WO2016199094A1

WO2016199094A1 - Mirna aberration(s) in squamous cell carcinoma of head and neck (hnscc) and applications thereof

Info

Publication number: WO2016199094A1
Application number: PCT/IB2016/053444
Authority: WO
Inventors: Binay PANDA
Original assignee: Genomics Applications And Informatics Technology (Ganit) Labs
Priority date: 2015-06-10
Filing date: 2016-06-10
Publication date: 2016-12-15

Abstract

The present disclosure relates to aberrations in microRNAs as indicators/biomarkers of head and neck squamous cell carcinomas (HNSCC), method of analysing the role of said indicators/biomarkers in HNSCC, corresponding methods of detection of HNSCC, use and kits thereof. In particular, the present disclosure relates to aberrations in microRNAs in head and neck squamous cell carcinomas (HNSCC), specifically in squamous cell carcinoma of larynx and/or hypopharynx which serve as indicators/biomarkers for such carcinomas and associated methods.

Description

"miRNA ABERRATION IN SQUAMOUS CELL CARCINOMA OF HEAD AND

NECK (HNSCQ AND APPLICATIONS THEREOF"

TECHNICAL FIELD

The present disclosure relates to the field of Oncology, Genomics, Molecular biology and Bioinformatics. The present disclosure relates to indicators/biomarkers of head and neck squamous cell carcinomas (HNSCC), method of analysing role of said indicators/biomarkers in HNSCC, method of detecting HNSCC with the help of these indicators/biomarkers, method of detecting the indicators/biomarkers in a sample, and corresponding kits thereof. In particular, the present disclosure relates to aberrations in microRNAs in head and neck squamous cell carcinomas (HNSCC), specifically in squamous cell carcinoma of larynx and/or hypopharynx which serve as indicators/biomarkers for such carcinomas and associated methods. BACKGROUND OF THE DISCLOSURE

Head and neck squamous cell carcinomas (HNSCC) are a diverse group of tumors that originate from anatomically different locations, including nasal cavity, sinuses, lips, mouth, salivary glands, and throat. Cancers of the upper aero-digestive tracts (oral cavity, pharynx and larynx) are the sixth most common cancer worldwide. In addition to tobacco, lifestyle factors, dietary deficiencies, gastroesophageal reflux and infection with human papilloma virus (HPV) are other reported risk factors for larynx and hypopharynx cancers.

Large-scale molecular characterizations of HNSCC have been performed in recent years for different subsites. Despite the presence of a large body of information, molecular biomarkers are not currently used in the detection, treatment/ management of patients for this group of cancer.

Earlier expression studies, in HNSCC, particularly larynx and hypopharynx cancers, using immunohistochemistry (IHC), quantitative-PCR (q-PCR) and cDNA microarray linked genes to processes like cell adhesion, cell proliferation, differentiation, migration, apoptosis, transcriptional regulation and/or angiogenesis. Additionally, overexpression of MDM2 and ERB2 were described as predictors of loco-regional failure of chemoradiation in larynx carcinoma. However, the drawbacks of such gene expression studies in HNSCC (particularly, larynx and hypopharynx squamous cell carcinomas) are that the gene expression studies were focused on few genes. They were not genome-wide and not integrative with other alterations in the genome. Therefore, the earlier studies missed certain genes and their altered expressions playing an important role in these cancers.

Therefore, there exists a need for a holistic approach to analyze/identify specific indicators/molecular biomarkers linked to squamous cell carcinomas of head and neck, in particular squamous cell carcinoma of larynx and hypopharynx, and employ such biomarkers and corresponding methods for understanding and practical management of HNSCC.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to a method of detecting head and neck squamous cell carcinoma (HNSCC) in a sample having or suspected of having the HNSCC, said method comprising step of detecting aberration of miR-139, miR-203, miR424 and miR-503 in the sample to detect said HNSCC.

In an embodiment, the present disclosure relates to aberration of miR-139, miR-203, miR-424 for detecting HNSCC in a sample having or suspected of having the HNSCC.

In another embodiment, the present disclosure relates to use of aberration of miR-139, miR-203, miR-424 for detecting HNSCC in a sample having or suspected of having the HNSCC.

In another embodiment, the present disclosure relates to a kit for detecting HNSCC in a sample having or suspected of having the HNSCC, said kit comprising agent for detecting aberration of miR-139, miR-203, miR-424 and miR-503, individually, wherein the agent is selected from a group comprising primer, probe, antibody and nanoparticle, or any combination thereof corresponding to miR-139, miR-203, miR-424 and miR-503.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

Figure 1 depicts the cellular processes/pathways viz. viral carcinogenesis (Figure 1A), p53 signalling pathway (Figure IB), cell cycle (Figure 1C) and pathways in cancer (Figure ID) which are altered due to aberrations in miRNA such as hsa-miR-139, hsa- miR-203, hsa-mir-424 and hsa-mir-503.

DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to indicators/ biomarkers of head and neck squamous cell carcinomas (HNSCC). Since there is a need of molecular biomarkers of head and neck squamous cell carcinoma (HNSCC), the present disclosure studies 'miRNA' as biomarkers of HNSCC. miRNAs are small non-coding RNA molecules found in plants, animals, and some viruses, which functions in RNA silencing and post-transcriptional regulation of gene expression. Further, aberrations in miRNAs affect gene regulation, cell cycle processes/mechanisms and metabolic pathways. Thus, the role of miRNAs in head and neck squamous cell carcinoma is analysed in the present disclosure. More particularly, the present disclosure analyses the aberrations in miRNAs as biomarkers/indicators of HNSCC.

The present disclosure relates to a method of analysing the role of miRNA in HNSCC, said method comprising steps of:

a) screening/examination of sample having or suspected of having the HNSCC, optionally screening/examination of a sample without having the HNSCC patients;

b) isolating nucleic acid and performing quality control analysis;

c) preparing nucleic acid library;

d) sequencing the nucleic acid library of the tumor sample(s), optionally comparing the sequenced nucleic acid of the tumor sample and the nucleic acid sequences of normal sample;

e) performing sequencing data quality control analysis; and f) performing statistical analyses on reads to analyse the role of miRNA in HNSCC.

In another embodiment, the present disclosure relates to a method of analysing the role of miRNA in HNSCC, said method comprising steps of:

a) screening/examination of tumor sample(s) and corresponding matched normal sample(s) of HNSCC patients;

b) isolating nucleic acid and performing quality control analysis;

c) preparing nucleic acid library;

d) sequencing the nucleic acid library and comparing the sequences of tumor sample(s) and normal sample(s);

e) performing sequencing data quality control analysis; and

f) performing statistical analyses on reads to analyse the role of miRNA in HNSCC.

In an embodiment of the present disclosure, the analysing of the role of miRNAs in HNSCC involves both qualitative and quantitative analysis. In another embodiment of the present disclosure, the above method determines aberrations in miRNA and provides miRNAs as indicators/biomarkers in HNSCC, more particularly, larynx and/or hypopharynx carcinoma. In a preferred embodiment, the above method analyses alteration in miRNA expression and thereby determines the role of miRNAs in HNSCC.

In yet another embodiment of the present disclosure, the screening/examination of step (a) involves clinical/pathological screening or screening based on habits of the patient, or a combination thereof. In still another embodiment of the present disclosure, according to step d), whole transcriptome analyses are performed using sequencing experiments to profile miRNA expression landscape in HNSCC, in particular, larynx and/or hypopharynx carcinoma samples. In an embodiment, the expression of microRNAs of larynx and/or hypopharynx tumors is profiled using high-throughput sequencing experiments.

In an embodiment of the present disclosure, whole transcriptomes of larynx and/or hypopharynx tumors are sequenced using sequencing technology and significantly and unique differentially expressed miRNAs are identified in such tumors. Sequencing-by- ligation chemistry is employed to produce paired-end color-space 75x35 reads for all tumor and normal samples are matched. Details of the read QC and mapping statistics (total number of mapped reads, reads mapped to exonic, intronic and intergenic regions) are provided in Table 2. Across all samples, total reads are filtered by mapping to tRNA, rRNA, adaptor and repeat sequences, 9-18% of total reads do not map to the reference sequence primarily due to the presence of low quality of reads while 40-75%> reads get mapped to the reference genome (Table 2). Further, the QC-filtered reads are used to analyse expression changes. Thereafter, using target genes for these microRNAs, some of the pathways linked to cell cycle, p53 signaling, and viral carcinogenesis are also identified.

The present method identifies altered expression of miRNA-139, miRNA-203, miRNA- 424 and miRNA-503 in HNSCC, particularly in larynx and/or hypopharynx carcinomas. The present methods also link target genes for these miRNAs to processes like cell cycle, p53 signaling and viral carcinogenesis in HNSCC (larynx and/or hypopharynx carcinoma) also.

In an exemplary embodiment, the method identifies aberration in miR-139, miR-203, miR-424 and miR-503 in HNSCC.

In a preferred exemplary embodiment, the above method identifies aberrations in hsa- miR-139, hsa-miR-203, hsa-mir-424 and hsa-mir-503 in HNSCC. In a specific embodiment, the above method identifies the down regulation in the expression of hsa- miR-139, down regulation in the expression of hsa-miR-203 and up regulation in the expression of hsa-mir-424 and up regulation in the expression of hsa-mir-503.

