WO2016199109A1

WO2016199109A1 - Gene signature in squamous cell carcinoma of head and neck (hnscc) and applications thereof

Info

Publication number: WO2016199109A1
Application number: PCT/IB2016/053479
Authority: WO
Inventors: Binay PANDA
Original assignee: Genomics Applications And Informatics Technology (Ganit) Labs
Priority date: 2015-06-12
Filing date: 2016-06-13
Publication date: 2016-12-15

Abstract

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 and method of detecting the indicators/biomarkers in a sample. In particular, the present disclosure relates to 8-gene signature in head and neck squamous cell carcinomas (HNSCC), specifically in squamous cell carcinoma of larynx and hypopharynx which serve as indicators/biomarkers for such carcinomas and associated methods.

Description

"GENE SIGNATURE IN SQUAMOUS CELL CARCINOMA OF HEAD AND NECK (HNSCC) AND APPLICATIONS THEREOF"

TECHNICAL FIELD

The present disclosure relates to the field of Oncology, Molecular Biology, Genomics and Bioinformatics. The present disclosure relates to gene signature as indicators/biomarkers of head and neck squamous cell carcinomas (HNSCC), method of analysing role of said indicators/biomarkers in HNSCC, corresponding methods of detection and kits thereof. In particular, the present disclosure relates to analyzing gene signature in head and neck squamous cell carcinomas (HNSCC), specifically in squamous cell carcinoma of larynx and hypopharynx which serve as indicators/biomarkers for such carcinomas and associated methods/applications.

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 a handful of 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 providing improved/reliable indicators/molecular biomarkers of squamous cell carcinomas of head and neck, in particular squamous cell carcinoma of larynx and hypopharynx, and employ such biomarkers for understanding and practical management of HNSCC. The present disclosure tries to address the above mentioned drawbacks of prior art.

STATEMENT OF THE DISCLOSURE

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:

BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIF1 genes, or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIF1, wherein BRDT and MAGEC2 are mandatory, optionally along with detecting gene fusion selected from CLTC-VMP1, CTBS-GNG5, or a combination thereof, in the sample to detect said HNSCC; aberration of: BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIF1 genes, or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIF1, wherein BRDT and MAGEC2 are mandatory,

optionally along with gene fusion selected from CLTC-VMPl, CTBS-GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC; use of aberration of BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIF1, or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIF1 wherein BRDT and MAGEC2 are mandatory, optionally along with gene fusion selected from CLTC-VMPl, CTBS-GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC; a kit for detecting HNSCC in a sample having or suspected of having the HNSCC, said kit comprising agent for detecting aberration of: BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1 genes, or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory, optionally along with gene fusion selected from CLTC-VMP1, CTBS-GNG5, or a combination thereof, individually, wherein the agent is selected from a group comprising primer, probe, antibody, nanoparticle and combinations thereof corresponding to said genes; and agent for use in detecting aberration of: BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1 genes, or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory, optionally along with gene fusion selected from CLTC-VMP1, CTBS-GNG5, or a combination thereof, for detecting FINSCC in a sample having or suspected of having the FINSCC, wherein the agent is selected from a group comprising primer, probe, antibody, nanoparticle and combinations thereof corresponding to said genes.

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: shows CLTC-CMP1 fusion gene in larynx and hypopharynx tumors.

A. Reads mapping the junction of CLTC (blue) and VMP1 genes (green).

B. Junction of CLTC and VMP1 gene.

C. Exon structures of CLTC and VMP1 genes.

D. Chromosome 17 map with the location of CLTC-VMP1 gene. Figure 2: shows promoter methylation study using Q-MSP for the gene WIF 1.

A. Products on an agarose gel after qMSP, SCCHP: squamous cell carcinoma of hypopharynx, SCCL: squamous cell carcinoma of larynx, OSCC: squamous cell carcinoma of oral cavity, N: matched normal, T: tumor, C M: control DNA used with methylated primer, C UM: control DNA used with unmethylated primer, arrow representing the amplified amplicon (210bp).

B. Grey color intensity represents the extent of methylation (positive control being the highest), green: hyper-methylation, red: hypo-methylation, numbers inside the boxes are the AAct values normalized against the internal beta-actin controls. The relative changes in gene expression and quantifying those changes using real-time PCR instruments requires certain assumptions. The AAct method is one of the methods used to calculate relative changes in gene expression determined from any real-time quantitative PCR experiments. Here, positive control and no-template controls are used to assess the accuracy of results from the real-time PCR experiments.

Figure 3: shows the efficiency of different gene combinations as markers of HNSCC.

DESCRIPTION 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:

BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1 genes; or

two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory, optionally along with detecting gene fusion selected from CLTC-VMP1, CTBS-GNG5, or a combination thereof, in the sample to detect said HNSCC. In an embodiment of the present disclosure, 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 HNSCC;

quantifying total RNA using kit selected from a group comprising Qubit RNA Assay Kit, Agilent Bioanalyzer, Nanodrop and any other kit for measurement of RNA;

subjecting the RNA to rRNA depletion using Ribominus Eukaryote Depletion system v2, or any other rRNA removal method, or a combination thereof;

quantifying the RNA using kit selected from a group comprising Qubit RNA Assay Kit, Agilent Bioanalyzer, Nanodrop and any other kit for measurement of RNA; preparing total RNA library using kit selected from SOLiD Total RNA seq kit or any other total RNA library preparation kit;

quantifying the RNA using kit selected from a group comprising Qubit HS Kit, Nanodrop, DNA 1000 Bioanalyzer and combinations thereof;

sequencing of RNA library using sequencer selected from SOLiD 5500x1 sequencer or any other RNA library sequencer to obtain sequenced data;

analysing the sequenced data to detect the aberration, wherein the analysis is selected from a group comprising read QC, alignment, read counts generation or any combination thereof.

