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Year : 2022  |  Volume : 65  |  Issue : 4  |  Page : 761-765
HIF – 1α as a marker of risk assessment for malignant transformation in oral submucous fibrosis: An immunohistochemical study

1 Department of Oral Pathology and Microbiology, Divya Jyoti College of Dental Sciences and Research, Uttar Pradesh, India
2 Department of Oral Pathology and Microbiology, I.T.S. College of Dental Sciences and Research, Uttar Pradesh, Kanpur, Uttar Pradesh, India
3 Department of Oral Pathology and Microbiology, Rama Dental College, Kanpur, Uttar Pradesh, India
4 Department of Internal Medicine, JPNA Trauma Centre, AIIMS, New Delhi, India
5 Department of Radiation Oncology, Maulana Azad Medical College, New Delhi, India

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Date of Submission11-Feb-2021
Date of Decision20-Mar-2021
Date of Acceptance18-Apr-2021
Date of Web Publication25-May-2022


Background: Oral submucous fibrosis (OSMF) is a potentially malignant disorder associated with habit of chewing betel quid containing arecanut. Morphological features of OSMF especially fibrosis suggests a possibility of the hypoxic environment in diseased tissues. The adaptation of cells to hypoxia appears to be mediated via hypoxia inducible factor-1α (HIF-1α) which is also said to be associated with malignant transformation of epithelial cells in various other carcinomas like prostate and cervical carcinoma. Therefore, the main objective of this study was to investigate the role of HIF-1α in progression and malignant transformation of OSMF. Materials and Methods: The study group consisted of histo-pathologically diagnosed 30 cases of oral submucous fibrosis and 10 cases of OSCC were taken as control. The immunohistochemistry was carried out on neutral buffered formalin-fixed paraffin-embedded tissue sections by using the monoclonal antibody of HIF-1α. Statistical analysis was done using SPSS software version 2.0. Results: A gradual and significant rise in the expression of HIF-1α was observed in various grades of OSMF and OSCC cases. HIF 1α expression was increased in cases showing hylanization and constricted blood vessels. A cut off value of 39.6% of HIF-1α positive cells was determined statistically to categorize the cases into high risk and low risk group for malignant transformation. Conclusion: Overexpression of HIF-1α may contribute to the progression and malignant transformation of OSMF. Cases expressing more than 40% of HIF-1α positive cells are at a greater risk for malignant transformation.

Keywords: HIF, high risk cases, hypoxia, malignant transformation, oral submucous fibrosis.

How to cite this article:
Sharma K, Shetty DC, Rathore AS, Juneja SA, Katyal S, Krishnatre A. HIF – 1α as a marker of risk assessment for malignant transformation in oral submucous fibrosis: An immunohistochemical study. Indian J Pathol Microbiol 2022;65:761-5

How to cite this URL:
Sharma K, Shetty DC, Rathore AS, Juneja SA, Katyal S, Krishnatre A. HIF – 1α as a marker of risk assessment for malignant transformation in oral submucous fibrosis: An immunohistochemical study. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Dec 7];65:761-5. Available from:

   Introduction Top

Oral submucous fibrosis (OSMF) is a chronic debilitating disease and a premalignant condition of the oral cavity with malignant transformation rate of about 2–8%.[1] The OSMF progression is biphasic, involving both the fibroblastic and keratinocytic lineages. The epithelial malignancy is considered to be a sequel of connective tissue changes.[2]

Excessive fibrosis in OSMF leads to constriction of the existing blood vessels resulting in restricted supply of nutrients to the epithelium. In addition, the epithelium undergoes constant insult from the varied carcinogens resulting in compromised healing and repair of the epithelium. This results in accumulation of cellular and nuclear alterations leading to changes in epithelial cells proliferation, maturation and metabolism.[3]

Association between hypoxia and fibrosis has been established in other organs of the body.[4] Also, hypoxia may up regulate molecules like Hypoxia Inducible Factor - 1α (HIF-1α) which is said to play a key role in carcinogenesis. Its increased expression has been reported in premalignant lesions of various other tumours such as colonic adenoma, breast ductal carcinoma in situ, Cervical intraepithelial neoplasia (CIN) and prostate intraepithelial neoplasia.[5],[6],[7],[8]

