial layers of the epithelium.
The slides were also analysed for pattern distribution of the positively stained nuclei across the epithelial strata in the p63 immunostained slides.
The staining pattern across the groups showed a gradual increase in the number of cells with positive nuclei and also in the epithelial strata from the basal to the superficial layer. This finding denotes a pattern wherein the expression of p63 is higher as the grade of OSMF progressed from early to advanced as compared to normal oral epithelium [Figure 1]: B1, B2, B3].
We found that in Group I, almost all the cases exhibited uniform nuclear positivity of the basal layer and few cells of the suprabasal layer. In Group II, majority of the cases >60% showed nuclear positivity of basal, suprabasal, spinous and few cells of granular cell layer. In the Group III > 60% cases showed strong positivity of the cells extending upto the superficial strata of the epithelium. ∼ 35% of cases showed uniform staining upto the spinous layer with few positive granular cell nuclei [Figure 3]. A positive correlation was obtained between the groups with a significant P-value of <0.001* using Chi-square test [Table 1].
|Figure 3 Depiction of the percentage of positive nuclei in cases across the three groups with p63 staining.|
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|Table 1 Comparison of p63 marker expression for nuclear staining intensity and epithelium staining pattern between different grades of OSMF cases using Chi-square test|
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Group I exhibited 60% Pattern I and 40% cases Pattern II; whereas in Group II, the pattern was distributed between I, II and III almost equally. ∼25% showed positive p63 expression of all the epithelial strata including few corneal cells as well (Pattern III). Few cases (13%) exhibited Pattern I staining as given in [Figure 4]. Though differences in pattern distribution of p63 staining across grades were observed, it was not statistically significant with p-value of 0.32 [Table 1].
|Figure 4 Comparison of p63 marker expression for epithelium staining pattern between different grades of OSMF.|
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p63 expression was compared between the three grades of OSMF using Chi-square test. Statistical significance was observed between Group I and II and between Group I and III; whereas comparison between Groups II and III did not show a significant variation [Table 2].
|Table 2 Multiple comparison of P63 marker expression for nuclear staining intensity between 03 grades of OSMF|
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From this we could infer that there was a definite increase in nuclear expression from the basal and parabasal layer to prickle cell layer [Group I to II, [Figure 1]: B1] and from basal/parabasal upto the superficial layers of the overlying epithelium in OSMF cases [Group I to III, [Figure 1]: B2]. Though there was increase in p63 positive nuclei from stratum spinosum upto stratum corneum [Group II to III, Figure 1: B3]; the p63 expression in all the cases was not uniform and therefore it was not statistically significant.
The total number of juxta-epithelial vessels adjacent to the fixed length of epithelium was counted and percentage was calculated based on whether the vessels were normal, constricted or dilated [Figure 5].
In Group I, most of the vessels (∼60%) showed constriction on CD31 staining and around 30% showed dilation [Figure 1: C1]. The number of constricted vessels were highest in this group owing to the histologically evident hyalinization.
Group II showed more number of vessels (∼52%) of normal diameter and these were more evident in those cases which showed epithelial hyperplasia [Figure 1: C2]. ∼21% of the vessels in this group were constricted and ∼26% showed dilation.
Group III showed highest number of dilated vessels (>70%) as compared to Group I & II and < 30% of constricted vessels [Figure 1: C3]. The dilated vessels positive for CD31 were more evident in those cases showing hyperplasia and dysplasia of the overlying epithelium, depicted in [Table 3]. There was a marked difference in the percentage of vessels showing diameter variation across the groups which was statistically significant (P-value 0.02*) [Table 3].
|Table 3 Comparison of CD 31 marker expression for predominant pattern of blood vessel staining between different grades of OSMF cases using Chi-square test|
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The groups were individually compared with each other using Chi-square test, in which Group II and Group III showed statistical significance with P-value of 0.02*, whereas comparison between Group I and II and Group I with Group III was not statistically significant [Table 4].
|Table 4 Multiple comparison of CD 31 marker expression for pattern of blood vessel staining between three grades of OSMF|
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We also looked at the Mean Vessel Density of the CD31 positive vessels and found that Group I showed the least number of vessels per unit area. Most cases exhibited atrophic epithelium [Figure 1: A1]. Group II showed the highest vessel density especially in cases with epithelial proliferation. In Group III most of the cases showed almost equal number of CD31 positive vessels per unit area in [Figure 6]. Kruskal-Wallis test was used to evaluate the mean number of vessels present adjacent to a fixed length of epithelium. (0.4 mm at x100 magnification). The mean number of vessels and SD group-wise did not show any statistical significance [Table 5].