The present disclosure further relates to aberration of:

BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1 genes; or

two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1,

IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory, optionally along with gene fusion selected from CLTC-VMP1, CTBS-GNG5, or a combination thereof, for detecting FINSCC in a sample having or suspected of having the HNSCC. The present disclosure further relates to use of aberration of BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WTFl; or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1 wherein BRDT and MAGEC2 are mandatory,

optionally along with gene fusion selected from CLTC-VMP1, CTBS-GNG5, or a combination thereof, for detecting FINSCC in a sample having or suspected of having the HNSCC.

The present disclosure also relates to a kit for detecting FINSCC in a sample having or suspected of having the FINSCC, said kit comprising agent for detecting aberration of: BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1 genes; or

two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory, optionally along with gene fusion selected from CLTC-VMP1, CTBS-GNG5, or a combination thereof, individually, wherein the agent is selected from a group comprising primer, probe, antibody, nanoparticle and combinations thereof corresponding to said genes.

The present disclosure also relates to an agent for use in detecting aberration of:

BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1 genes;

or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory, optionally along with gene fusion selected from CLTC-VMP1, CTBS-GNG5, or a combination thereof, for detecting FINSCC in a sample having or suspected of having the FINSCC, wherein the agent is selected from a group comprising primer, probe, antibody, nanoparticle and combinations thereof corresponding to said genes.

In an embodiment of the present disclosure the aberration is selected from a group comprising up regulation in expression, down regulation in expression, DNA methylation, histone modification, non-coding RNA (ncRNA)-associated gene silencing, chromosomal aberration, amplification, mutation, loss of heterozygosity, copy number variation, structural variation, allelic expression and combinations thereof.

In another embodiment of the present disclosure, the aberration is up regulation or down regulation in expression of the genes MAGEC2, BRDT and TFF1; down regulation in expression of the genes ACPP, DSC1 and WIF1; and up regulation in expression of the genes IFIT3 and MX 1.

In yet another embodiment of the present disclosure, the up regulation or down regulation in expression of the genes MAGEC2, BRDT and TFF1; down regulation in expression of the genes ACPP, DSC1 and WIF1; up regulation in expression of the genes IFIT3 and MX1; and differential methylation of the gene WTFl, detects UNSCC in the sample having or suspected of having the UNSCC. In still another embodiment of the present disclosure, the log2FC values of up regulation or down regulation in expression of the genes MAGEC2, BRDT and TFF1 ranges from about -7 to +7, the log2FC values of down regulation in expression of the genes ACPP, DSC1 and WIF1 ranges from about -1 to -5, the log2FC values of up regulation in expression of the genes IFIT3 and MX1 ranges from about +1 to +3. In another embodiment, the change in methylation (differential methylation) of the gene WIF1 ranges from about 3% to 60%.

In yet another embodiment of the present disclosure, 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.

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 'gene- signature' as biomarker of HNSCC. A gene signature is a group of genes in a cell whose combined expression pattern is uniquely characteristic of a biological phenotype or medical condition. A gene expression signature is defined as a combined gene expression/alteration/aberration with validated specificity in terms of diagnosis, prognosis or prediction of therapeutic response. Thus, the role of gene signatures in head and neck squamous cell carcinoma is analysed in the present disclosure. Additionally, gene fusion events in HNSCC are also studied and accordingly, gene signature and gene fusion events individually or in combination are provided as markers of HNSCC.

Accordingly, the present disclosure relates to a method of analysing the role of genes 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 analysis on reads to analyse the role of genes in HNSCC.

In an embodiment of the present disclosure, the analysing of the role of genes in HNSCC involves both qualitative and quantitative analysis. In another embodiment of the present disclosure, the above method determines gene signatures as indicators/biomarkers in HNSCC, more particularly, larynx and/or hypopharynx carcinoma. In a preferred embodiment, the above method analyses aberration/alteration in genes to determine their role in HNSCC.

In another exemplary embodiment, the above method in addition to determining gene signature, also determines gene fusions in HNSCC and accordingly provides indicators/biomarkers for HNSCC, more particularly, larynx and/or hypopharynx carcinoma.

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 step c) and step d) of the method as disclosed above, whole transcriptome analyses are performed using sequencing experiments to profile gene expression landscape in HNSCC, in particular, larynx and/or hypopharynx carcinoma samples.

In an embodiment, the gene signature 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 unique/differentially expressed genes 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 for the whole transcriptome study, between 5- 30% of 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 gene fusion events as well as gene expression changes. The present method identifies a list of 296 genes (as recited in Table 4a) which have a role in HNSCC. More particularly, the present disclosure identifies 8 genes in said list of 296 genes to be aberrated in FINSCC, particularly larynx and/or hypopharynx carcinomas which serve as biomarker/indicator in FINSCC. Additionally, 2 genes amongst the said 8 genes are found to be critical and significantly involved in FINSCC. Thus, said 2 genes (BRDT and MAGEC2) optionally along with one or more the remaining 6 genes (ACPP, DSCl, IFIT3, MXl, TFFl and WIFl) can act as biomarkers of FINSCC, particularly larynx and/or hypopharynx carcinomas.

Thus, in an exemplary embodiment, the above method identifies 8-gene signature, wherein the 8-gene signature consists of a combination of ACPP, BRDT, DSCl, IFIT3, MAGEC2, MXl, TFFl and WIFl.

In another exemplary embodiment, the above method identifies a 2 gene signature - BRDT and MAGEC2. In an embodiment, said 2 genes can be optionally employed with one or more of the remaining 6 genes of the 8-gene signature to serve as indicators/biomarkers of FINSCC.

The aforementioned method further identifies gene fusion selected from a group comprising CLTC-VMP1, CTBS-GNG5, or a combination thereof in FINSCC, particularly in larynx and/or hypopharynx carcinomas.

