WO2016199107A1

WO2016199107A1 - Gene aberration(s) in squamous cell carcinoma of head and neck (hnscc) and applications thereof

Info

Publication number: WO2016199107A1
Application number: PCT/IB2016/053473
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 aberration in gene(s) 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, kits for detection of HNSCC, use of gene aberration for detection of HNSCC. In particular, the present disclosure relates to analyzing gene expression changes and gene fusion events 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/applications.

Description

GENE ABERRATION(S) 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 aberrations in genes 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 and kits thereof. In particular, the present disclosure relates to analyzing gene expression changes and gene fusion events 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/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 they are focused on a handful of genes, not genome-wide. Further, the analysis in the prior art methods is not integrative with other alterations in the genome. Therefore, the prior art methods missed certain genes and their expression alterations which play 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 FINSCC. The present disclosure tries to address the above mentioned drawbacks of prior art.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to a method of detecting head and neck squamous cell carcinoma (FINSCC) in a sample having or suspected of having the FINSCC, said method comprising step of detecting aberration of:

MMP, ENDOU, CRNN, KRT4, KRT13, SPFNK5, TGM3, SCEL, ANXA9,

CAPN14 and SLURPl; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPFNK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, optionally along with detecting gene fusion or aberration of WIF1, or a combination thereof, wherein the gene fusion is selected from a group comprising CLTC-VMP1 and CTBS- GNG5, or a combination thereof, in the sample to detect said FINSCC.

The present disclosure further relates to aberration of:

MMP, ENDOU, CRNN, KRT4, KRT13, SPFNK5, TGM3, SCEL, ANXA9, CAPN14 andSLURPl; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPFNK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, optionally along with gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMPl and CTBS-GNG5, or a combination thereof, for detecting FINSCC in a sample having or suspected of having the FINSCC.

The present disclosure also relates to the use of aberration of MMP, ENDOU, CRNN, KRT4, KRT13, SPFNK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl; or one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPFNK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1, optionally along with gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMPl and CTBS-GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC.

The present disclosure also 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:

MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9,

CAPN14 and SLURP1; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN,

KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1, optionally along with detecting gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMPl and CTBS-GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC.

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

MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 andSLURPl; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN,

KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1. optionally along with detecting gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMPl and CTBS-GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC.

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 wherein: Figure 1 depicts 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 depicts quantitative PCR (qPCR) validation of gene expression changes. Red: down-regulation, Green: Up-regulation, shades of color is representative of the extent of expression changes in tumor samples compared to the matched normal tissues after normalization with internal beta-actin gene. Log fold change (Log₂FC) for the tumor samples (T) followed by validation from the larynx tumors from TCGA (TCGA L) are also given in the last two columns.

Figure 3 shows promoter methylation study using Q-MSP for the gene WIF1.

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 have been used to assess the accuracy of results from the real-time PCR experiments.

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 improved molecular biomarkers of head and neck squamous cell carcinoma (HNSCC), the present disclosure studies aberrations such as gene expression changes and gene fusions in HNSCC which serve as biomarkers for HNSCC. In particular, Matrix metalloproteinases (MMPs) (zinc-dependent endopeptidases) belonging to metzincin superfamily (proteases) play an important role in various physiological and/or pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis, and metastasis. Aberrations/altered expression of MMPs lead to imbalance in said physiological and/or pathological processes. Additionally, there are several protein-coding genes which are known to regulate cellular processes/mechanisms & metabolic pathways, wherein aberrations in them lead to imbalance in such processes. Accordingly, the present disclosure exploits said aspects and analyses the role of gene aberrations in head and neck squamous cell carcinoma (HNSCC). More particularly, the present disclosure analyses the gene expression changes of MMP coding genes and/or other protein-coding genes to provide biomarkers/indicators of HNSCC. Additionally, gene fusion events in HNSCC are also studied and accordingly, gene expression changes and gene fusion events individually or in combination are provided as markers of HNSCC.

Accordingly, the present disclosure provides a method of analysing gene aberrations 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 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 followed by validation to determine the gene aberration(s) in HNSCC.