In an embodiment, has-miR refers to homo sapien microRNA. For instance hsa-miR-139 is homo sapiens microRNA-139, hsa-miR-203 is homo sapiens microRNA-203, hsa-miR- 424 is homo sapiens microRNA-424 and hsa-miR-503 is homo sapiens microRNA-503.

As used in the present disclosure, head and neck squamous cell carcinomas (HNSCC) refers to cancers including but not limiting to cancers of oral cavity including the inner lip, tongue, floor of mouth, gingivae, and hard palate, nasopharyngeal cancer, oropharyngeal squamous cell carcinomas (OSCC), cancer of hypopharynx, laryngeal cancer and cancer of trachea. The said terms/phrases are used interchangeably in the present disclosure and should be construed accordingly.

In an exemplary embodiment, the method of analysing the aberration in miRNAs in HNSCC specifically involves the following steps:

a) performing screening by collecting details pertaining to clinical/pathological aspects such as staging, HPV, epidemiology, and habits of the patients from whom the samples are obtained;

b) isolating small RNA from tumor sample, optionally from normal samples, using Small RNA isolation kit selected from a group comprising Ambion's Mir Vana kit or Qiagen's miRNeasy Mini Kit;

c) preparing small RNA library by carrying out steps of hybridization, ligation, reverse transcription, purification of cDNA, gel size selection, amplification of size selected cDNA and purification;

d) quantifying the small RNA library using kit selected from group comprising Qubit HS kit, nanodrop and Agilent Bioanalyzer, or any combination thereof;

e) analysing size distribution of the library using analyzer selected from group comprising Agilent Bioanalyzer and nanodrop, or a combination thereof;

f) pooling tumor sample, optionally normal samples, into the small RNA library and enriching the pooled library;

g) sequencing the small RNA library using genetic analyser system- SOLiD 5500x1 genetic analyser system;

h) performing down-stream analysis of the sequenced data using software tool selected from a group comprising Lifescope and DeSeq2, or a combination thereof;

i) analysing pooled miRNA expression using web server- DESeq2 R package and determining the aberrations in miR-139, miR-203, miR-424 and miR-503.

In another exemplary embodiment, the aforementioned method of analysing aberration in miRNAs in HNSCC specifically involves the following steps:

a) performing screening by collecting details pertaining to clinical/pathological aspects such as staging, HPV, epidemiology, and habits of the patients from whom the samples are obtained; b) isolating small RNA from tumor and normal samples using Small RNA isolation kit selected from a group comprising Ambion's Mir Vana kit or Qiagen's miRNeasy Mini Kit;

f) pooling tumor/normal samples into the small RNA library and enriching the pooled library;

i) analysing pooled miRNA expression using web server- DESeq2 R package and determining the aberrations in miR-139, miR-203, miR-424 and miR-503..

Accordingly, the present disclosure specifically relates to microRNAs (miRNAs) as indicators/ biomarkers of HNSCC. In an embodiment of the present disclosure, aberrations in microRNAs serve as biomarkers/indicators of HNSCC.

In another embodiment of the present disclosure, aberration in microRNAs is observed in homo sapiens microRNA-139 (hsa-miR-139), homo sapiens microRNA-203 (hsa-miR- 203), homo sapiens microRNA-424 (hsa-miR-424) and homo sapiens microRNA-503 (hsa-miR-503) in HNSCC and serve as biomarkers of HNSCC.

As used in the present disclosure, the term "aberration" includes but is not limiting to alteration in expression including up-regulation/over expression or down-regulation/under expression, amplification, mutation, loss of heterozygosity, copy number variations, structural variations, gene fusion events, allelic expression, chromosomal abberations, epigenetic changes including DNA methylation, histone modification and non-coding RNA (ncRNA)-associated gene silencing or any combination of aberrations thereof. In still another embodiment of the present disclosure, "mutations" include but are not limiting to epigenetic mutation, transgenetic mutation, deletion, substitution and insertion or any combination thereof. In specific embodiments of the present disclosure, "aberrations" include up-regulation and down-regulation of miRNAs.

In an exemplary embodiment of the present disclosure, the miRNAs hsa-miR-139 and hsa-miR-203 are down regulated and miRNA hsa-mir-424 and hsa-miR-503 are up regulated in laryngeal and/or hypopharyngeal cancers. The present disclosure therefore detects aberrations in hsa-miR-139, hsa-miR-203, hsa-mir-424 and hsa-miR-503 as biomarkers of HNSCC, more particularly in squamous cell carcinoma of larynx and/or hypopharynx.