As used in the present disclosure, head and neck squamous cell carcinomas (FINSCC) 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 aforementioned method of analysing the role of gene signature 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 total RNA from the samples using RNA isolation kit selected from a group comprising PureLink RNA mini kit, Qiagen RNA isolation kit, or any other total RNA isolation kit;

c) quantifying the isolated RNA using RNA quantification kit selected from a group comprising Qubit RNA Assay Kit, Agilent Bioanalyzer and Nanodrop, or any other kit for measurement of RNA or spectrophotometric measurement of RNA; d) subjecting the isolated RNA to rRNA depletion using rRNA depletion system selected from a group comprising Ribominus Eukaryote Depletion system v2, or any other ribosomal RNA removal method, or any combination thereof;

e) quantifying the RNA post rRNA depletion using RNA quantification kit selected from a group comprising Qubit RNA kit, Agilent Bioanalyzer and Nanodrop, or any other kit for spectrophotometric measurement of RNA;

f) preparing total RNA library (whole transcriptome) using Library preparation kit such as SOLiD Total RNA seq kit;

g) quantifying the RNA post library preparation, using quantification kit selected from a group comprising Qubit HS kit, Nanodrop and DNA 1000 Bioanalyzer, or any combination thereof;

h) pooling and enrichment of libraries followed by sequencing using sequencer such as SOLiD 5500x1 sequencerto obtain sequenced data;

i) carrying out analysis of the sequenced data wherein the analysis is selected from a group comprising read QC, alignment, read counts generation or any combination thereof to determine aberration/ gene fusion; and wherein tools for carrying out said analysis is selected from a group comprising Lifescope (v2.5-r2.5.1) and DESeq R package (R-3.1.0, DESeq-1.16.0), or any statistical analysis methods, or any combination thereof; and

j) performing random forest analysis using data from TCGA portal and using tool such as DESeq and determining the role of gene signature in HNSCC.

Accordingly, the present disclosure specifically relates to 8-gene signature as indicators/ biomarkers of HNSCC. In an embodiment of the present disclosure, 8-gene signature serves as biomarkers/indicators of HNSCC. In an exemplary embodiment of the present disclosure, aberrations include differential expression of genes and/or gene fusions in HNSCC.

In another embodiment of the present disclosure, the 8-gene signature is a combination of ACPP, BRDT, DSCl, IFIT3, MAGEC2, MXl, TFFl and WIFl genes which are aberrant in HNSCC and serves as a biomarker of HNSCC. More particularly, in the pooled interactions the aberrations of said genes comprises of:

(i) down regulation in expression of ACPP, TFFl, DSC land WIFl; and

(ii) up regulation in expression of IFIT3, MXl, BRDT and MAGEC2 genes.

Further, the present disclosure provides a 2-gene signature - BRDT and MAGEC2 as indicators/ biomarkers of HNSCC. In an embodiment of the present disclosure, said 2- gene combination individually or along with one or more from the group of 6 genes - ACPP, DSCl, IFIT3, MXl, TFFl and WIFl act as biomarkers of HNSCC, particularly larynx and/or hypopharynx carcinomas. In an exemplary embodiment of the present disclosure, aberrations include differential expression of genes and/or gene fusions in HNSCC.

In another embodiment of the present disclosure, the pooled interactions of the aberrations of 2-gene signature optionally along with said 6 genes comprises of:

(i) up regulation or down regulation in expression of BRDT and MAGEC2;

(ii) down regulation in expression of ACPP, DSC land WIFl;

(iii) up regulation in expression of IFIT3 and MXl genes; and

(iv) up regulation or down regulation in expression of TFFl. 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 some embodiments 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/or down-regulation of the genes of the 8-gene signature, wherein the 8-gene signature consists of a combination of ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, TFF1 and WIF1; up-regulation and/or down-regulation of the genes from 2-gene signature BRDT and MAGEC2; and up-regulation and/or down-regulation of the genes in combinations comprising the 2-gene signature along with one or more genes selected from the 6 genes ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1.

The present disclosure relates to a method of detecting HNSCC in a sample having or suspected of having HNSCC, wherein said method comprises determining aberration(s) in 8 genes or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory, which are identified by the present disclosure to be critical in HNSCC. In an embodiment of the present disclosure, determination of aberration(s) in 8 genes, or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory, includes analysing expression levels of the genes that constitutes said signatures of the present disclosure. In a preferred embodiment, up-regulation and/or down-regulation of the genes of the 8-gene signature is determined to detect the HNSCC in a sample having or suspected of having HNSCC. In another embodiment, the 8-gene signature consists of a combination of genes- ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, WIF1 and TFF1. In another embodiment, up-regulation and/or down-regulation of two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory is determined to detect the HNSCC in a sample having or suspected of having HNSCC. In another embodiment, said two or more gene combination is selected from the 2-gene signature consisting of BRDT and MAGEC2, or signatures comprising said 2-gene signature along with one or more genes selected from ACPP, DSC1, IFIT3, MXl, WIFl and TFFl. In an 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 the 8-gene signature consisting of a combination of ACPP, BRDT, DSC1, IFIT3, MAGEC2, MXl, TFFl and WIFl genes, or, two or more genes selected from a group comprising BRDT, MAGEC2,

ACPP, DSC1, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory; and

(b) detecting the HNSCC based on step (a) wherein aberration(s) in the 8-gene signature, or, two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MXl, TFFl and WIFl, wherein BRDT and

MAGEC2 are mandatory correlates to the presence of HNSCC in said sample or vice-versa.

In an embodiment of the method as described above, the aberration(s) in the 8-gene signature relates to at least one of the following:

(i) down regulation in expression of ACPP, TFFl, DSC land WIFl genes; and

(ii) up regulation in expression of IFIT3, MXl, BRDT and MAGEC2 genes.

In another embodiment of the method as described above, the aberration in the 8-gene signature relates to down regulation in expression of ACP, MAGEC2, DSC1, BRDT and WIFl genes and up regulation in expression of IFIT3, MXl and TFFl genes.

In yet another embodiment of the method as described above, the aberration(s) in the two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory relates to at least one of the following:

(i) up regulation or down regulation in expression of BRDT and MAGEC2;

(ii) down regulation in expression of ACPP, DSC land WIFl; (iii) up regulation in expression of IFIT3 and MXl genes; and

(iv) up regulation or down regulation in expression of TFFl.