In an embodiment of the present disclosure, analysing the gene aberrations in HNSCC involves both qualitative and quantitative analysis. In an embodiment of the present disclosure, gene aberrations are analysed in HNSCC wherein said gene aberrations act as indicators/biomarkers in HNSCC. In an exemplary embodiment of the present disclosure, the above method determines genes which are differentially expressed (gene expression changes) in HNSCC and accordingly provide indicators/biomarkers for HNSCC, more particularly, larynx and/or hypopharynx carcinoma. In a preferred embodiment, the above method analyses differentially expressed genes and thereby determines the role of aberrations/altered gene expression in HNSCC. In another exemplary embodiment, the above method in addition to determining gene expression changes, also determines gene fusions in HNSCC and accordingly provides indicators/biomarkers for HNSCC, more particularly, larynx and/or hypopharynx carcinoma.

In an embodiment of the method as disclosed above, the screening/examination of step (a) involves clinical/pathological screening or screening based on habits of the patient, or a combination thereof. In an 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 expression of genes of larynx and/or hypopharynx tumors is profiled using high-throughput sequencing experiments.

In an embodiment of the present method, 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. In another embodiment of the method described above, the gene aberrations identified are further validated. In an exemplary embodiment, differential gene expression results are validated using technique selected from a group comprising quantitative PCR (qPCR), Microarray, Northern Blot, Dot Blot, In-situ Hybridization and any other hybridization-based protocol, or any combination thereof. The present method identifies a list of about 296 genes as recited in Table 5 which have a role in HNSCC. In particular, one or more genes in said list of 296 genes have altered expression and serve as biomarkers in HNSCC, particularly larynx and/or hypopharynx carcinomas. The present method further analyses said 296 genes and shortlists a set of about 47 genes as recited in Table 4 which individually or in any combination have altered expression in HNSCC and serve as biomarkers of HNSCC, particularly larynx and/or hypopharynx carcinomas. This list of 47 differentially expressed genes satisfies the significance criteria (padj<0.05) and have biological importance. This set of about 47 genes is further studied to identify most significantly altered genes that individually or in any combination act as primary biomarkers of HNSCC, particularly larynx and/or hypopharynx carcinomas. Accordingly, this study identified 14 genes selected from a group comprising matrix metalloproteases (MMPs), CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination thereof having an important role in HNSCC.

Thus, the present method identifies genes selected from a group comprising matrix metalloproteases (MMPs), CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination thereof to have significantly altered expression in HNSCC, particularly larynx and/or hypopharynx carcinomas. In another embodiment, the matrix metalloproteases (MMPs) are selected from a group including but not limiting to MMP1, MMP10, MMP11 and MMP12, or any combination thereof.

In a specific embodiment of the present disclosure, when the expression of genes MMPl, MMP10, MMP11 and/or MMP12 are altered in HNSCC, particularly larynx and/or hypopharynx carcinomas, such alteration is over expression/up regulation of said genes.

In another specific embodiment of the present disclosure, when the expression of genes CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14, and/or SLURPl are altered in HNSCC, particularly larynx and/or hypopharynx carcinomas, such alteration is under expression/down regulation of said genes.

The aforementioned method further identifies gene fusion selected from a group comprising CLTC-VMP1, CTBS-GNG5, or a combination thereof in HNSCC, particularly in larynx and/or hypopharynx carcinomas. 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 aforementioned method of analysing gene aberrations 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 HNSCC 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, 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 sequencer to obtain sequenced data; and

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/differential gene expression/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.

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

The present method accordingly analyses 296 genes and shortlists a set of about 47 genes as recited in Table 4 which individually or in any combination have altered expression in HNSCC and serve as biomarkers of HNSCC, particularly larynx and/or hypopharynx carcinomas. This list of 47 differentially expressed genes satisfies the significance criteria (padj<0.05) and have biological importance. This set of about 47 genes is further studied to identify most significantly altered genes that individually or in any combination act as primary biomarkers of HNSCC, particularly larynx and/or hypopharynx carcinomas. Accordingly, this study identifies 14 genes selected from a group comprising matrix metalloproteases (MMPs), CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination thereof having an important role in HNSCC.

