Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 13  |  Issue : 2  |  Page : 96-104

Histoscore and Discontinuity Score − A Novel Scoring System to Evaluate Immunohistochemical Expression of COX-2 and Type IV Collagen in Oral Potentially Malignant Disorders and Oral Squamous Cell Carcinoma


1 Department of Oral and Maxillofacial Pathology, AECS Maaruti College of Dental Sciences and Research Centre, Bangalore, Karnataka, India
2 Department of Oral and Maxillofacial Pathology, Employees State Insurance Corporation Dental College and Hospital, Kalaburagi, Karnataka, India

Date of Submission18-Jun-2021
Date of Acceptance27-Oct-2021
Date of Web Publication14-Jan-2022

Correspondence Address:
Dr. P. Sharada
Department of Oral and Maxillofacial Pathology, AECS Maaruti College of Dental Sciences and Research Centre, Bangalore 560076, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jofs.jofs_141_21

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  Abstract 


Introduction: Cyclooxygenase 2 (COX-2) expression in oral potentially malignant disorders (OPMDs) such as oral submucous fibrosis (OSMF) and oral squamous cell carcinomas (OSCCs) has revealed inconclusive reports. Studies on loss of type IV collagen expression in oral epithelial dysplasias (OEDs) and OSCCs were subjective and lacked systemic approach. To evaluate the immunohistochemical (IHC) expression of COX-2 and type IV collagen in OED, OSMF, and OSCC. Materials and methods: IHC expression of COX-2 and type IV collagen on paraffin-embedded tissue section of 10 cases each in normal oral mucosa, mild OED, moderate OED, and severe OED, OSMF, and OSCC were evaluated using mean H score and discontinuity Score (DS) designed grades for every group, respectively. Mean H score of COX-2 was compared within and between the groups using analysis of variance (ANOVA), and DS designed specifically for type IV collagen expression was compared using Kruskal–Wallis ANOVA. Pairwise comparison between the groups were performed using Tukey multiple posthoc procedure and Mann–Whitney U test for COX-2 and type IV collagen, respectively. Results: Mean H scores of COX-2 expression increased significantly (P = 0.0001) as disease progressed from mild OED to severe OED. But COX-2 in OSCC was less than that observed in mild OED (P = 0.0001). Expression of COX-2 in OSMF was more than that observed in moderate OED. Type IV collagen expression decreased as disease progressed from OED to malignancy (P = 0.0001). OSMF indicated a variation in grades of loss of type IV collagen expression. Conclusion: Expression of COX-2 in OED, OSMF, and OSCC and DS for type IV collagen expression in our study could be effectively applied to assess the malignant potential of OPMDs. However, further studies need to be implemented on a larger sample size to conclude the above findings.

Keywords: COX-2, discontinuity score, mean h score, oral epithelial dysplasias, oral submucous fibrosis, oral squamous cell carcinomas, type IV collagen


How to cite this article:
Sharada P, Swaminathan U, Nagamalini BR, Vinod Kumar K, Ashwini BK. Histoscore and Discontinuity Score − A Novel Scoring System to Evaluate Immunohistochemical Expression of COX-2 and Type IV Collagen in Oral Potentially Malignant Disorders and Oral Squamous Cell Carcinoma. J Orofac Sci 2021;13:96-104

How to cite this URL:
Sharada P, Swaminathan U, Nagamalini BR, Vinod Kumar K, Ashwini BK. Histoscore and Discontinuity Score − A Novel Scoring System to Evaluate Immunohistochemical Expression of COX-2 and Type IV Collagen in Oral Potentially Malignant Disorders and Oral Squamous Cell Carcinoma. J Orofac Sci [serial online] 2021 [cited 2022 Jan 22];13:96-104. Available from: https://www.jofs.in/text.asp?2021/13/2/96/335840




