ABSTRACT
PURPOSE: Study was aimed to quantify plasma level of total, short and long fragmented cell-free DNA (cfDNA) along with DNA integrity in patients with oral cancer, oral precancer and tobacco users without lesions and normal controls. In addition, study evaluated the correlation of cfDNA with clinicopathologic parameters of oral cancer. METHODOLOGY: Plasma samples were collected preoperatively from 44 patients with oral cancer, 40 patients with oral precancer, 40 tobacco users without any oral lesion and 40 healthy controls without any tobacco habit. cfDNA extraction was carried out from the plasma followed by quantitative and qualitative assessment of extracted DNA. Quantity of short and long fragmented DNA was assessed by using PCR with two different primer sets for the beta-actin gene, amplifying short (102 bp) and long (253 bp) products. The DNA integrity index was measured by calculating the ratio of quantity of long fragmented to short fragmented DNA. All quantitative cfDNA parameters were statistically analyzed to verify their correlation with clinicopathologic parameters. RESULTS: Results showed that total cfDNA level, short and long fragmented cfDNA concentration and DNA integrity was significantly higher in oral cancer group as compare to other (p=0.0001). Study demonstrated that there is no correlation total, short and long cfDNA and DNA integrity with tumor size and histologic type or grading. But positive correlation of total cfDNA was found with nodal metastasis (p=0.001) and clinical stages (p=0.006). CONCLUSION: Quantitative analysis of total cfDNA may be applied as a screening marker for early detection of precancer and cancer as well as for prognostication of oral cancer. Additionally, plasma levels of short and long fragmented cfDNA and DNA integrity index can be applied for early detection of oral cancer.
Subject(s)
Biomarkers, Tumor/blood , Carcinoma, Squamous Cell/secondary , Circulating Tumor DNA/blood , DNA/blood , Mouth Neoplasms/pathology , Precancerous Conditions/pathology , Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/blood , Carcinoma, Squamous Cell/genetics , Case-Control Studies , Circulating Tumor DNA/genetics , DNA/genetics , Follow-Up Studies , Humans , Lymphatic Metastasis , Mouth Neoplasms/blood , Mouth Neoplasms/genetics , Precancerous Conditions/blood , Precancerous Conditions/genetics , PrognosisABSTRACT
BACKGROUND: Methods of diagnostic molecular biology are routinely applied on formalin-fixed, paraffin-embedded tissues processed via conventional method. Recently, there has been a growing interest to use microwave technology in histopathology laboratories to overcome the deficiencies of the conventional processing method. Thefore, this study was aimed to compare and analyze the quality and quantity of DNA obtained from tissues processed by conventional and microwave tissue processing techniques and to further ascertain the applicability of the latter for PCR (polymerase chain reaction based research). METHODS: Thirty fresh tissues of oral squamous cell carcinoma (OSCC) were included, and each sample was cut into two equivalent halves. One tissue half was processed by conventional manual method whereas the other half was processed using a domestic microwave oven. DNA was obtained from all the tissues which were then subjected to Polymerase chain reaction (PCR) to evaluate GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene expression. RESULTS: The results revealed better DNA yield from microwave processed tissue while the quality of the DNA was alike from both the techniques. CONCLUSION: On the basis of the results obtained, it can be concluded that DNA produced by microwave processed tissues was similar to that obtained by conventional processing technique in terms of quantity and quality. Thus, microwave processed tissue samples can be successfully used for further molecular studies and researches.
