DESC:This Patent application is a cognate application of an earlier application No. 202121036456 dated 12/08/2021 and an earlier application no.202221035137 dated 20/06/2022 filed by applicant of the present application.

FIELD OF THE INVENTION
The present invention relates to in vitro assay, a kit and a method for oral cancer prognosis and diagnosis. More particularly the present invention relates to the in vitro assay, its method and the kit for detection of biomarkers specifically, salivary micro RNAs (miRNAs). Even more particularly, the present invention relates to identification of miRNAs signatures including miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p derived from salivary exosomes as potential biomarkers for early detection of oral cancer which infers to prognosis of the disease, survival rate prediction of OSCC patients, prediction of the aggressiveness of the tumor and detection of the sensitivity to chemotherapeutic drugs for deciding line of treatment in oral cancer patients.

BACKGROUND OF THE INVENTION AND DESCRIPTION OF THE PRIOR ART
Oral Squamous Cell Carcinoma (OSCC) is one of the major causes of morbidity and mortality worldwide with its predominance in the Indian subcontinent owed to the increased consumption of chewing tobacco. In spite of recent advancements, the 5-year survival rate has been disappointingly low. One of the major reasons for this is late stage diagnosis as there are no disease-specific biomarkers available for the early risk prediction of OSCC. Moreover, the conventional diagnostic strategies include repetitive invasive biopsies which are quite challenging, costly, painful, potentially risky for the patient, difficult when accessibility to deeper sites is required, and unable to predict tumour heterogeneity. Above all, the inadequacy of tissue and difficulty in accessibility of deeper tumor sites are some of the major limitations of tissue biopsy which increase the possibility of metastatic spread and requirement of repetitive biopsies to monitor mutational changes.

Tumour-derived exosomes (40-150 nm in diameter) are secreted in various body fluids like blood, saliva, urine, cerebrospinal fluid, amniotic fluid and are useful sources of liquid biopsies. These exosomes mediate intercellular communication through transmission of active biomolecules (mRNA, microRNA (miRNA), DNA, proteins, and lipids) into the tumour microenvironment. These exosomes are an attractive target for early diagnosis and risk prediction of cancer as they represent the molecular and mutational landscape of the tumour bulk. Given that saliva has close proximity to the oral cavity and can be collected using convenient non-invasive and cost effective methods, it is a good source of exosomes produced in oral cancers compared to other body fluids. Salivary exosomes preserve biomolecules like DNA, RNA and proteins within themselves and thus it is easier to obtain good quality intact RNA from salivary exosomes compared to whole saliva.

It is believed that in case of oral carcinoma, the genomic mutational landscape at the tissue level is inherently prone to be reflected in saliva as the malignant cells tend to shed exosomes comprising of biomolecules such as miRNAs from the tumor bulk into the saliva. However, till date, there is a lack of substantial research that focuses on using OSCC patient’s saliva derived exosomal miRNA profile for diagnosis.

The current solutions are heavily based on studies conducted on cell line derived exosomes or in tumor derived exosomes and emphasize on subtypes like nasopharyngeal or oropharyngeal cancers and etiological factors such as smoking tobacco which are not the primary sites or etiological patterns. Moreover, existing studies have focussed on identifying new biomarkers through Next Generation Sequencing techniques but have not explored their functional role in tumour progression thereby making it difficult to understand the mechanisms that regulate cell- cell interaction, gene expression and eventually the molecular basis of these miRNAs in oral carcinoma.

microRNAs (miRNAs) are small, conserved, non-coding RNAs that are involved in regulation of up to 60% of human protein coding genes in essentially all eukaryotic organisms. miRNAs from exosomes are emerging as a promising tool for non-invasive approaches due to their efficacy, stability and ability to reflect tumor expression patterns. These circulatory miRNAs have been isolated from the plasma or serum samples of some malignancies and have a potential role in early diagnosis, assessment of disease progression, relapse and real-time monitoring of therapies.

microRNAs (miRNAs) are molecules with regulatory function and marked tissue specificity that can modulate multiple targets belonging to several pathways. They are frequently deregulated in cancer and could constitute a new class of blood-based biomarkers useful for cancer detection and prognosis definition because, for their nature, they seem to remain rather intact and stable and are detectable with simple assays like quantitative real-time PCR (qRT-PCR). Certain studies showed that plasma levels of specific miRNAs have remarkable sensitivity and specificity to distinguish cancer patients from healthy subjects.
The biological function/role of miR-1307 is still undefined. Functional studies of miR-1307 are still very limited, current evidence supports that miR-1307 is associated with tumor cell proliferation, differentiation, and possibly tumorigenesis. It has been associated with chemoresistance in ovarian cancer and breast cancer.

Reference may be made to US20090317820A1 entitled as “Micro-RNA profile in human saliva and its use for detection of oral cancer”. The invention provides the methods and kits of detecting micro-RNA i.e. miR-200a or miR-125a in human saliva for diagnosis of oral cancers and the correlation between such micro-RNA and oral cancers. The main objective of the invention is to provide the method for diagnosis, prognosis, monitoring efficacy of a treatment, identification of disease state, and providing probes or primers for use in detecting miRNA in saliva.

Reference may be made to US8088591B2, which related to the biomarkers for detection and diagnosis of head and neck squamous cell carcinoma. The invention involves novel, sensitive and specific markers and methods for diagnostics and monitoring of head and neck squamous cell carcinoma (HNSCC). The disclosed method is based on detection of combination or individual biomarkers like CD44, hyaluronic acid (HA) and hyaluronidase (HAase) in saliva, useful for early detection of HNSCC. The objective of the invention is to detect/diagnose HNSCC, monitoring of effectiveness of the treatment, indicating of the severity of HNSCC, predicting of increased likelihood of recurrence.