|Figure 6 Comparison of mean number of blood vessels stained using CD31 between different grades of OSMF.|
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|Table 5 Comparison of mean number of blood vessels stained using CD 31 between different grades of OSMF using Kruskal Wallis test|
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We could observe that most of the vessels were constricted in Grade I lesions, showed dilation in Group II and Group III, especially in those cases which showed epithelial hyperplasia and dysplasia; and also showed a steady increase in the number of vessels per unit area across the groups.
| Discussion|| |
OSMF is a premalignant disorder with a malignant transformation rate of 2–8%. Epithelial changes that occur in this lesion are a harbinger of carcinomatous change. Reports have shown that OSMF cases are at a higher risk of developing leukoplakia and OSCC.
Though OSMF is primarily a connective tissue disorder, the risk of its epithelium accumulating dysplastic changes and further converting into oral squamous cell carcinoma cannot be ignored. The mechanism underlying such a transformation however is still yet to be established.
Areca nut and its alkaloids have been proved time and again to have a direct role in the etiology of OSMF. The availability and increased consumption of commercial areca nut preparations amongst the young adults maybe a reason for detecting oral cancer in younger age groups in the past few decades—bimodal peak., The fact that the oral epithelium is subject to continuous assault by the alkaloids and carcinogenic chemicals found in these preparations and therefore may harbour changes not yet visible clinically should be seriously considered.
Therefore, in order to identify such changes at the cellular level, we used p63, belonging to the p53 family; purported to have an oncogenic role in OSCC,, and CD31 which is a highly specific endothelial cell marker and has a modulatory role in angiogenesis and inflammation in an attempt to elucidate their role in the pathogenesis and malignant transformation of OSMF in this study.
The immunohistochemical expression of p63 in the ascending histologic grades of OSMF was studied and compared. We found considerably higher expression of p63 positive cells in the superficial layers of epithelium of the lesional epithelium as compared to normal. The nuclear expression significantly increased with the severity of the grade of dysplasia and was statistically significant with a P-value < 0.001* which was in accordance with other studies.,,,,
The p63 gene present on chromosome 3q27e29 comprises of 2 functional isoforms—TAp63 and ΔNp63. These are further subdivided into (TAp63a, TAp63b and TAp63g) and (ΔNp63a, ΔNp63b and ΔNp63g).  It has been found that the full length of the TA isoform is functionally and structurally akin to the wild type p53, that is possessing tumour suppressor activity. ΔNp63 isoforms are frequently overexpressed in epithelial cancers, including OSCC due to which it is considered as an oncogene. 
Studies have shown that p63 has an important functional role during growth and development of the craniofacial skeleton in proliferation and differentiation of the epithelial cells. p63 proteins have been implicated in stratification of the epithelium, preservation of the proliferative potential of epithelial stem cells and tissue homeostasis. According to Yang et al. and Ramasubramanian et al., presence of p63 positive cells in the superficial layers of epithelium may possibly be an indicator of higher proliferative capacity and lower rate of maturity of the cells,  which was a constant finding in our cases [Figure B1, B2, B3].
Koster et al. established that ΔNp63 aids in maintenance of basal cell phenotype of the epithelium by inhibiting the process of differentiation. Accordingly, its expression in normal oral epithelium should be low and should seldom be detected in the superficial layers. However, in epithelial dysplasia, the superficial keratinocytes tend to retain their proliferative capacity and thus continue to exhibit ΔNp63 activity and as a result, cause architectural disarray. Takeda et al showed that the disruption of normal distribution of ΔNp63-positive cells in epithelial dysplasia might play a role in oral tumorigenesis.
Chen et al. found that study groups of moderate and severe dysplastic lesions with ΔNp63-positive cells underwent malignant transformation within 5 years of follow-up. p63 immunoreactivity was observed in various squamous malignancies, suggestive of its tumorigenic potential.
p63 overexpression in the superficial strata can be attributed to the presence of higher amounts of ΔNp63 isoform as was observed in our cases. p63 labeling found evenly higher across histologic grades irrespective of overlying epithelial dysplasia, may underline a change that is occurring at a molecular level and therefore not apparent as a histomorphological feature.
Molecular studies also have determined that ΔNp63α is the isoform overexpressed in SCCs using RT-PCR validated with Western blot analysis. ΔNp63 mRNA expression was a 100-fold more than TAp63 mRNA., An important finding from RT-PCR studies was that p63 isoforms were upregulated in squamous dysplasia.,,
Though anti-human p63 IHC marker is a cocktail of all the isoforms of p63, it is a fair assumption that its upregulation indicates the increased expression or activity of ΔNp63, especially α-isoform in cases showing dysplastic changes of the epithelium. Moreover, Zangen et al reported that ΔNp63α expression has a direct correlation with poor response to cisplatin in HNSCC.