In another embodiment of the present disclosure, aberration in gene selected from a group comprising matrix metalloproteases (MMPs), CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination of said genes serves as biomarker of HNSCC, more particularly larynx and/or hypopharynx carcinomas. In a specific embodiment, the MMP is selected from a group including but not limiting to MMPl, MMP10, MMPl 1 and MMP 12, or any combination thereof. In an exemplary embodiment, when the genes MMPl, MMP 10, MMPl 1 and/or MMP 12 are aberrated in HNSCC, particularly larynx and/or hypopharynx carcinomas, such aberration is over expression/up regulation in the expression of said genes, which serves as biomarkers of HNSCC. In another exemplary embodiment, when the genes CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14, and/or SLURPl are aberrated in HNSCC, particularly larynx and/or hypopharynx carcinomas, such aberration is under expression/down regulation in the expression of said genes, which serves as biomarkers of HNSCC.

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

The present disclosure further relates to a method of detecting HNSCC in a sample having or suspected of having HNSCC, wherein said method comprises determining gene aberration. In an embodiment of the present disclosure, said determination of gene aberration includes analysing expression levels of one or more of 296 genes as recited in Table 5. In an exemplary embodiment, said determination of gene aberration includes analysing expression levels of one or more of 47 genes (a subset of 296 genes) as recited in Table 4. In a preferred embodiment, the aforesaid gene aberration employed for detecting HNSCC is up-regulation and/or down-regulation in expression of said genes. The present disclosure also relates to a method of detecting HNSCC in a sample having or suspected of having HNSCC, wherein said method comprises determining gene aberration. In an embodiment of the present disclosure, said determination of gene aberration includes analysing expression levels of gene selected from a group comprising MMP, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination of said genes. In an exemplary embodiment, up-regulation and/or down- regulation of gene selected from a group comprising MMPs, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination thereof is determined to detect the HNSCC in a sample having or suspected of having said HNSCC. In an embodiment, the MMP is selected from a group including but not limiting to MMP1, MMP 10, MMP11 and MMP 12, or any combination thereof.

In an embodiment, 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:

MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9,

CAPN14 and SLURP1; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1, optionally along with detecting gene fusion or aberration of WIF1, or a combination thereof, wherein the gene fusion is selected from a group comprising CLTC-VMP1 and CTBS- GNG5, or a combination thereof, in the sample to detect said HNSCC.

In an embodiment, the method of detecting aberration or gene fusion or combination thereof 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.

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 gene aberration wherein the gene is selected from a group comprising MMP, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination of said genes; and

(b) detecting the HNSCC based on step (a) wherein gene aberration correlates to the presence of HNSCC in said sample or vice-versa.

In an embodiment of the present disclosure, the fold-change (log2FC) values of up regulation in expression of the MMPs are MMPl l (2.38), MMP12 (3.61), MMP10 (4.7) and MMPl (5.42); and the fold-change (log2FC) values of down regulation in the expression are - ENDOU (-4.93), CRNN (-7.36), KRT4 (-6.31), KRT13 (-4.82), SPINK5 (-4.06), TGM3 (- 5.48), SCEL (-4.32), ANXA9 (-3.84), CAPN14 (-5.4), and SLURPl (-5.71). In another embodiment, the change in methylation (differential methylation) of the gene WIF1 ranges from about 3% to 60%.

In an embodiment of the method as described above, gene aberration correlated to the presence of HNSCC includes at least one of the following:

(i) up regulation in expression of MMPl, MMP10, MMPl l and MMP 12, or any combination thereof;

(ii) down regulation in expression of CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14, and SLURPl, or any combination thereof; and

(iii) gene fusion CLTC-VMP1, CTBS-GNG5, or a combination thereof.

In an exemplary embodiment of the method as described above, the gene aberration is determined for any combination of genes selected from a group comprising MMPs, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl. In specific embodiments, the gene aberrations are determined for gene combinations selected from a group comprising (i) MMPl, MMP10, MMPl l & MMP 12, (ii) MMPl, MMP10, MMPl l, MMP 12, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 & SLURPl, (iii) CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 & SLURPl, (iv) SCEL, ANXA9, CAPN14 & SLURPl, and (v) MMPl, MMP10, MMPl l, MMP 12, SCEL, ANXA9, CAPN14 & SLURPl. In some embodiments, aberration in any one or more of the aforementioned gene combinations 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 method as disclosed above, gene aberrations 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 a gene or corresponding gene product, or any combination of agent thereof based on solid support or solution based assays, in order to detect HNSCC. In a preferred embodiment, said agent is employed for determining aberrations in gene selected from a group comprising MMP, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP 1, or any combination of genes thereof. 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, gene aberration is determined 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 expression 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 still another embodiment, the solution-based assays to detect/measure gene expression 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 gene expression 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 method disclosed above, 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 differentially expressed gene(s) or gene fusion(s)], or a biological material/fluid/cell which do not possess tumor/cancer [or differential gene expression or gene fusion(s)]. Further, a sample may be derived from humans and/or mammals, or the sample may be any biological fluid/ biological material/cells prepared/obtained in a laboratory.