The present disclosure relates to a method of detecting HNSCC in a sample having or suspected of having HNSCC, wherein said method comprises determining aberrations in miRNAs such as miR-139, miR-203, miR424 and miR-503. In an embodiment of the present disclosure, determination of miRNA aberration includes analysing expression levels of the miRNAs. In a preferred embodiment, up-regulation and down-regulation of miRNA expression is determined to detect the HNSCC in a sample having or suspected of having HNSCC. In preferred embodiment, down regulation and up regulation in the expression of hsa-miR-139, hsa-miR-203, hsa-miR-424 and hsa-miR-503, respectively is determined to detect HNSCC.

In another exemplary embodiment of the present disclosure, the method of detecting HNSCC in a sample having or suspected of having HNSCC comprises acts of:

(a) contacting the sample with an agent or performing steps of a biomarker detection technique to determine aberration in hsa-miR-139, hsa-miR-203, hsa-miR-424 and hsa-miR-503; and

(b) detecting the HNSCC based on step (a) wherein aberration(s) in the hsa-miR-139, the hsa-miR-203, the hsa-miR-424 and the hsa-miR-503correlates to the presence of HNSCC in said sample or vice-versa.

In an embodiment of the method as described above, the aberration in miRNA for detecting HNSCC in a sample having or suspected of having the HNSCC relates to:

(i) down regulation in expression of hsa-miR-139;

(ϋ) down regulation in expression of hsa-miR-203; (iii) up regulation in expression of hsa-mir-424; and

(iv) up regulation in expression of hsa-miR-503.

In another embodiment of the present disclosure, aberration(s) in the hsa-miR-139, the hsa-miR-203, the hsa-miR-424 and the hsa-miR-503 are determined with the help of an agent selected from a group comprising primer, probe, antibody, nanoparticle, and a suitable interacting protein/biological agent capable of interacting with the hsa-miR-139, the hsa-miR-203, the hsa-miR-424 and the hsa-miR-503, individually in order to detect presence or absence of aberrations in the said miRNA, based on solid support or solution based assays/techniques. In a preferred embodiment, said agent is employed for determining aberration in the hsa-miR-139, the hsa-miR-203, the hsa-miR-424 and the hsa-miR-503.

In another embodiment of the present disclosure, the aberrations in the miRNA are identified by employing techniques selected from a group comprising but not limiting to solution- based assays, solid support based assays, PCR, Northern Blotting, ELISA, in- situ hybridization, luminescence/chemiluminescence-based assays, electrochemical assays, fluorescence- based assays, microarray and sequencing, or any combination thereof.

In yet another embodiment, the solution-based assays to detect miRNA aberration is selected from a group comprising but not limiting to Solution hybridization, PCR and luminescence- based assay, or any combination thereof. In still another embodiment, the solid support based assays employed to detect miRNA aberration is selected from a group comprising but not limiting to Northern Blot, fluorescence- based assays, ELISA and Microarray, or any combination thereof.

In still an embodiment of the present disclosure, HNSCC is selected from group comprising cancers of oral cavity including the inner lip, tongue, floor of mouth, gingivae, and hard palate, nasopharyngeal cancer, oropharyngeal squamous cell carcinomas (OSCC), cancer of hypopharynx, laryngeal cancer and cancer of trachea. In an exemplary embodiment of the present disclosure, the cancer is squamous cell carcinoma of larynx and/or hypopharynx. As used herein, the term 'sample' refers to any biological material/fluid/cell having or suspected of having tumor/cancer or miRNA aberration(s), or a biological material/fluid/cell which is not affected with tumor/cancer or miRNA aberration(s). Further, a sample may be derived from humans and/or mammals, or the sample may be any biological fluid prepared/obtained in a laboratory.

The present disclosure further relates to a method of detecting aberrations in miRNA in a sample having or suspected of having UNSCC, wherein the miRNA is detected in hsa- miR-139, hsa-miR-203, hsa-miR-503 and hsa-miR-424.

In an embodiment, said method of detecting miRNA aberrations comprises act of contacting the sample with an agent to determine aberration in hsa-miR-139, hsa-miR- 203, hsa-miR-424 and hsa-miR-503. In another embodiment, the method of detecting miRNA aberrations comprises act of performing steps of a biomarker detection technique to determine aberration in hsa-miR-139, hsa-miR-203, hsa-miR-424 and hsa-miR-503.

In an exemplary embodiment of the method of detecting aberrations in miRNA as described above for detecting UNSCC, constitutes the following:

(i) down regulation in expression of hsa-miR-139;

(ii) down regulation in expression of hsa-miR-203;

(iii) up regulation in expression of hsa-mir-424; and

(iv) up regulation in expression of hsa-miR-503.

In an embodiment, the method of detecting UNSCC in a sample having or suspected of having the UNSCC detects down regulation in the expression of miR-139 in the range about 2 fold to 7 fold, down regulation in the expression of miR-203 in the range about 2 fold to 7 fold, up regulation in the expression of miR-424 in the range about 2 fold to 7 fold and up regulation in the expression of miR-503 in the range about 2 fold to 7 fold.

