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  • UNC1999 One of the main reason for poor


    One of the main reason for poor survival of oral cancer patients is late detection. Clinical examination of the oral cavity and biopsy of the suspected lesion followed by histological analysis are generally used for diagnosis. Biopsy is an invasive procedure in which a portion of the suspected malignant tissue is obtained and further subjected to specialized and sophisticated histopathology or UNC1999 procedures. Biopsy is gold standard till date in detecting the histopathological type of a neoplasms and its degree of differentiation, and has been in practice since the 11th century [6,7]. Sometimes it becomes difficult to procure biopsy material due to inaccessibility of certain tumors, physical pain involved after the procedure, surgical complications, financial burden and lack of trained clinicians. Cell free nucleic acids (cfNAs), such as cell free DNA and RNA are present in body fluids of all individuals [8,9]. The expression profile of cfNAs varies between different disease states, so liquid biopsy has potential as a minimally invasive method for detection of disease, including malignant neoplasms [10,11].
    Serum/plasma miRNA as a biomarker in oral cancer Circulating miRNAs have been demonstrated to show different levels in the body fluids of OSCC patients compared to healthy individuals [18,19]. MiRNAs are highly stable in the serum or plasma leading to rising hopes for their future use as cancer biomarkers [20]. Circulating miRNAs are not digested by RNase and are stable at high pH, after boiling and after multiple freeze-thaw cycles [21,22]. While bound to the argonaute protein, miRNAs are stable in the extracellular environment whether encapsulated inside the extracellular vesicles such as exosomes or freely circulating [23,24]. Exosome-miRNAs are reported to represent a subset of about 3% of the entire amount of cell-free miRNAs [24]. In the last few years, several miRNAs have been reported to show differential expression levels in the serum or plasma of OSCC patients compared to healthy controls, although there is considerable variation in the types identified (Table 1). Yan et al. studied the expression of miRNAs in the plasma among OSCC patients from Denmark and China [25]. Next generation sequencing in Danish cohort identified increased levels of miR-26a-5p, miR-148a-3p, miR-21-5p and reduced levels of miR-486-5p [25]. On the contrary, quantitative analysis performed on the Chinese cohort identified lower levels of miR-375, miR-92b-3p and miR-486-5p, but did not show significant changes in the miRNAs that were found to be higher in the plasma of Danish cohort [25]. Since the method of plasma collection and handling of specimens in both cohort was uniform, the differences may be attributed to the population per se: to genetic inheritance or exposure to different risk factors for oral cancer. Another study on a Canadian population reported increased levels of 15 miRNAs and reduced levels of 7 miRNAs in the serum of OSCC patients compared to healthy controls [18]. Meanwhile Ayaz et al. reported a different set of miRNAs in the plasma of OSCC patients among a Turkish population [19]. Since both these studies involved a majority of patients having a Caucasian ethnicity, the differences in expression might be due to the sample source or method of sample processing. A detailed list of the expression profiles of miRNAs in the serum or plasma of OSCC patients is presented in Table 1. Despite the stability of circulating miRNAs and many encouraging results, these miRNAs have not found routine use as cancer biomarkers. Inconsistent results of miRNA profiles in serum and plasma have been reported [26]. Serum has been reported to show higher miRNA concentrations than corresponding plasma, suggesting that the coagulation process may affect the total amount of miRNA present [27]. Other issues relate to differences in sample handling and processing [28]. For example variations in speed and duration of centrifugation may influence the number of platelets and microvesicles remaining in the supernatant, and platelets may in turn unleash their miRNA leading to a variation in total amount of cell free-miRNAs [29]. Thus, whether studies are carried out on blood, plasma or on serum, each procedure should aim at reducing the risk of hemolysis [20]. For example, miR-451 and miR-16 are reported to show higher levels in OSCC serum [18]: both these miRNAs are present at high levels in RBCs, so hemolysis may result in elevated concentrations of these miRNAs in serum [30]. To reduce this risk, it has been suggested to centrifuge samples within 2 h from the time of blood collection [31]. Prolonged storage should also be avoided since that has been demonstrated to decrease the concentration of non-exosomal miRNA [31]. The incubation between sample collection and serum isolation is a critical factor for the stability of cell free nucleic acids. Furthermore, the type of anticoagulant in the collection tube can also affect result [32]. Samples can be screened for hemolysis by visual screening or by spectrophotometric quantification at 415 nm [26]. Exosomal purification before extraction of miRNA would minimize the effect of hemolysis. The volume of sample used was also seen to influence the outcome, as presence of inhibitors might affect synthesis of complementary DNA (cDNA) [33].