  Introduction Top


Oral squamous cell carcinoma (OSCC) is the 11th most common cancer in the world,[1] 92.8% of all oral malignancies appears to be OSCC.[2] India has the highest incidence of oral cancer(OC) in the world, with the annual death rate in India being close to 46,000.[3] International Agency for Research on Cancer has predicted that India’s incidence of cancer will be approximately 1.7 million in 2035.[4] Many of the OSCCs develop from pre-existing oral potentially malignant disorders (OPMDs), most prevalent being oral leukoplakia and oral submucous fibrosis (OSMF).[3] Incidence of OPMDs in India lies between 6/1000 and 30.2/1000.[5] The malignant transformation rate of oral leukoplakia ranges from 15% to 20% and OSMF approximately 7.6% over a 10-year-period incidence in India.[6]

In the 19th century, Rudolph Virchow first proposed the theory of correlation between inflammation and cancer. The microenvironment of neoplastic tissues consists of inflammatory signaling molecules that promote tumor growth and metastases.[7] Prostaglandins and lipid autacoids derived from arachidonic acid by the action of cyclooxygenase 2 (COX) enzymes are known to function as mediators of inflammation.[8] Two isoforms of COX–COX-1 and COX-2 exists. COX-1 is constitutively expressed, whereas COX-2 is inducible.[9] COX-2 is also termed as prostaglandin-endoperoxidase synthase. It is located on chromosome 1 locus of 1q25.2-q25.3. It is 8.0 kbp in size with 10 exons and 9 introns. It is composed of 604 amino acid residues.[10]

COX-2 expression is absent in normal tissues and organs under physiologic conditions but can be activated in response to stimuli such as cytokines and growth factors. Several inflammatory pathways play a vital role in a microenvironment of carcinogenesis and COX-2/prostaglandin E2 (PGE2) is the most important one. This COX-2-mediated pathway results in sustained cell survival, amplified cell proliferation, and migration and neoangiogenesis. Hence, COX-2 pathways are targets for therapeutic intervention.[10] Studies have reported the upregulation of COX-2 expression with increase in the severity of dysplasia to OSCC but other studies indicate reduced expression in OSCCs and thus, COX-2 expression in oral epithelial dysplasia (OED) and OSCC remains obscure.

The characteristic feature of carcinomas is the invasion of malignant cells into the underlying connective tissue by breakdown of basement membrane. Basement membranes are composed of collagen, glycoproteins, and proteoglycans.[11] The major molecules present in the basement

membrane are type IV collagen, laminin, perlecan, and eutactin.[2] Type IV collagen consists of six α-chains (α1–α6). Three α-chains assemble into triple-helical molecules that self-associate to form supramolecular networks. The major molecular form of type IV collagen in basal lamina is [α1(IV)]2α2(IV).[12]

The breakdown of basement membrane during progression to malignancy from neoplastic stage has been attributed to the actions of matrix metalloproteinases (MMPs). This loss of type IV collagen has been reported to increase as the disease progresses to OED to malignancy. These studies presented in the literature are subjective and lack systematic approach. Thus, the present study was conducted to evaluate the expression of COX-2 in OED, OSMF, and OSCC and explore the novel discontinuity score grading system to assess the loss of type IV collagen in OED, OSMF, and OSCC.


  Materials and Methods Top


An in vitro case–control study was performed on 60 specimens obtained from the archives of the Department of Oral and Maxillofacial Pathology. The study group was divided into 6 groups with 10 specimens in each group on the basis of histopathologic findings consisting of group I − mild OED (n = 10), group II − moderate OED (n = 10), group III (n = 10) − severe OED, group IV (n = 10) − OSMF, group V (n = 10) − normal oral mucosa (NOM), and group VI (n = 10) − OSCC. All the archival specimens were sectioned and stained with hematoxylin and eosin stain to reconfirm the diagnosis. Ethical approval for this study (AECS/MDC/611/2014-15) was provided by the Institutional Ethical Committee of Maaruti College of Dental Sciences and Research Centre, Bangalore, on March 6th 2015.