In an embodiment, down regulation in the expression of miR-139 in the range about 2 fold to 7 fold, down regulation in the expression of miR-203 in the range about 2 fold to 7 fold, up regulation in the expression of miR-424 in the range about 2 fold to 7 fold and up regulation in the expression of miR-503 in the range about 2 fold to 7 fold in a sample having or suspected of having UNSCC, detects the UNSCC in the sample. In another embodiment of the present disclosure, aberration(s) in the miRNA are determined with the help of an agent selected from a group comprising primer, probe, antibody, nanoparticle and a suitable interacting protein/biological agent capable of interacting with miRNA/detecting miRNA, or any combination thereof. In a preferred embodiment, said agent is employed for determining aberration in hsa-miR-139, hsa- miR-203, hsa-miR-424 and hsa-miR-503.

In another embodiment of the present disclosure, the aberrations in the miRNA are identified by employing techniques selected from a group comprising but not limiting to solution- based assays, solid support based assays, PCR, Northern Blotting, ELISA, in- situ hybridization, luminescence/chemiluminescence-based assays, electrochemical assays, fluorescence- based assays, microarray and sequencing, or any combination thereof. In yet another embodiment, the solution-based assays to detect miRNA aberration is selected from a group comprising but not limiting to Solution hybridization, PCR and luminescence- based assay, or any combination thereof.

In still another embodiment, the solid support based assays employed to detect miRNA aberration is selected from a group comprising but not limiting to Northern Blot, fluorescence- based assays, ELISA and Microarray, or any combination thereof.

The present disclosure further relates to a kit for detecting HNSCC in a sample having or suspected of having the HNSCC, wherein the kit comprises agents selected from a group comprising primer, probe, antibody and nanoparticle, wherein the agent detects aberration in miR-139, miR-203, miR-424 and miR-503. Particularly the agent of the kit detects down regulation in the expression of miR-139, down regulation in the expression of miR- 203, up regulation in the expression of miR-424 and up regulation in the expression of 503, individually.

In another embodiment, the present disclosure relates to a kit for detecting miRNA aberration in a sample having or suspected of having HNSCC, wherein the miRNA is hsa-miR-139, hsa-miR-203, hsa-mir-424 and hsa-mir-503. In an embodiment, said kit comprises suitable agent(s) to detect aberration in hsa-miR-139, hsa-miR-203, hsa-mir- 424 and hsa-mir-503, optionally along with an instruction manual thereof. In another embodiment, the agent is selected from a group comprising primer, probe, antibody and nanoparticle, or any combination thereof.

In an embodiment, the kit for detecting HNSCC in a sample having or suspected of having the HNSCC detects down regulation in the expression of miR-139 in the range about 2 fold to 7 fold, down regulation in the expression of miR-203 in the range about 2 fold to 7 fold, up regulation in the expression of miR-424 in the range about 2 fold to 7 fold and up regulation in the expression of miR-503 in the range about 2 fold to 7 fold. In an embodiment, down regulation in the expression of miR-139 in the range about 2 fold to 7 fold, down regulation in the expression of miR-203 in the range about 2 fold to 7 fold, up regulation in the expression of miR-424 in the range about 2 fold to 7 fold and up regulation in the expression of miR-503 in the range about 2 fold to 7 fold, detected by the agents of the kit in a sample having or suspected of having HNSCC, detects the HNSCC in the sample.

In another embodiment, the present disclosure relates to aberration of miR-139, miR-203, miR-424 and miR-503 for detecting HNSCC in a sample having or suspected of having the HNSCC.

The aberration of miR-139, miR-203, miR-424 and miR-503 is detected by the method of the present disclosure or the kit of the present disclosure, or a combination thereof, wherein the aberration is selected from a group comprising up regulation in expression, down regulation in expression, amplification, mutation, loss of heterozygosity, copy number variation, structural variation, gene fusion, allelic expression, DNA methylation, histone modification and gene silencing, or any combination thereof.

In an embodiment, the aberration is down regulation in the expression of the miR-139 in the range of about 2 fold to 7 fold, down regulation in the expression of miR-203 in the range of about 2 fold to 7 fold, up regulation in the expression of miR-424 in the range of about 2 fold to 7 fold and up regulation in the expression of miR-503 in the range of about 2 fold to 7 fold, wherein the said aberration detects HNSCC in the sample having or suspected of having the HNSCC. In another embodiment, the present disclosure relates to use of aberration of miR-139, miR-203, miR-424 and miR-503 for detecting HNSCC in a sample having or suspected of having the HNSCC, wherein the aberration is selected from a group comprising up regulation in expression, down regulation in expression, amplification, mutation, loss of heterozygosity, copy number variation, structural variation, gene fusion, allelic expression, DNA methylation, histone modification and gene silencing, or any combination thereof.