The present disclosure further relates to a method of detecting gene aberrations in a sample having or suspected of having such aberration, wherein said gene includes one or more genes from 296 genes as recited in Table 5. In an exemplary embodiment, said determination of gene aberration includes analysing gene aberration of one or more of 47 genes (a subset of 296 genes) as recited in Table 4. In a preferred embodiment, the aforesaid gene aberration is up-regulation and/or down-regulation in expression of said genes. The present disclosure also relates to a method of detecting gene aberrations in a sample having or suspected of having such aberration, wherein said gene is selected from a group comprising MMP, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14, SLURPl, CLTC, VMP1, CTBS and GNG5, or any combination of genes thereof. In a preferred embodiment, the gene aberration includes differential gene expression, gene fusion, or a combination thereof. In another embodiment, said method of detecting gene aberration comprises act of contacting the sample with an agent to determine aberration in genes selected from a group comprising MMP, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination thereof. In yet 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-VMPl, CTBS-GNG5, or a combination thereof. In still another embodiment, the method of detecting gene aberration comprises act of performing steps of a biomarker detection technique to determine the expression of gene selected from a group comprising MMP, CRNN, KRT4, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination thereof. 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 gene(s) as described above, the aberration includes at least one of the following:

(i) up regulation in expression of MMP1, MMP10, MMPl l and MMP 12, or any combination thereof;

(ii) down regulation in expression of CRNN, KRT4, SPINK5, TGM3, SCEL, ANXA9, CAPN14, and SLURPl, or any combination thereof; and

(iii) gene fusion CLTC-VMP1, CTBS-GNG5, or a combination thereof.

In an exemplary embodiment of the method as described above, the gene aberration constitutes altered expression of any combination of genes selected from a group comprising MMP, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl. In specific embodiments, the aberrations are determined for gene combinations selected from a group comprising (i) MMP1, MMP10, MMPl l & MMP 12, (ii) MMP1, MMP10, MMPl l, MMP 12, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 & SLURPl, (iii) CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 & SLURPl, (iv) SCEL, ANXA9, CAPN14 & SLURPl, and (v) MMP1, MMP10, MMPl l, MMP 12, SCEL, ANXA9, CAPN14 & SLURPl. In some embodiments, detection of aberration in any one or more of the aforementioned gene combinations are combined with gene fusion event CLTC-VMP1, CTBS-GNG5, or a combination thereof.

In an exemplary embodiment, the present disclosure relates to aberration of:

MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 andSLURPl; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, optionally along with gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMPl and CTBS-GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC. In an embodiment, the present disclosure further relates to an agent for use in detecting aberration of:

MMP, E DOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl. optionally along with detecting gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMP1 and CTBS-GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC.

In another embodiment of the method as described above, the agent employed to determine aberration in genes is selected from a group comprising primer, probe, antibody, nanoparticle and a suitable interacting protein/biological agent capable of interacting with a gene or corresponding gene product, or any combination of agents thereof based on solid support or solution based assays. In a preferred embodiment, said agent is employed for determining expression levels of genes selected from a group comprising MMP, CRNN, ENDOU, KRT4, SPINK5, KRT13, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination of genes thereof. In another preferred embodiment, said agent is employed to determine gene fusion selected from a group comprising CLTC-VMP1, CTBS-GNG5, or a combination thereof.

In another embodiment of the method as described above, gene aberration is determined 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, 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 still another embodiment, the solution-based assays to detect/measure gene expression 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 gene expression 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 gene aberration in a sample having or suspected of having aberration in one or more of 296 genes as recited in Table 5 or 47 genes (a subset of 296 genes) as recited in Table 4. In an embodiment, said kit comprises suitable agent(s) to detect expression in one or more of 296 genes as recited in Table 5 or 47 genes (a subset of 296 genes) as recited in Table 4; and an instruction manual thereof. In another embodiment, the agent is selected from a group comprising primer, probe, antibody and nanoparticle, or any combination thereof.