The reference may be made to JP2020068673, which relates to an oral cancer determination device, method, program and kit that can determine oral cancer with low invasion and with high accuracy. The disclosed device is based on the expression level of microRNA like hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-183-5p, hsa-miR-423-5p, hsa-miR-5100 and hsa-miR-144 in a body fluid sample. The main objective of the invention is determine the presence or absence of oral cancer by comparing the expression level of the microRNA with a reference value, determination of prognosis of oral cancer based on expression level of microRNA.

There is increasing evidence pointing towards circulating miRNA profiling from direct saliva of oral cancer patients. However none of these approaches have been translated into routine clinical use. Hence there remains a dire need to identify biomarkers able to predict prognosis of the disease that can be used for predicting the survival of OSCC patients, prediction of aggressiveness of the tumor and detection of the sensitivity to chemotherapeutic drugs for devising a personalized drug therapy.
Therefore, the present invention identifies miRNAs signature from salivary exosomes as novel target that is exclusively expressed in OSCC samples by a comprehensive comparative analysis. The present invention provides in vitro assay for salivary miRNAs as a biomarker in oral cancer detection, a method and a kit development. The data demonstrates that miRNA including miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p are useful biomarkers for predicting disease progression and prognosis. Additionally, the present invention proposes a mechanism by which miRNAs regulate chemoresistsance and progression of OSCC.
OBJECTIVES OF THE INVENTION
Primary object of the present invention is to provide an in vitro assay for salivary miRNAs particularly miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p as a biomarker in oral cancer detection, an assay method and a kit development.

Yet another object of the present invention is to provide an in vitro assay for salivary miRNAs,particularly miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p detection, an assay method and a kit development for early diagnosis of oral cancer.

Still another object of the present invention is to provide an in vitro assay for salivary miRNAs,particularly miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p detection and anassay method and a kit development for monitoring chemo sensitivity of the tumor.

Still another object of the present invention is to provide an in vitro assay for salivary miRNAs, particularly miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p detection, an assay method, and a kit development for early prediction of relapse in oral cancer.

Still another object of the present invention is to provide an in vitro assay for salivary miRNAs, particularly miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p detection and an assay method and a kit development for predicting the survival rate of OSCC patients.

Still another object of the present invention is to provide an in vitro assay for salivary miRNAs particularly miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p detection and an assay method and a kit development for predicting progression of the tumor.

An additional object of the present invention is to develop a kit for effective prognosis of oral cancer based on salivary miRNAs, particularly miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p expression. The kit is helpful for a physician to predict the progression of leucoplakia to active tumor in case of patients with OSCC.

Another object of the present invention is to provide quantification method for miRNAs inside exosomes from the saliva.

Another object of present invention is to elucidate the plausible mechanism by which miRNA regulates mRNA targets (THOP1, EHF, RNF4, GET4, RNF114) thereby inducing disease aggressiveness and therapeutic refractoriness in OSCC patients.

SUMMARY OF THE INVENTION
The present invention deploys usage of miRNA-mRNA regulatory networks for identification of miRNA signature from salivary exosomes of oral cancer patients for early detection of tumours. The five miRNAs (miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p) are differentially expressed in salivary exosome samples from OSCC patients compared to controls. These miRNAs showed significantly higher sensitivity, specificity and association with disease progression, recurrence, and chemo-resistance in OSCC patients. Out of the five expressed in the salivary exosomes, miR-1307-5p is found to be an independent risk predictor, prognosticator for OSCC patients and demonstrated a significant clinical association with disease progression, locoregional aggressiveness and chemotherapeutic refractoriness.