In an exemplary embodiment of the method as described above, the expression of BRDT and MAGEC2 genes is up regulated.

In some embodiments, aberration in the 8-gene signature, or, two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory are combined with gene fusion event CLTC-VMP1, CTBS-GNG5, or a combination thereof to serve as biomarker(s) for detection of HNSCC.

In another embodiment of the present disclosure, aberration in the 8-gene signature, or, two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory is determined with the help of an agent selected from a group comprising primer, probe, antibody, nanoparticles and a suitable interacting protein/biological agent capable of interacting with genes of the 8-gene signature, or any combination thereof, in order to detect presence or absence of aberrations. In a preferred embodiment, said agent is employed for determining aberration(s) in 8-gene signature, consisting of a combination of ACPP, BRDT, DSC1, IFIT3, MAGEC2, MXl, TFFl and WIFl genes. In another preferred embodiment, said agent is employed for determining aberration(s) in two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory. In another preferred embodiment, said agent is employed for determining gene fusion selected from a group comprising CLTC-VMP1, CTBS-GNG5, or a combination thereof.

In another embodiment of the present disclosure, aberration(s) in the 8-gene signature, or, two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory is identified by employing techniques selected from a group comprising but not limiting to solution- based assays and solid-support based assays, or a combination thereof. In yet another embodiment of the present disclosure, gene aberration is determined by employing techniques selected from a group comprising but not limiting to Sequencing, Reporter gene technique, PCR, Northern Blotting, Western blotting, ELISA, fluorescence- based assays, luminescence/chemiluminescence-based assays, in-situ hybridization, Serial analysis of gene expression (SAGE), microarrays, tiling array, RNA Sequencing/Whole Transcriptome Shotgun Sequencing (WTSS) and electrochemical assays, or any combination of techniques thereof.

In yet another embodiment, the solution-based assays to detect aberration(s) in the 8-gene signature, or, two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory 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 aberration(s) in the 8-gene signature, or, two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory 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, UNSCC 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.

In still another embodiment of the present disclosure, the expression of BRDT and MAGEC2 in the 2-gene signature or said genes BRDT and MAGEC2 of the 8-gene signature are altered in UNSCC selected from group comprising cancer of larynx, hypopharynx and oral cavity, or any combination thereof. As used herein, the term 'sample' refers to any biological material/fluid/cell having or suspected of having tumor/cancer (or 8-gene signature or gene fusion(s)), or a biological material/fluid/cell which is not affected with tumor/cancer. 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 aberration(s) in 8-gene signature or signature including two or more genes in a sample having or suspected of having aberration(s) in 8-gene signature, wherein the 8-gene signature consists of a combination of ACPP, BRDT, DSCl, IFIT3, MAGEC2, MXl, TFFl and WIFl genes, and the two or more genes are selected from a group comprising BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory. In an embodiment, said method of detecting aberration(s) in the 8-gene signature comprises act of contacting the sample with an agent to determine aberration in genes of the 8-gene signature, consisting of a combination of ACPP, BRDT, DSCl, IFIT3, MAGEC2, MXl, TFFl and WIFl genes. In yet an embodiment, said method of detecting aberration(s) in the signature comprising two or more genes comprises act of contacting the sample with an agent to determine aberration in two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory. In still another embodiment, said method of detecting gene aberration comprises act of contacting the sample with an agent to determine gene fusion selected from a group comprising CLTC-VMP1, CTBS-GNG5, or a combination thereof. In another embodiment, the method of detecting aberration in gene signatures comprises act of performing steps of a biomarker detection technique to determine aberration in the genes of the said signatures, wherein the 8-gene signature consists of a combination of ACPP, BRDT, DSCl, IFIT3, MAGEC2, MXl, TFFl and WIFl genes, and the two or more genes are selected from a group comprising BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory. In still another embodiment, the method of detecting gene aberration comprises act of performing steps of a biomarker detection technique to determine gene fusion selected from a group comprising CLTC-VMP1, CTBS-GNG5, or a combination thereof. In an exemplary embodiment of the method of detecting aberration in the 8-gene signature as described above, the aberration relates to at least one of the following:

(i) down regulation in expression of ACP, TFF1, DSC1 and WIF1; and

(ii) up regulation in expression of IFIT3, MX1, BRDT and MAGEC2.

In another exemplary embodiment of the method as described above, the aberration in the 8-gene signature constitutes down regulation in the expression of ACP, MAGEC2, DSC1, BRDT and WIF1 genes and up regulation in the expression of IFIT3, MX1 and TFF genes.

In yet another exemplary embodiment of the method of detecting aberration in the two or more genes as described above, the aberration relates to at least one of the following:

(i) up regulation or down regulation in expression of BRDT and MAGEC2;

(ii) down regulation in expression of ACPP, DSC land WIF1;

(iii) up regulation in expression of IFIT3 and MX1 genes; and

(iv) up regulation or down regulation in expression of TFF 1.

In some embodiments, detection of aberration in the aforementioned gene signatures is combined with gene fusion event CLTC-VMP1, CTBS-GNG5, or a combination thereof.

In another embodiment of the present disclosure, aberration in the 8-gene signature, or, the two or more genes as described above is determined with the help of an agent selected from a group comprising primer, probe, antibody, nanoparticles and a suitable interacting protein/biological agent capable of interacting with genes/detecting the genes, or any combination of agent thereof based on solid support or solution based assays. In a preferred embodiment, said agent is employed for determining aberration in genes of the 8-gene signature, wherein the 8-gene signature consists of a combination of ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, TFF1 and WIF1 genes. In another preferred embodiment, said agent is employed for determining aberration in the two or more genes, wherein the said genes are selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, and wherein BRDT and MAGEC2 are mandatory. In yet another preferred embodiment, said agent is employed for determining gene fusion selected from a group comprising CLTC-VMP1, CTBS-GNG5, or a combination thereof. In another embodiment of the present disclosure, the aberration in the 8-gene signature, or, the two or more genes is identified by employing techniques selected from a group comprising but not limiting to solution- based assays and solid support based assays, or a combination thereof. In yet another embodiment of the present disclosure, aberration in the 8-gene signature, or, the two or more genes is determined by employing techniques selected from a group comprising but not limiting to Sequencing, Reporter gene technique, PCR, Northern Blotting, Western blotting, ELISA, fluorescence- based assays, luminescence/chemiluminescence-based assays, in-situ hybridization, Serial analysis of gene expression (SAGE), microarrays, tiling array, RNA Sequencing/Whole Transcriptome Shotgun Sequencing (WTSS) and electrochemical assays, or any combination of techniques thereof.

