Biopsy tissue analysis
Diagnostic Testbiopsy samples for IHC staining and PCR
NCT Number: NCT03732872
Oral Sub mucous Fibrosis (OSMF) is essentially an imbalance between collagen metabolism and wound healing mechanism induced by arecanut chewing habit. Clinically the disease progresses in stages with patients presenting with burning sensation, intolerance to spicy food, vesicles particularly on the palate, ulceration and dryness of the mouth , fibrosis of the oral mucosa, leading to lips, tongue, and palate rigidity and finally trismus. As the disease is progressively debilitating and has potential to turn in to malignant cancer a study was designed to assess if there any tissue or saliva markers that can be assessed for early diagnosis and indicate malignant transformation if any.
Participants who had OSMF and habit history, patients without OSMF but habit history formed the case group where as normal patients without OSMF and no habit history were in control group. Eligible candidates who consented to participate in study were subjected to biopsy procedure and also their saliva samples were collected. Biopsy samples were subjected to immunohistochemistry (IHC) and polymerase chain reaction (PCR) to assess the EMT markers like vimentin, e-cadherin and collagen IV. miRNA copies were extracted from saliva and were subjected RT-PCR.
Research question was:
1. Is EMT a positive signature in OSMF. 2. Does histopathological grading and dysplasia in OSMF have any correlation with EMT. 3. Can aberrant EMT markers be a reliable indicator for risk assessment of early malignant transformation. 4. Can expression of mi RNA 21 in saliva predict the disease severity and more importantly assess risk of early malignant transformation in OSMF.
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Notify Me18 year and older
All sexes
Observational
Epithelial-mesenchymal transition (EMT) describes the process whereby cells shed their epithelial properties and adopt a more mesenchymal and invasive phenotype. A continuous molecular cross talk between epithelial and mesenchymal cells is required during embryonic development (epithelial mesenchymal transition) and probably plays an important role in pathologic process like wound healing and tumour progression. The development of carcinomas is due to the genetic changes within the target epithelium. As there is conversion of normal epithelium to potentially malignant and to squamous cell carcinoma, the stroma also changes from normal to activated or tumor associated called as the stromal reaction. 10 EMT can generate mesenchymal cells that express α-smooth muscle actin (αSMA), as seen in myofibroblasts. It is, however, unclear whether functional myofibroblasts seen in fibrosis or cancer derived from epithelial or endothelial cells
Hence EMT markers in OSMF were assessed in this study. the following markers chosen as:
Hence lower expression of E- cadherin is a sign of loss of epithelial integrity
Healthy volunteers accepted: Yes
Only the study team can determine whether someone qualifies for participation.
Inclusion criteria
Exclusion criteria
biopsy samples for IHC staining and PCR
Saliva samples for miRNA 21 expression analysis
Time frame: 10 days from sample collection
For IHC: The criteria used to define E-cadherin positive cells was brown staining in cell membrane of epithelial cells; the percentage of positively stained cells were scored as -
Percentage of POSITIVE CELLS SCORE No cells 0 ≤ 10% 1 11% -50 % 2 51%-80% 3
≥81% 4
Staining Intensity was also assessed in comparison with the positive control tissue and scored accordingly
INTENSITY SCORE Negative 0 Weak 1 Moderate 2 Strong 3
Time frame: 10 days from sample collection
vimentin positive cells was brown staining in cytoplasm of epithelial cells; the percentage of positively stained cells were scored as -
Percentage of POSITIVE CELLS SCORE No cells 0 ≤ 10% 1 11% -50 % 2 51%-80% 3
≥81% 4
Staining Intensity was also assessed in comparison with the positive control tissue and scored accordingly
INTENSITY SCORE Negative 0 Weak 1 Moderate 2 Strong 3
Time frame: 10 days from sample collection
collagen IV positive cells was brown staining in basement membrane cells; the percentage of positively stained cells were scored as -
Percentage of POSITIVE CELLS SCORE No cells 0 ≤ 10% 1 11% -50 % 2 51%-80% 3
≥81% 4
Staining Intensity was also assessed in comparison with the positive control tissue and scored accordingly
INTENSITY SCORE Negative 0 Weak 1 Moderate 2 Strong 3
Time frame: 10 days from sample collection
Considering the expression of miRNA21 in normal sample (N) as 1, the expression of all the samples were calculated The formula used is: number of copies = (amount * 6.022x1023) / (length * 1x109 * 650)
Time frame: 20 days after sample collection
After extraction of DNA from biopsy samples the primers for E-cadherin were designed using Primer Express software, synthesized and HPLC purified for real time PCR for quantification.
Time frame: 20 days after sample collection
After extraction of DNA from the biopsy samples the primers for Vimentin were designed using Primer Express software, synthesized and HPLC purified for real time PCR for quantification
Time frame: 15 days from sample collection
E cadherin level in tissue samples with respect to different Clinical expression of disease according to Kerr et al classification system.
Time frame: 15 days from sample collection
E cadherin level in tissue samples with respect to different Clinical expression of disease according to Kanna and Andrade et al classification system.
Time frame: 15 days from sample collection
Vimentin level in tissue samples with respect to different Clinical expression according to Kerr et al classification system.
Time frame: 15 days from sample collection
Vimentin level in tissue samples with respect to different histological expression according to Khanna and Andrade classification system.
Time frame: 15 days from sample collection
Collagen-IV level in tissue samples with respect to different Clinical expression category according to Kerr et al classification system.
Time frame: 15 days from sample collection
Collagen-IV level in tissue samples with respect to different histological expression according to Khanna and Andred et al classification system.
Time frame: 15 days from sample collection
miRNA 21 level in saliva samples with respect to different Clinical expression of OSMF according to Kerr et al classification system.
Time frame: 15 days from sample collection
miRNA 21 level in saliva samples with respect to different histological expression according to Khanna and Andred et al classification system.
The Oxford Dental College, Hospital and Research Center, Bangalore, India
Other
Risk Assessment of Malignant Transformation in Oral Submucous Fibrosis Using Epithelial Mesenchymal Transition (EMT) Signatures in Tissue Samples and Saliva
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