The present invention relates to the in vitro assay, its method and the kit for detection of biomarkers specifically, salivary micro RNAs (miRNAs), particularly miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p derived from salivary exosomes as a potential biomarker for early detection of oral cancer which infers to prognosis of the disease, survival rate prediction of OSCC patients, prediction of the aggressiveness of the tumor and detection of the sensitivity to chemotherapeutic drugs.
According to one embodiment of present invention, the assay method for isolation of miRNA from salivary exosomes of OSCC patients involves the steps comprising of collecting a tumor and a leukoplakia tissue, buccal scrapings and saliva sample from OSCC patients, followed by freezing and storing simultaneously at a temperature ranging between -75ºC to -85ºC; pooling samples from buccal scrapping and salivary exosomes of healthy volunteer to generate reference sequence; sequencing the samples from the tumor and leukoplakia tissue, the buccal scrapings and saliva samples by small RNA and mRNA sequencing; validating the sequenced samples by real time PCR; isolating the exosomes from saliva of the OSCC patients and healthy individuals; characterizing the size of the exosomes by Nanoparticle Tracking Analysis (NTA), Transmission electron microscopes (TEM) and flow cytometry; extracting the RNA from the exosomes as well as tissue samples; sequencing of miRNAs from the extracted total RNA of the samples via Illumina NextSeq500 platform; profiling of transcriptome of the tumour tissues, the salivary exosomes from OSCC patients and library generation of transcriptome sequencing, and predicting gene targets of miRNA by TargetScan v8.0 prediction algorithm.
According to another embodiment of present invention, based on the differentially expressed genes, protein-protein interaction networks are generated using the STRING database. Network visualization is performed using the open-source software platform Cytoscape (v3.6.1). Further, these differentially expressed genes are also imported into Ingenuity Pathways Analysis software (IPA) (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA) and are subjected to regulatory network analysis. Significant pathways based on differentially expressed genes are identified. For canonical pathway analysis, the -log (p) >2 is taken as the threshold, and Z-score >2 is defined as the threshold of significant activation, whereas Z-score <-2 is defined as the threshold of significant inhibition.
According to one aspect of present invention, involves identification of miRNA-mRNA gene regulatory networks that contributes to the early diagnosis of oral carcinoma by establishing the molecular mechanisms responsible for transforming a normal epithelial phenotype into a malignant one. The invention involves isolation of salivary exosomes from patients diagnosed with leukoplakia and OSCC and healthy controls, characterization of exosomes by NTA, TEM, and flow cytometry. The NTA results shows, an exosomes isolated from OSCC patients saliva contained 10-fold more exosomes than that of the controls. A TEM result represents sphere-shaped vesicles with a mean radius of 40-50 nm, which is consistent with the NTA profiles. Further, flow cytometry analysis indicated that the presence of the tetraspanins CD9, CD63, and CD81 (86-88% exosomes expressed these markers) in salivary exosomes from both patients and healthy counterparts, thus confirming that the isolated vesicles comprise pure exosomal population.
According to another aspect of present invention, the RNA extraction from the exosomes is performed by modifying the existing method of Prendergast et al. The extraction step involves addition of 750µL TRIzol LS reagent and 200µL chloroform to each salivary exosome sample (40µL); mixing thoroughly by vortexing for 30S, incubating for 10 min at room temperature, phase separation by centrifuging at 12,000 × g at 4 °C for 15 min. The upper aqueous phase is collected followed by addition of sodium acetate (3 M, pH 5.5) equal to 10% of the sample volume, addition of 5µL glycogen and absolute ethanol, mixing of sample, incubation overnight at -80 °C, centrifuging at 16,000 × g at 4 °C for 30 min to pellet the RNA. The pellets are washed with 70% ethanol, centrifuged at 16,000 × g at 4 °C for 5 min, air drying of pellet and resuspension in 32µL of nuclease-free water to get purified RNA pellets.
According to another aspect of present invention, involves identification of differentially expressed miRNAs enriched in both salivary exosomes and primary tumours from OSCC patients by miRNA profiling. The invention involves RNA-Seq analysis of small RNAs to identify differentially expressed miRNAs in tumours and salivary exosomes derived from OSCC patients compared to those derived from age and sex-matched controls. The results from this study shows that 188 miRNAs are differentially expressed (log2 fold change + 2; adj p-value < 0.05) in tumour tissues, 181 miRNAs in leukoplakia samples, 316 miRNAs in salivary tumour exosomes and 69 miRNAs in salivary leukoplakia exosomal samples compared to those in their normal counterparts. Amongst these, 47 miRNAs are found to be differentially expressed across all sample types (saliva and tissue) and stages, including pre-cancerous lesions (Figure 3a). A comparison of our results with the data from The Cancer Genome Atlas (TCGA) database showed that 27 of these differentially expressed miRNAs exhibited significantly differential expression between both datasets. Based on a stringent cut-off for adj p-value (p 2), 12 miRNAs (10 downregulated and 2 upregulated) are found to be robustly differentially expressed across all sample types. These 12 miRNAs are further validated by means of PCR with a validation set comprising a larger patient cohort (Table 1). Seven miRNAs (miR-140, miR143, miR-145, miR-30a, miR-21, let-7i, and miR-423) are found to have significant differential expression in both tissue and salivary exosome samples derived from OSCC patients compared to those derived from controls. This indicates that differentially expressed miRNAs enriched in salivary exosomal fractions could reflect the miRNA profile of the primary tumour site.
According to another aspect of the present invention, involves identification of robust miRNA signatures, particularly miR-30a, miR-140, miR-143, miR-145 and miR-1307-5p for the early detection of oral cancers using the liquid biopsy approach.
In accordance with one aspect of the present invention, involves conducting ROC analysis on seven significantly expressed miRNAs, particularly miR-140, miR 143, miR-145, miR-30a, miR-21, let-7i, miR-423 both individually and collectively to determine their utility as potential early diagnostic markers and generate a predictive model based on miRNA expression and verify its sensitivity and specificity.

In accordance with one aspect of the present invention, the potential utility of this 4-miRNA model i.e miR-30a, miR-140, miR-143, and miR-145 having similar efficacy irrespective of the sample type (saliva or tissue) in effectively differentiating the cancerous and pre-cancerous patients from the control counterparts. Thus exosome-derived miRNAs is used to differentiate between non-cancerous and cancerous cohorts. on assessing various combinations of miRNAs, miR-30a, miR-140, miR-143, and miR-145 showed an AUC of 0.92 (p < 0.0001) with 92% sensitivity and 98% specificity in tissue samples as compared to the seven miRNAs in the validation set of tissue samples from both OSCC and leukoplakia patients, which had an AUC of 0.8 (p < 0.0001) with 80% sensitivity and 85% specificity. Similarly, ROC analysis of salivary exosomes revealed an AUC of 0.97 (p < 0.0001) with 95% sensitivity and 98% specificity for the 4-miRNA combination model, which is higher than that of the 7-miRNA model and in accordance with the findings for the tissue samples.

In accordance with one aspect of the present invention, four-miRNA signature i.e.miR-30a, miR-140, miR-143, and miR-145 correlates with disease progression, therapeutic refractoriness, and relapse. Expression levels of the four significantly expressed miRNAs (miR-140, miR-143, miR-145, and miR-30a) are correlated with OSCC tumour stages using a student’s t-test to assess the feasibility and effectiveness of these miRNAs as biomarkers. The significant upregulation is observed in the expression of miR-30a, miR-140, and miR-145 in tissue samples and of miR-30a, miR-140, and miR-143 in salivary exosomal samples in early stages (stage-I and II; n = 20) compared to that in late-stage patients (stage-III and IV; n = 30) in tissue as well as saliva samples (p<0.05). Also miR-140, miR-145 and miR-143 shows significantly downregulated expression (p<0.05) in recurrent OSCC tumour tissue (n = 8) samples as compared to that in non-recurrent samples (n = 8). Similarly, all the four miRNAs included in our predictive model has significantly downregulated expression in the chemo-resistant (n=5) cohort compared to that in the chemo-sensitive patient cohort (n = 5; p <0.05).