In yet another embodiment, the solution-based assays to detect/measure aberration in the 8-gene signature, or, the two or more genes 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/measure aberration in the 8-gene signature, or, the two or more genes 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 aberration in 8-gene signature or, the two or more genes in a sample having or suspected of having said aberration, wherein the 8-gene signature consists of a combination of ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, TFF1 and WIF1 genes; and the two or more genes are selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory. In an embodiment, said kit comprises suitable agent(s) to detect aberration in 8-gene signature, or, the two or more genes as described above; and an instmction manual thereof. In another embodiment, said kit comprises suitable agent(s) to detect gene fusion selected from a group comprising CLTC-VMP1, CTBS-GNG5, or a combination thereof; and an instruction manual thereof. In yet another embodiment, the agent is selected from a group comprising primer, probe, antibody, nanoparticle, suitable interacting protein/biological agent capable of interacting with genes of the 8-gene signature, or, the two or more genes, or any combination thereof.

The present disclosure also provides a kit for detecting HNSCC in a sample having or suspected of having the HNSCC. In an embodiment, said kit comprises suitable agent(s) to determine aberration in 8-gene signature, or, the two or more genes, wherein the 8- gene signature consists of a combination of ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, TFF1 and WIF1 genes; and the two or more genes are selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory, and an instruction manual thereof which provides step-wise protocol of detecting HNSCC and correlates the aberration in the aforesaid gene signature with HNSCC detection. In another embodiment, the agent is selected from a group comprising primer, probe, antibody, nanoparticle, suitable interacting protein/biological agent capable of interacting with genes of the 8-gene signature/the two or more genes, or any combination thereof.

Accordingly, the present disclosure also relates to the aforementioned agents such as primer, probe, antibody and nanoparticles, suitable interacting protein/biological agent capable of interacting with genes of the 8-gene signature/the two or more genes or any combination thereof for use in detecting aberration in said gene signatures and/or correlation of such aberrations to detect HNSCC.

The present disclosure also provides aberration in WIF1 gene as a biomarker for detection of HNSCC, particularly larynx and/or hypopharynx carcinomas. In an embodiment, said aberration in WIF1 includes alteration in expression and/or methylation in WIF1. In an exemplary embodiment, the aberration includes alteration in WIF1 expression which may be linked to WIF1 promoter methylation. In some embodiments, the aforesaid aberration in WIFl expression which may be linked to WIFl promoter methylation along with aberration in 8-gene signature or, the signature comprising two or more genes, wherein the 8-gene signature consists of a combination of ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, TFF1 and WIFl genes, and the two or more genes are selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIFl, wherein BRDT and MAGEC2 are mandatory serve as biomarker in detection of UNSCC, particularly larynx and/or hypopharynx carcinomas.

In other embodiments, any combination of aberration selected from:

(a) alteration in WIFl expression, or WIFl expression which may be linked to WIFl promoter methylation;

(b) aberration in 8-gene signature, wherein the 8-gene signature consists of a combination of ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, TFF1 and WIFl genes;

(c) aberration in two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIFl, wherein BRDT and MAGEC2 are mandatory; and

(d) gene fusion in CLTC-VMP1, CTBS-GNG5, or a combination thereof,

serve as biomarkers in detection of UNSCC, particularly larynx and/or hypopharynx carcinomas.

Predicting larynx and pharynx carcinoma-specific minimal gene signature

In order to find a specific gene signature that distinguishes between the carcinoma of larynx and pharynx from those of the other subsites in the head and neck region, random forest (RF) analysis is performed using gene expression data from this study and that from The Cancer Genome Atlas (TCGA) dataset for validation. Random forest algorithm operates by constructing multiple decision trees based on a training set, and outputs the best prediction for the prediction set. Also, a method is devised to calculate the overall score, an indication of strength of the signature. The score is calculated based on multiple factors including sensitivity (max possible = 100), number of iterations (max possible = 500), and number of genes in a set (min possible = 2). Since UNSCC samples in the TCGA study belong to multiple different subsites of the head and neck region, the samples are classified into 2 types: those in larynx and/or hypopharynx sites (TCGA L) and those in the oral cavity (TCGA O). Each type is used independently with the samples in this study for training and predicting using the same matrix of samples and genes (Table 5). The best prediction set contains 8 genes, ACPP. BRDT. DSC1. IFIT3. MAGEC2. MX1. TFF1 and WIF1 with a score of nearly 500 when larynx and/or hypopharynx samples (including the larynx samples from TCGA) are used both as training and as prediction sets. When random forest analysis is performed using larynx, hypopharynx and oral cavity samples from TCGA as both training and prediction sets, a two gene signature is found, with genes BRDT and MAGEC2, come up with a score of 17200 indicating the possibility of specificity of these two genes in all three groups of cancer. Hence, apart from determining the 8-gene signature, the random forest analysis also shows that BRDT and MAGEC2 genes of the 8-gene signature are aberrated in cancers of larynx, hypopharynx and oral cavity.