HIF-1α is a cytoplasmic protein which in well oxygenated cells is continuously degraded by the ubiquitin-proteasome system. Under hypoxic conditions, HIF-1α subunits translocate to the nucleus, heterodimerize with HIF-1β subunits and form an active HIF-1 protein that binds to specific hypoxic response elements present in target genes, ultimately activating transcription of these genes.[9] A number of target genes like erythropoietin, VEGF, transferrin, various glycolytic enzymes are activated by HIF-1α whose protein products are involved in angiogenesis, energy metabolism, erythropoiesis, cell proliferation and viability, vascular remodelling, and vasomotor responses.[10]

It has become clear over the past years that hypoxia inducible factor 1α (HIF-1α) is the intrinsic survival factor of tumour cells to overcome oxygen and nutrition deficits during proliferation and progression.[11] However, its role has not been explored much in OSMF. Therefore the present study was designed to assess the role of HIF-1α in the progression and malignant transformation of OSMF. The present study also correlated HIF-1α expression with the clinical and histopathological findings and attempted to identify the high-risk cases based on the HIF-1α expression.

   Material and Method Top

The retrospective study was conducted in the Department Of Oral and Maxillofacial Pathology and Microbiology, ITS-CDSR, Murad Nagar, Ghaziabad, using tissue samples that were submitted for histopathological evaluation. Study samples consisted of 30 cases of histo-pathologically confirmed OSMF. Ten cases of OSCC were taken as control. Clinical data such as age, gender, site, habit, history, signs and symptoms were obtained for each case. The expression levels of HIF1α were studied by immunohistochemistry. Ethical approval and informed consent from patients were obtained for the study (ethical committee was obtained dated 7th November, 2014).

Grading and stage of the disease

Histopathological grading of 30 OSMF cases was done according to the classification proposed by Utsunomiya et al. (2005)[12] into early, intermediate and advanced OSMF. Study group consist of 10 cases each of early, intermediate and advanced OSMF.


Formalin-fixed paraffin-embedded tissue sections were cut to 5 μm thickness. Poly -L-lysine coated slides were used for the proper adherence of tissue sections to the glass slides. The immunohistochemical staining for HIF-1α was carried out in the Department of Oral Pathology and Microbiology. Sections were hydrated with increasing grades of alcohol and transferred to distilled water and treated with hydrogen peroxide to eliminate endogenous peroxidase activity. Then antigen retrieval with 0.01 M tri-sodium citrate buffer for HIF-1α was carried out in Biogenex antigen retriever system V.2.1. The tissue was incubated sequentially with primary antibody HIF-1α (Quadrett Inc.; Cat. No: 1-HY033-07; Lot No. 354716), HRP labelled polymer detection system and 3, 3, Diaminobenzidine substrate solution. This leads to the formation of a brown coloured precipitate at the site of tissue antigen. Counterstaining with hematoxylin was done to aid in visualization.

All the immune-stained slides were viewed under the research light microscope. Positive staining for HIF-1α was nuclear as well as cytoplasmic brown staining. Quantitative analysis was done by selecting five random fields with minimum of 200 cells per field at 400x magnification. Minimum of 1000 cells were evaluated per case and percentage of nuclear expression was calculated.

Qualitative analysis was done by observing tissues under light microscope at 100x and 400x. Homogenous dark brown staining was considered as strong (score 3) and light faint staining considered as mild (score 1) and cases in between the two extremes were graded into the moderate (score 2) category. Cases with no staining were given score 0. Each of these assessments was independently carried out by three investigators. Interpretation of agreement of numerical scoring of cytoplasmic and nuclear expression of HIF-1α was done using kappa analysis. Statistical evaluation was done using SPSS software version 20.

   Results Top

Present study shows that the mean percentage HIF-1α positive cells significantly increase from early (10.91% ± 9.03) to intermediate (27.42% ± 8.55) to advance OSMF (44.07% ± 11.3). A gradual statistically significant increase in the qualitative score was noted from early to intermediate to advance OSMF. Significant difference was also reported in the expression of HIF-1α in OSMF and OSCC group [Table 1] and [Figure 1].
Table 1: Immuno-Scoring of HIF-1α Expression in Study and Control Cases

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Figure 1: a. Early oral submucous fibrosis showing weak cytoplasmic immunopositivity of HIF-1α. b. Intermediate oral submucous fibrosis showing nuclear and cytoplasmic immunopositivity of HIF-1α. c. Advanced oral submucous fibrosis showing intense nuclear immunopositivity of HIF-1α d. Oral squamous cell carcinoma showing intense immunopositivity of HIF-1α (40x magnification)