The present disclosure further relates to a kit for detecting gene aberration in a sample having or suspected of having aberration in gene(s), wherein said gene is selected from group comprising MMP, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14, SLURPl, CLTC, VMPl, CTBS and GNG5, or any combination thereof. In an embodiment, said kit comprises suitable agent(s) to detect expression in gene selected from a group comprising MMP, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, or any combination of said genes; and an instruction 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 and nanoparticle, or any combination thereof. The present disclosure further relates 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 one or more of 296 genes as recited in Table 5 or 47 genes (a subset of 296 genes) as recited in Table 4; and an instruction manual thereof which provides stepwise protocol of detecting HNSCC and correlates the aberration in gene with HNSCC detection. In another embodiment, the agent is selected from a group comprising primer, probe, antibody, nanoparticle, or any combination thereof.

The present disclosure also provides a kit for detecting HNSCC in a sample having or suspected of having the HNSCC, said kit comprising suitable agent(s) to determine aberration in gene selected from group comprising MMP, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14, SLURP1, CLTC, VMP1, CTBS and GNG5, or any combination thereof; and an instruction manual thereof which provides step-wise protocol of detecting HNSCC and correlates the aberration in gene with HNSCC detection. In another embodiment, the agent is selected from a group comprising primer, probe, antibody and nanoparticle, or any combination thereof.

Accordingly, the present disclosure also relates to the aforementioned agents such as primer, probe, antibody and nanoparticle, or any combination thereof for use in detecting aberrations in genes and/or correlation of such aberrated genes to detect HNSCC. In an exemplary embodiment, the gene aberration is altered/differential expression of gene(s), gene fusion variant(s), or a combination thereof.

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, aforesaid aberration in WIF1 expression along with aberration in one or more of 296 genes of Table 5, OR one or more of 47 genes of Table 4, OR one or more genes selected from group comprising MMP, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14, SLURP1, CLTC, VMP1, CTBS and GNG5, or any combination of genes thereof, OR gene fusion in CLTC-VMPl, CTBS-GNG5, or a combination thereof, serve as biomarker in detection of HNSCC, particularly larynx and/or hypopharynx carcinomas.

In other embodiments, any combination of aberration selected from:

(a) alteration in WIF1 expression which may be linked to WTFl promoter methylation; (b) gene expression changes in one or more of 296 genes of Table 5;

(c) gene expression changes in one or more of 47 genes of Table 4;

(d) gene expression changes in gene selected from group comprising MMP1, MMPIO, MMP12, MMPl l, CRNN, ENDOU, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1, or any combination thereof; and

(e) gene fusion in CLTC-VMPl, CTBS-GNG5, or a combination thereof,

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

In exemplary embodiments, any combination of aberration selected from:

(i) down-regulation in expression of SCEL, ANXA9, CAPN14, SLURP1, or any combination thereof,

(ii) down-regulation in expression of SCEL, ANXA9, CAPN14, SLURP1, MMP1, MMP10, MMP11, MMP12, or any combination thereof,

(iii) down-regulation in expression of KRT4, SPINK5, TGM3, CRNN, or any combination thereof,

(iv) down-regulation in expression of KRT13, SPINK5, TGM3, CRNN, or any combination thereof,

(v) down-regulation in expression of KRT3, KRT13, SPINK5, TGM3, CRNN, or any combination thereof,

(vi) up-regulation in expression of MMP1, MMP10, MMPl l, MMP12, or any combination thereof and down-regulation in expression of CRNN, KRT4, SPINK5, TGM3 or any combination thereof,

(vii) up-regulation in expression of MMPIO and down-regulation of CRNN, KRT4, SPINK5, TGM3, or any combination thereof,

(viii) up-regulation in expression of MMPl l and down-regulation in expression of CRNN, KRT4, SPINK5, TGM3, or any combination thereof, and