In exemplary embodiment, the use of aberration for detecting HNSCC in a sample having or suspected of having the HNSCC is down regulation in the expression of the miR-139 in the range of about 2 fold to 7 fold, down regulation in the expression of miR-203 in the range of about 2 fold to 7 fold, up regulation in the expression of miR-424 in the range of about 2 fold to 7 fold and up regulation in the expression of miR-503 in the range of about 2 fold to 7 fold.

In another embodiment, the present disclosure relates to an agent for use in detecting aberration of miR-139, miR-203, miR-424 and miR-503 for detecting HNSCC in a sample having or suspected of having the HNSC, wherein the agent is selected from a group comprising primer, probe, antibody and nanoparticle, or any combination.

In exemplary embodiment, the agent detects HNSCC in a sample having or suspected of having the HNSCC by detecting down regulation in the expression of the miR-139 in the range of about 2 fold to 7 fold, down regulation in the expression of miR-203 in the range of about 2 fold to 7 fold, up regulation in the expression of miR-424 in the range of about 2 fold to 7 fold and up regulation in the expression of miR-503 in the range of about 2 fold to 7 fold.

The present disclosure also relates to detection of pathways/molecular mechanisms which are significantly altered in HNSCC, particularly larynx and hypopharynx carcinomas. In an exemplary embodiment, said detection of significantly altered pathways in HNSCC comprises steps of:

(a) analysing miRNAs aberrations in HNSCC; and

(b) performing pathway analysis of target genes of the aberrant miRNA to detect the altered pathway(s) in HNSCC. In an embodiment of the above method, the aberrant miRNAs are hsa-miR-139, hsa-miR- 203, hsa-mir-424 and hsa-mir-503 in HNSCC. In another embodiment of the above method, pathway analysis of miRNA target genes is performed using tool DIANA mirPath web server, (http://diana.imis. athena- innovation.gr/DianaTools/index. php?r=mirpath/index) along with using pathway information from KEGG database (Vlachos et al, Nucleic Acids Research 2012, doi:10.1093/nar/gks494).

In yet another embodiment of the above method, the important pathways altered in HNSCC are selected from a group comprising but not limiting to cell cycle pathways, p53 signalling pathways, pathways related to viral carcinogenesis, PI3K-Akt signaling pathway and pathways of cancer.

MicroRNA expression:

As described in methods disclosed above, four miRNAs with significantly altered expression levels in the sequenced HNSCC (particularly, larynx and/or hypopharynx) tumors are found. They are hsa-miR-139, hsa-miR-203, hsa-miR-424 and hsa-miR-503. Out of these, microRNAs miR-424 and miR-503 belong to the same cluster (miR- 424/503).

Further, pathway analysis of the miRNA target genes using mirPath is performed. As described in the methods disclosed above, four different KEGG pathways viz. cell cycle, p53 signaling, viral carcinogenesis and pathways of cancer are detected to be significantly altered (with -value 10^"13, 10^"9, 10^"7 and 10^"6 respectively). A list of all miRNA target genes, pathways, and gene annotations are provided in Table 4 (Examples). The methods of the present disclosure also provide that some of the aberrant miRNAs detected are related to regulation of cancer stem cell-like cells related markers affecting invasion and metastasis.

The technology of the instant application is further elaborated with the help of following examples, tables and figures. However, the examples, tables and figures should not be construed to limit the scope of the present disclosure. EXAMPLES:

MATERIALS AND METHODS

Informed consent and ethics approval:

Informed consent is obtained voluntarily from each patient enrolled in the study. Ethics 5 approval is obtained from the Institutional Ethics Committee of the Mazumdar Shaw Cancer Centre.

EXAMPLE 1

Patient samples used in the study, screening for clinical parameters and habits

10 The study involves 10 biological samples consisting of 5 tumor samples and 5 matched normal samples from the same patients (i.e. 5 pairs of samples). Details of the tumor specimens and matched normal samples collected and used in the study are presented in Table 1. Only those patients with histologically confirmed squamous cell carcinoma that had at least 70% tumor cells in the specimen are recruited for the study. Patients included

15 in this study underwent staging according to AJCC criteria at the Mazumdar Shaw Cancer Centre and the samples are accrued for the study. The treatment and surveillance are carried out as per NCCN guidelines (http://www.nccn.org/). Post-treatment surveillance is carried out by clinical and radiographic examinations as per the NCCN guidelines. All tissue samples are collected in RNAlater® solution (tissue storage reagent) at the time of

20 resection and stored at - 800 °C until further processing.

EXAMPLE 2

Library preparation and sequencing

Sequencing libraries are prepared for small RNA.