Immunohistochemical technique

A 3-μm-thick section of samples and controls (Skin − Type IV Collagen, Colon − COX-2) were obtained and mounted onto aminopropyltriethoxysilane-coated slides. The sections were deparaffinized, using three changes of xylene, and were rehydrated through decreasing grades of isopropyl alcohol. Endogenous peroxide activity was blocked by immersing the tissue sections in peroxidase block for 20 minutes. Antigen retrieval was achieved by heat-induced epitope retrieval using Tris ethylenediamminetetraacetate buffer (pH 8) in a pressure cooker at 150°C for 65 minutes. The sections were washed with Tris buffer and covered with protein block for 15 minutes to eliminate nonspecific staining. The sections, which required to be immunostained with type IV collagen, were covered with type IV collagen primary mouse monoclonal antibody (PathnSitu; Lot no.: R02201TA, EXP DT: 7/2018), whereas sections that required COX-2 immunostaining were covered with COX-2 primary rabbit monoclonal antibody (BioGenex; Lot no.: AN4640613, MFG DT: 01/2016) for a period 60 and 45 minutes, respectively. The sections were then washed with Tris buffer and incubated with poly horseradish peroxidase for 30 minutes. The antigenic sites demonstrated using freshly prepared diaminobenzidine (DAB). The nuclei were counterstained with Mayer hematoxylin. The sections were cleared using xylene and mounted with dibutyl phthalate xylene. For each batch of staining, positive controls were run simultaneously with the study specimens. The presence of brown-colored end product (DAB positivity) was indicative of positive immunoreactivity. The intensity of immunohistochemical (IHC) expression of positive controls [Figure 1] was utilized as a reference to grade the intensity of IHC expression.
Figure 1 Positive controls: (A) Colon − demonstrating immunohistochemical (IHC) expression of COX-2 (×100). (B) Skin − demonstrating IHC expression of type IV collagen (×100 magnification).

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Interpretation of IHC expression

COX-2 IHC expression

The intensity of COX-2 IHC expression was determined using the intensity score criteria as described by Allred et al.,[13] and the scores were interpreted as: 0–no positive cells; 1+ − mild intensity; 2+ − moderate intensity; and 3+ − strong intensity. For semiquantitative analysis of immunohistochemistry, H score as described by Hinsch[14] was used. The H score was calculated for every sample and tabulated using the formula as shown below:

1x (% cells 1+) + 2x (% of cells 2+) + 3x (% of cells 3+)

Type IV collagen IHC expression

Type IV collagen expression was assessed as described by Tosios et al.[15] for all the groups. A regular continuous brown line beneath the basal layer of epithelium consistent of basement membrane zone was considered as positive. Any alterations in this continuity, when viewed with scanner objective, were defined as gaps/discontinuity.[15] The study of Tosios et al.[15] described type IV collagen as continuous or discontinuous expression and lacked to define the extent of discontinuity. Hence, the study attempted to define the extent of discontinuity reported in type IV collagen expression in OED and OSCC. Hence, we propose a new score, discontinuity score (DS) grading system for interpreting type IV collagen expression. Any gaps/discontinuities arising in the entire stretch of basement membrane for type IV collagen expression were expressed as percentage of loss of expression. This percentage of loss of expression was graded as DS which are as follows: grade 1: 1% to 10%; grade II: 11% to 20%; grade III: 21% to 30%; grade IV: 31% to 40%; grade V: 41% to 50%; grade VI: 51% to 60%; grade VII: 61% to 70%; grade VIII: 71% to 80%; grade IX: 81% to 90%; and grade X: 91% to 100%. On the other hand, no gaps/discontinuities arising in the entire stretch of basement membrane was considered as 100% expression.