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HIF-1α immuno-scoring was also correlated with histological features in the OSMF group. HIF-1α expression was significantly higher in OSMF cases with hyalinised type of fibrosis followed by dense fibrosis and loose fibrosis in the connective tissue. Cases with constricted blood vessels demonstrated significantly higher number of HIF-1α positive cells followed by mixed, normal and dilated type of blood vessels. Type of epithelium did not correlate significantly with HIF-1α expression [Table 2].
Table 2: Correlation of Histological Features with HIF-1α Immune-Scoring in the OSMF

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Receiver operating characteristic (ROC) curve analysis was performed to select the optimal cut off value of HIF-1α expression in OSMF group. The area under the ROC curves was 0.892 with a 95% confidence interval (CI) of 0.787 – 0.997 with P value = 0.000. A cut off value of 39.6% of HIF-1α percentage positivity in OSMF group presented a sensitivity of 70% and a specificity of 73.3% [Figure 2]. Based on the cut-off values the OSMF cases were classified into high risk and low risk categories and then HIF-1α expression was compared between the two categories. There was statistically significant difference in the mean percentage positivity of HIF-1α between high risk and low risk OSMF cases [Table 3]. High risk and low risk categories are further correlated with Mouth Opening (MO) in OSMF group. High risk group in OSMF consists of total 8 cases out of which 7 cases had MO less than 25 mm [Figure 3]. The low risk group consists of total 22 cases maximum cases having MO more than 25 mm (n = 18) and 4 cases with Mouth Opening between 15 to 25 mm. This difference was statistically significant (P value ≤ 0.05).
Figure 2: ROC curve showing cut off value for HIF-1α in OSMF

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Figure 3: Graph showing correlation between Risk group and mouth opening in OSMF

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Table 3: Correlation of Risk Groups with HIF-1α Immuno-Scoring in the Study Cases

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   Discussion Top

A gradual increase in HIF-1α expression was observed as the stage of OSMF advances similar to the findings of Chaudhary et al.[13] Contradictory to this, Tilakaratne et al.,[14] was not able to decipher statistically significant difference between the disease stage and staining density of HIF-1α in the OSMF epithelium. In present study, HIF-1α expression increased as the density and thickness of collagen fibres increase. Norman JT et al.,[15] suggested that hypoxia and HIF 1α co-ordinately up-regulates matrix production and decreases turnover in renal fibroblasts. They provided evidence for direct hypoxic effects on expression of genes regulating fibrogenesis and concluded that hypoxia plays important role in the pathogenesis of fibrosis.

The present study also demonstrates that HIF-1α expression correlated significantly with type of blood vessels seen in the histological sections of OSMF. It was maximum in the cases with constricted blood vessels followed by cases of mixed blood vessels and dilated blood vessels [Table 2]: To best of our knowledge no study has correlated type of collagen fibre bundles and type of blood vessels to HIF-1α expression in different histopathological grades of OSMF.

Fibrosis in OSMF is mostly mediated via transforming growth factor (TGF)-β as a result of arecoline challenge.[16] TGF β promotes deposition of collagen in the connective tissue which may lead to the constriction of Blood vessels. Compromised blood supply to overlying epithelium leads to overexpression of HIF - 1α in epithelial cells.[13] Recent literature shows that HIF-1α is associated with the upregulation of various growth factors including TGF-β.[14] Thus, a vicious cycle is formed which support our hypothesis that hypoxia plays a role in progression of fibrosis in OSMF once the disease process is initiated by arecoline in betel quid [Figure 4].
Figure 4: Role of HIF-1α in progression of Oral Submucous Fibrosis

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Present study reported significant difference in the expression of HIF 1α in OSMF and OSCC cases. Chaudhary et al.,[13] reported a gradual rise in HIF-1α intensity from OSMF to OSCC and to OSCC with OSMF group. A study done by Higgins DF et al.,[17] demonstrated that HIF-1α in renal diseases enhanced epithelial-to mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes. During EMT, epithelial cells dedifferentiate and express mesenchymal markers displaying phenotypic alterations. This results in the development of a more motile fibroblast-like cell.