In yet another aspect of the invention entails RNA-seq analysis-based identification of differentially expressed genes in tissues from OSCC patients compared to those in tissues from normal controls. A total of 5868 mRNAs that are significantly expressed (log2 fold change > 2; adj p-value < 0.01) across these datasets irrespective of the varying clinicopathological status of patients and differences in sequencing techniques are observed.

In yet another aspect of the invention, the present invention elaborates prediction of miRNA targets and identification of regulatory miRNA-mRNA networks. The identified 4-miRNA signature i.e. miR-30a, miR-140, miR-143, and miR-145 seems to target these 16 hub genes to modulate major effector functions, and cellular and biological mechanisms responsible for oral cancer progression.

In one aspect of present invention, the present invention involves Identification of miRNA-mRNA regulatory networks modulate signalling pathways responsible for oral cancer progression. The top canonical pathways which are statistically significant are activation of IL-15 production, activation of MSP-RON signalling in cancer cells, activation of FAK signalling, and inhibition of PTEN signalling pathways, the epithelial-mesenchymal transition (EMT) pathways which are implicated to play a vital role in oral cancer disease progression, aggressiveness, resistance to conventional therapy, and relapse.

In still another aspect of the invention, the differential expression profiles of the 16 hub genes clearly indicates 16 identified hub genes controlled by the four miRNAs predicts disease progression and poor prognosis of oral cancer patients.

It has been found according to this invention it is indicative of the fact that the said Small RNA sequencing analysis identifies miR-1307-5p, a miRNA exclusively expressed in tissues and salivary exosomes of OSCC patients. Further the validation by using Real time PCR indicates upregulation of miR-1307-5p in OSCC tissue (FC:10.3±7.9, p-value: 0.02) and salivary exosomal samples (FC:7.3 ± 4.5, p-value: 0.0083). The receiver operator characteristic (ROC) curves and area under the curve (AUC) for miR-1307-5p showed statistically significant diagnostic strength of this miRNA with sensitivity and specificity of 99.99% across all sample types (AUC:0.99, 95 % CI, p<0.001). Also miR-1307-5p is able to predict poor overall survival rate more effectively and significantly (50% patients demonstrated mortality within 17 months) compared to miR-21-5p (p-value<0.001).
A further advantageous of the present invention is miR-1307-5p demonstrates clinical association with disease progression, aggressiveness and therapeutic refractoriness. Increased levels of miR-1307-5p are observed in patients with high grade tumours (III/IV) in tissues (FC = 14.45 ± 9.76, p-value: 0.04) compared to low grade tumours (I/II) (FC = 5.37 ± 3.25). These findings are consistent in salivary exosomal samples of high grade (FC = 10.9 ± 5, p-value: 0.02) patients as compared to low grade OSCC patients (FC = 3.1 ± 1.2) (Figure 3(a)). Moreover, elevated levels of miR-1307-5p is observed in patients with locoregional aggressiveness (N1, N2, N3) in both tumour tissues (FC:17.5 ± 10, p-value: 0.03) and salivary exosomal samples (FC:13 ± 4, p-value: 0.0037) in comparison to those who do not report lymph node involvement (N0) (tissue FC = 7.5 ± 4.5; salivary exosome FC = 5 ± 2.7).

An additional advantage of the present invention is expression levels of miR-1307-5p in chemoresistant OSCC patients compared to chemosensitive patients showed complete disease remission post treatment. Thus the presence of miR-1307-5p is clinically associated with disease progression, local aggressiveness and chemotherapeutic refractoriness, thus making it an ideal prognosticator for OSCC patients.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Reference will be made to embodiments of the invention, examples of which may be illustrated in accompanying figures. These figures are intended to be illustrative, not limiting. Although the invention is generally described in the context of these embodiments, it should be understood that it is not intended to limit the scope of the invention to these particular embodiments.
Figure 1 and Figure 2 represents an assay method for identification of miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC)
Figure 3 represents characterisation of exosomes based on size, concentration, and expression of antigens
Figure 4 represents a differential expression of miRNAs in OSCC patients compared to healthy controls
Figure 5 represents differentiation between cancerous and non-cancerous sample by four miRNA panel with very good sensitivity and specificity
Figure 6 represents association of identified four miRNA signature with various clinicopathological parameters
Figure 7 represents differential expression of miR-1307-5p and poor survival rate prediction in oral cancer patients
Figure 8 represents expression level of miR-1307-5p in late stage tumours and refractory, chemoresistant tumours
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a comprehensive comparative analysis of miRNA and mRNA profiles of tumour tissue and salivary exosomes derived from leukoplakia (pre-malignant) and oral cancer patients and normal controls. The present invention involves identification of miRNA-mRNA gene regulatory networks that play an important role in the early diagnosis of oral carcinoma. The results provide insights into molecular mechanisms responsible for transforming a normal epithelial phenotype into a malignant one. This is the first study to explore the correlation between saliva and tumour derived miRNA-mRNA regulatory networks, which eventually facilitates early diagnosis of oral cancer. Further, the invention identifies miR-1307-5p as a novel candidate miRNA that is exclusively expressed in OSCC samples. The data demonstrates that miR-1307-5p is a useful biomarker for predicting disease progression and prognosis. Additionally, the present invention proposes the mechanism via which miR-1307-5p regulates chemoresistance and progression of OSCC.
According one embodiment of present invention, an assayfor identification and quantification of the salivary miRNAs involves collection of salivary sample and oral squamous tumour tissue followed by small RNA sequence analysis. The process is represented in Figure 1 and detailed in Figure 2. This analysis shows unregulated expression for the miRNAs like miR-1307-5p and the panel of four miRNAs i.e. miR-30a, miR-140, miR-143, and miR-145. The levels of the miR-1307-5p and the panel of four miRNAs are correlated with disease progression, aggression and chemotherapeutic resistance. Further the transcriptome sequencing shows down regulation of target genes which modulate important cellular processes like cell proliferation, apoptosis, angiogenesis, and maintenance of cancer stem cells and regulated by miR-1307-5p and the panel of four miRNAs.