Statistical analysis

Mean H score of COX-2 expression for each group was calculated and was used to compare and correlate within and between the groups. Kolmogorov–Smirnov test, which was performed to assess the distribution of samples within the six groups for COX-2 showed a normal distribution pattern. Therefore, parametric test was applied to compare and correlate the findings within and between the groups for COX-2. The parametric test considered was one-way analysis of variance (ANOVA) to compare mean H score of COX-2 expression between the six groups and within the groups and pairwise comparison with Tukey multiple posthoc was used to locate any statistically significant difference between the groups. On the other hand, for type IV collagen, a nonparametric test, Kruskal–Wallis ANOVA, was used to compare and correlate the findings within and between the groups and pairwise comparison was performed using Mann–Whitney U test to locate any statistically significant difference between the groups.


  Results Top


The mean H scores (mean ± standard deviation) of COX-2 expression in groups NOM, mild OED, moderate OED, severe OED, OSMF, and OSCC were 0.0 ± 0.0, 98.0 ± 16.2, 115.5 ± 20.7, 158.0 ± 30.6, 139.5 ± 24.9, and 84.0 ± 7.7, respectively [Table 1] and [Figure 2]. COX-2 expression was predominantly expressed in basilar and parabasilar layers in mild OED and involved the superficial layers of epithelium in moderate OED, severe OED, and OSMF. COX-2 expression also increased with the increase in the grades of dysplasia. However, in OSCC cases, COX-2 expression was less than that of mild OED [Figure 3] and was observed as a patchy expression either in basilar or parabasilar areas or more discreetly in superficial cells, whereas in epithelial islands, the COX-2 expression was intense, as indicated in [Figure 3]. When mean H score of COX-2 expression was compared using ANOVA, a statistical significant difference was observed between the groups and within the groups with a P-value of 0.0001. Further, Tukey multiple posthoc procedure showed a statistical significant difference between mild OED and severe OED (P = 0.0001), mild OED and OSMF (P = 0.0004), mild OED and NOM (P = 0001), and moderate OED and severe OED (P = 0.0003). Moderate OED and NOM (P = 0.0001), moderate OED and OSCC (P = 0.0089), severe OED and NOM (P = 0.0001), severe OED and OSCC (P = 0.0001), OSMF and NOM (P = 0.0001), OSMF and OSCC (P = 0.0001), and NOM and OSCC (P = 0.0001), as indicated in [Table 2] and Graph 1.
Table 1 Comparison of COX-2 mean H score between the groups using analysis of variance

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Figure 2 Immunohistochemical expression of COX-2 in study groups (×100 magnification).

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Figure 3 Immunohistochemical expression of type IV collagen in study groups (×100 magnification).

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Table 2 Pairwise comparison of COX-2 mean H score between the groups using Tukey multiple posthoc procedure

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Type IV collagen expression was observed as a regular continuous brown line beneath the basal layer of epithelium indicating basement membrane zone. The novel grading system, when used, showed 90% of cases in mild OED group to be in grade I and remaining 10% in grade II. Fifty percent of cases in moderate OED group showed grade II loss and remaining 50% showed grade I loss, whereas 80% of severe OED showed grade III loss and 20% showed grade IV loss. OSMF group demonstrated 40% of cases with grade V, 30% of cases with grade III, 20% of cases with grade IV, and 10% of cases with grade VI loss of type IV collagen expression. NOM showed 100% type IV collagen expression in basement membrane, whereas OSCC group showed 60% of cases with grade VIII, 20% of cases with grade VII and 20% with grade IX loss [Table 3] and Graph 2]. Kruskal–Wallis ANOVA when used to compare all the six groups with Discontinuity Grades showed a statistical significant (P = 0.0001) difference between and within the groups. Mann–Whitney U test showed a statistical significant difference between (P<0.05), Mild Oral Epithelial Dysplasia vs. severe Oral Epithelial Dysplasia, Mild Oral Epithelial Dysplasia vs. Oral submucous fibrosis, Mild Oral Epithelial Dysplasia vs. Normal oral mucosa, Mild Oral Epithelial Dysplasia vs. oral squamous cell carcinoma Moderate Oral Epithelial Dysplasia vs. severe Oral Epithelial Dysplasia, Moderate Oral Epithelial Dysplasia vs. oral submucous fibrosis, Moderate Oral Epithelial Dysplasia vs. NOM, Moderate Oral Epithelial Dysplasia vs. oral squamous cell carcinoma, Severe Oral Epithelial Dysplasia vs. Normal oral mucosa, Severe Oral Epithelial Dysplasia vs oral squamous cell carcinoma , oral sub mucous fibrosis vs Normal oral mucosa, oral submucous fibrosis vs. oral squamous cell carcinoma, Normal oral mucosa vs. oral squamous cell carcinoma and Severe Oral Epithelial Dysplasia vs. oral sub mucous fibrosis.
Table 3 Comparison of discontinuity grades of type IV collagen expression between the groups using Kruskal–Wallis analysis of variance