Detecting Gene fusions

As disclosed in the above methods, splice-finder module in Lifescope is used to detect gene fusion variants with a Junction Confidence Value (JCV) that aids in the detection of false positives. A number of gene fusions detected involve small nuclear/RNA genes (Table 3). In one sample (a6f41n), fusion between CLTC-VMPl genes is detected, supported by 16 reads perfectly mapping across the breakpoint (Figure 1A). The observed CLTC-VMPl fusion transcript is a result of fusions between the first 15 exons of CLTC and the last 2 exons of VMP1 gene (Figure 1). In addition to CLTC-VMPl fusion, an intra-chromosomal fusion event (JCV=68.52) involving CTBS-GNG5 genes is also found in a tumor sample (Table 3). The genes involved in the fusion event are functionally important genes and thus the fusion events identified in tumors act as important marker for HNSCC detection in isolation or along with differentially expressed gene(s) and/or aberration in WIF1 expression which may be linked to WIF1 promoter methylation as described above.

Epigenetic silencing of WIF1

WIF1 is an extracellular wnt antagonist that functions as a tumor suppressor and is involved in regulation of cancer sternness and senescence. In order to understand the promoter hypermethylation of WIF1 and to understand whether such a mechanism is in operation in the HNSCC, particularly larynx and/or hypopharynx carcinoma, quantitative methylati on-specific PCR (Q-MSP) is performed for the WIFl and the extent of methylation in the samples is calculated. In the results of qMSP experiment, fifty percent of the hypopharynx samples are shown to have promoter hyper-methylation at WIFl promoter and the rest have hypo methylation at the same locus. In all samples, WIFl gene is altered or aberrated which may be linked to WIFl promoter methylation and the same can be employed as a biomarker in hypopharynx and/or larynx tumors. The WIFl gene is also a part of the minimal gene signature from the random forest analysis.

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.

In an embodiment of the present disclosure, the technology of the instant application is further elaborated with the help of following examples and figures. However, the examples should not be construed to limit the scope of the disclosure.

EXAMPLES

MATERIALS AND METHODS

Informed consent and ethics approval:

Informed consent is obtained voluntarily from each patient enrolled in the study. Ethics 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

The present study is conducted using a set of patients, wherein the significant alteration/aberration in gene expression is analysed [discovery/experimental set, N (tumor samples- 13 (larynx-9, hypopharynx-4), matched normal samples-5) =18]]. 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 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. In the discovery set, ten treatment-naive (primary) and three recurrent patient samples are used for sequencing study. Out of the eighteen samples used in the discovery set, thirteen are tumor samples and five are normal samples.. Clinical details along with the treatment details for all samples are provided in Table 1. All tissue samples are collected in RNAlater® solution at the time of resection and stored at - 800C until further processing.

Table 1: Clinical details of the tumors used for discovery and validation set

EXAMPLE 2

Library preparation and sequencing

Sequencing libraries are prepared for whole transcriptome.

Whole transcriptome: Total RNA is isolated using Purelink RNA mini kit (Ambion, Life technologies) following manufacturer's instructions. Isolated total RNA is quantified using Qubit RNA Assay Kit (Invitrogen) before being used in the sequencing library preparation. For SOLiD whole transcriptome library preparation, two micrograms of total RNA is subjected to rRNA depletion using Ribominus Eukaryote Depletion System v2 (Ambion, Life technologies). Post rRNA depletion, the samples are quantified by Qubit RNA kit again and used for library preparation using SOLiD Total RNA seq kit, following the manufacturer's instructions. In brief, rRNA-depleted RNA is fragmented, cleaned, hybridized to adaptors, reverse transcribed, purified, size selected by AMPure beads over two rounds (to remove any

fragments below 150bp), amplified, and finally, purified again. It is then quantified using Qubit HS kit (Invitrogen) and DNA 1000 Bioanalyzer (Agilent, 2100). Post library preparation, all the libraries are pooled in equimolar concentration and 0.6pM of the pooled library is used for the ePCR and enrichment step, as per the protocol. Following enrichment, the libraries are 3' modified, loaded onto 4 lanes of the SOLiD flow cell and sequenced on SOLiD 5500x1 sequencer. Data is obtained for 75x35 bp paired end reads (forward and reverse) in XSQ format.

Read QC, alignment, read counts generation and gene-fusion detection

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 (details of QC and mapping statistics)

RESULTS: Across all samples for the whole transcriptome study, between 5-30% of total 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 5 quality of reads while 40-75% reads get mapped to the reference genome. The QC- filtered reads are used further to find gene fusion variants, analyze expression changes, and perform further statistical analysis using random forest. All genes show differential expression/aberration with 95% confidence in all tumor samples.

10 EXAMPLE 3

Detection of Gene fusion variants

Splice-finder module in Lifescope is used to detect gene fusion variants with a Junction Confidence Value (JCV) that aids in the detection of false positives. A number of gene fusions detected involve small nuclear/RNA genes (Table 3). In one sample (a6f41n), fusion is detected between CLTC-VMPl genes, supported by 16 reads perfectly mapping across the breakpoint (Figure 1A). The observed CLTC-VMPl fusion transcript is a result of fusions between the first 15 exons of CLTC and the last 2 exons VMPl gene (Figure 1). In addition to CLTC-VMPl fusion, an intra-chromosomal fusion event (JCV=68.52)

5 is found involving CTBS-GNG5 genes in a tumor sample (Table 3).

Table 3 (a to c): Gene fusions found in larynx and hypopharynx samples.

Table 3 (a):

Table 3 (b):

Table 3 (c):

Note. Tables 3(a) to 3(c) are to be read in conjunction in specific sequence 3(a) followed by 3(b) and 3(c) respectively. In other words, columns Gl to El-end of Table 3(a) is followed by columns E2 to E2-RPKM of Table 3(b) followed by columns Exon-distance to E2-all-genes of Table 3(c) for sample names - LP8 N, LP8 T, LP9 N, LP9 T, LP 1 O N, LP 10_T, LP7 N

LP7 T, LP6 N, LP6 T, LP3 T, LP2 T, LP1 T, LP4 T, LP12 T, LP5 T, LP11 T and LP13 T respectively.

INFERENCE: A fusion event involving the gene VMP1 is found. Specifically, CLTC- VMP1 fusion is identified which serves as a marker for HNSCC. Further, there is a relationship between the CLTC-VMP1 fusion detected in the present example and tumor invasion and proliferation. Additionally, CTBS-GNG5 fusion is detected which also serve as marker for HNSCC. EXAMPLE 4

Differential gene expression 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.