The way cells change their metabolism under hypoxia shares many similarities to the way tumours cells change their metabolism to survive and proliferate.[18] A hallmark of malignant tumours is the elevated uptake of glucose even under normal oxygen conditions, known as aerobic glycolysis or the “Warburg effect.” It has been proposed that cancer cells have increased glycolytic rates despite the presence of oxygen because these cells have irreversible damages to OXPHOS. These alterations leads to distinct transformations like the upregulation of HIF-1α that help the neoplastic cell to survive in adverse conditions.[13] Our findings show that HIF-1α over-expression may indicate a role of hypoxia in malignant transformation of OSMF similar to other tissues such as the breast and prostate.[18] Association of HIF-1α with increased expression of proliferation markers, growth factors, anti-apoptotic molecules in other tissues further enhances the evidence for increased survival and possible malignant transformation of oral epithelium in the background of fibrosis.[19]

To best of our knowledge this is the first study to infer cut-off value for HIF-1α expression in OSMF. The cut-off of 39.6% HIF-1α positive cells suggest that those cases of OSMF having more than 39.6% of HIF-1α positivity are at higher risk of malignant transformation [Figure 3]. In addition, our study also shows that the high-risk cases correlated significantly with the reduced mouth opening in OSMF cases deciphering that patient with Mouth opening less than 25 mm are at higher risk of malignant transformation.

Jayasooriya PR et al.,[20] in their study revealed a significant increase in the incidence of epithelial dysplasia as the thickness of fibrosis increases in OSMF and concluded that lesions that showed increased fibrosis in OSMF were more likely to present with epithelial dysplasia [Box]. Combining the result of our study with this study we can hypothesize that more the expression of HIF-1α more will be the fibrosis and more will be the chances of dysplasia in the overlying epithelium and hence increased possibility of malignant transformation.

   Conclusion Top

The present study revealed that progression and malignant transformation in OSMF is mediated by HIF-1α in the background of fibrosis. Over expression of HIF-1α may occur even before histologic evidence of angiogenesis or invasion. Hence, it can be used as a predictor for malignant transformation in OSMF.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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Utsunomiya H, Tilakratne WM, Oshiro K, Maruyama S, Suzuki M, Ida-Yonemochi H, et al. Extracellular matrix remodelling in oral submucous fibrosis: Its stage specific modes revealed by immunohistochemistry and in situ hybridization. J Oral Pathol Med 2005;34:498-507.  Back to cited text no. 12
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[PUBMED]  [Full text]  
Tilakaratne WM, Iqbal Z, Teh MT, Ariyawardana A, Pitiyage G, Cruchley A, et al. Upregulation of HIF-1alpha in malignant transformation of oral submucous fibrosis. J Oral Pathol Med 2008;37:372-7.  Back to cited text no. 14
Norman JT, Clark IM, Garcia PL. Hypoxia promotes fibrogenesis in human renal fibroblasts. Kidney Int 2000;58:2351-66.  Back to cited text no. 15
Rai A, Ahmad T, Parveen S, Parveen S, Faizan MI, Ali S. Expression of transforming growth factor beta in oral submucous fibrosis. J Oral Biol Craniofac Res 2020;10:166-70.  Back to cited text no. 16
Higgins DF, Kimura K, Bernhardt WM, Shrimanker N, Akai Y, Hohenstein B, et al. Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition. J Clin Invest 2007;117:3810-20.  Back to cited text no. 17
Apte SP, Semenza GL, Sarangarajan R. Cellular Respiration and Carcinogenesis. 1st ed. New York: Springer-Verlag; 2008. p. 73-4.  Back to cited text no. 18
Tilakaratne WM, Iqbal Z, Teh MT, Ariyawardana A, Pitiyage G, Cruchley A, et al. Upregulation of HIF-1alpha in malignant transformation of oral submucous fibrosis. J Oral Pathol Med 2008;37:372-7.  Back to cited text no. 19
Jayasooriya PR, Jayasinghe N, Arachchige K, Mudiyanselage Tilakaratne W. Relationship between thickness of fibrosis and epithelial dysplasia in oral submucous fibrosis. J Investig Clin Dent 2011;2:171-5.  Back to cited text no. 20

Correspondence Address:
Kanika Sharma
Flat No. 706, A.R. Reflections, Raj Nagar Extention, Ghaziabad, Uttar Pradesh - 201 017
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijpm.ijpm_160_21

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