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COX-2 expression increased as the disease progressed with increasing grades of OED. However, this was not evident in OSCC as the mean H score of COX-2 was less than the expression reported in mild OED. Expression of COX-2 in OSMF was more than that observed in moderate OED. A stepwise increase in grades of loss of type IV collagen expression as the disease progressed from NOM to increasing grades of OED to malignancy was observed. Loss of type IV collagen expression in OSMF was variable.


  Discussion Top


COX-2 is an inducible isoform of cycloxygenase and is undetectable in most normal tissues but is rapidly induced in response to inflammatory or mitogenic stimuli including cytokines, growth factors, and tumor promoters.[16] COX-2 is regulated at transcriptional and posttranscriptional levels and can also be regulated by rate of protein synthesis and/or degradation. Studies report that COX-2 has a significant role in carcinogenesis and is overexpressed in transformed cells, premalignant, and malignant tissues.[16]

The contribution of COX-2 overexpression to tumorogenesis is related to increased production of mutagens such as malondialdehyde leading to DNA damage, suppression of apoptosis, involvement of angiogenesis contributing to the production of proangiogenic factors such as vascular endothelial growth factor, platelet-derived growth factor, and endothelin-1, and its response to oncogenes v-src, v-Ha-ras, Her-2/neu, and wnt.[16]

In the present study, COX-2 expression was not observed in NOM, whereas cases of mild OED showed expression in basal and parabasilar layers. Superficial layers of epithelium also showed COX-2 expression in moderate OED, severe OED, and OSMF. These findings of the study are consistent with the studies reported by Prado et al.[16] and Nagatsuka et al.[17] Further, COX-2 expression in the present study significantly increased with increasing grades of OED. However, in OSCC group, the expression of COX-2 was less than that of mild OED and the expression was observed at the periphery of tumor islands and within the keratin pearls. This may be attributed to loss of COX-2 expression due to advanced cytodifferentiation of tumor cells and result of alteration of cell surface molecules.[17] These findings in our study were in accordance with the findings in studies by Sawhney et al.,[18] Prado et al.,[16] Anirchaghmaghi et al.,[19] Nagatsuka et al.,[17] Aruldoss et al.,[9] and Fang Chiu et al.[20]

Increased production of COX-2 causes increased production of prostaglandins leading to increased cell proliferation and decreased apoptosis mediated by PGE2 and its E-prostanoid receptor (EP1-4).[21] PGE2 and EP1 interaction might acts through protein kinase C-δ (PKCδ), C-Src, c-jun, and AP-1 pathway to induce intercellular adhesion molecule-1 (ICAM-1) activation in human OC cells,[9] thus contributing to the transformation of OPMDs to malignancy.