5 Small RNA: Small RNA is isolated from tumor and matched normal samples using Ambion's MirVana kit following manufacturer's instructions. Small RNA library is prepared for 5 tumor-normal pairs and its quality is analyzed on RNA6000 nanochip using Agilent Bioanalyzer. Small RNA SOLiD sequencing libraries are prepared following manufacturer's instructions (Life Technologies). In brief, the library preparation involves hybridization and ligation, reverse transcription, purification of cDNA using Minelute PCR purification kit (Qiagen , gel size selection, amplification of the size selected cDNA and purification using Purelink PCR micro kit (Life technologies). For the gel size selection, the Novex TBE urea 10% - PAGE gel system (Life technologies) is used to excise bands between 60-80 nucleotides. Each gel piece is cut into 4 portions and two such portions are used for the process of amplification. Thus, for each sample the cDNA library amplification is done in duplicates, with one gel piece in each tube. The final amplification of the size selected cDNA library is done for 15-18 cycles. Each of the libraries is labelled with a different barcode using SOLiD RNA Barcoding Kit, Module 1-16, which enables multiplexing. The libraries are quantified using Qubit HS kit (Invitrogen) and the size distribution is analyzed using DNA HS chip on Agilent Bioanalyzer. Post library preparation, the five tumor/normal pairs (10 samples) are pooled together in equimolar concentration into a single library and 0.4pM of the pooled library is used for the ePCR, using e80 scale, and enrichment, as per manufacturer's instructions. Following enrichment, the library is 3' modified, loaded onto a single lane of the SOLiD -61ane flowchip cell and sequenced on SOLiD 5500x1 genetic analyzer system. Sequenced data is obtained (35 bp single-end reads) in the XSQ format and further used for downstream analysis using Lifescope. EXAMPLE 3

Read QC, alignment, and read counts generation

For all the tumor and normal samples, reads are filtered against tRNA, rRNA, adaptor and repeat sequences using Lifescope (v2.5-r2.5.1). The remaining reads are aligned to the hgl9 reference sequence with default options. Of these, only primary alignments with minimum mapping quality of 10 are counted and the output is stored in a tab-delimited file, containing all gene annotations and their raw read counts. Details of the read QC and mapping statistics (total number of mapped reads, reads mapped to exonic, intronic and intergenic regions) are provided in Table 2. Table 2: Read statistics (read QC and Mapping) for miRNA

RESULTS: Across all samples, low quality reads are filtered by mapping to tRNA, rRNA, adaptor and repeat sequences, 9-18% of total reads does not map to the reference sequence due primarily to the presence of low quality of reads while 40-75%> reads get mapped to the reference genome. The QC-filtered reads are further used to analyze miRNA expression changes.

EXAMPLE 4

Differential miRNA expression and miRNA pathway analysis

The gene-wise read counts of all the tumor and normal samples are pooled, and only those genes with a non-zero read count in at least one sample are selected.

Normalization of the raw counts is done using the DESeq R package (R-3.1.0, DESeq- 1.16.0) and tested for differential expression with the following combinations: 1) all tumors vs their matched normal samples (referred as TN P), 2) all tumor vs normal samples (matched & unmatched, TN UP), and 3) pair-wise analyses for the individual tumor-normal pairs (T P). From the DESeq output, all genes with a differential expression significance (padj) threshold of 0.05 (95% significance) in any of the three interpretations are selected as the genes of interest.

Pooled miRNA expression analysis is carried out using DESeq2 R package (www.bioconductor.org) to detect upregulated and downregulated miRNAs (Table 3). miRNA functional analyses using individual cancer-related pathway information are analyzed using the DIANA mirPath web server that uses KEGG as the underlying database.

Table 3: Significantly up- and down-regulated miRNAs in laryngeal and/or

hypopharyngeal tumors (Pooled miRNA analysis)

Based on the above results of table 3, it is evident that has_mir_139 and has_mir_203 are down regulated whereas hsa_miR_424 and hsa miR 503 are up regulated in HNSCC, particularly in laryngeal and/or hypopharyngeal carcinomas. Pathway analysis of the miRNA target genes using mirPath is also performed. It is found that four different KEGG pathways, cell cycle, p53 signaling, viral carcinogenesis and pathways of cancer are significantly altered (with -value 10^"13, 10^"9, 10^"7 and 10^"6 respectively). The adjusted significance (P value) is obtained from the statistical test as described previously (Love, Huber and Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 2014, 15:550 doi:10.1186/sl3059-014-0550-8). Briefly, the significance is calculated by using a Wald test that uses the shrunken estimate of the logarithmic fold change of expression between tumor and matched normal dividing by its standard error, resulting in a z-statistic that is in turn compared to a standard normal distribution. The Wald test P values from the subset of the miRNA that pass a filtering step are adjusted for multiple testing using the Benjamini and Hochberg method (Benjamini and Hochberg, Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B Methodol 1995, 57:289-300). A list of all miRNA target genes, pathways, and gene annotations are provided in Table 4. It is further identified that some of the miRNAs identified in the present study are related to regulation of cancer stem cell-like cells related markers affecting invasion and metastasis.