(ix) up-regulation in expression of MMP12 and down-regulation in expression of CRNN, KRT4, SPINK5, TGM3, or any combination thereof,

optionally along with gene fusion or aberration of WIF1 or a combination thereof, wherein the gene fusion is selected from a group comprising CLTC-VMPl and CTBS- GNG5, or a combination thereof, serve as biomarkers in detection of HNSCC, particularly larynx and/or hypopharynx carcinomas. In an embodiment, the present disclosure further relates to use of aberration of MMP, E DOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1; or one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1, optionally along with gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMPl and CTBS-GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC. Differentially expressed genes and experimental validation of genes in additional tumors

As described in methods disclosed above, sequencing data analysis involving significance testing of the tumor vs normal FC values to find differentially expressed genes in the larynx and hypopharynx samples is performed, selecting for those with a corrected p-va\ue (padj) of at most 0.05 in either pooled or paired interpretations. A complete list of 296 genes that are significantly and differentially expressed in tumors in either pooled or individual interpretations is provided in Table 5. A list of 47 differentially expressed genes satisfying the significance criteria (padj<0.05) and genes that are reported to play biological importance is given in Table 4. Most genes that meet the criteria are down-regulated in tumor. Based on the results, 14 biologically important genes (MMP1, MMP10, MMP 12, MMP11, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14, SLURP1) are selected for validation in additional tumors. For validation, qPCR primers (Table 6) are designed and the expression profiles of these 14 genes are measured in a validation set with 18 tumors (Figure 2). The overall changes in gene expression among these validation set samples correlate well with the sequencing data analysis results (Figure 2). Taken together, the present disclosure provides the most important and improved expression markers in larynx and hypopharynx cancers.

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-VMP1 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 UNSCC detection in isolation or along with differentially expressed gene(s) and/or alteration in WIFl expression with possible link to WIFl promoter methylation as described above.

Epigenetic silencing of WIFl

WIFl 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 WIFl and to understand whether such a mechanism is in operation in the UNSCC, particularly larynx and/or hypopharynx carcinoma, quantitative methylation- 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 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 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 two sets of patients, one where the significant alteration in gene expression is analysed [discovery/experimental set, N (tumor samples) =13] and the other where the results are validated [validation set, N (tumor samples) =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 thirteen samples used in the discovery set, nine are tumors of the larynx and four of the hypopharynx. In the validation set of 18 tumors that included the discovery set, twelve samples are from larynx; three are from hypopharynx; and three other pharyngeal sites. Clinical details along with the treatment details for all samples are provided in Table 1. All tissue samples are collected in RNAlater® solution (tissue storage reagent) at the time of resection and stored at - 800 °C until further processing.

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 was 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, 5 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, and read counts generation and gene-fusion detection

10 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. Additionally, putative gene-fusions in all

15 samples are detected using the 'splice finder' module in Lifescope. A junction confidence value (JCV) ranging from 1 to 100 is specified with each predicted fusion event. The closer the JCV value to 100, the likelier the fusion event is to be real. 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.

20

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 quality of 5 reads while 40-75% reads get mapped to the reference genome. The QC-filtered reads are used further to find gene fusion variants and analyze gene expression changes. The QC filtered reads are used to perform statistical analysis of differential gene expression and gene fusion finding in the larynx and hypopharynx tumors. All genes show differential expression with 95% confidence in all tumor samples. 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-VMP1 genes, supported by 16 reads perfectly mapping across the breakpoint (Figure 1A). The observed CLTC-VMP1 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-VMP1 fusion, an intra-chromosomal fusion event (JCV=68.52) 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, LP10 N, LP10 T, LP7 N LP7 T, LP6 N, LP6 T, LP3 T, LP2 T, LP1 T, LP4 T, LP12 T, LP5 T, LPl I T and LP13 T respectively.

INFERENCE: A fusion event involving the gene VMP1 is found. Specifically, CLTC-VMPl fusion is identified which serves as a marker for HNSCC. Further, there is a relationship between the CLTC-VMPl 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. A complete list of 296 genes that are significantly and differentially expressed in tumors in either pooled or individual interpretations is provided in Table 5. A list of 47 differentially expressed genes satisfying both the statistical significance criteria (padj<0.05) and genes that are reported to play biological importance is given in Table 4. Table 4: Top over- and under-expressed genes (47 genes) in laryx and/or hypopharynx

tumors.