Figure 2 relates to an assay method for identification of miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC).
Figure 3 relates to the characterisation of exosomes based on size, concentration, and expression of antigens. According to one embodiment of present invention,the size and the concentration of salivary exosomes from healthy volunteers and OSCC patients is estimated using Nanoparticle Tracking Analysis (NTA), transmission electron microscope and flow cytometry. The Figure 3 (a-b) representsthe size and concentration of salivary exosomes from healthy volunteers and OSCC patients using Nanoparticle Tracking Analysis (NTA). The x-axis indicates the size distribution of particles and the y-axis shows the relative concentration (particles/ml) in NTA;the NTA results show a single peak for the concentration of exosomes in the size range of 30 - 40 nm. Salivary exosomes derived from OSCC patients has a mean size of 31.5 ± 1.9 nm and a concentration of 7.31 × 108 particles/mL. The control exosomes has an average particle size of 39.8 ± 4.2 nm and a concentration of 6.67 × 107 particles/mL. The exosomes isolated from OSCC patients saliva contained 10-fold more exosomes than that of the controls. Figure 3 (c-d) represents TEM images of exosomes from control volunteer - and OSCC patient, (Scale: 200nm); the TEM results represents sphere-shaped vesicles with a mean radius of 40-50 nm, which is consistent with the NTA profiles. Figure 3(e-f) Representative plots of salivary exosomes stained with anti-CD63-Alexa fluor 488, anti-CD81-PE, and anti-CD9-APC antibodies analysed by flow cytometry.Flow cytometry analysis indicated the presence of the tetraspanins CD9, CD63, and CD81 (86-88% exosomes expressed these markers) in salivary exosomes from both patients and healthy counterparts (Figure 2 e–i), thus confirming that the isolated vesicles comprise pure exosomal population.
According to one embodiment of present invention, relatesto the RNA-Seq analysis of small RNAs to identify differentially expressed miRNAs in tumours and salivary exosomes derived from OSCC patients compared to those derived from age- and sex-matched controls. Figure 4 relates to a differential expression of miRNAs in OSCC patients compared to healthy controls. For tumour tissue samples from OSCC patients, the mean RNA integrity value is 7.6 with a 260/280 ratio ranging from 1.87–2.12 (average 2.06) and an average RNA concentration of 655.02 ng/µL across all samples. However, salivary exosomes has a mean RNA integrity value of 5.6 (260/280 ratio: 1.68-2.01) and an average concentration of 57.31 ng/µL. The low integrity value of RNA extracted from salivary exosomes may be due to the RNA-degrading enzymes present in saliva. An average of 25.4 million reads per sample were obtained for all tissue and salivary exosomes samples. After pre-processing, adapter sequence trimming and filtering low quality reads, the mean Q20 values for tissue-based libraries and salivary exosome-derived libraries are 97.6% (2,31,53,568) and 93.40%, respectively. Reads shorter than 15 nucleotides in length and a quality score <30 (reads less than 30) are excluded to ensure high quality of the sequencing results. Amongst these, an average of 68% (range 61-75%) of the reds are mapped to the reference human genome (hg38) with high confidence. 188 miRNAs are differentially expressed (log2 fold change + 2; adj p-value < 0.05) in tumour tissues, 181 miRNAs in leukoplakia samples, 316 miRNAs in salivary tumour exosomes and 69 miRNAs in salivary leukoplakia exosomal samples compared to those in their normal counterparts. Amongst these, 47 miRNAs are found to be differentially expressed across all sample types (saliva and tissue) and stages, including pre-cancerous lesions (Figure 4a). A comparison of our results with the data from The Cancer Genome Atlas (TCGA) database showed that 27 of these differentially expressed miRNAs exhibited significantly differential expression between both datasets (Figure 4b). Based on a stringent cut-off for adj p-value (p 2), 12 miRNAs (10 downregulated and 2 upregulated) are found to be robustly differentially expressed across all sample types. These 12 miRNAs are further validated by means of PCR with a validation set comprising a larger patient cohort. Seven miRNAs (miR-140, miR143, miR-145, miR-30a, miR-21, let-7i, miR-423) are found to have significant differential expression in both tissue and salivary exosome samples derived from OSCC patients compared to those derived from controls (Figure 4c). This indicates that differentially expressed miRNAs enriched in salivary exosomal fractions could reflect the miRNA profile of the primary tumour site.
Yet another embodiment of present invention relates to the establishment of an early diagnostic predictive model based on miRNA expression. Figure 5 represents differentiation between cancerous and non-cancerous sample by four miRNA panel with very good sensitivity and specificity. The seven significantly expressed miRNAs are subjected to ROC analyses both individually and collectively to determine their utility as potential early diagnostic markers and generate a predictive model based on miRNA expression and verify its sensitivity and specificity. On assessing various combinations of miRNAs, miR-30a, miR-140, miR-143, and miR-145 shows an AUC of 0.92 (p < 0.0001) with 92% sensitivity and 98% specificity in tissue samples as compared to the seven miRNAs in the validation set of tissue samples from both OSCC and leukoplakia patients, which has an AUC of 0.8 (p < 0.0001) with 80% sensitivity and 85% specificity (Figure 5a). Similarly, ROC analysis of salivary exosomes reveals an AUC of 0.97 (p < 0.0001) with 95% sensitivity and 98% specificity for the 4-miRNA combination model, which is higher than that of the 7-miRNA model and in accordance with the findings for the tissue samples (Figure 5b). These results indicate the potential utility of this 4-miRNA model having similar efficacy irrespective of the sample type (saliva or tissue) in effectively differentiating the cancerous and precancerous patients from the control counterparts. Further, PCA models are constructed for both salivary exosomes as well as tissue derived from oral cancer patients compared to their healthy counterparts using miR140, miR143, and miR145 and miR30a expression levels. These PCA plots demonstrate that leukoplakia and tumour miRNA profiles in both exosomes and tissues are clustered differently compared to those in control samples. Interestingly, among all profiled miRNAs, patient-derived miRNAs clustered separately. Thus, exosome-derived miRNAs are used to differentiate between non-cancerous and cancerous cohorts (Figure 5c).
Another embodiment of present invention depicts the correlation between four-miRNA signature and disease progression, therapeutic refractoriness, relapse. Figure 6 represents association of identified four miRNA signature with various clinicopathological parameters. Expression levels of the four significantly expressed miRNAs (miR-140, miR-143, miR145, and miR-30a) are correlated with OSCC tumour stages using a student’s t-test to assess the feasibility and effectiveness of these miRNAs as biomarkers. Characteristic stage-dependent variation in the expression levels of tissue miRNAs and salivary exosomal miRNAs is analysed between various stages (I, II, III, and IV) of OSCC. Significant upregulation is observed in the expression of miR-30a, miR-140, and miR-145 in tissue samples (Figure 6a) and of miR-30a, miR140, and miR-143 in salivary exosomal samples (Figure 6b) in early stages (stage-I and II; n = 20) compared to that in late-stage patients (stage-III and IV; n = 30) in tissue as well as saliva samples (p < 0.05). We further examined whether differential expression of the identified miRNAs has any clinical association with the relapse and chemo-resistance in patients using Student’s t-test. The results showed that miR-140, miR-145 and miR-143 significantly downregulated expression (p< 0.05) (Figure 5d). Among these, the expression of miR-140, miR-143, and miR-145 possess the potential to predict disease progression, recurrence, and therapeutic effectiveness in OSCC.
According to another embodiment of the present invention, miR-1307-5p is significantly overexpressed in salivary exosomes and tumour tissues derived from OSCC patients as compared to their non-cancerous counterparts. The miR-1307-5p acts as a potential prognosticator in OSCC patients, also predicts poor overall survival rate more effectively and significantly. The Figure 7 indicates (a) Box plots representing the expression of miR-1307-5p in 15 salivary exosomal samples of OSCC patients and 19 OSCC tissue samples compared to healthy controls. The expression levels of miRNAs are estimated using real-time PCR. The combined measure of sensitivity and specificity miR-1307-5p is 99.99% across (b) tissue and (c) exosome samples (AUC: 0.99, 95 % CI, p<0.001) Diagonal reference line acts as a performance measure of the diagnostic test. Note: AUC: Area Under the Curve, CI: Confidence Interval. (d) Kaplan–Meier plot of overall survival of miR-1307-5p and miR-21 generated from data available on TCGA_HNSC and data obtained from literature. The expression levels of miRNAs are estimated using real-time PCR. The data are normalized with U6 values, and the relative expression of miRNAs is analyzed using the ddCt method.Buccal scrapings and saliva samples obtained from healthy controls are used to calculate the relative expression of miR-1307-5p in OSCC tissue and salivary exosomes respectively. Error bars represent mean ± SD of three independent experiments (*p = 0.05; **p = 0.05; ***p =0.001).
According to another embodiment of present invention depicted enhanced expression of miR-1307-5p in tumour tissues and exosomes as an independent risk predictor, prognosticator and non-invasive prognostic biomarker for OSCC patients. miR-1307-5p demonstrated to be involved in disease progression, locoregional aggressiveness and chemotherapeutic refractoriness. Figure 8 represents expression level of miR-1307-5p in late stage tumours and refractory, chemoresistant tumours. Representative box plots depict relative expression patterns of miR-1307-5p in (a) early stages (Stage I/II) vs. late stages (Stage III/IV) of OSCC patient-derived tissue samples and salivary exosomal samples, (b) OSCC tissue and salivary exosome samples of patients with no nodal metastasis (N0) vs. those with metastasis (N1/2/3), and (c) salivary exosomal samples of OSCC patients with recurrent tumours and those with complete remission. (d) Expression of miR-1307-5p in CD44+ and CD44- cells determined by qRT-PCR. The data is normalized with U6 values, and the relative miRNA levels are analyzed using the ddCt method. Buccal scrapings and saliva samples obtained from healthy controls are used to calculate the relative expression of miR-1307-5p in OSCC tissue and salivary exosomes respectively. Expression of miR-1307-5p in OECM-1, CD44-, and CD44+ samples are compared to buccal scrapings obtained from healthy controls. Error bars represent mean ± SD of three independent experiments (*p = 0.05; **p = 0.01; ***p =0.001).
EXAMPLES
The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention in any manner.