RESULTS: It is found that different KEGG pathways are altered. Further, four different KEGG pathways- cell cycle, p53 signaling, viral carcinogenesis and pathways of cancer show significant alterations (with -value 10^"13, 10^"9, 10^"7 and 10^"6 respectively).

Claims

We Claim:

1. A method of detecting head and neck squamous cell carcinoma (HNSCC) in a sample having or suspected of having the HNSCC, said method comprising step of detecting aberration of miR-139, miR-203, miR424 and miR-503 in the sample to detect said HNSCC.

2. The method as claimed in claim 1, wherein detecting the aberration comprises:

isolating nucleic acid from the sample having or suspected of having the HNSCC, optionally along with isolating nucleic acid from a sample not having the HNSCC;

preparing small RNA library from the isolated nucleic acid by methods selected from a group comprising hybridization, ligation, reverse transcription, purification of cDNA, gel size selection and amplification of size selected cDNA, and purification;

quantifying the small RNA library by at least one of Qubit HS kit, nanodrop, Agilent Bioanalyzer, or any combination thereof; analyzing size distribution of the library by at least one of Agilent Bioanalyzer, nanodrop, or a combination thereof;

sequencing the small RNA library by at least one of SOLiD 5500x1 sequencing by ligation, single molecule sequencing and pyrosequencing; performing downstream analysis of the sequenced small RNA library by at least one of Lifescope, DeSeq2, or a combination thereof; and detecting the aberration of the miR-139, miR-203, miR-424 and miR-503 by at least one of DESeq2, Edgar, or a combination thereof.

3. Aberration of miR-139, miR-203, miR-424 and miR-503 for detecting HNSCC in a sample having or suspected of having the HNSCC.

4. Use of aberration of miR-139, miR-203, miR-424 and miR-503 for detecting HNSCC in a sample having or suspected of having the HNSCC.

5. A kit for detecting HNSCC in a sample having or suspected of having the HNSCC, said kit comprising agent for detecting aberration of miR-139, miR-203, miR-424 and miR-503, individually, wherein the agent is selected from a group comprising primer, probe, antibody and nanoparticle, or any combination thereof corresponding to miR-139, miR-203, miR-424 and miR-503.

6. Agent for use in detecting aberration of miR-139, miR-203, miR-424 and miR- 503 for detecting HNSCC in a sample having or suspected of having the HNSCC.

7. The agent as claimed in claim 6, wherein said agent is selected from a group comprising primer, probe, antibody and nanoparticle, or any combination thereof.

8. The method as claimed in claim 1, or the kit as claimed in claim 5, or the aberration as claimed in claim 3, or the use as claimed in 4, or the agent as claimed in claim 6, wherein the aberration is selected from a group comprising up regulation in expression, down regulation in expression, amplification, mutation, loss of heterozygosity, copy number variation, structural variation, gene fusion, allelic expression, DNA methylation, histone modification and gene silencing, or any combination thereof.

9. The method as claimed in claim 1, or the kit as claimed in claim 5, or the aberration as claimed in claim 3, or the use as claimed in 4, or the agent as claimed in claim 6, wherein the aberration is down regulation in expression of the miR-139, down regulation in expression of the miR-203, up regulation in expression of the miR424 and up regulation in expression of the miR-503.

10. The method or the kit or the aberration or the use or the agent as claimed in claim

9, wherein down regulation in expression of miR-139, down regulation in expression of miR-203, up regulation in expression of miR424 and up regulation in expression of miR-503 detects HNSCC in the sample having or suspected of having the HNSCC.

11. The method or the kit or the aberration or the use or the agent as claimed in claim

10, wherein the down regulation in expression of miR-139 ranges from about 2 fold to about 7 fold, the down regulation in expression of miR-203 ranges from about 2 fold to 7 fold, the up regulation in expression of miR-424 ranges from about 2 fold to 7 fold, and up regulation in expression of in miR-503 ranges from about 2 fold to 7 fold.

12. The method as claimed in claim 1, or the kit as claimed in claim 5, or the aberration as claimed in claim 3, or the use as claimed in 4, or the agent as claimed in claim 6, wherein the HNSCC is selected from a group comprising cancer of hypopharynx, laryngeal cancer, cancer of oral cavity, nasopharyngeal cancer, oropharyngeal squamous cell carcinomas and cancer of trachea.