The significantly upregulated and downregulated genes in laryngeal and/or hypopharyngeal tumors and genes of the 8-gene signature which are aberrated in FINSCC are provided in the below tables 4a and 4b respectively. Table 4a: Significantly up- and down-regulated genes in laryngeal and/or hypopharyngeal tumors

Table 4(b) : Upregulated and downregulated genes of the 8-gene signature

RESULTS: The results presented in the above tables indicate that 296 genes are differentially expressed/ aberrated in HNSCC. Particularly, within the 8-gene signature, the genes- ACP, TFF1, DSCl, and WIF1 are downregulated whereas, the genes- IFIT3, MX1, BRDT and MAGEC2are upregulated when pooled sample statistical analysis is performed.

EXAMPLE 5

Random Forest analysis

From the TCGA portal (https://tcga-data.nci.nih.gov/tcga/), raw RNA-seq read counts for matched tumor-normal TCGA FLNSCC samples are downloaded and normalized using DESeq. For each sample pair, gene-wise tumor vs normal ratios of normalized counts, called fold changes (FC), are calculated. Genes with fold change calculations of less than 1 (FC<1) are taken as down-regulation (D), genes with FC>1 are taken as up-regulation (U) and FC=1 meant no change (X) in the expression of the gene in tumor with respect to matched normal. Furthermore, based on the subsite, all the above TCGA samples are classified into 2 types - TCGA L (larynx subsite, N=l l pairs), TCGA O (all subsites in the oral cavity, N=29 pairs). Finally, for the set of significantly differentially expressed genes from SCCL and SCCFIP samples, FC values (in D, U or X notations) and TCGA L or TCGA O samples are used as input to the machine learning algorithm random forests. The computation is run with different seeds (1-500) that controlled for the randomness in the experiment and calculated a RF score (R) by using a formula:

where S is the sensitivity of detection, ni is the number of iterations in which a predictive set of genes is repeated in a set of 500 and ng is the number of genes in the predictive set. The highest score achievable using this method is 25000. The results of random forest analysis is provided in the below table 5.

Table 5: Results of random forest analysis

RESULT: The results presented in the above table indicates that the best prediction set contain 8 genes, ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, TFF1 and WIF1 with a score of nearly 500 when larynx and hypopharynx samples are used (including the larynx samples from TCGA) both as training and as prediction sets. When random forest analysis is performed using larynx, hypopharynx and oral cavity samples from TCGA as both training and prediction sets, a two gene signature, with genes BRDT and MAGEC2, comes up with a score of 17200 indicating the specificity of these two genes in all three groups of cancer. Hence, in addition to determining the 8-gene signature, the random forest analysis also shows that BRDT and MAGEC2 of the 8-gene signature are aberrated in cancers of larynx, hypopharynx and oral cavity. Accordingly, aberration in BRDT and MAGEC2 as a 2-gene signature or signature comprising said BRDT and MAGEC2 along with one or more genes from ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1 can be employed for detecting FINSCC.

EXAMPLE 6

Quantitative Methylation-Specific PCR (qMSP)

Genomic DNA is isolated from normal and tumor samples using DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer's instructions. Genomic DNA (500 ng) is bisulfite converted using EZ DNA Methylation Kit (Zymo Research), and amplified using two primer sets, one set specific for methylated and the other for non-methylated DNA. Methylation-specific primers are designed using MethPrimer tool (http://www.urogene.org/methprimer/). Sequences of the methylation-specific primers, annealing temperature and amplicon size is provided in Table 6. Table 6: Methylation- specific primer sequences (WIFl) used for validation experiment

Quantitative-MSP reactions are carried out using Kapa SYBR Fast Kits (Kapa Biosystems). The reaction mix is denatured at 95°C for 3 minutes and amplified for 40 cycles at 95 °C for 3 seconds, annealing temperature depending on the primer for 30 seconds, followed by extension at 72°C for 1 minute. The amplification is followed by dissociation curve analysis. Universal Methylated/Un-methylated Human DNA Standards (Zymo Research) are used as positive and negative assay controls for qMSP. Quantitative methylation in each sample is normalized using the methylated and un-methylated primers for beta-actin. Post-qMSP, the products are loaded on a gel for visualizing the methylation difference in WIF 1 promoter. RESULTS: The result of this experiment is provided in Figure 2. Based on the qMSP experiment, fifty percent of the hypopharynx samples have promoter hyper-methylation at WIFl promoter and the rest have hypo methylation at the same locus. In all samples, WIFl gene is altered or aberrated which may be linked to WIFl promoter methylation. For example, in the tumor sample LP6, the gene is down-regulated with log2FC of 5.3 but only 3% of the WIFl promoters are methylated. The extent of down-regulation of WIFl in the tumor LP7 is much smaller (Log2FC = 0.7) but WIFl promoters are methylated to a larger extent (10%) than the LP6 tumor. In another tumor sample LP 10, the log2FC for WIFl is 2.7 while 16% of the WIFl Promoters are hypo-methylated. As a control, a tumor from oral cavity (OT6) is used to study WIFl promoter methylation. Very strong WIFl hyper methylation (52% methylation, Figure 2) is found. The results showcase that the mechanism of WIFl silencing in hypopharynx and larynx tumors may be linked to its promoter methylation. Thus, alteration or aberration in WIFl expression may be linked to WIFl promoter methylation serve as a marker in HNSCC, especially in larynx and hypopharynx tumors. EXAMPLE 7

Efficiency of gene signature of the present invention

Genes are grouped together on X-axis for comparing different combination of genes and finding the efficient signatures amongst them. The 8-gene signature and the signatures comprising 2 or more genes of the present invention (with BRDT and MAGEC2 being mandatory) obtained high scores from the machine learning statistical approach. Further, the gene combination No. 3 (genes consisting of ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, TFF1 and WIF1) obtained the highest score.