Table 5: Significantly up- and down-regulated genes in laryngeal and/or hypopharyngeal

tumors

EXAMPLE 5

Validation of genes with significant expression change

Based on the results of Example 4, 14 biologically important genes (MMPl, MMP10, MMP12, MMPl l, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14, SLURPl) are selected for validation in additional tumors. For validation, qPCR primers (Table 6) are designed and the expression profiles of these 14 genes are measured in a validation set with 18 tumors (Figure 2). The overall changes in gene expression among these validation set samples correlate well with the sequencing data analysis results (Figure 2).

Table 6: Primer sequences used for validation experiment (qPCR)

RESULTS: Taken together, 14 genes viz. MMP1, MMP10, MMP12, MMP11, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl are validated to have significantly altered expression wherein ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl are significantly up regulated and MMP1, MMP10, MMP12 and MMP11 are significantly down regulated. Thus, these aberration/altered expression of these genes individually or in combinations serve as most important and improved expression markers in HNSCC, particularly larynx and/or hypophary nx cancers .

EXAMPLE 6

Ouanitative 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. Methylati on- 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 7. Table 7: 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 about 95°C for about 3 minutes and amplified for about 40 cycles at about 95 °C for about 3 seconds, annealing temperature depending on the primer for 30 seconds, followed by extension at about 72°C for about 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 WIFl promoter.

RESULTS: The results of this experiment is provided in Figure 3. 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 LP10, 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 3) 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.

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:

MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1, optionally along with detecting gene fusion or aberration of WIF1, or a combination thereof, wherein the gene fusion is selected from a group comprising CLTC-VMPl and 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 or gene fusion or a combination thereof, 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 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.

3. Aberration of:

MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 andSLURPl; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1, optionally along with gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMP1 and CTBS- GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC.

4. Use of aberration of MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl; or one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, optionally along with gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMP1 and 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:

MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURPl, optionally along with detecting gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMPl and CTBS-GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC.

6. Agent for use in detecting aberration of:

MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 andSLURPl; or

one or more genes selected from a group comprising MMP, ENDOU, CRNN, KRT4, KRT13, SPINK5, TGM3, SCEL, ANXA9, CAPN14 and SLURP1. optionally along with detecting gene fusion or aberration of WIF1, or a combination thereof, wherein gene fusion is selected from a group comprising CLTC-VMPl and CTBS-GNG5, or a combination thereof, for detecting HNSCC in a sample having or suspected of having the HNSCC.

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 MMP is selected from a group comprising MMPl, MMP10, MMP 12 and MMPl 1, or any combination 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 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.

9. The method or the aberration or the use or the kit, or the agent as claimed in claim 8, wherein the aberration is up regulation in expression of the MMP selected from a group comprising MMPl, MMP10, MMP12 and MMP11, or any combination thereof; down regulation in expression of the ENDOU, the CRNN, the KRT4, the KRT13, the SPINK5, the TGM3, the SCEL, the ANXA9, the CAPN14 and the SLURP 1; and differential methylation of the WIF1.

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

up regulation in expression of the MMP selected from a group comprising MMP1, MMP 10, MMP 12 and MMP11, or any combination thereof; down regulation in expression of the E DOU, the CRNN, the KRT4, the KRT13, the SPINK5, the TGM3, the SCEL, the ANXA9, the CAPN14 and the SLURP 1; differential methylation of the WIF1; the gene fusion selected from a group comprising CLTC-VMP1 and CTBS-GNG5, or combination thereof; or any combination thereof detects HNSCC in the sample having or suspected of having the HNSCC.

11. 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 aberration is selected from a group comprising:

(ii) down-regulation in expression of SCEL, ANXA9, CAPN14, SLURP1, MMP1, MMP 10, MMP11, MMP 12, or any combination thereof,

(vi) up-regulation in expression of MMP1, MMP10, MMP11, MMP 12, or any combination thereof and down-regulation in expression of CRNN, KRT4, SPINK5, TGM3 or any combination thereof,

(vii) up-regulation in expression of MMP 10 and down-regulation of CRNN, KRT4, SPINK5, TGM3, or any combination thereof, (viii) up-regulation in expression of MMP11 and down-regulation in expression of CRNN, KRT4, SPINK5, TGM3, or any combination thereof, and

optionally along with gene fusion or aberration of WIF1 or a combination thereof, wherein the gene fusion is selected from a group comprising CLTC-VMP1 and CTBS- GNG5, or a combination thereof.

12. 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.