The breakdown of basement membrane marks the early event in malignant transformation of OPMDs.[18],[22] It is believed that most of the events are brought about by the action of MMPs leading to destruction of type IV collagen in the basement membrane zone.[23] Further, studies have reported the morphologic alterations of type IV collagen expression in basement membrane from preneoplastic state to cancer in breast,[24] ovarion surface epithelium,[25] pancreas,[26] and oral mucosa.[25],[27] In addition to this, variations in α-chains also have been explored in basal cell carcinoma, breast cancer,[28] bronchoalveolar carcinoma,[29] adenoid cystic carcinoma,[30] colorectal cancer,[31] and OSCC.[32]

Besides this, it is hypothesized that chemopreventive activity of COX-2 inhibitors in epithelial cancers is related to the integrity of the epithelial basement membranes,[33] as prostaglandins, the products of COX-2 activation, activate collagenase and proteolysis and decrease the synthesis of the basement membrane components in ovarian granulosa and surface epithelial cell.[34] Supplementary to this, there exists scientific data pertaining to applicability of inhibitors of COX-2, as chemopreventive agents in a number of epithelial cancers, including colon,[35],[36],[37] mammary,[38],[39] esophageal,[40] lung,[41] and oral cavity.[42],[43] In OSCC, the overexpression of COX-2 promotes the release of PGE2, which acts on its various cell surface receptors and facilitates the development of OSCC in advanced stages of disease.[44] Moreover, the aggressive nature in OCs is often correlated with increased MMP expression, which is known to degrade type IV collagen, a major component of basement membrane.[45] Furthermore, loss of basement membrane collagen IV α-chain correlated with gain of expression for MMP-2 and MMP-9 during progression of OC.[12] Hence, COX-2/PGE2 may increase cell invasiveness via MMPs, which may be reflected on type IV collagen expression.

Further, studies of Davies et al.,[24] Agarwa and Ballabh,[2] Tosios et al.,[15] and Tamamura et al.[12] have shown a strong and well-defined linear pattern of expression of type IV collagen in basement membrane of NOM and gradual increase in frequency of discontinuity from normal to malignant states. The present study also demonstrated similar findings as that Davies et al.,[24] Agarwal and Ballabh,[2] Tosios et al.,[15] and Tamamura et al.,[12] wherein type IV collagen was completely expressed in NOM group and with increased frequency in loss of expression as the disease progressed from dysplasia to malignancy.

These observations, in all the above mentioned studies, rely on the continuous and discontinuous status of type IV collagen expression, which appears to be arbitrary and subjective. Hence, there is a lack of systematic approach to assess the loss of type IV collagen expression as disease progresses from dysplasia to malignancy. A study by Zhang et al.,[46] which applied the grading system to assess the expression pattern of type IV collagen in OED and OSCC, was subjective and inconclusive. Therefore, present study devised and evaluated a novel grading system to predict malignant transformation potential of OPMDs. These grades of type IV collagen expression loss, increased significantly as the disease progressed from increasing grades of epithelial dysplasia to malignancy. The grading system, significantly (P = 0.0001) differentiated mild OED from severe OED, NOM, OSMF, and OSCC; moderate OED from severe OED, OSMF, NOM, and OSCC; severe OED from OSCC and NOM. These findings of the novel grading system to assess the loss of type IV collagen expression have shown promising results to effectively segregate various grades of OED from OSCC. Therefore, we propose for its application in predicting malignant transformation. The present study did not categorize the histopathologic grades of OSCC and thereby limits the study to define the expression pattern of COX-2 and type IV collagen in OSCC, restricting the assessment of the progression of disease.


  Conclusion Top


In the present study, COX-2 expression significantly increased with increasing grades of OED; and reduced expression in OSCC may be attributed to the loss of COX-2 expression due to advanced cytodifferentiation of tumor cells. The novel grading system applied to assess loss of type IV collagen expression significantly differentiated various grades of OED and also between dysplasia and malignancy. The present study also suggests that grade VI and above of DS should be cautiously viewed for potential transformation to microinvasive carcinoma. Hence, COX-2 and type IV collagen have potential ability to predict malignant transformation in OPMDs. However, the observations should be studied on a larger sample size and correlated with clinical findings and habits.

Acknowledgment

The authors are grateful to Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka for providing grant for this study.

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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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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