Example 1, Sample collection from patient cohort and details for extraction of miR-1307-5p
Samples of resected tumour tissue and unstimulated saliva are collected from patients diagnosed with oral squamous cell carcinoma (OSCC), freezed and stored at -80ºC. Brush biopsies and unstimulated whole saliva of healthy individuals (matched for age and gender with the patients) with no etiological history of tobacco chewing and no clinically detectable oral lesions are used as normal controls, freezed and stored at -80ºC. The invention involves division of the participants into a discovery cohort, consisting of 4 buccal scrapings (pooled) and 3 salivary exosomes from controls (pooled), 12 OSCC tissue samples and 8 salivary exosome samples from OSCC patients.
Example 2, Exosomal isolation for extraction of miR-1307-5p
Saliva of OSCC patients and healthy controls is diluted with PBS (1:1) followed by centrifugation at 2000g for 10 minutes at room temperature. Exosomes are precipitated from the supernatant using Invitrogen™ Total Exosome Isolation Reagent from other body fluids kit. The precipitated exosomes are re-suspended either in TRIzol LS reagent or in PBS depending on the subsequent analysis to be performed.
Example 3, Exosome Characterization
The number of exosomes and their sizes are determined by conducting Nanoparticle Tracking Analysis (NTA) on NanoSight LM10 (Malvern Instruments Ltd) after diluting the exosome pellet in a physiologic solution (1:500). Imaging of negatively stained EVs (NanoVan, Nanoprobes, Yaphank, NY, USA) is carried out via transmission electron microscopy (TEM) using the Jeol JEM 1010 electron microscope (Jeol, Tokyo, Japan). Isolated exosomes are also characterised by flow cytometry using the vFC EV Analysis kit (Cellarcus Biosciences, San Diego, CA).The samples are further processed for further for staining. The instrument is then calibrated using a vesicle size standard of synthetic 50 nm liposomes. The samples (at optimal dilution) are then incubated with PE tagged antibodies against tetraspanins (TS; CD9, CD63, and CD81) (Cellarcus Biosciences, CA, USA) and APC tagged CD47 antibody (Thermo Scientific, Waltham, MA, USA) for one hour at room temperature. Samples are acquired on the CytoFLEX-LX (Beckman Coulter, Brea, CA, USA) flow cytometer. Thresholding is set on the violet laser side scatter (v-SSC) and voltages are set as per instructions in the Cellarcus protocol. All samples are acquired using the high flow rate for 2 minutes. The invention used a hierarchical gating strategy to identify the exosomes expressing CD47 and tetraspanins. Since our exosomes expressed CD47 strongly, CD47 and vFRed are used as primary discriminators. Double positive events are then analysed for expression of tetraspanins and events stained with vFRed, CD47 and tetraspanins are considered as exosomes. NTA suggests that salivary exosomes appeared as a homogenous population with mean size of 32.9 nm and concentration of 3.66 x 108 particles/ml.Further TEM results represent the sphere-shaped morphology of the vesicles with a size ranging from 40-50 nm which is consistent with NTA profiles. Salivary exosomes shows a similar violet-SSC and vFRed staining pattern as compared to the Lipo-50 synthetic liposomes indicating a size around 50 nM. These exosomes shows a strong expression of CD47. The expression of the tetraspanins CD9, CD63 and CD81 characteristic of exosomes are detected. Collectively, these findings substantiated that the vesicles derived from patients and healthy volunteers derived saliva constituted a pure exosomal population.
Example 4, RNA Extraction from exosomes and tissue sample:
Exosomes- RNA from salivary exosomes are isolated using the method described by Prendergast et al. RNA is extracted by adding 750µL TRIzol LS reagent (Thermo Fisher Scientific) and 200µL chloroform to 40µL exosomal sample. 5µL glycogen (5 mg/mL, Sigma Aldrich, St. Louis, MO) is used instead of 3µL in the original protocol. The RNA pellet is resuspended in 32µL of nuclease-free water. The final concentration of the purified RNA is determined using Qubit Fluorometer 4. Tissue- TRIzol reagent is used to extract total RNA from OSCC tissue (as per manufacturer's protocol). Purity and yield of extracted RNA is determined using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States) and Qubit Fluorometer 4 (Thermo Fisher Scientific, Waltham, MA, United States). Thus results indicate that miR-1307-5p is significantly overexpressed in salivary exosomes and tumour tissues derived from OSCC patients as compared to their non-cancerous counterparts.
Example 5, miRNA Sequencing The extracted total RNA from the samples is submitted for miRNA sequencing via Illumina NextSeq500 platform. Small RNA libraries are prepared using the QIAseq® miRNA Library Kit (Cat: 331502). The ligation of 3’ and 5’ adapters to the total RNA are followed by reverse transcription and amplification of the RNA using polymerase chain reaction (PCR) which resulted in the enrichment and barcoding of cDNA. Fragment size distribution of the libraries is followed by sequencing (12-15 million single end reads per run, per sample). FastQC is used to assess the overall quality of raw sequencing reads and Cutadapt (v. 1.8) is used for trimming the reads (11, 12). The filtered reads are mapped to the human reference genome (GRCh, version 38) using miRDeep2 (v0.0.7). The same toolkit is used to annotate potential novel miRNAs (cut-off: 6) and validate the predicted miRNAs from miRbase. DESeq2 is used to conduct statistical analysis of genes with read counts in = 30 of the samples. A likelihood ratio test is conducted to contrast the conditions (leucoplakia-control)-(tumor-control) and obtain differentially expressed miRNAs among 3 levels (log2 FC>2; adjusted P<0.01). An upregulation of miR-1307-5p is observed in OSCC tissue (FC:10.3±7.9, p-value: 0.02) and salivary exosomal samples (FC:7.3 ± 4.5, p-value: 0.0083). The survival analysis of the miR-21-5p with miR-1307-5p, suggested that miR-1307-5p could predict poor overall survival rate more effectively and significantly (50% patients demonstrated mortality within 17 months) compared to miR-21-5p (p-value<0.001). Collectively, these results suggest that miR-1307-5p indicates predictive power and patient outcome with high accuracy, and can efficiently function as a novel and independent prognostic biomarker for OSCC patients. Increased levels of miR-1307-5p are observed in patients with high grade tumours (III/IV) in tissues (FC = 14.45 ± 9.76, p-value: 0.04) compared to low grade tumours (I/II) (FC = 5.37 ± 3.25). These findings are consistent in salivary exosomal samples of high grade (FC = 10.9 ± 5, p-value: 0.02) patients as compared to low grade OSCC patients (FC = 3.1 ± 1.2). Moreover, elevated levels of miR-1307-5p are observed in patients with locoregional aggressiveness (N1, N2, N3) in both tumour tissues (FC: 17.5 ± 10, p-value: 0.03) and salivary exosomal samples (FC: 13 ± 4, p-value: 0.0037) in comparison to those do not report lymph node involvement (N0) (tissue FC = 7.5 ± 4.5; salivary exosome FC = 5 ± 2.7). The expression levels of miR-1307-5p in chemoresistant OSCC patients compared to chemosensitive patients that showed complete disease remission post treatment.
Example 6, Transcriptome sequencing
RNA extracted from tumour tissues and salivary exosomes of OSCC patients is subjected to mRNA-sequencing. cDNA libraries are generated using Illumina TruSeq RNA Sample Preparation v2 Kit (Lot# RS-122-2001, RS-122-2002) followed by paired-end sequencing on the Illumina HiSeq 2500 (2x150bp). Raw sequencing reads are subjected to quality assessment using FastQC followed by subsequent cleaning via Cutadapt. The refined reads are compared with a reference genome (GRCh, version 38) using Subread aligner. A total of 17.8-28.5 million reads are obtained from tissue samples. Further, an average of 62% of the reads mapped with the reference human genome (hg38) with high confidence. A total of 10660 differentially expressed mRNAs from tumour patient samples are identified as compared to their non-cancerous counterparts. On comparing these with the TCGA datasets, we found that a total of 5868 mRNAs are significantly expressed (log2FC= + 2; p<0.01) across these datasets irrespective of the varying clinico-pathological status of patients and discrepancy in sequencing techniques.
Example 7, miRNA Target Prediction
In order to identify the gene targets of miRNA, TargetScan v8.0 prediction algorithm is used. Genes are considered differentially expressed if FC = 2 and confidence is measured using predictive relative KD = -2. This study reports the association of THOP1, RNF4, GET4 and RNF114 with oral cancers. miRNAs regulation of gene expression results in an inverse correlation between miRNA and its target.