1. ACPP BRDT DSC1 IFIT3 KRT15 MAGEA11 MAGEC2 MX1 TFF1 WIF1

2. ACPP BRDT DSC1 IFIT3 KRT15 MAGEC2 MX1 SAA1 TFF1 WIF1

3. ACPP BRDT DSC1 IFIT3 MAGEC2 MX1 TFF1 WIF1

4. BRDT DSC1 IFIT3 KRT15 MAGEC2 MX1 TFF1 WIF1

5. ACPP BRDT DSC 1 IFIT3 KRT 15 MAGEC2 MX1 WIF 1

6. BRDT DSC1 IFIT3 KRT 15 MAGEC2 MX1 SAA1 WIF1

7. ACPP BRDT DSC1 IFIT3 MAGEC2 MX1 SAA1 WIF1

8. BRDT DSC1 IFIT3 KRT 15 MAGEA11 MAGEC2 MX1 WIF1

9. BRDT DSC1 IFIT3 MAGEC2 MX1 SAA1 TFF1 WIF1

10. BRDT DSC1 IFIT3 MAGEC2 MX1

Statistical analysis and score calculation (Figure 3):

Data from gene expression analyses were used as input to the machine learning algorithm random forests. Computation was run with different seeds (1-500) that controlled for the randomness in the experiment and calculated a RF score or Score by using the following formula:

where S is the sensitivity of detection, ni is the number of iterations in which a predictive set of genes is repeated in a set of 500 and ng is the number of genes in the predictive set.

Out of the 8-gene set (Nos. 3-9), it is found that BRDT and MAGEC2 are the essential components and can be retained as a minimal number of genes, with a sensitivity 91.17 % (see Table 5) when tested with an independent dataset derived from The Cancer Genome Analysis using larynx tumors only (n = 11 pairs). In other words, BRDT and MAGEC2 needs to be mandatorily employed in the signatures of the present invention comprising 2 or more genes, wherein said BRDT and MAGEC2 can be combined with one or more genes selected from a group of ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1. Further, the 8-gene combination (No. 3) consisting of ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, TFF1 and WIF1 provides the best score amongst all including 5 gene combination and 10 gene combinations. Thus, the present example showcases the importance of 8 genes ACPP, BRDT, DSC1, IFIT3, MAGEC2, MX1, TFF1 and WIF1 wherein all 8 genes can be used as combination providing an 8-gene signature, or two or more genes from the said 8 genes can be employed as signature in which BRDT and MAGEC2 are mandatory.

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:

BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIFl genes; or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP,

DSCl, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory, optionally along with detecting gene fusion selected from CLTC-VMP1, CTBS- GNG5, or a combination thereof, 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 HNSCC; quantifying total RNA using kit selected from a group comprising Qubit RNA Assay Kit, Agilent Bioanalyzer, Nanodrop and any other kit for measurement of RNA;

quantifying the RNA using kit selected from a group comprising Qubit RNA Assay Kit, Agilent Bioanalyzer, Nanodrop and any other kit for measurement of RNA;

preparing total RNA library using kit selected from SOLiD Total RNA seq kit or any other total RNA library preparation kit;

quantifying the RNA using kit selected from a group comprising Qubit HS Kit, Nanodrop , DNA 1000 Bioanalyzer and combinations thereof;

3. Aberration of:

BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIFl genes; or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory, optionally along with gene fusion selected from CLTC-VMPl, CTBS-GNG5, or a combination thereof, for detecting UNSCC in a sample having or suspected of having the HNSCC.

4. Use of aberration of BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIFl; or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIFl wherein BRDT and MAGEC2 are mandatory,

optionally along with gene fusion selected from CLTC-VMPl, CTBS-GNG5, or a combination thereof, 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:

DSCl, IFIT3, MXl, TFFl and WIFl, wherein BRDT and MAGEC2 are mandatory, optionally along with gene fusion selected from CLTC-VMPl, CTBS-GNG5, or a combination thereof, individually, wherein the agent is selected from a group comprising primer, probe, antibody, nanoparticle and combinations thereof corresponding to said genes.

6. Agent for use in detecting aberration of:

BRDT, MAGEC2, ACPP, DSCl, IFIT3, MXl, TFFl and WIFl genes; or two or more genes selected from a group comprising BRDT, MAGEC2, ACPP, DSC1, IFIT3, MX1, TFF1 and WIF1, wherein BRDT and MAGEC2 are mandatory, optionally along with gene fusion selected from CLTC-VMP1, CTBS-GNG5, or a combination thereof, for detecting FINSCC in a sample having or suspected of having the FINSCC, wherein the agent is selected from a group comprising primer, probe, antibody, nanoparticle and combinations thereof corresponding to said genes.

7. The method as claimed in claim 1, or the aberration as claimed in claim 3, or the use as claimed in 4, or the kit as claimed in claim 5, 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, DNA methylation, histone modification, non-coding RNA (ncRNA)-associated gene silencing, chromosomal aberration, amplification, mutation, loss of heterozygosity, copy number variation, structural variation, allelic expression and combinations thereof.

8. The method as claimed in claim 1, or the aberration as claimed in claim 3, or the use as claimed in 4, or the kit as claimed in claim 5, or the agent as claimed in claim 6, wherein the aberration is up regulation or down regulation in expression of the genes MAGEC2, BRDT and TFF1; down regulation in expression of the genes ACPP, DSC1 and WIF1; and up regulation in expression of the genes IFIT3 and MX1.

9. The method as claimed in claim 1, or the aberration as claimed in claim 3, or the use as claimed in 4, or the kit as claimed in claim 5, or the agent as claimed in claim 6, wherein up regulation or down regulation in expression of the genes MAGEC2, BRDT and TFF1; down regulation in expression of the genes ACPP, DSC1 and WIF1; up regulation in expression of the genes IFIT3 and MX1; and differential methylation of the gene WIF1, detects FINSCC in the sample having or suspected of having the FINSCC.

10. The method as claimed in claim 1, or the aberration as claimed in claim 3, or the use as claimed in 4, or the kit as claimed in claim 5, 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.