Evaluation of our slides revealed that the higher the histologic grade of OSMF, the higher was the nuclear positivity of p63, staining almost the full thickness of the epithelium in advanced grade, depicting a linear increase in the expression of p63. Once dysplasia sets in, the altered keratinocytes present in the suprabasal and superficial layers are able to express p63 and therefore induce an increased proliferative capacity of dysplastic cells. The finding that even those cases of OSMF, which lacked epithelial dysplasia, also showed a high p63 index indicate that molecular changes predisposing toward malignant transformation do take place in the OSMF epithelium, even in the absence of morphologically apparent dysplastic changes. Hence increase in p63 expression in superficial strata of epithelium can be an indicator of the grade of epithelial dysplasia and of future malignant change.
CD31 has been identified as one of the most specific immunohistochemical markers to detect endothelial cells lining vascular channels. Platelet endothelial cell adhesion molecule (PECAM-1/CD31) is a member of the immunoglobin super family of cell adhesion molecules with important roles in angiogenesis and inflammation. It modulates pro-angiogenic properties of endothelial cells. It is present on the gene locus of chromosome 17q23.
CD31 is a primary constituent of endothelial cell-cell junctions in endothelial cells and its expression pattern is regulated during development and angiogenesis.
In our study, we observed that Group I cases on CD31 staining showed majority of vessel constriction as given in [Table 3]. The initial constriction of the blood vessels due to fibrosis in early OSMF leads to tissue hypoxia, which in turn causes the recruitment and accumulation of pro-angiogenic factors to this niche area. This is in accordance with Tilakaratne et al. who demonstrated that hypoxia-induced mechanisms act in OSMF and have a role in its malignant transformation.
We observed that as the grade of dysplasia increased, the vessel diameter enlarged causing dilation of the microvasculature as seen in [Figure 1: C1, C2, C3]. This plausibly increased the blood supply to the overlying epithelium, which exhibited hyperplastic change. This could be suggested as a crucial change aiding transformation of the overlying epithelium. Ekanayaka et al. opined that tissue hypoxia is an innate reaction which follows constriction of blood vessels that reduce blood and oxygen to tissues. Sustenance of this hypoxia is difficult, hence; in response, the blood vessels undergo dilation. Many researchers have observed that vessels in OSMF tissues do not always show constriction. This finding that blood vessels of different sizes were observed in OSMF cases was supported by Rajendran et al., who suggested that as the connective tissue gets denser; physical and biochemical restriction to the microvasculature occurs, which in turn induces angiogenic activity. Therefore the probability that the dilated blood vessels are closely associated with angiogenesis cannot be ruled out. Such cases warrant further study and follow-up in close correlation with histologic grade of OSMF.Angiogenesis is a vital process in carcinogenesis, that promotes growth, maintenance, and spread of tumour. A range of angiogenic factors have been demonstrated in OSMF which have a role in maintenance of its vascularity. Therefore, it can be speculated that while other factors induce epithelial atrophy, the blood vessels in the connective tissue of OSMF have a role in inducing hyperplasia and aiding conversion of the dysplastic epithelium into OSCC; supporting its development and dissemination.
In the present study, CD31 showed a significant and linear increase in Microvascular diameter with increasing severity of disease. A significant difference between normal oral mucosa and advanced OSMF could be observed. Comparison between the groups was significant with a P-value of 0.02*. This was in accordance with Surendran et al., who in their study, indicated that patients with increasing levels of CD31 were associated with high risk oral premalignant disorders and carcinoma.
From our observations, we could infer that in those cases showing epithelial changes, the vasculature showed increase in number and in diameter, providing angiogenic support to the proliferative epithelium harbouring hyperplastic and dysplastic changes. Therefore this staining pattern may be predictive of those lesions that might undergo malignant transformation.
| Conclusion|| |
Increased expression of p63 and CD31 in cases of OSMF can be considered as definitive quantitative markers in the prediction of the malignant transformation of OSMF. Therefore, patients showing the overexpression of these biomarkers are at considerable risk of malignant transformation and should be closely monitored. More so because, during the last decade, OSCC arising from OSMF is being considered as a distinct entity with higher incidence in younger individuals and bearing a better prognosis with better histologic differentiation, lower nodal metastases and extracapsular spread and better staging for OSCC.
Financial support and sponsorship
We are grateful to the Rajiv Gandhi University of Health Sciences, Bangalore for providing us with financial support for this original work.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]