ADVANTAGES OF THE INVENTION
• Non-invasive, rapid and highly sensitive alternative to tissue biopsies
• Easy assay method to replicate in diagnostic lab equipped with RT-PCR equipment.
• Convenience in carrying out repeated assay for disease monitoring or monitoring response to therapy
• Assist in an improved patient monitoring, prediction of chemo-resistance and timely intervention if the patient is not responding to a particular form of therapy.
• Highly sensitive and specific for identification of miRNA-1307-5p in salivary exosomes for prognosis of the disease prediction of the survival of OSCC patients, prediction of aggressiveness of the tumor.
• The present invention provides a non invasive and point of care diagnostic method for oral cancer
• Point of care diagnostic method for oral cancer
,CLAIMS:We Claim,
1. An assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC), comprising the steps:
a. Collecting tumor tissue and saliva sample from OSCC patients, tissue and saliva samples from leukoplakia patients and buccal scrapings and saliva samples from non-cancerous volunteers followed by freezing and storing simultaneously at a temperature ranging between -75ºC to -85ºC;
b. Isolating the exosomes from saliva of the OSCC patients and healthy individuals;
c. Characterizing the size of the exosomes;
d. Pooling samples from buccal scrapping and salivary exosomes of healthy volunteer to generate reference sample;
e. Extracting the RNA from the exosomes as well as from tissue samples;
f. Sequencing the samples from the tumor and leukoplakia tissue, the buccal scrapings and saliva samples by small RNA and mRNA sequencing;
g. Validating the sequenced samples by real time PCR and,
h. Predicting gene targets of miRNA.

2. The assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC) as claimed in claim 1, wherein the exosomes isolated from the saliva of the OSCC patients having size ranging between 25 to 50 nm and tenfold more concentrated ranging between 5x107 - 5x108 particles/mL than that of control having size ranging between 25-50nm and concentration ranging between 1x107 particles/mL to 7x107 particles/ml respectively.

3. The assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC) as claimed in claim 1, wherein the exosomes isolated from the saliva of the OSCC patients consist of expression of CD47, CD9, CD63, and CD81.

4. The assay, a kit and a method for identification of salivary miRNA-based found in Oral Squamous Cell Carcinoma (OSCC) as claimed claim 1, wherein miRNA particularly miR-140, miR-143, miR-145, miR-30a, miR-21, let-7i, and miR-423 are differentially expressed in tissues and salivary exosomes of OSCC patients compared to non-cancerous controls.

5. The assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC) as claimed in claim 1, wherein the miR-1307-5p is an independent prognostic biomarker exclusively expressed in tissues and salivary exosomes of OSCC patients.

6. The assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC) as claimed in claim 1, wherein the sensitivity and the specificity of the combination of miR-140, miR-143, miR-145, miR-30a ranges between 90% to 100% across all sample types.

7. The assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC) as claimed in claim 1, wherein the sensitivity and the specificity of miR-1307-5p ranges between 95% to 100% across all sample types.

8. The assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC), as claimed in claim 1, wherein the levels of miR-1307-5p are significantly increased in the chemoresistant cohort compared to patients with incomplete remission.

9. The assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC) as claimed in claim 1, wherein the levels of miR-1307-5p are associated with tumour progression and aggressiveness.

10. The assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC) as claimed in claim 1, wherein the levels of a combination of miR-140, miR-143, miR-145, miR-30a are associated with tumour progression and aggressiveness in OSCC patients.

11. The assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC) as claimed in claim 1, wherein the levels of miR-1307-5p are associated with a higher chance of disease relapse.

12. The assay, a kit and a method for identification of salivary miRNA-based biomarkers found in Oral Squamous Cell Carcinoma (OSCC) as claimed in claim 1, wherein the levels of a combination of miR-140, miR-143, miR-145, miR-30a are associated with a higher chance of disease relapse.